Our major research interests are to elucidate molecular mechanisms of patho-genicity and species specificity of minus and single strand RNA viruses(Mononegavirales), and to control viral diseases. For these purposes, we arestudying virus replication and identifying viral and host factors important for the ex-pression of pathogenicity using a novel reverse genetics technique. We are alsodeveloping new virus vaccines and virus vectors by genetic engineering. In theanimal research center, more than 30,000 mice, mainly transgenic or knockout,are kept for research of IMSUT, and the technical staff support their breeding, fro-zen storage of eggs and microbiological cleaning.

The nonstructural proteins of Nipah virusplay a key role in pathogenicity in experimen-tally infected animals.

Misako Yoneda, Vanessa Guillaume1, HirokiSato, Kentaro Fujita, Marie-Claude Georges-Courbot2, Fusako Ikeda, Mio Omi, Yuri Muto-Terao, T. Fabian Wild1, Chieko Kai: 1InstitutNational de la Sante et de la Recherche médi-cale, U758, Lyon, France, 2Laboratory P4 IN-SERM Jean Mérieux, Lyon, France.

Nipah virus (NiV) P gene encodes phospho-protein (P) and three accessory proteins (V, Cand W). It has been reported that all four Pgene products have IFN antagonist activitywhen the proteins were transiently expressed.However, the role of those accessory proteins innatural infection with NiV remains unknown.We generated recombinant NiVs lacking V, C orW protein, rNiV(V-), rNiV(C-), and rNiV(W-),respectively, to analyze the functions of theseproteins in infected cells and the implications inin vivo pathogenicity. All the recombinants

grew well in cell culture, although the maxi-mum titers of rNiV(V-) and rNiV(C-) werelower than the other recombinants. The rNiV(V-), rNiV(C-) and rNiV(W-) suppressed the IFNresponse as well as the parental rNiV, therebyindicating that the lack of each accessory proteindoes not significantly affect the inhibition of IFNsignaling in infected cells. In experimentally in-fected golden hamsters, rNiV(V-) and rNiV(C-)but not the rNiV(W-) virus showed a significantreduction in virulence. These results suggestthat V and C proteins play key roles in NiVpathogenicity, and the roles are independent oftheir IFN-antagonist activity. This is the first re-port that identifies the molecular determinantsof NiV in pathogenicity in vivo.

Peroxiredoxin 1 is Required for the EfficientTranscription and Replication of Measles Vi-rus

Akira Watanabe, Misako Yoneda, FusakoIkeda, Hiroki Sato, and Chieko Kai

Affiliated Facilities

Laboratory Animal Research Center実験動物研究施設

Professor Chieko Kai, D.V.M., Ph.D.Associate Professor Misako Yoneda, D.V.M., Ph.D.Assistant Professor Hiroki Sato, Ph.D.Assistant Professor Tomoyuki Honda, M.D., Ph.D.

教 授 農学博士 甲 斐 知恵子准教授 農学博士 米 田 美佐子助 教 理学博士 佐 藤 宏 樹助 教 医学博士 本 田 知 之

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Measles is a highly contagious human diseasecaused by the measles virus (MeV). In thisstudy, by proteomic analysis, we identified per-oxiredoxin 1 (Prdx1) as a host factor that bindsto the C -terminal region of the nucleoprotein(NTAIL) of MeV. GST pull-down experimentsshowed that the Prdx1-binding site overlappedwith the MeV P protein-binding site on NTAIL,and that Prdx1 competed the binding to NTAIL

with the P protein, which is a component ofRNA-dependent RNA polymerase (RdRp). Fur-thermore, RNA interference for Prdx1 resultedin a significant reduction in MeV growth inHEK293-SLAM cells. Minigenome assay indi-cated that Prdx1 suppression affected the viralRNA transcription and/or replication step.Quantitative real-time (QRT) PCR analysisshowed that Prdx1 suppression not only re-duced viral RNA transcription and replicationbut also enhanced polar attenuation in viralmRNA transcription. Furthermore, surface plas-mon resonance analysis showed that the bindingaffinity of Prdx1 to MeV-N was 40-fold lowerthan that of MeV-P to MeV-N, which suggestedthat Prdx1 might be involved in the early stageof MeV infection, when the expression level ofPrdx1 was much higher than tht of MeV-P.Prdx1 was required for efficient viral RNA syn-thesis regardless of vaccine or wild-type strain.These results indicate that Prdx1 is the first ex-ample of an inherent host factor implicated inMeV RNA synthesis.

CD147/EMMPRIN acts as a functional entryreceptor for measles virus on epithelial cells.

Akira Watanabe, Misako Yoneda, FusakoIkeda, Yuri Terao-Muto, Hiroki Sato andChieko Kai

The wide tissue tropism of MeV suggests thatit involves ubiquitously expressed molecules be-sides signaling lymphocytic activation molecule(SLAM) known as a cellular receptor for morbil-liviruses, We identified cyclophilin B (CypB) ashost factors binding to MeV-N by proteomicanalysis. Western blot analysis of purified viral

particles showed that CypB was incorporated inviral virions through the binding to N protein.As CypB is known as a ligand for CD147/EMMPRIN (extracellular matrix metalloprotein-ase inducer), a cell surface glycoprotein, we ex-amined whether CD147 acts as a receptor forMeV. Anti-CD147 antibody or recombinantCypB inhibited MeV-infection on HEK293 cellswhich are SLAM-negative epithelial cells, whileoverexpression of human CD147 on CHO cellsenhanced MeV-infection. Furthermore, preven-tion of CypB-incorporation in virions signifi-cantly reduced their infectivity to SLAM-negative HEK293 cells, while it had no effect ontheir infectivity to SLAM-positive HEK293-SLAM cells. These results indicated that CD147is used as an entry receptor for MeV through in-corporated CypB in the virions on SLAM-negative cells.

Comparative and mutational analyses of pro-moter regions of rinderpest virus.

Chieko Imai, Kentaro Fujita, Fusako Shimizu,Akihiro Sugai, Misako Yoneda, Chieko Kai

Comparative and mutational analysis of pro-moter regions of rinderpest virus was con-ducted. Minigenomic RNAs harboring thegenomic and antigenomic promoter of the lap-inized virulent strain (Lv) or an attenuated vac-cine strain (RBOK) were constructed, and theexpression of the reporter gene was examined.The activities of the antigenomic promoters ofthese strains were similar, whereas the activityof the genomic promoter (GP) of the RBOKstrain was significantly higher than that of theLv strain, regardless of cell type and the sourceof the N, P and L proteins. Increased replication(and/or encapsidation) activities were observedin the minigenomes that contained RBOK GP.Mutational analysis revealed that the nucleo-tides specific to the RBOK strain are responsiblefor the strong GP activity of the strain. It wasalso demonstrated that other virulent strains ofRPV (Kabete O, Saudi/81 and Kuwait 82/1)have weaker GPs than that of the RBOK strain.

Publications

Imai, C., Fujita, K., Shimizu, F., Sugai, A.,Yoneda, M. and Kai, C. Comparative and mu-tational analyses of promoter regions ofrinderpest virus. Virology, 396: 169-177, 2010.

Watanabe, A., Yoneda, M., Ikeda, F., Terao-Muto, Y., Sato, H., Kai, C. CD147/EMMPRINacts as a functional entry receptor for measlesvirus on epithelial cells. J. Virol., 84(9), 4183-

4193, 2010.Yoneda, M., Guillaume, V., Sato, H., Fujita, K.,

Georges-Courbot, M-C., Ikeda, F., Omi, M.,Muto-Terao, Y., Wild, F. and Kai, C. The non-structural proteins of Nipah virus play a keyrole in pathogenicity in vivo. PLoS ONE, 5(9),e12709(1-8), 2010.

Omi-Furutani, M., Yoneda, M., Fujita, K., Ikeda,

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F. and Kai, C. Novel phosphoprotein-interacting region in Nipah virus nucleocapsidprotein and its involvement in viral replica-tion. J. Virol., 9793-9799.

Satoh, M., Saito, M., Tanaka, K., Iwanaga, S.,Sale -Ali, S.N.E., Seki, T., Okada, S., Kohara,M., Harada, S., Kai, C. and Tsukiyama-Kohara, K. Evaluation of a recombinant mea-sles virus expressing hepatitis C virus enve-lope proteins by infection of human PBL-NOD/Scid/Jak3null. Comp. Immunol. Microb.,2010, e80-e88.

Watanabe, A., Yoneda, M., Ikeda, F., Sugai, A.,Sato, H. and Kai, C. Peroxiredoxin 1 is Re-quired for the efficient transcription and repli-cation of measles virus. J. Virol ., in press.

Kodama, A., Yanai, T., Kubo, M., Habashi, N.,Kasem, S., Sakai, H., Masegi, T., Fukushi, H.,Kuraishi, T., Yoneda, M., Hattori, S. and Kai,C.: Cynomolgus monkey (Macaca fascicularis)may not infected with equine herpesvirus 9. J.Med. Primatol. In press, 2010.

Takano T., Kohara M, Kasama, Y., Nishimura,T., Saito, M., Kai, C. and Tsukiyama-Kohara,K. Translocase of outer mitochondrial mem-brane 70 expression in sinduced by hepatitisC virus and is related to the apoptotic re-sponse. J. Med. Virol . In press.米田美佐子，甲斐知恵子 ニパウイルス，ヘンドラウイルス（特集：種の壁を越える感染症-EpidemiologyとEpizootiology-)，臨床と微生物，近代出版 ３７�：１３３―１３８，２０１０．

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The Amami Laboratory of Injurious Animals was established in 1965 at Setouchi-cho in Amami-oshima Island in order to study on endemic diseases involvingparasite, arthropods, and venomous snakes in the tropics or subtropics.The Amami-oshima Island belongs to the Nansei (Southwest) Islands and thefauna is quite different from that in other islands of Japan. Since establishment ofthe laboratory, trials have been carried out to utilize small mammals found uniquein the Amami islands as experimental animals in addition to studies on preventionof Habu bites. As well known, successful eradication of filariasis from this island isone of the monumental works of the laboratory. Our present works are as follows:

1. Research on the Habu control

Shosaku Hattori, Takeshi Kuraishi, MotonoriOhno1, Naoko Oda-Ueda2, Takahito Chijiwa1,Aichi Yoshida3, Yoshihiro Hayashi4, MichihisaToriba5 and Tomohisa Ogawa6,: 1Departmentof Applied Life Science, Faculty of Bioscience,Sojo University, 2Department of Biochemistry,Faculty of Pharmaceutical Science, Sojo Uni-versity, 3School of Health Science, Faculty ofMedicine, Kagoshima University, 4Departmentof Veterinary Anatomy, Faculty of Agriculture,University of Tokyo, 5The Japan Snake Insti-tute, 6Faculty of Agriculture, Tohoku univer-sity.

Snake bites by the venomous snake Habu,Protobothrops flavoviridis , have been reported an-nually about 60 cases in the population of100,000 in the Amami Islands. Moreover, thereis no indication that the population of the Habuitself has decreased, despite a campaign for cap-ture of snakes by the Kagoshima PrefecturalGovernment. Rat-baited box traps have been in-troduced to catch the snakes and found to bequite effective. However, maintenance of live

rats requires man power and its cost is expen-sive. Therefore, our effort has been focused onthe development of attractant for Habu. The at-tractant extracted from rats seems ineffective ifcompared with use of live rats.

It was known that the Habu survived the in-jection of the Habu venom since early times, be-cause some proteins in the serum of the Habublood combine to the elements of the Habuvenom. The research of these binding proteinshas been initiated with an objective of clinicaltrials. Phospholipase A2 and its isozymes iso-lated from Habu venom have myonecrotic activ-ity and hemorrhagic activity, and metal proteasehas hemorahagic activity. The binding proteinsisolated from serum of Habu inhibit myone-crotic activity of phospholipase A2 and itsisozymes. We found that protein-HSF andpeptide-AHP isolated from the Habu serum ef-fectively control the hemorrhage caused byvenom of the Habu, Ovophis okinavensis ,Agkistrodon blomhoffi brevicaudus, Calloselasmarhodostoma, Bitis arietans , Bothrops asper, and,Trimeresurus stejnegeri .

Further, a statistics analysis and the simula-tion were done with the snakes captured by the

Affiliated Facilities

Amami Laboratory of Injurious Animals奄美病害動物研究施設

Professor Chieko Kai, D.V.M., Ph.D.Associate Professor Shosaku Hattori, D.V.M., Ph.D.

教 授 農学博士 甲 斐 知恵子准教授 農学博士 服 部 正 策

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Government, and the analysis of population dy-namics of Habu was attempted. As a result ofinvestigating the individual measurement dataof the captured Habu over 9 years, we wereable to obtain the generous age composition ofthe Habu. From analyzing of the age pyramid ofthe Habu and the result of questionnaire sur-veys for the inhabitant in the Amami-oshima Is-land, the total population of the Habu whichlives in this island was estimated at about80,000. By the analysis of the measured data oflast nine years, the snake sizes were miniatur-ized, and the population of young snakes de-creased. According to these investigations, thepopulation of the Habu is expected to decreasein the near future.

These studies are supported by grants fromthe Ministry of Land, Infrastructure and Trans-port and the Kagoshima Prefectural Govern-ment.

2. Unique structural characteristics and evo-lution of a cluster of venom phospholipaseA2 isozyme genes of Protobothrops flavo-viridis snake

Naoki Ikeda1, Takahito Chijiwa1, Kazumi Mat-subara7, Naoko Oda-Ueda1, Shosaku Hattori,Yoichi Matusda7, Motonori Ohno1: 7Division ofBiological Scinece, Graduate School of Science,Hokkaido University.

Protobothrops flavoviridis (Crotalinae) venomgland phospholipase A2 (PLA2) isozyme geneshave evolved in an accelerated manner to ac-quire diverse physiological activities in theirproducts. For elucidation of the multiplicationmechanism of PLA2 genes, a 25,026 bp genomesegment harboring five PLA2 isozyme genes wasobtained from Amami-Oshima P. flavoviridisliver and sequenced. The gene PfPLA2 encoded[Lys49]PLA2 called BPII, the gene PfPLA4 neuro-toxic [Asp49]PLA2 called PLA-N, the genePfPLA5 basic [Asp49]PLA2 called PLA-B, andPfPLA1(ψ ) and PfPLA3(ψ ) were the inactivatedgenes. The 5’ truncated reverse transcriptase(RT) elements, whose intact forms constitutelong interspersed nuclear elements (LINEs),were found in close proximity to the 3’ end ofPLA2 genes and named PLA2 gene-coupled RTfragments (PcRTFs). The facts that PcRTFs havethe stem-loop and repetitive sequence in the 3’untranslated region (UTR) which is characteris-tic of CR1 LINEs suggest that PcRTFs are the

debris of P. flavoviridis ancestral CR1 LINEs, de-noted as Pf CR1s. Since the associated pairs ofPLA2 genes and PcRTFs are arranged in tandemin the 25,026 bp segment, it is thought that anancestral PLA2 gene-Pf CR1 unit (PfPLA-Pf CR1)which was produced by retrotransposition ofPf CR1 by itself to the 3’ end of PLA2 gene du-plicated several times to form a multimer ofPfPLA-Pf CR1, a cluster of PLA2 genes, in the pe-riod after Crotalinae and Viperinae snakesbranched off. Recombinational hot spot of a 37bp segment, named Scomb, was found in the re-gion 548 bp upstream from the TATA box ofPLA2 genes. Thus, it could be assumed that mul-tiplication of PfPLA-Pf CR1 occurred by unequalcrossing over of the segment, -Scomb-PfPLA-Pf CR1-Scomb-. The Pf CR1 moieties were after-ward disrupted in the 5’ portion to PcRTFs. Thedetection of two types of PcRTFs different inlength which were produced by elimination oftwo definitive sequences in Pf CR1 moiety possi-bly by gene conversion clearly supports suchprocess but not multiplication of the PLA2 gene-PcRTF unit. (Abstract, Gene, 461: 15-25, 2010)

3. Reproduction of squirrel monkeys.

Shosaku Hattori, Takeshi Kuraishi, KumikoIkeda, Hazuki Yoshimura and Chieko Kai

The squirrel monkey, Saimiri sciurea, is widelydistributed in the tropical rainforest in Centraland South America between 10 degrees N and17 degrees S of latitudes. The advantage of us-ing this species for medical researches resides inits small size and gentle behavior. In this labora-tory, about 3～7 newborns are given annuallyby 24 adult females.

The aim is to optimize the use of the non-human primate model in future the AmamiLaboratory research activities. The laboratorynewly established experimental infection sys-tems which require or can be adapted to thesquirrel monkey model, particularly the study ofhuman falciparum malaria. Development ofparasites, immune response to malaria parasitesand pathological changes were investigated inin-vivo condition, further more, in vitro analysisof cell and molecular level was performed. It isalso investigating the mechanisms of infection inimmunology, vector development, a vaccineproduction program, and a clinical trials pro-gram.

Publications

Ikeda, N., Chijiwa, T., Matsubara, K., Oda-Ueda, N., Hattori, S., Matsuda, Y., and Ohno, M.

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Unique structural characteristics and evolu-tion of a cluster of venom phospholipase A2

isozyme genes of Protobothrops flavoviridissnake. Gene, 461: 15-25, 2010.服部正策，倉石 武．病理実験部会報告．平成２１年度奄美ハブ毒免疫機序研究報告書．（鹿児島県）．pp.４６―５９，２０１０．倉石 武．間接ELISA法を用いたハブ毒特異的

IgEの測定．平成２１年度奄美ハブ毒免疫機序研究報告書．（鹿児島県）．pp.７３―７８，２０１０．服部正策．生態査部会報告．平成２１年度ハブ動態制御研究報告書．（鹿児島県）．pp.９―４７，２０１０．服部正策，倉石 武．奄美大島の林道の夜間動物調査．平成２１年度ハブ動態制御研究報告書．（鹿児島県）．pp.５５―５８，２０１０．

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This laboratory has two main activities, development of efficient expression vectorsfor gene therapy, especially for anti-cancer, and supporting the researchers by ad-vising on recombinant DNA technology and on biohazards under the safety guide-lines.

The purposes of our laboratory are concernedabout not only research but also support for allresearchers in this institute. Our supporting ac-tivity is involved in advising service on gene-manipulation experiments and on biohazardsunder the safety guidelines and laws. For the re-search part, we intend to develop novel meth-ods or new experimental systems leading in thefield of gene expression and its regulation. Weare concentrating mainly on developing efficientadenovirus expression vectors aiming at genetherapy. We are maintaining more than 20 col-laborations within and outside of this institute.In these collaborations, we offer and supply ourefficient method to construct adenovirus vector(AdV) expressing various genes efficiently. Andrecently we developed the new cosmid cassettefor AdV construction using a full-length viralgenome with intact viral termini (Fukuda. et al .,Microbiol. Immunol. 50: 643-654, 2006). Thisnew cassette is available from Takara Bio andNippon Gene. We have also developed amethod for ON/OFF switching of gene expres-sion in mammalian cells using a combination ofadenovirus vector and Cre / loxP system(Kanegae et al ., Nucleic Acids Res. 23: 3816-3821, 1995; Kanegae et al ., Gene 181: 207-212,

1996) as well as FLP/frt system (Nakano et al .,Nucleic Acids Res. 29: e40, 2001; Kondo et al .,Nucleic Acids Res. 31: e76, 2003; Kondo et al .,Microbiol. Immunol., 50: 831-843, 2006; Kondoet al., J. Molec. Biol., 2009). These methods con-tinuously promote studies of various fields ofmolecular biology and medicine. We recentlyidentified adenovirus pIX gene as a main causeof inflammation observed in AdV infection(Nakai et al ., Hum. Gene Ther. 18: 925-936,2007). The research activities in 2010 wereshown below.

1. The positions and orientations of the ex-pression unit are important to obtain highexpression in adenovirus vector

Naoki Goda, Mariko Suzuki, Yumi Kanegaeand Izumu Saito

Adenovirus vectors (AdV) are valuable be-cause of high efficiency of transduction and ex-pression to broad range of cell types. In thefirst-generation AdV a foreign expression unit issubstituted for viral E1A and E1B genes (calledE1 substitution). The right orientation of the ex-pression unit is used worldwide simply because

Affiliated Facilities

Laboratory of Molecular Genetics遺伝子解析施設

Professor Izumu Saito, M.D., D.M.Sc.Assistant Professor Yumi Kanegae, D.M.Sc.Assistant Professor Saki Kondo, D.M.Sc.

教 授 医学博士 斎 藤 泉助 教 医学博士 鐘ヶ江 裕 美助 教 医学博士 近 藤 小 貴

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original E1 genes are located in this orientationin the adenovirus genome. However, we previ-ously showed that viral titers are sometimes lowwhen an expression unit containing a potentCAG promoter was located in this orientationand therefore we always recommend the leftorientation for E1 substitution (Miyake et al .,PNAS 93: 1320-1324, 1996). Besides E1 substitu-tion, E3 substitution and E4 insertion (Saito etal, J. Virol., 58: 544-560, 1986) have been used asthe position of the expression unit. Difference ofexpression level among the three substitution/insertion sites, if any, are very important if aweak, specific promoter is used. Such informa-tion is probably very important, for example, es-pecially for construction of a cancer-specificreplication-competent AdV.

To examine influences on position and orien-tation of transgene expression, we constructedsix different AdVs containing the “Switch unit”of Cre gene under the control of α-fetoprotein(AFP) promoter. A small amount of expressedCre turned on a potent EF1α promoter in the“Target unit” and a high-level of dsRed was ex-pressed (double infection method). The resultshowed that the expression level was quite dif-ferent depending on the site and the orientation.The order of expression level was: E1/left＝E1/right＞E4/left＝E3/left＞E4/right＞E3/right.Because the specificity of AFP promoter waslow when using at E1 site in the right orienta-tion, we concluded that E1/left is the best site/orientation for use of a specific promoter inAdV. The results possibly give valuable infor-mation for users of AdV bearing cancer-specificpromoter.

2. Cancer-targeting gene therapy using anovel adenovirus vector: a mouse modelof disseminated hepatocarcinoma

Yumi Kanegae, Naoki Goda, Miho Terashimaand Izumu Saito

Development of curing method against dis-seminated and recurrent microtumors is impor-tant for the next generation of anti-cancer treat-ment. Tissue/cancer specific promoter is re-garded as a variable tool. We have developedthe method of an “excisional expression” ade-novirus vector (AdV). This vector, which pos-sesses Cre driven by tissue/cancer specific pro-moter as “Switch unit” and the expression unitof a purpose gene by EF1α promoter under con-trol of Cre as “Excisional unit”, can express ahigh level of purpose-gene while maintainingvery strict specificity; using α-fetoprotein (AFP)promoter, it can express 40-90 fold more thepurpose-gene in hepatocellular-carcinoma cells

(HuH-7 and HepG2) than the AdV bearing theauthentic AFP promoter.

We improved the “excisional expression” vec-tor, because the AdV genome has position effectfor the purpose-gene expression level and theprevious insertion site of “Switch unit” wasworse than E1-substitution region. We newlyconstructed “E1L-type excisional expression”vector and were able to increase the expressionlevel of herpes thymidine-kinase (TK). Thesehigh-level expression and strict specificity of thisvector have been shown in cultured cells but ap-propriate animal models for disseminatedhepatocellular-carcinoma hardly reported.

As a breakthrough, we have established sucha mouse model in collaboration with CLEA Ja-pan Inc. We injected HuH-7 cells highly-expressing the luciferase from the portal veininto nude mice and succeeded in generating dis-seminated liver tumors in a high efficiency. Themice can be used for in vivo imaging and cansupply from CLEA Japan. The model will bevaluable for study of disseminated hepatocarci-noma and could also be useful for assay ofchemical anti-cancer substances.

3. Keratinocyte growth factor gene transduc-tion ameliorates pulmonary fibrosis in-duced by bleomycin in mice

Seiko Sakamoto1, Takuya Yazawa2, YasukoBaba1, Hanako Sato2, Yumi Kanegae, ToyohiroHirai3, Izumu Saito, Takahisa Goto1, KiyoyasuKurahashi1: 1Department of Anesthesiology andCritical Care Medicine, 2Department of Pathol-ogy, Yokohama City University GraduateSchool of Medicine, and 3Department of Respi-ratory Medicine, Graduate School of Medicine,Kyoto University.

Pulmonary fibrosis has high rates of mortalityand morbidity, but there is no established ther-apy at present. We investigated whetherbleomycin-induced pulmonary fibrosis in micewould be improved by intratracheal administra-tion of keratinocyte growth factor (KGF)-expressing adenovirus vector (Ad-KGF). Pro-gressive pulmonary fibrosis was created by con-tinuous administration of 120 mg/kg of bleomy-cin subcutaneously using an osmotic pumptwice from Day 1 to 7 and Day 29 to 35. Thosemice initially experienced subpleural fibrosisthen advanced fibrosis in the parenchyma of thelungs. These histopathological changes were ac-companied by reduced lung compliance (0.041±0.011 vs. 0.097±0.004; p＜0.001), reduced surfac-tant protein D messenger expression (0.409±0.119 vs. 1.006±0.081; p＜0.009), and reducedKGF messenger expression in the lungs at 4

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weeks compared with naïve group. Intratrachealinstillation of Ad-KGF at 1 week after the ad-ministration of bleomycin increased KGF mRNAexpression in the lungs compared with the fi-brosis induced mice that were instilled salinealone. This phenotype was associated with al-veolar epithelial cell proliferation, increased pul-

monary compliance (0.062±0.005 vs. 0.041±0.011; p＝0.023), and decreased mortality (sur-vival rate on Day 56: 68.8％ vs. 0％; p＝0.002),compared with those received only the salinevehicle. These observations suggest the thera-peutic utility of a KGF-expressing adenoviralvector for pulmonary fibrosis.

Publications

Kanegae Y, Terashima M, Kondo S, Fukuda H,Maekawa A, Pei Z, and Saito I. High-level ex-pression by tissue/cancer-specific promoterwith strict specificity using a single adenoviralvector. Nucleic Acids Res. 39: e7, 2010

Sakamoto S, Yazawa T, Baba Y, Sato H,

Kanegae Y, Hirai T, Saito I, Goto T, and Kura-hashi K. Keratinocyte growth factor genetransduction ameliorates pulmonary fibrosisinduced by bleomycin in mice. Am. J. Respir.Cell Molec. Biol. in press, 2010.

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The mission of our laboratory is to develop technologies for protein research thatenable us to analyze complex cellular systems leading to a variety of diseasessuch as cancer and infection. We mainly focus on the researches based on ad-vanced technologies regarding mass spectrometry and electron microscopy forprecise measurement of dynamic behaviors of functional protein networks.We are also engaged in collaborative researches regarding electron microscopy,mass spectrometry, peptide synthesis and purification of proteins and their func-tional analyses and have made a substantial contribution to many scientificachievements.

‹Group I›1. Biology of calcium-dependent proteases in

Caenorhabditis elegans

Caenorhabditis elegans, a free-living nematodein the soil, consists of no more than 1,000 cells,but retains various functions similar to those ofhigher-order animals. We have been interestedin biology of vertebrate calpain, an intracellularproteolytic enzyme activated with calcium ions.Genes related to mammalian calpain are alsopresent in C. elegans, and they are expected tobe translated into proteinous forms, but theirfunction have not been characterized biochemi-cally.

a. Identification of cysteine proteases inCaenorhabditis elegans

Yohei Kato, Nozomi Ichikawa and ShinobuImajoh-Ohmi

E64c, [L-3-trans-carbonyloxirane-2-carbonyl]-

L-leucine(3-methylbutyl)amide, is a synthetic in-hibitor for cysteine proteases such as cathepsinsB, H, L and calpain. To inhibit intracellular cys-teine proteases E64d, [L-3-trans-ethoxycarbony-loxirane-2-carbonyl]-L-leucine(3-methylbutyl)am-ide, a membrane-permeable derivative of E64c isused instead of E64c. E64d penetrates into thecell membranes where endogenous esterasesconvert it to enzyme-inhibitable E64c that cova-lently binds to the SH group of active center incysteine proteases. Thus, anti-E64c antibody is auseful probe for in vivo analysis of cysteine pro-teases.

We have succeeded in making an antibody toE64c. First, we tried to establish an antibodyagainst E64c-bound calpain. A peptide corre-sponding to the active center of calpain wassynthesized by means of the multiple-antigenpeptide system using Fmoc chemistry. E64c waschemically introduced into the SH group of ac-tive center cysteine under reducing conditions.Rabbits were immunized with the E64c-conjugated calpain-derived peptide without fur-

Affiliated Facilities

Medical Proteomics Laboratory疾患プロテオミクスラボラトリー

Professor Jun-ichiro Inoue, Ph.D.Professor Kouhei Tsumoto, Ph.D.Associate Professor Shinobu Imajoh-Ohmi, D. Sc.Associate Professor Masaaki Oyama, Ph.D.Assistant Professor Hiroshi Sagara, Ph.D.

教 授 薬学博士 井 上 純一郎教 授 工学博士 津 本 浩 平准教授 理学博士 大 海 忍准教授 医学博士 尾 山 大 明助 教 医学博士 相 良 洋

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ther conjugation with a carrier protein. Unex-pectedly, an antibody thus prepared reacted notonly with E64c-inactivated calpain but also withE64c-bound other cysteine proteases such as pa-pain and cathepsins. Low antigenicity of peptideregion in the immunogen may result in suchbroad specificity of the antibody. Our antibodyis expected to be used for identification of E64c-targeted novel proteases. When cells weretreated with E64d, cell growth was suppressedand several proteins were labeled by E64c thatis visualized with this antibody on immunoblot-ting. Structural analysis of these proteins maylead identification of novel cysteine proteases.

Homogenates of C. elegans were treated withE64c in the presence or absence of calcium ion,and subjected to electrophoresis/immunoblot-ting using an anti-E64c antibody. A 55-kDapolypeptide (p55) was labelled with E64c in acalcium ion-dependent manner. In C. elegansseveral calpain-related gene products were iden-tified at the mRNA level, but their physiologicalfunction remains to be elucidated.

2. Development of novel antibodies as toolsavailable for in situ analyses of post-translational modification of proteins

After biosynthesis proteins undergo variouspost-translational modifications, and their func-tions are modulated. In order to understandsuch biochemical reactions in a single cell, wehave been making modification-specific antibod-ies as probes for such in situ analyses; cleavage-site-directed antibodies for proteolysis, phos-phorylation-site-specfic antibodies, myristoilatedpeptide-specific antibodies , ubiquitination-specific antibodies, inhibitor-bound enzyme-specific antibodies etc. These antibodies shouldbe useful tools for research in cellular biochem-istry.

a. Evaluation of polyclonal cleavage-site di-rected antibodies and their fractionationinto more easy-to use probes

Tsuyoshi Katagiri, Chidzuko Takamura, No-zomi Ichikawa and Shinobu Imajoh-Ohmi

Cleavage-site directed antibodies are conven-ient tool for in situ analysis of proteolysis, sincethey do not bind unproteolyzed native proteinsthat retain the same sequence internally. To ob-tain such antibodies, peptides corresponding tothe terminal regions around the cleavage arechemically synthesized and used for haptens,where molecular design of the peptides is criti-cal for quality of the antibodies. Too short pep-tide results in generation of useless antibodies

recognizing the short peptide but not the termi-nus of cleaved proteins. On the other hand,when a longer sequence is selected for immuno-genic peptide, antibodies raised bind unprote-olyzed proteins as well as the cleaved ones.Thus, an evaluation system is necessary forcleavage-site directed antibodies. Phage displaylibraries were used for evaluation of antigenicspecificity of cleavage-site directed antibodies.Randomized sequences of synthetic oligonucleo-tide were introduced into phage DNA in orderthat a fusion protein with randomized se-quences of amino- or carboxyl-terminal region.A library was applied to immobilized antibod-ies, and phages bound were subjected to se-quence analysis for terminal regions. When anti-genic specificity of a cleavage-site directed anti-body was examined by this method, the anti-body was found to be a mixture of three ormore types of antibodies that bind to terminaland internal regions of the peptide used for im-munogen. Quality of the antibody was success-fully improved by affinity chromatography im-mobilized three peptides according to the evalu-ation method.

b. A novel method for hunting substrates ofcaspases in apoptotic cells

Maiko Okada, Chidzuko Takamura, HiroyukiFukuda, Masahiko Kato and Shinobu Imajoh-Ohmi

Caspases catalyze limited proteolysis of manyproteins in apoptotic cells. Hundreds of sub-strates have been identified as targets of cas-pases so far. Previously, nonmuscle myosinheavy chain-A and a component of DNA-dependent proein kinase, Ku80, are found to becleaved during apoptosis in human Jurkat Tcells. We used first a cleavage-site derected anti-body against the amino-terminal fragment ofcaspase 3/7-catalyzed calpastatin. Carboxyl-terminal region of caspase-proteolyzed frag-ments resemble each other, and such antibodiesare expected to misrecognize the target mole-cules. We further investigated the apoptotic Jur-kat cells for the anitobody-stained polypeptides.Cells were selectively extracted with salt- anddenaturant-containing buffers, and extracts weresubjected to two-dimensional gel electrophore-sis/immunoblotting. Candidate polypeptidesstained with antibodies were digested with tryp-sin and analyzed by mass spetrometer. Isoformsof ribonucleoprotein were thus identified.

3. Post-translational modification of proteinsduring apoptotic cell death

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Apoptotic cell death involves various bio-chemical reactions . Among them , post-translational modification of proteins is inten-sively investigated in this laboratory. First, intra-cellular proteolytic enzymes are activated priorto and during apoptosis. Caspases are now es-tablished as pivotal apoptosis-executing en-zymes that cleave various substrates. Endoge-nous or viral proteins and synthetic substancesinhibitory for caspases suppress the apoptoticcascade and rescue cells from cell death. On theother hand, proteasomes drive the cell cycle bydegrading cyclins etc., and also play importantparts in apoptosis, since proteasome inhibitorsinduce apoptotic cell death in growing cells butsuppress apoptosis of some cells that is in quies-cent state. Furthermore, in some specific cellssuch as polymorphonuclear leukocytes, otherproteases might be involved in cell death.

a. Limited proteolysis of actin in apoptoticneutrophils

Junko Ohmoto and Shinobu Imajoh-Ohmi

Neutrophil actin is proteolyzed to a 40-kDafragment during preparation/isolation from pe-ripheral blood. The truncated actin lacks amino-terminal region of native protein and presum-ably cannot copolymerize to F-actin. The 40-kDaactin-derived fragment is apparently related tospontaneous apoptosis of neutrophils. To inves-tigate the role of actin proteolysis, especiallycause-and-effect relationship to neutrophil apop-tosis, we have made a cleavage-site-directed an-tibody (#1090pAb) for the 40-kDa form of actinusing synthetic peptides as haptens. The anti-body reacted with the 40-kDa polypeptide butnot with unproteolyzed native actin which re-main abundant in the cell. Using this antibody,we have found that (1) the truncated actin isgenerated during isolation of neutrophils fromperipheral blood, (2) neutrophils without thetruncated actin can be prepared in the presenceof diisopropyl fluorophosphate, and (3) leuko-cyte elastase is possibly responsible for this lim-ited proteolysis.

Herein we analyzed cellular localization of thetruncated actin using #1090pAb. Confocal lasermicroscopic observation indicated that theplasma membrane of neutrophils were stronglystained with #1090pAb, but that intracellular re-gions near the membrane were sometimesstained weakly. We examined here whether ornot the amino-teriminal region of the 40-kDa ac-tin is on the cell surface of neutrophils using atthe same time established antibodies for compo-nents of superoxide-generating system com-posed of transmembranous cytochrome and cy-

tosolic activator proteins. Furthermore, flow-cytometric analysis revealed that #1090pAbstained the cell-surface antigen under the condi-tions that antibodies for cytosolic proteins didnot. Our findings suggest that the truncated ac-tin is, at least in its amino-terminal part, on thesurface of neutrophils. However, another anti-body against the amino-terminal region of na-tive actin did not stain neutrophils from outsidesuggesting that the cleavage site is inaccessibleto exogenous proteinases.

b. Fas, a death receptor, is polymerized tohigh-molecular weight forms during Fas-mediated apoptosis in Jurkat T cells

Hidehiko Kikuchi, Fotoshi Kuribayashi andShinobu Imajoh-Ohmi

An apoptotic receptor Fas mediates death sig-nal from Fas ligand. A cell death-inducing mon-oclonal antibody CH11 mimics Fas ligand andtriggers apoptotic signal mediated by Fas mole-cule. Plasma transglutaminases are found to in-volved in down-regulation of apoptosis inducedby a cytotoxic anti-Fas monoclonal antibody inJurkat cells. When cells were treated with theantibody in fetal calf serum-containing media,Fas was polymerized to higher-molecular-weight polypeptides as judged by immunoblot-ting. Under conditions where the transglu-taminase activity was eliminated or supprressed,the polymerization of Fas was not observed, andconcurrently cell death was hastened. Further-more, an antibody against blood coagulationfactor XIII strongly accelerated the Fas-mediatedapoptosis, indicating that plasma transglu-taminases catalyze polymerization of Fas anddown-regulate apoptotic cell death.

4. BRCA2 binds to motor domain of nonmus-cle myosin heavy chain IIC

Akira Nakanishi1,2, Yoshio Miki2,3, and ShinobuImajho-Ohmi1: 2Depertment of Molecular Ge-netics, Medical Research Institute, TokyoMedical and Dental University, 3Department ofGenetic Diagnosis, The Cancer Institute, Japa-nese Foundation for Cancer Research.

Cytokinesis is the final step of the M phase ofthe cell cycle and completed by scission of themidbody, a narrow intercellular bridge betweendaughter cells. We reported that human non-muscle myosin heavy chain (NMHC) IIC andphosphorylated BRCA2 by Plk1 was recruitedbetween both of the plus-ends of microtubulesof the midbody. Furthermore, when a cell lysatewas fractioned by a glycerol density gradient

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centrifugation, endogenous BRCA2 and NMHCIIC were observed to sediment at approximately700-800 kDa.

To examine the interactions BRCA2 of NMHCIIC, we performed coimmunoprecipitationanalysis and determined whether BRCA2-FLAGcan associate with HA-NMHC IIC in COS7 cells.Indeed, we found that the HA-NMHC IIC canbe coimmunoprecipitated with BRCA2-FLAG. Inreciprocal experiment, BRCA2-FLAG also copre-cipitates with HA-NMHC IIC. Next, we investi-gated which subunit of NMHC IIC associatedwith BRCA2, we divided the NMHC IIC intotwo fragments (IIC-N: motor domain (1-1000 a.a.) and IIC-C: coild-coil domain (887-2003 a.a.)).The lysates of COS-7 cells transfected with dele-tion mutants of NMHC IIC were incubated withBRCA2-FLAG or FLAG. As a result, BRCA2 in-teracted with IIC-N in the N-terminal region. Tofurther narrow down the binding region, we di-vided the IIC-N region into three fragments(IIC-N/1: 1-341 a.a., N/2: 331-670 a.a., N/3: 661-1000 a.a.). As a result, the interaction withBRCA2 was observed only in the IIC-N/1 re-gion near the N-terminal end of motor domain.These results indicate that BRCA2 associatedwith the N-termini of NMHC IIC.

Conversely, we tried to narrow down theNMHC IIC-binding region of BRCA2. A seriesof overlapping FLAG-fused deletion mutants ofBRCA2 (R1: 1-157 a.a., R2: 113-685 a.a., R3: 639-1508 a.a., R4: 1475-1620 a.a., R5: 1596-2280 a.a.,R6: 2241-2940 a.a., R7: 2611-3318 a.a., R8: 3119-3418 a.a.) were incubated with COS-7 celllysates transfected with HA-NMHC IIC. HA-NMHC IIC specifically associated with R3, R5, R6 and R7. We demonstrated that there wasstrong (R5 and R6), intermediate (R3) and weak(R7) binding to NMHC IIC. The results suggestthat the residues in the 1596-2940 a.a. regions ofBRCA2 appear to be required for its interactionwith NMHC IIC. EMSY is capable of silencingthe activation potential of BRCA2. The EMSY-binding resion (23-44 a.a.) of BRCA2 did notbind to NMHC IIC, suggesting that the NMHCIIC binding region did not overlap with theEMSY interaction surfaces.

‹Group II›1. Integrative analysis of phosphoproteome

and transcriptome dynamics defines drug-resistance properties of breast cancer

Masaaki Oyama, Takeshi Nagashima1, HirokoKozuka-Hata , Noriko Yumoto1 , YuichiShiraishi1, Kazuhiro Ikeda2, Yoko Kuroki1,Noriko Gotoh3, Satoshi Inoue2, Hiroaki Kitano4

and Mariko Okada-Hatakeyama1: 1RIKEN,2Research Center for Genomic Medicine, Sai-

tama Medical University, 3Division of SystemsBiomedical Technology, IMSUT, 4Sony Com-puter Science Laboratories, Inc.

Signal transduction system, in orchestrationwith subsequent transcriptional regulation,widely regulates complex biological events suchas cell proliferation and differentiation. There-fore, a comprehensive and fine description oftheir dynamic behavior provides a fundamentalplatform for systematically analyzing the regula-tory mechanisms that result in each biologicaleffect. Here we developed an integrated frame-work for time-resolved description of phospho-proteome and transcriptome dynamics based onthe SILAC-nanoLC-MS and GeneChip system.In this study, we analyzed cellular informationnetworks mediated by estrogen receptor/ErbB2pathways, which have long been implicated indrug response of breast cancer. Through shot-gun identification and quantification of phos-phorylated molecules in breast cancer MCF-7cells, we obtained a global view of the dynamicsregarding breast cancer-related signaling net-works upon estrogen (E2) or heregulin (HRG)stimulation. Comparative analysis of wild-typeand tamoxifen-resistant MCF-7 cells revealed al-tered behaviors of signaling hub dynamics, indi-cating distinct signaling network properties be-tween these two cell types. Pathway and motifactivity analyses using the transcriptome datasuggested that deregulated activation of GSK3βand MAPK1/3 signaling might be associatedwith altered activation of CREB and AP-1 tran-scription factors in tamoxifen-resistant MCF-7cells. Thus, our integrative analysis of phospho-proteome and transcriptome in human breastcancer cells revealed distinct signal-transcriptionprograms in tamoxifen-sensitive and insensitivetumor cells, which potentially defines drug-resistance properties against tamoxifen.

2. System-level analysis of CagA-dependentsignaling network dynamics by Helicobac-ter pylori infection

Hiroko Kozuka-Hata, Masato Suzuki5, KotaroKiga5, Shinya Tasaki, Jun-ichiro Inoue,Tadashi Yamamoto6, Chihiro Sasakawa5 andMasaaki Oyama: 5Division of Bacterial Infec-tion, Department of Microbiology and Immu-nology, IMSUT, 6Division of Oncology, Depart-ment of Cancer Biology, IMSUT.

The signal transduction system within a cellregulates complex biological events in responseto bacterial infection. The previous analyses ofcell signaling in Helicobacter pylori-infected gas-tric epithelial cells have revealed that CagA, a

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major virulence factor of Helicobacter pylori, isdelivered into cells via the type IV secretion sys-tem and perturbs signaling networks throughthe interaction with the key signaling moleculessuch as SHP-2, Grb2, Crk/Crk-L, Csk, Met, andZO-1. Although the biological activity oftyrosine-phosphorylated CagA has intensivelybeen studied, system-wide effects of the viru-lence factor on cellular signaling have yet to beanalyzed. Here we performed time-resolvedanalyses of phosphoproteome and CagA-interactome in human gastric AGS cells byCagA-positive/negative Helicobacter pylori in-fection. Our highly sensitive nanoLC-MS/MSanalyses in combination with the Stable IsotopeLabeling by Amino acids in Cell culture (SILAC)technology defined CagA-dependent perturba-tion of signaling dynamics along with a subsetof CagA-associated possible modulators on anetwork-wide scale. Our result indicated thatthe activation level of the phosphotyrosine-related signaling molecules in AGS cells wassuppressed overall in the presence of CagA dur-ing Helicobacter pylori infection. As Helicobac-ter pylori infection plays pivotal roles in theprogression of gastric diseases including car-cinogenesis, a comprehensive and fine descrip-tion of the signaling dynamics would serve as afundamental platform to theoretically explorefor the potential drug targets through analyzingthe regulatory mechanisms at the system-level.

3. Phosphoproteomics-based modeling de-fines the regulatory mechanism underlyingaberrant EGFR signaling

Shinya Tasaki, Masao Nagasaki7, HirokoKozuka-Hata, Kentaro Semba8, Noriko Gotoh3,Seisuke Hattori9, Jun-ichiro Inoue, TadashiYamamoto6, Satoru Miyano7, Sumio Sugano10

and Masaaki Oyama: 7Laboratory of DNA In-formation Analysis, Human Genome Center,IMSUT, 8Department of Life Science andMedical Bio-Science, Waseda University, 9De-partment of Biochemistry, School of Pharma-ceutical Sciences, Kitasato University, 10Labora-tory of Functional Genomics, Department ofMedical Genome Sciences, Graduate School ofFrontier Sciences, The University of Tokyo.

Mutation of the epidermal growth factor re-ceptor (EGFR) results in a discordant cell signal-ing, leading to the development of various dis-eases. However, the mechanism underlying thealteration of downstream signaling due to suchmutation has not yet been completely under-stood at the system level. Here, we report aphosphoproteomics-based methodology forcharacterizing the regulatory mechanism under-

lying aberrant EGFR signaling using computa-tional network modeling. Our phosphopro-teomic analysis of the mutation at tyrosine 992(Y992), one of the multifunctional docking sitesof EGFR, revealed network-wide effects of themutation on EGF signaling in a time-resolvedmanner. Computational modeling based on thetemporal activation profiles enabled us to notonly rediscover already-known protein interac-tions with Y992 and internalization property ofmutated EGFR but also further gain model-driven insights into the effect of cellular contentand the regulation of EGFR degradation. Our ki-netic model also suggested critical reactions fa-cilitating the reconstruction of the diverse effectsof the mutation on phosphoproteome dynamics.This is the first phosphoproteomics-drivenmathematical description of the regulatorymechanism of EGFR signaling networks, whichcould provide a systematic strategy toward con-trolling disease-related cell signaling.

4. Photo-cross-linking-based proteomics elu-cidates direct protein-protein interactionsinvolving a defined binding domain

Nobumasa Hino1, Masaaki Oyama, Aya Sato1,Takahito Mukai1 , Hiroko Kozuka-Hata ,Tadashi Yamamoto6, Kensaku Sakamoto1, andShigeyuki Yokoyama1

Signal transduction pathways are essentiallyorganized through the distribution of variousbinding domains in signaling proteins, witheach domain binding to its target molecules. Toidentify the targets of these domains, we devel-oped a novel proteomic approach, based onphoto-cross-linking and mass spectrometry.Through the use of an expanded genetic code, aphotoreactive amino acid, ptrifluoromethyl-diazirinyl-L-phenylalanine, was site-specificallyincorporated into the SH2 domain of the adap-tor protein GRB2 in human embryonic kidneycells. By exposing the cells to 365-nm light afteran EGF stimulus, the SH2 of GRB2 was cross-linked with the endogenous proteins directly in-teracting with it. These targets were identifiedby a comparative mass-spectrometric strategy.Thus, we discovered that GRB2-SH2 directlybinds to the GIT1 scaffold protein and the AF6protein, a putative effector of the RAS protein.Furthermore, heterogeneous nuclear ribonucleo-proteins F, H1, and H2 were found to be directtargets of GRB2-SH2.

5. Mass spectrometry-based annotation ofthe human short ORFeome

Masaaki Oyama, Hiroko Kozuka-Hata, Sumio

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Sugano10, Tadashi Yamamoto6 and Jun-ichiroInoue

In parallel with the human genome projects,human full-length cDNA data has also been in-tensively accumulated. Large-scale analysis oftheir 5’-UTRs revealed that about half of thesehad a short ORF upstream of the coding region.Experimental verification as to whether such up-stream ORFs are translated is essential to recon-sider the generality of the classical scanningmechanism for initiation of translation and de-fine the real outline of the human proteome.Our previous proteomics analysis of small pro-teins expressed in human K562 cells providedthe first direct evidence of translation of up-stream ORFs in human full-length cDNAs(Oyama et al., Genome Res, 14: 2048-2052, 2004).In order to grasp an expanded landscape of thehuman short ORFeome, we have performed anin-depth proteomics analysis of human K562and HEK293 cells using a two-dimensionalnanoLC-MS/MS system. The results led to theidentification of eight protein-coding regions be-sides 197 small proteins with a theoretical massless than 20 kDa that were already annotatedcoding sequences in the curated mRNA data-base. In addition to the upstream ORFs in thepresumed 5’-untranslated regions of mRNAs,bioinformatics analysis based on accumulated5’-end cDNA sequence data provided evidenceof novel short coding regions that were likely tobe translated from the upstream non-AUG startsite or from the new short transcript variantsgenerated by utilization of downstream alterna-tive promoters. Protein expression analysis ofthe GRINL1A gene revealed that translationfrom the most upstream start site occurred onthe minor alternative splicing transcript ,whereas this initiation site was not utilized onthe major mRNA, resulting in translation of thedownstream ORF from the second initiation co-don. These findings reveal a novel post-transcriptional system that can augment the hu-man proteome via the alternative use of diversetranslation start sites coupled with transcrip-tional regulation through alternative promotersor splicing, leading to increased complexity ofshort protein-coding regions defined by the hu-man transcriptome (Oyama et al., Mol Cell Pro-teomics, 6: 1000-1006, 2007).

‹Group III›1. Analysis of neural circuits in the retina us-

ing electron microscopic tomography andx-ray microscopy

Hiroshi Sagara and Haruo Mizutani1: 1SienceIntegration Program, Organization for Inter-

disciplinary Research Projects, University ofTokyo.

Neural circuits in the central nervous systemare the basis of various high-order brain func-tions. It is also true in case of retin. In the retina,six main classes of cells connect each other sys-tematically to make up complex neural circuits.Characteristics of the retinal functions have beenexamined precisely by the electrophysiologicalmethods and models of cell connectivity havebeen proposed. But morphological studies of theactual neural connection, which constitute thephysiological properties of higher order neu-rons, still not enough. In this study, we are try-ing to reveal the actual neural circuit morpho-logically by using electron microscopic com-puted tomography (CT) and X-ray microscopy.This year, we improved the specimen prepara-tion procedure of the conventional electron mi-croscopy to gain higher membrane contrast andexamined by electron microscopic tomographyand x-ray microscopy. Either of the techniquesrevealed nerve fibers and organelles includingmitochondria and synapses in the 200nm thicksections of neural tissue. In the future, we willutilize that information to begin deciphering thewiring diagram of the retina.

2. Collaborative and supportive researchesas electron microscope core-laboratory

This group is also engaged in collaborative re-searches using electron microscope. We offersupports for the research projects those needelectron microscopic analysis. The services avail-able in this group are the conventional thin sec-tion transmission electron microscopy, immuno-electron microscopy, negative staining tech-niques and scanning electron microscopy. By us-ing these individual technique or combination ofsome of these, we can offer direct visual evi-dence that cannot be acquired by other meth-ods. This year, 19 projects in 13 laboratorieswere performed as core-laboratory works.

a. Thin section electron microscopy

Thin section electron microscopy is the mostwidely used technique to observe the innerstructure of cells and tissues. In this method,samples are fixed and embedded in epoxy resin,thin sections with about 70nm thickness are cutand observed in the electron microscope. In caseof immuno-electron microscopy, thin sectionsare obtained by similar procedure, and the anti-gen epitopes exposed on the surface of the sec-tions are marked by sequentially reacted withappropriate primary antibodies and colloidal

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gold labeled secondary antibodies. This year,thin section electron microscopy combined withimmuno-electron microscopy were used inmany collaborative works.

a-1. Ultrastructural analysis of entry and as-sembly of Herpes Simplex Virus

We have been performing several studies withresearch groups in Dr. Kawaguchi2’s laboratory:2Department of Infectious Disease Control, Inter-national Research Center for Infectious Diseases,regarding the infection/replication processes ofherpes simplex virus (HSV). This year, serialthin section electron microscopy was applied toa single target cell cultured on the surface of acover slip to figure out the virus forming com-partment observed by confocal lazer fluorescentmicroscope. In early stages of infection, the com-partments were revealed to be the intracellularstructure. But in later stages, the compartmentsobserved by confocal laser microscpe were re-vealed to be the accumulation of viruses in theextracellular space. This indicates that the vi-ruses are first formed in the intracellular spacesand then exocytosed to the extracellular space.

a-2. Morphological and immuno-electron mi-croscopic analysis of the mucosal M-cells

We have been performing several studies alsowith research groups in Dr. Kiyono3’s labora-tory: 3Division of Mucosal Immunology, Depart-ment of Microbiology and Immunology. In thesestudies, we analyzed the ultrastructure of theM-cells in the intestinal epithelium by thin sec-tion transmission electron microscopy and scan-ning electron microscopy. Molecular characteris-tics of the M-cells were analyzed usingImmuno-electron microscopy. In another study,several species of proteins were induced to ex-press in rice and examined the localizationwithin the cell with immuno-electron micros-copy. We revealed that the expressed proteins

were accumulated in the different compartmentsdepending on the kind of the expressed protein.

Some other collaborative research works usingthin section electron microscopy and / orimmuno-electron microscopy were performedwith Dr. Noda4 et al in 4Division of Virology,Department of Microbiology, regarding thestructure of the influenza viruses and ebola vi-rus, Dr. Masaike5 et al , in 5Integrated ResearchInstitute, Tokyo Institute of Technology (ref.Masaike et al ), Dr. Hoshina6 in 6Division of On-cology, regarding the structure of the synapses,Dr. Ma7 in 7Department of Pediatric Hematol-ogy/Oncology, regarding the morphology of theeosinophilic leucocytes induced from ES/iPScells and so on.

b. Negative staining techniques

Negative staining techniques are simple andquick method to observe the morphology of themacro molecules. This year, the negative stain-ing techniques were used in collaborative workwith Dr. Noda4 et al . In this study, negativestaining techniques were used to analyze themorphology of the ebola virus nucleoprotein-RNA complex (ref. Noda4 et al ).

c. Scanning electron microscopy

Scanning electron microscopy is a techniqueused to examine the surface structure of thecells, tissues or other non-biological materials.The collaborative works using scanning electronmicroscopy were done with Dr. Nishizumi-Tokai6 et al , regarding the surface structure ofthe egg cells. Other works are in progress withDr. Sanada8 et al , 8Department of GerontologicalNursing, Division of Health Science and Nurs-ing, Graduate School of Medicine, to analyze theeffects of diabetes or bacterial infection duringwound repair. Scanning electron microscopywas also used to analyze the non-biological ma-terials as a collaborative work with Dr. Cheng9

in 9Olympus Co.

Publications

‹Group II›Tsujita K, Itoh T, Kondo A, Oyama M, Kozuka-

Hata H, Irino Y, Hasegawa J, and TakenawaT. Proteome of acidic phospholipid-bindingproteins: Spatial and temporal regulation ofcoronin 1A by phosphoinositides. J BiolChem, 285: 6781-6789, 2010.

Fujita T, Kozuka-Hata H, Uno Y, Nishikori K,Morioka M, Oyama M, and Kubo T. Func-tional analysis of the honeybee (Apis mellifera

L.) salivary system using proteomics. BiochemBiophys Res Commun, 397: 740-744, 2010.

Matsumura T, Oyama M, Kozuka-Hata H,Ishikawa K, Inoue T, Muta T, Semba K, andInoue J. Identification of BCAP-(L) as a nega-tive regulator of the TLR signaling-inducedproduction of IL-6 and IL-10 in macrophagesby tyrosine phosphoproteomics. Biochem Bio-phys Res Commun, 400: 265-270, 2010.

Arii J, Goto H, Suenaga T, Oyama M, Kozuka-

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Hata H, Imai T, Minowa A, Akashi H, AraseH, Kawaoka Y, and Kawaguchi Y. Non-muscle myosin IIA is a functional entry recep-tor for herpes simplex virus 1. Nature, 467:859-862, 2010.

Tasaki S, Nagasaki M, Kozuka-Hata H, SembaK, Gotoh N, Hattori S, Inoue J, Yamamoto T,Miyano S, Sugano S, and Oyama M.Phosphoproteomics-based modeling definesthe regulatory mechanism underlying aber-rant EGFR signaling. PLoS One, 5: e13926,2010.

Oyama M＊#, Nagashima T＊, Suzuki T, Kozuka-Hata H, Yumoto N, Shiraishi Y, Ikeda K,Kuroki Y, Gotoh N, Ishida T, Inoue S, KitanoH, and Okada-Hatakeyama M#. (＊; equal con-tribution) (#: corresponding authors) Inte-grated quantitative analysis of the phospho-proteome and transcriptome in tamoxifen-resistant breast cancer. J Biol Chem, in press.

Hino N＊, Oyama M＊, Sato A, Mukai T, Iraha F,Hayashi A, Kozuka-Hata H, Yamamoto T,Yokoyama S, and Sakamoto K. (＊; equal con-tribution) Genetic incorporation of a photo-crosslinkable amino acid reveals novel protein

complexes with GRB2 in mammalian cells. JMol Biol, in press.

Oyama M, Tasaki S, and Kozuka-Hata H.Tyrosine-Phosphoproteome Dynamics In Sys-tems Biology for Signaling Networks (ed. Choi,S), Springer, 447-454, 2010.

‹Group III›Masaike Y, Takagi T, Hirota M, Yamada J, Ishi-

hara S, Yung TM, Inoue T, Sawa C, Sagara H,Sakamoto S, Kabe Y, Takahashi Y, YamaguchiY and Handa H. Identification of dynamin-2-mediated endocytosis as a new target of os-teoporosis drugs, bisphosphonates. Mol Phar-macol, 77: 262-269, 2010.

Noda T, Hagiwara K, Sagara H and Kawaoka Y.Characterization of the Ebola virusnucleoprotein-RNA complex. J Gen Virol, 91:1478-1483, 2010.

Kikuchi, H.; Kuribayashi, F.; Takami, Y.; Imajoh-Ohmi, S.; Nakayama, T.: GCN5 regulates theactivation of PI3K/Akt survival pathway in Bcells exposed to oxidative stress via control-ling gene expressions of Syk and Btk: Bio-chem Biophys Res Commun (2011) 405, 657-61

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