Osaka University

Research Institute forMicrobial Diseases

2010

Contents
History & Outline

1

Organization2

Former Directors & Professors

3

Department Heads

4

Faculty & Students

5

Research & Activities
Division of Infectious Diseases……………………………………… 6
Division of Host Defense ……………………………………………… 14
Division of Cellular and Molecular Biology …………………………… 24
Research Center for Infectious Disease Control ……………………… 30
Genome Information Research Center ………………………………… 34
International Research Center for Infectious Diseases ……………… 40

Office of Combined Program on Microbiology and immunology

49

Thailand-Japan Research Collaboration Centeron Emerging and Re-emerging Infections

51

Endowed Chair

54

Global COE Program

56

Research Facilities

60

Immunology Frontier Research Center

66

Accounts & Building Area

68

Map & Access70

Photo by Hiroshi Nojima.

History & Outline

The Research Institute for Microbial Diseases was originally established as a ﬁve-department“Research Center for Communicable Diseases” in February, 1934 after a donation from Mr. Gendo

History & Outline

Research Institute for Microbial Diseases

Yamaguchi at Dojima in Osaka City. At that time, the Institute was located on Nakanoshima
campus of Osaka Medical School, but then became a part of Osaka University in September, 1934.
It was moved to its present site on Suita campus in 1967, in accordance with the university's
relocation program.
Until 1993, the Institute had grown to include nineteen departments spanning a wide range ofdiverse subjects, such as infectious disease, immunology, cancer and molecular biology. Additionally, it had three special research facilities, a central laboratory and a library. In 1997, the ResearchCenter for Emerging Infectious Diseases was added to the Institute. In 2005, the Institute wasreorganized to constitute three Research divisions; the “Division of Infectious Disease”, the “Division of Host Defence” and the “Division of Cellular and Molecular Biology”, which represent 15
departments together with three attached centers for specialized research on infectious disease
and genome information. Moreover, the Research Collaboration Center on Emerging and Reemerging Infections was founded in Bangkok in collaboration with the National Institute of Health,Thailand, to defend people against possible emerging and re-emerging infections.
Basic research on infectious disease, immunology, and cell biology is the Institute's principal
focus. The results of research in these ﬁelds at the Institute have contributed considerably to thediagnosis, prevention, and treatment of infectious diseases, immunological diseases and cancer, as
well as to progress in basic biomedical science.
The Institute was selected as one of “the 21st
century center of excellence programs” on thetheme of “combined program on microbiology
and immunology” in 2003. Also, it was certiﬁed as

joint usage / research center

by

MEXT (the Ministry of education, culture,
sports, science and technology) in 2009, andstarted the mission in April, 2010. Moreover,the institute newly established the Center for
The copper plate recalling that the Research Institutefor Microbial Diseases was originally establishedby a donation from Mr. Gendo Yamaguchi.
(At the entrance hall of the main building)

Genetic Analysis of Biological Responses

toconserve gene resources and to protect intellectual property.
The Institute accepts and trains Master andPh.D. candidates in the medical and biological

Mr. Gendo Yamaguchi

Born in Onomichi City in 1863 as the eldest
son of a physician, he moved to Osaka at theage of 15 and became one of westernJapan s most successful businessmen. He
retired in 1917, and devoted the rest of hislife to religion and the tea ceremony. He
donated most of the proceeds of his estate to
public enterprise, shrines and temples.

sciences. At present, more than 200 full,
associate, and assistant professors, research
associates, graduate students and research
fellows pursue studies in microbiology, oncology and molecular biology in state of the art
facilities at the Institute.
1

Organization
Director
Faculty Meeting

Delegate Assembly

Research Division
Division of Infectious Diseases
Department of Molecular Bacteriology
Department of Viral lnfections
Department of Molecular Virology
Department of Pharmacotherapy
Department of Pathology
Division of Host Defense
Department of Molecular Immunology
Department of Immunoregulation
Department of Host Defense
Department of Cell Biology
Department of Immunochemistry
Division of Cellular and Molecular Biology
Department of Molecular Microbiology
Department of Molecular Genetics
Department of Oncogene Research
Department of Signal Transduction
Department of Cellular Regulation

Special Research Facilities
Research Center for Infectious Disease Control
Department of Bacterial Infections
Department of Molecular Protozoology
Department of Virology
Genome Information Research Center
Department of Experimental Genome Research
Department of Genome Informatics
Department of Infection Metagenomics
International Research Center for Infectious Diseases
Department of Special Pathogens
Department of Infectious Disease Control
Pathogenic Microbes Repository Unit
Animal Resource Center for Infectious Diseases
DNA-chip Development Center for Infectious Diseases
Center for Genetic Analysis of Biological Responses

Oﬃce of Combined Program on Microbiology and Immunology
Research Promotion Group
Education Promotion Group

Research Collaboration Center in Overseas
Thailand-Japan Research Collaboration Center on Emerging and Re-emerging Infections
Section of Bacterial Infections
Section of Viral Infections

Endowed Chair
Laboratory of Molecular Biology (Life Technologies Co.- Endowed Chair)

Common Research Facilities
Central Instrumentation Laboratory
Radioisotope Laboratory
Central Laboratory for Biological Hazardous Microbes
Library

Administration
General Aﬀairs Section
Accounting Section
Research Cooperation Section

Related Institution
2

World Premier International Research Center
Immunology Frontier Research CenterFormer Directors & Professors

Yashiro Kotake, M.D., Professor

1934.９−1940. 6

Michiaki Takahashi, M.D., Professor

1984.４−1986. 3

Arao Imamura, M.D., Professor

1940.８−1943. 7

Toshio Miwatani, M.D., Professor

1986.４−1988. 3

Tenji Taniguchi, M.D., Professor

1943.７−1955. 3

Takeo Kakunaga, D.Pharm., Professor 1988.４−1988. 9

Tsunesaburo Fujino, M.D., Professor

1955.４−1958. 3

Hajime Fujio, M.D., Professor

1988.11−1990.10

Juntaro Kamahora, M.D., Professor

1958.４−1964. 3

Kumao Toyoshima, M.D., Professor

1990.11−1993.10

Tsunehisa Amano, M.D., Professor

1964.４−1968. 3

Akira Hakura, D.Sc., Professor

1993.10−1997.10

Yoshiomi Okuno, M.D., Professor

1968.４−1972. 3

Yoshitake Nishimune, M.D., Professor 1997.10−2001.10

Mitsuo Hori, M.D., Professor

1972.４−1976. 3

Takeji Honda, M.D., Professor

Junichi Kawamata, M.D., Professor

1976.４−1980. 3

Taroh Kinoshita, D.Med.Sc., Professor 2003.10−2007.10

Shiro Kato, M.D., Professor

1980.４−1984. 3

Hitoshi Kikutani, M.D., Professor

Organization, Former Directors & Professors

Former Directors

2001.10−2003.10
2007.10−Former Professors
Yashiro Kotake, M.D., Professor

Toshio Nakabayashi, M.D., Professor

Sadao Yoshida, M.D., Professor

Takahisa Yamanouchi, M.D., Professor

Arao Imamura, M.D., Professor

Toshio Miwatani, M.D., Professor

Yukichi Satani, M.D., Professor

Michiaki Takahashi, M.D., Professor

Tenji Taniguchi, M.D., Professor

Hajime Fujio, M.D., Professor

Kota Sera, M.D., Professor

Tetsuo Taguchi, M.D., Professor

Tatsunori Masayama, M.D., Professor

Aizo Matsushiro, D.Sc., Professor

Shohei Otani, M.D., Professor

Atsuo Nakata, D.Sc., Professor

Teishiro Seki, M.D., Professor

Hiroto Okayama, M.D., Professor

Masami Suda, M.D., Professor

Kumao Toyoshima, M.D., Professor

Kaoru Morishita, M.D., Professor

Teruo Kitani, M.D., Professor

Hisashi Yamaguchi, M.D., Professor

Shin-ichiro Takai, M.D., Professor

Tsunesaburo Fujino, M.D., Professor

Morihiro Matsuda, M.D., Professor

Masakazu Ito, M.D., Professor

Takashi Kurimura, M.D., Professor

Juntaro Kamahora, M.D., Professor

Koichi Yamanishi, M.D., Professor

Shinji Nishimura, M.D., Professor

Akira Hakura, D.Sc., Professor

Mitsuhiko Kato, M.D., Professor

Tetsu Akiyama, D.Sc., Professor

Masahiko Yoneyama, M.D., Professor

Takeshi Kurata, M.D.,D.Med.Sci., Professor

Shigeru Shiba, M.D., Professor

Shigeharu Ueda, M.D.,D.Med.Sci., Professor

Shozo Inoki, M.D., Professor

Kazunori Shimada, M.D.,D.Med.Sci., Professor

Mitsuo Hori, M.D., Professor

Chihiro Sasakawa, M.D., Professor

Yoshiomi Okuno, M.D., Professor

Akio Sugino, D.Sci., Professor

Shigeyuki Ishigami, M.D., Professor

Hiroshi Kiyono, D.D.S.,Ph.D., Professor

Tsunehisa Amano, M.D., Professor

Yoshitake Nishimune, M.D., Professor

Junichi Kawamata, M.D., Professor

Toru Nakano, M.D., D.Med. SC., Professor

Yoshio Okada, M.D., Professor

Hideo Shinagawa, D.Sc., Professor

Mitsuo Torii, D.Sc., Professor

Shin-ichi Tamura, Ph.D., Professor

Konosuke Fukai, M.D., Professor

Michiyuki Matsuda, M.D., D.Med. SC., Professor

Tatsuo Mori, M.D., Professor

Takeshi Honda, M.D., Ph. D.

Tonetaro Ito, M.D., Professor

Naoyuki Taniguchi, M.D., Ph. D.

Takeo Kakunaga, D.Pharm., Professor

Tamotsu Yoshimori, M.D., Ph. D.

Shiro Kato, M.D., Professor

Kazuyuki Tanabe, M.D., Ph. D.

3

Department Heads
2010,04,01

Director
Vice Director
Division of Infectious Diseases
Department of Molecular Bacteriology
Department of Viral Infections
Department of Molecular Virology
Department of Pharmacotherapy
Department of Pathology
Division of Host Defense
Department of Molecular Immunology
Department of Immunoregulation
Department of Host Defense
Department of Cell Biology
Department of Immunochemistry
Division of Cellular and Molecular Biology
Department of Molecular Microbiology
Department of Molecular Genetics
Department of Oncogene Research
Department of Signal Transduction
Department of Cellular Regulation

4

Professor Hitoshi Kikutani M. D., Ph.D.
Professor Eisuke Mekada Ph.D.

Professor Yasuhiko Horiguchi D. V. M., Ph.D.
Professor Tatsuo Shioda Ph.D.
Professor Yoshiharu Matsuura D. V. M., Ph.D.
Professor Atsushi Kumanogoh M. D., Ph.D.
Professor Hitoshi Kikutani M. D., Ph.D.
Professor Taroh Kinoshita Ph.D.
Professor Shizuo Akira M. D., Ph.D.
Professor Eisuke Mekada Ph.D.
Professor Hisashi Arase M. D., Ph.D.

Professor Hiroshi Nojima Ph.D.
Professor Masato Okada Ph.D.
Professor Nobuyuki Takakura M. D., Ph.D.

Research Center for Infectious Disease Control
Department of Bacterial Infections
Department of Molecular Protozoology
Department of Viorogy
Genome Infomation Research Center
Department of Experimental Genome Research
Department of Genome Informatics
Department of Infection Metagenomics
International Research Center for Infectious Diseases
Department of Special Pathogens
Laboratory of Clinical Research on Infectious Diseases
Laboratory of Infection Cell Biology
Laboratory of Viral Pathogenesis and Immunity
Department of Infectious Disease Control
Laboratory of Genomic Research on Pathogenic Bacteria
Laboratory of Malariology
Laboratory of Combined Research on Microbiology and Immunology

Head, Professor Toshihiro Horii Ph.D.

Animal Resource Center for Infectious Diseases
DNA-chip Development Center for Infectious Diseases
Center for Genetic Analysis of Biological Responses

Head, Professor Masaru Okabe Ph.D.
Head, Professor Hiroshi Nojima Ph.D.
Head, Professor Masaru Okabe Ph.D.

Oﬃce of Combined Program on Microbiology and Immunology
Research Promotion Group
Education Promotion Group

Head, Director Hitoshi Kikutani M. D., Ph.D.
Associate Professsor Yoshiko Murakami M. D., Ph.D.
Associate Professsor Hodaka Fujii M. D., Ph.D.

Research Collaboration Center in Overseas
Section of Bacterial Infections
Section of Viral Infections

Head, SA Professor Shigeyuki Hamada, D.D.S., Ph.D.
SA Professor Shigeyuki Hamada, D.D.S., Ph.D.
SA Professor Naokazu Takeda, Ph.D.

Laboratory of Molecular Biology (Life Technologies Co.- Endowed Chair)

Professor Fumio Imamoto Ph.D.

Central Instrumentation Laboratory
Radioisotope Laboratory
Central Laboratory for Biological Hazardous Microbes
Library

Head,Professor Masato Okada Ph.D.
Head,Professor Masato Okada Ph.D.
Head, Professor Tatsuo Shioda Ph.D.
Head, Professor Hisashi Arase M. D., Ph.D.

Administration

Head, Keiichi Kitomi

Professor Toshihiro Horii Ph.D.
Professor Kazuyoshi Ikuta Ph.D.
Head, Professor Teruo Yasunaga Ph.D.
Professor Masaru Okabe Ph.D.
Professor Teruo Yasunaga Ph.D.
Head, Professor Toshihiro Horii Ph.D.
SA Professor Kazunori Oishi M. D., Ph.D.
SA Associate Professor Yukako Fujinaga Ph.D.
SA Associate Professor Takaaki Nakaya M. D., Ph.D.
SA Professor Tetsuya Iida Ph.D.
SA Associate Professor Hiroki Nagai, Ph.D.

＊SA : Specially Appointed

Faculty & Students

2010,04,01
Professor

12

Endowed Chair Professor

1

SA Professor

5

Associate Professor

16

Endowed Chair Associate Professor

0

SA Associate Professor

4

SA Lecturer

2

Assistant Professor

27

Endowed Chair Assistant Professor

1

SA Assistant Professor

5

Educational Support Staﬀ

3

Technical Staﬀ

3

Administrative Staﬀ

17

SA Researcher

54

Research Collaborator

5

Part-time General & Technical staﬀ

43

Department Heads, Faculty & Students

Staﬀ

198

Total
SA：Specially Appointed

Graduate Students
2010,04,01

Doctor Course

Master Course

Graduate School of Medicine

57

5

Graduate School of Science4

7

Graduate School of Pharmaceutical Science2

6

Graduate School of Dentistry00

Graduate School of Frontier Biosciences

2

7

65

25

Total

Research Fellows & Research Students
2010,04,01

Special research students

1

Research Students

6

Visiting Research Scholars

1

JSPS Research Fellows

4

Total

12

5

Research & Activities

Division of Infectious Diseases

Department of Molecular Bacteriology
Research Group

Professor
Assistant Professor
Assistant Professor
Assistant Professor
SA Researcher

Yasuhiko Horiguchi, D. V. M., D. Agr. Sci.
Shigeki Kamitani, D. M. Sc.
Hiroyuki Abe, Ph. D.
Aya Fukui, Ph. D.
Hirono Toshima, Ph. D.

The objective of this department is to understand the molecular mechanisms by which pathogenic bacterial
virulence factors aﬀect host cell functions. Our present research interests include:(1) Analysis of the structure and function of bacterial protein toxins.
Bacterial protein toxins, which are the most poisonous substances on the earth, are known to act speciﬁcally ona particular cell and a particular biomolecule. To understand how bacterial toxins can act so powerfully andspeciﬁcally, we are analyzing their eﬀects on the host at the systemic, tissue, cellular and molecular levels. The
toxins currently under investigation are Bordetella dermonecrotic toxin, Pasteurella toxin, Clostridium perfringens
enterotoxin, and Escherichia coli cytotoxic necrotizing factor. We are also analyzing the steric structure andmolecular localization of the functional domains of these toxins. These approaches together will help to clarify
the structure and function of these bacterial toxins.
(2) Analysis of whooping cough pathogenesis.
Bordetella pertussis, a pathogenic bacteria, infects the human respiratory tract and causes whooping cough, which
is characterized by paroxysmal coughing. There are two signiﬁcant questions about the pathogenesis of B.
pertussis infection. First, why does B. pertussis infect humans but no other mammals? Second, how does thisbacterium induce the paroxysmal coughing? We are currently examining the pathology of the disease and thefunction of B. pertussis virulence factors by using an animal model of the infection.

Fig. 1: Bacterial protein toxins with various activities that inﬂuence particular cellular functions.
Many bacterial protein toxins exert their toxic eﬀects by modifying important functions of the hostcells. Signiﬁcantly, the relevant physiological functions of the cells can also be determined bydissecting the actions of the bacterial toxins.
6Research & Activities

Fig. 2: Overall structure of the intracellular
active region of Pasteurella multocida toxin,
which is composed of three domains andhas a Trojan horse-like shape.

Fig. 3: Bordetella pertussis, B. parapertussis,
and B. bronchiseptica are closely-related
pathogenic bacteria. B. bronchiseptica has thelargest genome and the broadest hostrange, whereas B. pertussis has the smallest
genome and the narrowest host range. It isbelieved that B. pertussis evolved from a B.
bronchiseptica lineage through the deletion
and/or translocation of a large number ofgenes.

Recent publications
１．Kimura J, Abe H, Kamitani S, Toshima H, Fukui A, Miyake M, Kamata Y, Sugita-Konishi Y, Yamamoto S, andHoriguchi Y. Clostridium perfringens enterotoxin interacts with claudins via electrostatic attraction. J Biol Chem.
2010 Jan 1;285(1):401-8.
２．Miyake M, Sakane S, Kobayashi C, Hanajima-Ozawa M, Fukui A, Kamitani S, and Horiguchi Y. A colorimetricassay for studying eﬀector secretion through the bacterial type III secretion system. FEMS Microbiol Lett. 2008
Jan;278(1):36-42.
３．Ohnishi H, Miyake M, Kamitani S, and Horiguchi Y. FEMS Microbiol Lett. 2008 Feb;279(2):174-9. The
morphological changes in cultured cells caused by Bordetella pertussis adenylate cyclase toxin. FEMS Microbiol
Lett. 2008 Feb;279(2):174-9.
４．Kitadokoro K, Kamitani S, Miyazawa M, Hanajima-Ozawa M, Fukui A, Miyake M, and Horiguchi Y. Crystal
structures reveal a thiol protease-like catalytic triad in the C-terminal region of Pasteurella multocida toxin. Proc
Natl Acad Sci U S A. 2007 Mar 20;104(12):5139-44.
５．Hanajima-Ozawa M, Matsuzawa T, Fukui A, Kamitani S, Ohnishi H, Abe A, Horiguchi Y, and Miyake M.
Enteropathogenic Escherichia coli, Shigella flexneri, and Listeria monocytogenes recruit a junctional protein, zonula
occludens-1, to actin tails and pedestals. Infect Immun. 2007 Feb;75(2):565-73.

7

Research & Activities

Division of Infectious Diseases

Department of Viral Infections
Research Group

Professor
Assistant Professor
Assistant Professor
SA Researcher
Postdoctoral Fellow
Postdoctoral Fellow

Tatsuo Shioda, D.Med.Sc.
Jun-ichi Sakuragi, D.Med.Sc.
Emi E. Nakayama, M.D., D.Med.Sc.
Sayuri Sakuragi, D.Med.Sc.
Ken Kono, D.Med.Sc.
Ayumu Kuroishi, D.Med.Sc.

The main focus in this department is to elucidate the molecular mechanisms of viral diseases including humanimmunodeﬁciency virus (HIV) infection. The following projects are currently underway.
(1) Anti-retroviral factors
HIV does not establish a productive infection in any other monkey except for the chimpanzee; this is thought tobe due to inhibitors in simian lymphocytes that act at the early stage (reverse transcription) of viral infection.
To date, TRIM5α and cyclophilin A have been identiﬁed as such restriction factors. We had shown that
diﬀerences in the amino acid sequences in the C-terminal domain of TRIM5α of diﬀerent monkey species aﬀect
the species-speciﬁc restriction of retrovirus infection (Fig.1, left). We also found that sequence variations in the
N-terminal half of the viral capsid protein (Fig. 1, right) determine viral sensitivity to TRIM5α-mediatedrestriction, which indicates that there is an interaction between TRIM5α and the virus capsid. In addition, we
showed that HIV-2 replication levels in infected individuals are associated with capsid variations, and wesuggested that viral sequence analysis can predict AIDS progression. Furthermore, we have succeeded in
improving the simian-tropic HIV-1 virus and the methods of monkey genome analysis. These new developments
greatly facilitate the generation of an HIV-1 animal model, which would be a highly useful tool in researchaiming to understand AIDS pathogenesis and to develop an eﬀective vaccine. We are also seeking to identify
the binding surface between the viral capsid protein and TRIM5α, as this may be useful for the development of
new anti-retroviral drugs.

The 3D structure model of viral capsid
protein. A single amino acid change
from P to A or Q radically aﬀected the
conﬁguration of the loop.

Structure of C-terminal SPRY domain of
TRIM5α.The amino acids that areimportant for viral restriction are locatedin the surface of SPRY domain. V1 andV2 denote the regions that vary between
the diﬀerent monkey species.

Fig. 1 Structural models of TRIM5α (left) and the viral capsid (right).
8Research & Activities(2) Host factors that participate in HIV pathogenesis and anti-retroviral drug side-eﬀects.
As an animal model of AIDS has not yet been established, we are utilizing epidemiological procedures tounderstand the mechanisms of AIDS pathogenesis. There are cases who are not infected despite repeatedexposure to HIV. There are also HIV-positive patients who do not develop symptoms of AIDS despite notreceiving any anti-retroviral treatment. These cases are suspected to bear a resistance-inducing factor (RIF)
against HIV. To characterize these RIFs, we have compared the genome sequences of the cases describedabove to those of HIV-infected patients and uninfected individuals. We found (a) the deletion mutant CCR5-893(-), which fails to produce a co-receptor that is needed for HIV entry, (b) a polymorphism in the promoter of thechemokine RANTES, and (c) a polymorphism in the promoter of IL4, which regulates the expression of theco-receptor. We then demonstrated that these mutations aﬀect susceptibility to HIV infection and the rate withwhich the disease progresses to AIDS.
At present, in collaboration with Thai groups, we are also focusing on the relationship between human genomic
variation and anti-retroviral therapy side-eﬀects, with the aim of establishing tailor-made therapies that will
improve the quality of life of HIV-infected patients.
(3) Molecular mechanisms of HIV particle formation.
The HIV genomic RNA always forms dimers in
the mature virion. It was suggested previouslythat the presence of the dimerized genome in
the virion is advantageous for survival, as itprovides an extra template that can be used
when one RNA molecule is damaged; it may also
endow the progeny with genetic variety. We
were able to identify the minimal HIV genome
region that is suﬃcient for genome dimerization.
Our data suggest that RNA dimerization is partof RNA packaging. We also found that HIV
genome dimerization aﬀects the early stage ofHIV replication after its entry into cells.
Fig. 2 HIV-1 genome RNA dimeriation.

Recent publications
１．Nakayama EE, ShiodaT. Anti-retroviral activity of TRIM5alpha. Rev Med Virol. 2010 Mar;20(2):77-92.
２．Likanonsakul S, Rattanatham T, Feangvad S, Uttayamakul S, Prosithsirikul W, Tunthanathip P, Nakayama EE,
Shioda T.HLA-Cw*04 allele associated with nevirapine-indued rash in HIV-infected Thai patients. AIDS Res Ther.
2009 Oct 21;6:22.
３．Nakajima T, Nakayama EE, Kaur G, Terunuma H, Mimaya JI, Ohtani H, Mehra N, Shioda T, Kimura A. Impact ofnovel TRIM5alpha variants, Gly110Arg and G176del, on the anti-HIV-1 activity and the susceptibility to HIV-1
infection. AIDS. 2009 Oct 23;23(16):2091-100.
４．Kuroishi A, Saito A, Shingai Y, Shioda T, Nomagushi M, Adachi A, Akari H, Nakayama EE. Modiﬁcation of a loop
sequence between alpha-helices 6 and 7 of virus capsid (CA) protein in a human immunodeﬁciency virus type 1(HIV-1) derivative that has simian immunodeﬁciency virus (SIVmac239) vif and CA alpha-helices 4 and 5 loop
improves replication in cynomolgus monkey cells. Retrovirology. 2009 Aug 3;6:70.
５．Sakuragi J, Sakuragi S, ShiodaT. Minimal region suﬃcient for genome dimerization in the humanimmunodeﬁciency virus type 1 virion and its potential role in the early stages of viral replication. J Virol. 2007
Aug;81(15):7985-92.

9Research & Activities

Division of Infectious Diseases

Department of Molecular Virology
Research Group

10

Professor
Associate Professor
SA Associate Professor
Assistant Professor
SA Assistant Professor
Postdoctoral Fellow
Postdoctoral Fellow
Postdoctoral Fellow

Yoshiharu Matsuura, D.V.M., Ph.D.
Kohji Moriishi, D.V.M., Ph.D.
Wataru Kamiya, Ph.D.
Takayuki Abe, Ph.D.
Hideki Tani, Ph.D.
Yuuki Kaname, Ph.D.
Takasuke Fukuhara, M.D., Ph.D.
Hiroshi Kato, D.V.M., Ph.D.

We are seeking to understand the molecular mechanisms by which hepatitis C virus (HCV) enters host cells,
replicates, escapes the immune system, and induces disease. This department is also currently developing anovel virus vector for gene delivery.
1. Studies on the molecular biology of HCV
replication and pathogenesis
HCV has infected more than 3% of the world s
population, 80% of whom will be persistently
infected. Persistent HCV infection often leads tochronic hepatitis, cirrhosis and hepatocellular
carcinoma. The incidence of hepatitis C in Japan
has decreased signiﬁcantly since a screening
system for anti-HCV antibodies was introduced in
1999. However, more than two million people inJapan are already infected with HCV. The
proportion of patients who achieve a sustained
virological response to therapy has been increasedby the use of pegylated-IFNα and ribavirin
combination therapy. However, half of the patientswith HCV genotype 1 fail to mount a response tothis combination therapy.
Since HCV exhibits quasispecies heterogeneity, it isdiﬃcult to determine the mechanisms by which
HCV infects the human host by using surrogate systems such as pseudotype and recombinant viruses derived
from a single HCV clone. The in vitro replication of genotype 2a HCV (HCVcc) was established recently andsurrogate viruses and HCVcc were used to identify several receptor candidates for HCV entry, including
hCD81, SR-BI, and Claudins. However, the sera of persistently infected patients contain high levels ofneutralization antibodies that recognize these artiﬁcial viruses, which suggests that these antibodies do not playa crucial role in the clearance of HCV. Our studies also showed that HCV NS3/4A protease cleaves adaptor
molecules involved in the TLR- and RIG-I‒dependent signaling pathways, that HCV particles are internalized
into cells through endocytosis, and that after uncoating, a viral RNA is translated into a large precursor
polyprotein composed of 3,000 amino acids. With regard to the latter viral polyprotein, it is then cleaved bysignal peptidase (SP), signal peptide peptidase (SPP) and virus-encoded proteases to generate at least 10 viralproteins. The open reading frame of the polyprotein is ﬂanked at both ends by highly conserved untranslated
regions (UTRs), which are required for viral RNA replication. The 5 -UTR harbors an internal ribosome entry
site (IRES) that is essential for Cap-independent translation of viral RNA.
Although there has been considerable successful
innovation in the drugs used in the clinic toeliminate the virus in patients with HCV,
drug-resistant viruses emerge easily. Therefore, themost ideal targets for the development of newchronic hepatitis C therapies that have a broad
spectrum and are unlikely to generate breakthroughviruses are host proteins that are indispensable for
HCV replication. To search for such molecules, wehave taken advantage of the fact that HCV belongs
to the family of Flaviviridae, which includes theﬂavivirus Japanese encephalitis virus (JEV). JEV
research is favored by the existence of a robust cell
culture system and a small animal model. We arethus investigating the replication and pathogenesisof JEV as a surrogate model of HCV.

Research & Activities

2. Development of baculoviral vectors
Viral vectors are essential tools for studies on replication-deﬁcient viral infectious diseases such as HCV.
Furthermore, the development of novel viral vectors is essential for future gene therapy. We are seeking toconvert the baculovirus Autographa californica nucleopolyhedrovirus (AcNPV) into a versatile viral vector thatwill mediate gene delivery both in vitro and in vivo. AcNPV is an insect virus that has a 134-kb double-stranded
circular DNA genome. Due to their strong promoters, baculoviruses are commonly used for the large-scale
production of recombinant protein in insect cells. Baculoviruses are also capable of entering a variety of
mammalian cells and, without replicating the viral genome, facilitating the expression of foreign genes under
the control of mammalian promoters. Therefore, baculoviruses are useful viral vectors, not only because they
can be used to induce the abundant expression of foreign genes in insect cells, but also because they caneﬃciently deliver genes to mammalian cells. AcNPV has a number of unique beneﬁcial properties as a viral
vector, including a large capacity for foreign gene incorporation, easy manipulation, and replication competence
in insect cells combined with incompetence in mammalian cells. Therefore, compared to other viral vectors thatare currently in use, AcNPV is much less likely to generate replication-competent revertants that express
baculoviral gene products and can induce harmful immune responses against mammalian cells. Furthermore,
we have shown that intranasal inoculation with AcNPV induces a strong innate immune response that protects
mice from lethal challenges with inﬂuenza viruses. In addition, we have demonstrated that AcNPV induces thisinnate immune response via a TLR9/MyD88-dependent pathway, and that this requires the internalization ofviral DNA via membrane fusion with the envelope glycoprotein in the endosome. This ﬁnding raises thepossibility that AcNPV can be harnessed therapeutically to induce host immune responses against various
infectious diseases, especially those that are caused by pathogens that invade from the respiratory tract.

Recent publications
１．Tani H, Shiokawa M, Kaname Y, Kambara H, Mori Y, Abe T, Moriishi K, Matsuura Y. Involvement of ceramide in
the propagation of Japanese encephalitis virus. J Virol. 2010 Mar;84(6):2798-807.
２．Taguwa S., Kambara H., Omori H., Tani H., Abe T., Mori Y., Suzuki T., Yoshimori T., Moriishi K., and Matsuura Y.
Co-chaperone activity of human butyrate-induced transcript 1 facilitates hepatitis C virus replication through an
Hsp90-dependent pathway. J. Virol., 83, 10427-10436 (2009).
３．Yamashita T., Mori Y., Miyazaki N., Cheng H.R., Yoshimura M., Unno H., Shima R., Moriishi K., Tsukihara T., Li
T.C., Takeda N., Miyamura T., and Matsuura Y. Biological and immunological characteristics of hepatitis E
virus-like particles based on the crystal structure. Proc. Natl. Acad. Sci. USA, 106, 12986-12991 (2009).
４．Kukihara H., Moriishi K., Taguwa S., Tani H., Abe T., Mori Y., Suzuki T., Fukuhara T., Taketomi A., Maehara Y.,
and Matsuura Y. Human VAP-C negatively regulates hepatitis C virus propagation. J. Virol., 83, 7959-7969 (2009).
５．Abe T., Kaname Y., Wen X., Tani H., Moriishi K., Uematsu S., Takeuchi O., Ishii K.J., Kawai T., Akira S., andMatsuura Y. Baculovirus induces type I IFN production through TLR-dependent and -independent pathways in acell type-speciﬁc manner. J. Virol., 83, 7629-7640 (2009).

11

Research & Activities

Division of Infectious Diseases

Department of Immunopathology
Research Group

Professor（SUP）
Atsushi Kumanogoh, M.D., Ph.D.
Associate Professor
Toshihiko Toyofuku, M.D., Ph.D.
Associate Professor（SUP） Masaru Ishii, M.D., PhD. *Assistant Professor（SUP） Tatsusada Okuno, M.D., Ph.D.
Assistant Professor（SUP） Hyota Takamatsu, M.D., Ph.D.

Regulation of immune responses by Immune Semaphorins
The semaphorin family contains soluble and membrane-bound proteins that were ﬁrst identiﬁed as axonal
guidance cues that function during neuronal development. However, cumulative ﬁndings suggest that
semaphorins also play diverse roles in processes that are unrelated to axon guidance, including organogenesis,
vascularization, angiogenesis, apoptosis and neoplastic transformation. After we discovered that CD100/Sema4D
participates in the immune system, we have found that a subset of semaphorins called the Immune
Semaphorins also function in the immune system. These include Sema4A, CD100/Sema4D, Sema6D, andSema7A. In addition, we found recently that Sema3A plays an essential role in the migration of dendritic cells(Fig. 2). We are currently studying the molecular mechanisms by which semaphorins and their receptors
regulate immune responses.

Figure 1. Representative immune semaphorins. The involvement of these semaphorins
in immune responses has been demonstrated by using gene-targeted mice.

12

Research & Activities

Figure 2. Sema3A is important in the migration of DCs.

Recent publications
１．Okuno T, Nakatsuji Y, Moriya M, Takamatsu H, Nojima S, Takegahara N, Toyofuku T, Nakagawa Y, Kang S,
Friedel RH, Sakoda S, Kikutani H, Kumanogoh A. Roles of Sema4D-plexin-B1 interactions in the central nervous
system for pathogenesis of experimental autoimmune encephalomyelitis. J Immunol. 2010 Feb 1;184(3):1499-506.
２．Toyofuku T, Yoshida J, Sugimoto T, Yamamoto M, Makino N, Takamatsu H, Takegahara N, Suto F, Hori M,
Fujisawa H, Kumanogoh A, Kikutani H.
Repulsive and attractive semaphorins cooperate to direct thenavigation of cardiac neural crest cells. Dev Biol. 2008 Sep 1;321(1):251-62.
３．Suzuki K, Kumanogoh A, Kikutani H. Semaphorins and their receptors in immune cell interactions. Nat Immunol.
2008 Jan;9(1):17-23.
４．Suzuki K, Okuno T, Yamamoto M, Pasterkamp J, Takegahara N, Takamatsu H, Kitao T, Takagi J, Rennert P,
Kolodkin A, Kumanogoh A, Kikutani H. Semaphorin 7A initiates T-cell mediated inflammatory responses through
α1β1 intergrin. Nature 2007 Apr 5;446(7136):680-4.
５．Takegahara N, Takamatsu H, Toyofuku T, Tsujimura T, Okuno T, Yukawa K, Mizui M, Yamamoto M, Prasad
DV, Suzuki K, Ishii M, Terai K, Moriya M, Nakatsuji Y, Sakoda S, Sato S, Akira S, Takeda K, Inui M, Takai T,
Ikawa M, Okabe M, Kumanogoh A, Kikutani H. Plexin-A1 and its interaction with DAP12 in immune responses
and bone homeostasis. Nat Cell Biol. 2006 Jun;8(6):615-22.
13

Research & Activities

Division of Host Defense

Department of Molecular Immunology
Research Group

Professor
Associate Professor
Assistant Professor
Postdoctoral fellow
Postdoctoral fellow
Postdoctoral fellow

Hitoshi Kikutani, M.D., Ph.D.
Teruhito Yasui, Ph.D.
Masayuki Mizui, M.D., Ph.D.
Olivia Simma, Ph.D.
Tomomi Morimoto, Ph.D.
Kentaro Morita, Ph.D.

Molecular mechanisms involved in the regulation of immune responses.
T cells are activated by MHC-bound antigenic peptides on antigen-presenting cells. Once activated, the T cells
diﬀerentiate into functional, helper, or eﬀector T cells. In contrast, antigen-stimulated B cells diﬀerentiate into
antibody-forming or memory B cells with the help of antigen-speciﬁc T cells. Thus, T- and B-cell diﬀerentiation
requires physiological interactions between T cells and antigen-presenting cells, and between T cells and B
cells, respectively. Such cell-cell interactions are mediated by a variety of costimulatory molecules, including
CD40, CD40 ligand, B-7 and CD28. In addition, it was revealed recently that several members of the semaphorin
family play crucial roles in immune cell interactions. We are currently studying how these molecules function in
the regulation of immune responses.
A) Mechanisms by which semaphorin molecules regulate immune responses:
The semaphorin family molecules were ﬁrst identiﬁed as axonal guidance factors that function during neuronaldevelopment. However, a series of studies by our laboratory has shown that several semaphorin molecules play
crucial roles at various stages of immune responses (Figure 1). For instance, Sema4D/CD100 is involved in theactivation of B cells and dendritic cells, while Sema4A participates in both T-cell priming and Th1
diﬀerentiation. The interaction between Sema6D and its receptor Plexin-A1 was also shown to participate incellular immune responses since it activates dendritic cells and promotes bone homeostasis by inducing
osteoclastgenesis. Furthermore, we demonstrated recently that Sema7A on activated T cells stimulates
macrophages, which then produce inﬂammatory cytokines; it also triggers inﬂammatory responses through α1β
1 integrin (Figure 2).

Fig. 1
Representative immune semaphorins
Semaphorins and their receptors have been shown by our
research group to participate in immune regulation.

Fig. 2. Accumulation of Sema7A and α1
integrin in the immunological synapse
between T cells and macrophages.

B) Elucidation of the molecular mechanism by which B cells survive and diﬀerentiate into eﬀector cells:
Eﬀective responses to the invasion of non-self antigen-bearing entities require that B cells diﬀerentiate into
antibody-secreting cells and memory B cells. B cell survival and diﬀerentiation are driven by B cell-antigen
receptor (BCR) signaling along with the signals of members of the TNF receptor family, such as CD40 and
BAFF-R, on the B cell surface. To date, our group has demonstrated the immunological signiﬁcance of themolecules that are involved in the signaling pathways downstream of CD40. In particular, we found that TRAF3,
which interacts with the cytoplasmic region of both CD40 and BAFF-R, plays a crucial role in B cell survival anddiﬀerentiation. Furthermore, we identiﬁed a PKC family member, PKN1, which is associated with the TRAF
family and serves as a negative regulator of Akt in BCR signaling. Our observations together suggest that PKN1
may be responsible for the immunological tolerance that eliminates autoreactive B cells.
14Research & Activities

Molecular mechanism by which Epstein-Barr virus (EBV) induces immunological disorders.
EBV is a human herpes virus that causes infectious mononucleosis in healthy donors and proliferative disorders inpatients who are immunosuppressed because of aging, immunosuppressive therapy, or HIV infection. It appears
that EBV infection may be associated with B cell malignancies such as Burkitt s lymphomas and Hodgkin s
lymphomas. It may also be linked to autoimmune diseases such as systemic lupus erythematosus (SLE) andmultiple sclerosis (MS). EBV infects B cells in a latent fashion and is prevalent worldwide. We are currently
studying EBV biology to determine how EBV leads to human carcinogenesis. The outcomes of this study may
also reveal attractive therapeutic strategies for EBV-associated immune disorders (Figure 3).
A) The molecular mechanism by which EBV infects the human host:
EBV infection induces B cell growth transformation and immortalization. The mechanism by which EBV invades
B cells involves multiple steps, namely virus entry, latency, and lytic infection. We are currently seeking toestablish a system that will allow us to trace in vitro the infection dynamics of EBV and the frequency of cell
growth transformation. This valuable system involves the production of recombinant EBV particles that carry thegene for GFP, which facilitates the visualization of EBV as it infects human peripheral B cells (Figure 4).

Fig.3. EBV and the host immune system.
The mechanism by which EBV induces human B cell growthtransformation is associated closely with the vulnerability of
the host immune system.

Fig.4. Immortalization of human peripheral
blood B lymphocytes by recombinant EBV
particles that carry the GFP gene.

Recent publications
１．Mizui M, Kumanogoh A, Kikutani H. Immune semaphorins: novel features of neural guidance molecules. J Clin
Immunol. 2009 Jan;29(1):1-11.
２．Mizui M, Shikina T, Arase H, Suzuki K, Yasui T, Rennert PD, Kumanogoh A, Kikutani H. Bimodal regulation of T
cell-mediated immune responses by TIM-4.Int Immunol. 2008 May;20(5):695-708.
３．Suzuki K, Okuno T, Yamamoto M, Pasterkamp RJ, Takegahara N, Takamatsu H, Kitao T, Takagi J, Rennert PD,
Kolodkin AL, Kumanogoh A, Kikutani H. Semaphorin 7A initiates T-cell-mediated inﬂammatory responses
through alpha1beta1 integrin. Nature. 2007 Apr 5;446(7136):680-4.
４．Mizui M, Kikutani H.Neuropilin-1: the glue between regulatory T cells and dendritic cells? Immunity. 2008
Mar;28(3):302-3.
５．Takegahara N, Takamatsu H, Toyofuku T, Tsujimura T, Okuno T, Yukawa K, Mizui M, Yamamoto M, Prasad
DV, Suzuki K, Ishii M, Terai K, Moriya M, Nakatsuji Y, Sakoda S, Sato S, Akira S, Takeda K, Inui M, Takai T,
Ikawa M, Okabe M, Kumanogoh A, Kikutani H. Plexin-A1 and its interaction with DAP12 in immune responses
and bone homeostasis. Nat Cell Biol. 2006 Jun;8(6):615-22.

15

Research & Activities

Division of Host Defense

Department of Immunoregulation
Research Group

Professor (SUP)
Associate Professor
Associate Professor (SUP)
Assistant Professor
SA Assistant Professor
Postdoctoral Fellow (SUP)
Postdoctoral Fellow
Postdoctoral Fellow (SUP)
Postdoctoral Fellow

Taroh Kinoshita, D. Med. Sc.
Yusuke Maeda, M.D., D. Med. Sc.
Yoshiko Murakami, M.D., D. Med. Sc.
Yasuhiro Morita, Ph. D.
Morihisa Fujita, D. Sc.
Yetao Wang, M.D., D. Med. Sc.
Noriyuki Kanzawa, D. Med. Sc.
Matthew Stokes, Ph. D.
Takeshi Fukuda, D. Eng.

1) Biogenesis, transport and remodeling of GPI-anchored proteins (GPI-APs).
Glycosylphosphatidylinositol (GPI) is a glycolipid that consists of phosphatidylinositol, glucosamine, mannoses
and phosphoethanolamines, and acts as a lipid anchor for various plasma-membrane proteins. GPI-APs play
important roles in host self-defense, intercellular signal transduction, and other important processes. In addition,some GPI-APs function as receptors for certain viruses and toxins. The GPI-anchor is widely distributed andconserved in various eukaryotes and is essential for the development of higher animals, as well as for thegrowth of yeasts and protozoan parasites. The modiﬁcation of proteins due to the attachment of the GPI-anchor
functions as a protein localization and sorting signal. Our current project is to identify and clarify the functionsof all the genes involved in the biosynthesis of the GPI-anchor in the ER (PIG genes; PhosphatidylInositol
glycan) and in the sorting and localization of GPI-APs after their anchorage with GPI (PGAP genes; Post
GPI-Attachment to Proteins). We expect that these studies will reveal why many proteins are modiﬁed with the
GPI-anchor.
Fig. 1 GPI-anchor biosynthesis and thetransport/remodeling of GPI-APs.
PIG genes are involved in thebiosynthesis of the GPI-anchor in the ER.
Thereafter, GPI-APs are transported tothe plasma membrane and enriched inrafts. PGAP genes are involved in theselater processes. PGAP1 and PGAP5,
which localize in the ER, and PGAP2 andPGAP3, which localize in the Golgi, areinvolved in the lipid or glycan
remodeling of the GPI-anchor. We foundthat the remodeling aﬀects the sorting of
GPI-APs because it alters the physicalcharacteristics of the GPI-anchor.

2) Molecular genetics of acquired (paroxysmal nocturnal hemoglobinuria, PNH) and inherited GPI deﬁciencies.
PNH is an acquired hematopoietic stem cell disorder in which clonal cells that are defective in GPI biosynthesis
are expanded. As a result, abnormal erythrocytes that lack CD59 and DAF/CD55 predominate. CD59 and
DAF/CD55 are widely distributed GPI-anchored proteins that inhibit the activation of complement on the hostcell surface, and their absence on erythrocytes makes these cells very sensitive to complement and lysis during
infections and other events. We are proposing a three-step model of PNH pathogenesis. Step 1 involves thegeneration of GPI-deﬁcient hematopoietic stem cells due to the somatic mutation of the PIG-A gene. Step 2
involves the immunological selection of GPI-deﬁcient hematopoietic stem cells. In this step, GPI-deﬁcient cells
not only survive, but they also proliferate much more frequently than usual to compensate for anemia. Thiselevated proliferation rate may increase the chance that additional genetic mutations are acquired, which leads
to Step 3, where a subclone bearing the growth phenotype is generated (Fig. 2). We identiﬁed HMGA2 as thecandidate gene for Step 3.
Along with our colleagues in England, we have also identiﬁed a novel disease that is characterized by venous
thrombosis and seizures, and is caused by a GPI deﬁciency that has been inherited in an autosomal recessive
manner. The patients have a point mutation in the promoter of PIG-M, a mannosyltransferase-encoding genethat plays an essential role in GPI biosynthesis. The point mutation severely reduces PIG-M expression andleads to partial GPI deﬁciency. While complete GPI deﬁciency is lethal, partial GPI deﬁciency could be caused
by a partial defect in one of the GPI biosynthesis genes, and the symptoms may vary depending on the defect.
16

Research & Activities

Fig. 2 Pathogenesis of PNH
Step 1 involves the generation of GPI-deﬁcient hematopoietic stem cells due to the somatic mutation of the
PIG-A gene. Step 2 involves the immunological selection of GPI-deﬁcient hematopoietic stem cells. In this step,
GPI-deﬁcient cells survive and proliferate much more frequently than usual to compensate for anemia. Thiselevated proliferation may increase the chance that additional genetic mutations occur. Step 3 involves thegeneration of a subclone bearing the growth phenotype.

3) Glycolipid biosynthesis in pathogens and its use in drug
development.
Our research focuses on elucidating the biosynthesis of GPIs in
mycobacteria and Trypanosoma brucei. T. brucei is the causative agent ofAfrican sleeping sickness while mycobacteria cause a number of
diseases, including tuberculosis. GPIs are located on the cell surface of
these pathogens and appear to play key roles in their evasion of hostimmune attack. In particular, GPI-like molecules found in mycobacteria
have anti-inﬂammatory activities and are thought to be important for
the establishment of the infection. We aim ﬁrst to identify the genesthat are involved in these GPI biosynthetic pathways, after which we
can create and characterize gene deletion/overexpression mutants.
This research will help us to understand the roles these GPI molecules
play at the molecular level in cell surface structure maintenance andhost immune response modulation. We are also seeking to determine
the key enzymes of the biosynthetic pathways and develop ahigh-throughput screening system that will help us to identify leadcompounds in drug libraries.

Fig. 3 Structure of the GPIs of pathogens

Recent publications
１．Sena CB, Fukuda T, Miyanagi K, Matsumoto S, Kobayashi K, Murakami Y, Maeda Y, Kinoshita T, Morita YS.
Controlled expression of branch-forming mannosyltransferase is critical for mycobacterial lipoarabinomannan
biosynthesis. J Biol Chem. 2010 Mar 9. doi: 10.1074/jbc.M109.077297
２．Kanzawa N, Maeda Y, Ogiso H, Murakami Y, Taguchi R, Kinoshita T. Peroxisome dependency of alkyl-containing
GPI-anchor biosynthesis in the endoplasmic reticulum. Proc Natl Acad Sci U S A. 2009 Oct 20;106(42):17711-6.
３．Fujita M, Maeda Y, Ra M, Yamaguchi Y, Taguchi R, Kinoshita T. GPI glycan remodeling by PGAP5 regulates
transport of GPI-anchored proteins from the ER to the Golgi. Cell. 2009 Oct 16;139(2):352-65.
４．Maeda Y, Ide T, Koike M, Uchiyama Y, Kinoshita T. GPHR is a novel anion channel critical for acidification andfunctions of the Golgi apparatus. Nat Cell Biol. 2008 Oct;10(10):1135-45.
５．Almeida AM, Murakami Y, Baker A, Maeda Y, Roberts IA, Kinoshita T, Layton DM, Karadimitris A. Targeted
therapy for inherited GPI deficiency. N Engl J Med. 2007 Apr 19;356(16):1641-7.

17

Research & Activities

Division of Host Defense

Department of Host Defense
Research Group

Professor（SUP）
Shizuo Akira
Associate Professor
Osamu Takeuchi
Associate Professor
Taro Kawai
Associate Professor（SUP） Satoshi Uematsu
Assistant Professor
Tatsuya Saito
SA Assistant Professor
Yutaro Kumagai

Our laboratory studies pathogen recognition by the innate immune system and the mechanisms that regulate
innate immune responses. The innate immune system senses invading microbial pathogens, such as bacteria,
viruses and parasites, and plays an essential role in inducing inﬂammatory responses and assisting adaptive
immune responses. Pattern-recognition receptors (PRRs) expressed on innate immune cells such as
macrophages and dendritic cells recognize pathogen-associated molecular patterns (PAMPs), which areconserved molecular features of microbial pathogens. We are seeking to clarify the complex regulatory
mechanisms of the innate immune system.
1) Characterization of the pathogen recognition by Toll-like receptors (TLRs) and their signaling pathways
TLR family members play essential roles in the recognition of pathogens by the innate immune system. Theirsignaling pathways also play an important role in the gene induction involved in inﬂammation and immune
responses. We have identiﬁed many TLR family members and their signaling molecules, and have characterized
their functions by generating knockout mice. As a result, we have identiﬁed most of the ligands for these TLR
family members and their signaling pathways (Figure 1). We also found that the stimulation of TLRs induces
not only proinﬂammatory cytokine genes, but also type I interferon genes. For example, theTRIF-TBK1/IKK-i-IRF-3 pathway plays an important role in the TLR3- and TLR4-mediated induction of IFN-β(Figure 2). Moreover, TLR7 and TLR9 are preferentially expressed in plasmacytoid dendritic cells (pDCs), which
produce large amounts of IFN-α upon viral infection. We identiﬁed a speciﬁc signaling pathway in pDCs that is
stimulated by TLR7 and TLR9 ligands and induces IFN-α expression (Figure 2). In summary, TLR signaling isregulated by distinct and complex mechanisms that operate in a ligand- and cell-type speciﬁc manner. We are
currently expanding our understanding of the in vivo functions of TLRs and their signaling pathways.

Figure 1: Pathogen recognition by TLRs. TLRs
recognize molecular patterns associated with a broad
range of pathogens, including bacteria, fungi, protozoa
and viruses.

18

Figure 2: TLR signaling pathways. All TLR family members
apart from TLR3 share a common pathway called the
MyD88-dependent pathway that induces inﬂammatory cytokine
production. Each TLR family member also has its own speciﬁcsignaling pathway. Thus, TLR3 and TLR4 operate via a
TRIF-dependent pathway while TLR7 and TLR9 act in pDCs via
a unique pathway to induce IFN-α expression.

2) Therapeutic applications of TLR agonists and antagonists
Appropriate agonist-induced stimulation of TLRs could stimulate an innate immune response that boosts hostresistance to cancer, allergy, and infectious diseases. This approach could also be used to promote thedevelopment of an adaptive immune response to a co-administered vaccine. TLR antagonists may also havetherapeutic potential, as they could prevent or ameliorate the inappropriate or exaggerated TLR stimulationthat leads to deleterious outcomes such as autoimmune diseases, sepsis or atherosclerosis.

Figure 3: Signaling pathways employed by anti-viral RNA
helicases. Viruses produce dsRNA during their replication in thehost cell cytoplasm. RIG-I and Mda5 recognize this viral RNA andinitiate antiviral signaling. In this signaling pathway, IPS-1
interacts with RIG-I and Mda5 via the CARD-like domain, andthis leads to the TBK1- and IKKi-dependent phosphorylation andactivation of IRF3 and IRF7. IPS-1 also activates NF-kB via
FADD/RIP1-dependent pathways. In addition, synthetic dsDNA
activates type I IFN promoters, although the receptor responsible
for the dsDNA recognition has not yet been identiﬁed.

Research & Activities

3) Investigation of pathogen recognition mechanisms by cytoplasmic receptors.
Infection with pathogens such as viruses induces type I IFNs in both a TLR-dependent and a TLR-independent
manner. RIG-I and MAD5, which are RNA helicases that recognize viral RNAs, recognize diﬀerent viruses andare important for host antiviral responses (Figure 3). We also identiﬁed a new adaptor molecule, IPS-1, whichplays an essential role in RIG-I- and MDA5-mediated antiviral responses (Figure 3). We are currently exploringthese TLR-independent mechanisms further by generating knockout mice.

Figure 4: Mechanisms that regulate the inﬂammatory responses
generated by TLR-inducible genes. TLR-inducible IkBz induces
the transcription of genes such as Il6 via NFkBp50. In contrast,
Zc3h12a, another TLR-inducible gene, functions as an Rnase that
degrades the mRNAs for Il6 and Il12, among others, andnegatively regulates inﬂammatory responses. TTP is also known
to be involved in the degradation of TNF mRNA.

4) Investigation of the mechanisms that regulate inﬂammatory responses.
The inﬂammatory responses that are elicited by the activation of innate immunity are properly regulated byvarious mechanisms. Our recent studies revealed that TLR signal-inducible molecules further positively andnegatively regulate inﬂammatory responses in response to infection. For example, the TLR-inducible nuclear
factor IkBz functions as a transcriptional modulator that is responsible for inﬂammatory cytokine production(Figure 4). In contrast, TLR-inducible Zc3h12a, an RNase, destabilizes a set of mRNAs such as Il6 and negatively
regulates inﬂammation. Mice that lack Zc3h12a spontaneously develop severe autoimmune inﬂammatory
diseases. Currently, we are examining the posttranscriptional regulation of inﬂammatory responses.

Recent publications
１．Takeuchi O, Akira S. Pattern Recognition Receptors and Inﬂammation. Cell. 2010 Mar 19;140(6):805-820.
２．Kawagoe T, Takeuchi O, Takabatake Y, Kato H, Isaka Y, Tsujimura T, Akira S. TANK is a negative regulator of
Toll-like receptor signaling and is critical for the prevention of autoimmune nephritis. Nat Immunol. 2009
Sep;10(9):965-72.
３．Matsushita K, Takeuchi O, Standley DM, Kumagai Y, Kawagoe T, Miyake T, Satoh T, Kato H, Tsujimura T,
Nakamura H, Akira S. Zc3h12a is an RNase essential for controlling immune responses by regulating mRNA
decay. Nature. 2009 Apr 30;458(7242):1185-90.
４．Saitoh T, Fujita N, Jang MH, Uematsu S, Yang BG, Satoh T, Omori H, Noda T, Yamamoto N, Komatsu M, Tanaka
K, Kawai T, Tsujimura T, Takeuchi O, Yoshimori T, Akira S. Loss of the autophagy protein Atg16L1 enhances
endotoxin-induced IL-1beta production. Nature. 2008 Nov 13;456(7219):264-8.
５．Uematsu S, Fujimoto K, Jang MH, Yang BG, Jung YJ, Nishiyama M, Sato S, Tsujimura T, Yamamoto M, Yokota
Y, Kiyono H, Miyasaka M, Ishii KJ, Akira S. Regulation of humoral and cellular gut immunity by lamina propria
dendritic cells expressing Toll-like receptor 5. Nat Immunol. 2008 Jul;9(7):769-76.

19

Research & Activities

Division of Host Defense

Department of Cell Biology
Research Group

Professor
Associate Professor
Assistant Professor
SA Researcher
SA Researcher

Eisuke Mekada, Ph.D.
Ryo Iwamoto, Ph.D.
Hiroto Mizushima, Ph.D.
Hiroki Moribe, Ph.D.
Takashi Nakamura, Ph.D.

We are studying cell growth and diﬀerentiation mechanisms that involve growth factors and adhesion
molecules presented at cell-cell contact sites. In particular, we are focusing on the mode of action of HB-EGF, amembrane-anchored EGF family of growth factors, and the molecules to which they bind, namely thetetraspanin family. These proteins function in morphogenesis and tissue maintenance and repair by regulatingcell proliferation, migration and adhesion. They are also involved in the growth, invasion and metastasis ofcancer cells.
1) Mode of action of HB-EGF
HB-EGF is a member of the EGF family of growth factors and binds to and activates EGFR and ErbB4. It issynthesized as proHB-EGF, a membrane-anchored precursor protein, and is cleaved on the cell surface to yield
the soluble growth factor (sHB-EGF). The conversion of proHB-EGF into the soluble form is critical for theactivity of this growth factor, and therefore this process is tightly regulated. HB-EGF is secreted by various
tissues and cells and functions in several physiological processes. For example, it maintains heart muscle
function, suppresses the cell proliferation involved in heart valve and lung alveolar development, promotes thecell migration that participates in wound healing and eyelid closure, supports blastocyst attachment to theuterus during implantation, and promotes the cell proliferation involved in skin hyperplasia. ProHB-EGF is not
only a precursor of the soluble form, it is also a biologically active molecule that regulates the growth ofneighboring cells in a juxtacrine fashion. How is the conversion of the membrane-anchored form into the soluble
form regulated? How does HB-EGF function in the manifold physiological processes that are dependent on thismolecule? What roles do sHB-EGF and proHB-EGF play in such physiological processes? Do they participate inpathological processes? These questions are currently being analyzed at the molecular level.
2) Development of anti-cancer drugs targeting HB-EGF
HB-EGF is involved in the growth, invasion and metastasis of various cancers. We are developing newanti-cancer drugs that target HB-EGF, and pre-clinical and clinical studies assessing an anti-HB-EGF monoclonal
antibody and a non-toxic mutant protein of diphtheria toxin CRM197 are currently in progress.
3) CD9 and tetraspanin function
CD9, a member of the tetraspanin superfamily, is a membrane protein with four transmembrane domains. It
associates with proHB-EGF and upregulates proHB-EGF function. CD9 is also involved in cell signaling, growth,motility, and adhesion, and in tumor cell metastasis and sperm-egg fusion. In addition, the Caenorhabditis
elegans tetraspanin TSP-15 is essential for the epidermal integrity of the worm. We are analyzing thephysiological activity of CD9 and other tetraspanins by using genetically engineered mice or C. elegans worms
that lack CD9 or other tetraspanins.

Fig. 1. Structure of proHB-EGF.

20

Fig. 2. Tumorigenesis in nude mice explanted with ovarian cancer
cells (left) and inhibition of this tumorigenesis by CRM197, a
non-toxic mutant of diphtheria toxin (right).

Research & Activities

Fig. 3. HB-EGF KO mice exhibit several tissue abnormalities. Unlike WT mice (a, c, e, g), KO mice(b, d, f, h) show abnormal phenotypes in the heart (a, b), heart valves (c, d), and lung alveoli (e, f), as
well as retinoid-induced skin hyperplasia (g, h). HB-EGF KO embryos also show defects in eyelid
closure because HB-EGF is normally expressed at the tip of the leading edge of migrating
epithelium (i).

Fig. 4. Expression of TSP-15 in C. elegans (left) and reduction of tsp-15
function in C. elegans by RNA interference (right), which induces
abnormalities of the hypodermis, including dissociation of the cuticle anddegeneration of the hypodermis.

Fig. 5. Entry mechanism of diphtheria toxin.

Recent publications
１．Koshikawa N, Mizushima H, Minegishi T, Iwamoto R, Mekada E, Seiki M. MT1-MMP cleaves oﬀ theNH2-terminal portion of HB-EGF and converts it into a heparin-independent growth factor. Cancer Res. 2010 inpress.
２．Iwamoto R, Mine N, Kawaguchi T, Minami S, Saeki K, Mekada E. HB-EGF function in cardiac valve
development requires interaction with heparan sulfate proteoglycans. Development 2010 in press.
３．Mizushima H, Wang X, Miyamoto S, Mekada E. Integrin signal masks growth-promotion activity of HB-EGF in
monolayer cell cultures. J Cell Sci. 2009 Dec 1;122(Pt 23):4277-86.
４．Miyado K, Yoshida K, Yamagata K, Sakakibara K, Okabe M, Wang X, Miyamoto K, Akutsu H, Kondo T,
Takahashi Y, Ban T, Ito C, Toshimori K, Nakamura A, Ito M, Miyado M, Mekada E, Umezawa A. The fusing
ability of sperm is bestowed by CD9-containing vesicles released from eggs in mice. Proc Natl Acad Sci U S A.
2008 Sep 2;105(35):12921-6.
５．Takeda Y, He P, Tachibana I, Zhou B, Miyado K, Kaneko H, Suzuki M, Minami S, Iwasaki T, Goya S, Kijima T,
Kumagai T, Yoshida M, Osaki T, Komori T, Mekada E, Kawase I. Double deﬁciency of tetraspanins CD9 andCD81 alters cell motility and protease production of macrophages and causes COPD-like phenotype in mice. J
Biol Chem. 2008 Sep 19;283(38):26089-97.

21

Research & Activities

Division of Host Defense

Department of Immunochemistry
Research Group

Professor（SUP）
Assistant Professor
Assistant Professor
Postdoctoral Fellow
Postdoctoral Fellow

Hisashi Arase, M.D., Ph.D., SUP
Tadahiro Suenaga, M.D., Ph.D.
Junji Uehori, Ph.D.
Fumiji Saito, Ph.D.
Kouyuki Hirayasu,Ph.D.

Our department analyzes how pathogens such as viruses acquire the ability to evade the immune system andhow host immune systems have acquired resistance to various pathogens. In particular, we are studying afundamental host defense mechanism that acts against various pathogens by employing various immune
regulatory receptors. Of particular interest are the paired receptors that are expressed on various immune
cells and consist of activating and inhibitory receptors (Figure 1). Our identiﬁcation of the host ligands andviral ligands that these paired receptors recognize has led us to propose that these paired receptors have
evolved in tandem with pathogens. In addition, we have found that these receptors are also involved in viralentry into cells. These studies will help to elucidate the fundamental mechanisms by which pathogens evadethe host immune system and the host factors that shape resistance to various infections. This research will
help to build the foundation that is required for the development of new vaccines and therapies for infectious
diseases.
(1) Analysis of the ligands recognized by paired receptors and the consequences of recognition.
Immune cells express various receptor pairs that consist of activating and inhibitory receptors that are highly
homologous to each other. The inhibitory receptors recognize self-antigens such as MHC molecules. In
contrast, the activating receptors generally do not recognize self-antigens and their ligands remain unclear atpresent. We have found that one of these paired receptors recognizes cytomegalovirus protein and we showedthat paired receptors play an important role in determining host resistance to pathogens (Figure 2). We arecontinuing to analyze the functions of these receptors to elucidate the interactions between pathogens and thehost immune system.

Figure 1. Paired receptors
Paired receptors consist of inhibitory
and activating receptors that arehighly homologous to each other.
The inhibitory receptors transduce
inhibitory signals via ITIM in theircytoplasmic domain, whereas theactivating
receptors
transduce
activating signals by associating with
ITAM-bearing adaptor molecules.

22

Figure 2. Recognition of cytomegalovirus-infected cells by inhibitory andactivating paired receptors
Viruses have acquired MHC-like molecules that serve as ligands forinhibitory receptors expressed on the NK cells of susceptible mice strains.
As a result, virus-infected cells are not killed by NK cells, even though
they express MHC at low levels (left). In contrast, NK cells from resistantmouse strains do not express inhibitory receptors that recognize virus
MHC-like molecules. Instead, they express activating receptors that dorecognize virus MHC-like molecules; therefore, these cells can eﬃciently
eliminate virus-infected cells (right) (Arase et al. Science 2002).

Research & Activities(2) Mechanisms by which viruses enter cells.
Several viruses that show persistent infection downregulate immune responses by expressing ligands that arerecognized by inhibitory receptors. Interestingly, we have found that some viruses also exploit these inhibitory
receptors to enter the cell. For example, such an interaction between immune receptors and viral proteins is
involved in the entry mechanism of herpes simplex virus (HSV) and varicella-zoster virus (VZV) (Figure 3).
Since other viruses may also use similar receptors to enter cells, we are continuing to investigate the molecularmechanisms by which viruses enter cells.

Figure 3. Mechanism by which viruses enter cells
Some viruses express ligands for inhibitory receptors and downregulate the immune response. We found that
PILRα, the inhibitory member of a receptor pair, recognizes herpes simplex virus (HSV)-infected cells. Molecularcloning of the ligands for PILRα revealed that PILRα associates with HSV glycoprotein B, which plays anessential role in HSV infection. Furthermore, the interaction between PILRα and glycoprotein B was found to
be involved in the entry of the virus into cells. We also found that glycoprotein B of varicella-zoster virus (VZV)
associates with myelin-associated glycoprotein (MAG, Siglec-4), one of paired receptors, and that this association
mediates the cell entry of VZV. Thus, paired receptors play important roles in both immune regulation and thehost cell entry of viruses.

Recent publications
１．Suenaga T, Satoh T. Somboonthum P, Kawaguchi Y, Mori Y, and Arase H. Myelin-associated glycoprotein
mediates membrane fusion and entry of neurotropic herpesviruses. Proc Natl Acad Sci USA. 2010 Jan 12; 107 (2):866-71.
２．Wang J, Fan Q, Satoh T, Arii J, Lanier LL, Spear PG, Kawaguchi Y, Arase H.Binding of herpes simplex virus
glycoprotein B (gB) to PILRα depends on speciﬁc sialylated O-linked glycans on gB. J Virol. 2009 Dec; 83 (24):13042-5.
３．Orr MT, Sun JC, Hesslein DG, Arase H, Phillips JH, Takai T, Lanier LL. Ly49H signaling through DAP10 isessential for optimal natural killer cell responses to mouse cytomegalovirus infection. J Exp Med. 2009 Apr 13; 206(4): 807-17.
４．Satoh T, Arii J, Suenaga T, Wang J, Kogure A, Uehori J, Arase N, Shiratori I, Tanaka S, Kawaguchi Y, Spear PG,
Lanier LL, Arase H. PILRα is a herpes simplex virus-1 entry co-receptor that associates with glycoprotein B. Cell.
2008 Mar 21; 132 (6): 935-44
５．Wang J, Shiratori I, Satoh T, Lanier LL, Arase H. An essential role of sialylated O-linked sugar chains in therecognition of mouse CD99 by paired immunoglobulin-like type 2 receptor (PILR). J Immunol. 2008 Feb 1; 180 (3):1686-9323

Research & Activities

Division of Cellular and Molecular Biology

Department of Molecular Genetics
Research Group

Professor
Hiroshi Nojima, Ph.D.
Assistant Professor
Norikazu Yabuta, Ph.D.
Assistant Professor（SUP） Daisuke Okuzaki, Ph.D.
Postdoctoral Fellow
Yoko Naito, Ph.D.

We are studying the eukaryotic cell cycle tounderstand the mechanism that is responsible for thechromosome instability in cancer cells. Chromosome
instability is observed in cancer cells, but not innormal cells. Indeed, many human cancer cells exhibit
mitotic defects (such as centrosome aberrations,abnormal spindle formation, and chromosome
missegregation), and the resulting chromosome
instability has been shown to be a major cause of
malignant tumor progression. We are focusing onfunctional analyses of the Ser/Thr kinases Lats (large
tumor suppressor) and GAK (cyclin G-associatedkinase). These kinases localize at the centrosome,Fig. 1: The functions of the Lats and GAK complexes
regulate mitotic progression in response to DNA
correlate closely.
damage, and cause chromosome instability when theirfunctions are disrupted. Both Lats
(Lats1 and Lats2) and GAK form
complexes with Mdm2. In turn,Mdm2 controls the stability of p53,
which is a transcriptional regulatorof the Lats2, cyclin G1 and Mdm2
genes. Thus, the Lats and GAK
Fig. 2: The structures of the Lats1 andcomplexes have intimate correlation in their function (Fig. 1).
Lats2 proteins are similar.
In addition, to inhibit spontaneous metastasis and the growth ofmalignant tumors by inhibiting connexin 26, our laboratory has
developed safe oleamide-derivative drugs that are associated withfew side eﬀects.

24

Our research subjects are as follows:(1) Lats Group: Lats1 and Lats2, which belong to the Lats kinase
family, are highly conserved across species and localize at thecentrosome during the cell cycle (Fig. 2). Two miRNAs, miRNA-372
and -373, function as potential novel oncogenes in testicular germ
cell tumors by inhibiting LATS2 expression, which suggests that
Lats2 is an important tumor suppressor (Voorhoeve et al., Cell, 2006).
Lats2 binds Mdm2, thereby inhibiting its E3 ligase activity andactivating p53; in turn, p53 rapidly and selectively upregulates Lats2
expression in G2/M cells. This positive feedback loop constitutes anovel checkpoint pathway that plays a critical role in themaintenance of proper chromosome numbers (Aylon et al., Gene
Dev., 2006). We have discovered the following: (A) Lats2 knockout
mice are embryonic lethal, which indicates the essential role ofLats2 in the development and diﬀerentiation of mammalian germ
Fig. 3: Loss of Lats2 leads to centrosome
fragmentation.
cells. (B) Lats2-/- mouse embryonic ﬁbroblasts (MEF) display anenhanced growth rate, centrosome fragmentation (Fig. 3),
misalignment of the chromosome at M phase, abnormal chromosome segregation, and aberrant cytokinesis.
These results indicate the essential role Lats2 plays in proper M phase progression. (C) Aurora-A
phosphorylates Lats2 on three distinct serines during mitosis; Lats2 localizes at the centrosome, the mitotic
spindle, or the spindle midzone during the cell cycle depending on which site is phosphorylated. (D)Down-regulation of Lats2 by siRNA causes the mislocalization of the chromosomal passenger complex (CPC)
during the metaphase/anaphase transition, with the consequence that cytokinesis fails. These observations
suggest that the Aurora-A-Lats2-CPC axis is a novel pathway that regulates proper cytokinesis. (E) Lats1/Lats2
knockout mice show arrested development at a very early stage of embryogenesis.

Research & Activities(2) GAK Group:
GAK is an association partner of clathrin
heavy chain (CHC) and is essential forclathrin-mediated membrane traﬃcking.
Unlike neuron-speciﬁc auxilin, which plays asimilar role in neural cells, GAK has a kinasedomain (Fig. 4) whose function has remained
Fig. 4: GAK is similar to auxilin except for bearing a kinase domain.
unclear. We have discovered the following:(A) GAK forms the KBG（KBG1 and KBG2）
complex with PP2A B' γ1 and cyclin G (cyclin G1 and cyclin G2), which regulate the dephosphorylation activityof PP2A. (B) GAK localizes not only in the cytoplasm but also at the nucleus, where it has two additional
functions, namely the maintenance of proper centrosome maturation and mitotic chromosome congression. (C)GAK knockdown by siRNA causes cell cycle arrest at the metaphase, which indicates that GAK is required forproper mitotic progression. This impaired mitotic progression was found to be due to the activation of thespindle assembly checkpoint (SAC), which senses protruding, misaligned, or abnormally condensed
chromosomes in knockdown cells. (Fig. 5) (D) CHC is involved in this regulatory process since GAK functions
cooperatively with clathrin during mitotic progression as well as during endocytosis. (Fig. 6)

Fig. 6: GAK knockdown by siRNA (Ki9) causes
the abnormal localization of CHC.
Fig. 5: GAK knockdown by siRNA (Ki9)generates abnormal chromosomes.

Recent publications
１．Shigehisa A, Okuzaki D, Kasama T, Tohda H, Hirata A, Nojima H. Mug28, a Meiosis-speciﬁc Protein of
Schizosaccharomyces pombe, Regulates Spore Wall Formation. Mol Biol Cell. 2010 Apr 21. In press.
２．Shimizu H, Nagamori I, Yabuta N, Nojima H. GAK, a regulator of clathrin-mediated membrane traﬃc, also controls
centrosome integrity and chromosome congression. J Cell Sci. 2009 Sep 1;122(Pt 17):3145-52.
３．Nishioka N, Inoue K, Adachi K, Kiyonari H, Ota M, Ralston A, Yabuta N, Hirahara S, Stephenson RO, Ogonuki N,
Makita R, Kurihara H, Morin-Kensicki EM, Nojima H, Rossant J, Nakao K, Niwa H, Sasaki H. The Hippo signaling
pathway components Lats and Yap pattern Tead4 activity to distinguish mouse trophectoderm from inner cellmass. Dev Cell. 2009 Mar;16(3):398-410.
４．Ohtaka A, Okuzaki D, Nojima H. Mug27 is a meiosis-speciﬁc protein kinase that functions in ﬁssion yeast meiosis
II and sporulation. J Cell Sci. 2008 May 1;121(Pt 9):1547-58.
５．Yabuta N, Okada N, Ito A, Hosomi T, Nishihara S, Sasayama Y, Fujimori A, Okuzaki D, Zhao H, Ikawa M, Okabe
M, Nojima H. Lats2 is an essential mitotic regulator required for the coordination of cell division. J Biol Chem. 2007
Jun 29;282(26):19259-71.
25

Research & Activities

Division of Cellular and Molecular Biology

Department of Oncogene Research
Research Group

Professor
Associate Professor
Assistant Professor
SA Researcher

Masato Okada, D.Sc.
Shigeyuki Nada, D.Sc.
Chitose Oneyama, D.Sc.
Kentaro Kajiwara, D.Sc.

Cancers arise, evolve and develop progressively due to the accumulations of mutations and/or modiﬁcations in
the genomic DNA. Loss-of-function mutations in tumor suppressor genes induce cell immortalization, while
gain-of-function mutations in proto-oncogenes induce cell transformation. Cell immortalization prevents theinduction of apoptosis and/or senescence, which is a defense mechanism against cancer development. Celltransformation involves the gain of autonomous cell growth, the loss of cell communication, morphologicalchanges, and the elevated production of matrix proteases and growth factors that participate in invasion,
metastasis and angiogenesis. These cellular events thus lead to the malignant conversion of cancer cells. The
primary focus of this department is to understand the molecular basis of the cell transformation that is inducedby the gain-of-function mutations in proto-oncogenes. As a representative proto-oncogene, we have focused on
the c-Src proto-oncogene, which encodes a non-receptor tyrosine kinase. To date, we have analyzed its
physiological roles in development and the mechanisms bywhich its speciﬁc regulators, such as Csk and Cbp,
regulate it. To obtain a full picture of the cell signaling
pathways that lead to c-Src‒mediated cell transformationand to search for new therapeutic targets that will blockc-Src‒mediated cancer progression, the following projects
are currently in progress:
I. Molecular mechanisms that suppress c-Src‒mediated celltransformation.
In normal cells, c-Src is present in an inactive form that is
phosphorylated by its negative regulatory kinase Csk.
Extracellular stimuli transiently activate it (Fig. 1), after
which it in turn activates downstream pathways such as
the MAPK pathway, thereby inducing the gene expressionthat is required for cell growth and the phenotypicchanges that are involved in cell transformation (Fig. 2).
While the c-Src gene is rarely mutated in human cancers,
its protein is frequently hyperactivated and overexpressed.
This aberrant activation of c-Src is suggested to contributeto cancer malignancy (Fig. 2).
Recently, we found a new system by which the aberrant
activation of c-Src could be suppressed. We previously
showed that the inactivation of c-Src is facilitated whenCsk is recruited to lipid rafts by the speciﬁc adaptor Cbp.
Further analysis then revealed that Cbp can recruit
activated c-Src to lipid rafts directly, and that this is
suﬃcient for suppressing cell transformation. Furthermore,
we found that the expression of Cbp is substantiallydownregulated in various human cancers, which suggeststhat the Cbp gene serves as a tumor suppressor gene (ref.
3, Fig. 3). Currently, we are analyzing the molecularmechanisms behind the downregulation of the Cbp gene incancer cells.
26

Fig. 1. Functions and regulation of c-Src.

Fig. 2. c-Src and human cancer.

Research & Activities

Fig. 3. Tumor-suppressing role of Cbp.

II. Cell signaling pathway of c-Src‒mediated cell transformation.
To elucidate the main pathway by which c-Src induces malignant transformation, we searched for new targetsof c-Src. Recently, one such potential c-Src target was found to be the novel adaptor protein p18, which is
exclusively localized at the lipid rafts of late endosomes. p18 can recruit a branch of MAPK pathway to lateendosomes by directly binding the p14/MP1 complex, which is known to be a speciﬁc scaﬀold of MEK1.
Analyses of p18 KO mice (embryonic lethal), p18 KO cells, and epidermis-speciﬁc p18 KO mice revealed thatp18 plays a pivotal role in endosome dynamics by regulating membrane fusion between endosomal vesicles.
More recently, we also found that the p18-dependent MAPK pathway is essential for the cell transformationinduced by Src, K-Ras and Pak1 (ref. 2, Fig. 4). We are currently analyzing the details of this pathway. We havealso started a project that seeks to identify anti-cancer agents that target the p18-dependent pathway.

Fig. 4. Roles played by
the p18-MAPK
pathway in endosome
dynamics and cancer
growth.

Recent publications
１．Oneyama C, Iino T, Saito K, Suzuki K, Ogawa A, Okada M. Transforming potential of Src family kinases is limitedby the cholesterol-enriched membrane microdomain. Mol Cell Biol. 2009 Dec;29(24):6462-72.
２．Nada S, Hondo A, Kasai A, Koike M, Saito K, Uchiyama Y, Okada M. The novel lipid raft adaptor p18 controls
endosome dynamics by anchoring the MEK-ERK pathway to late endosomes. EMBO J. 2009 Mar 4;28(5):477-89.
３．Oneyama C, Hikita T, Enya K, Dobenecker MW, Saito K, Nada S, Tarakhovsky A, Okada M. The lipid
raft-anchored adaptor protein Cbp controls the oncogenic potential of c-Src. Mol Cell. 2008 May 23;30(4):426-36.
４．Oneyama C, Hikita T, Nada S, Okada M. Functional dissection of transformation by c-Src and v-Src. Genes Cells.
2008 Jan;13(1):1-12.
５．Yagi R, Waguri S, Sumikawa Y, Nada S, Oneyama C, Itami S, Schmedt C, Uchiyama Y, Okada M. C-terminal Src
kinase controls development and maintenance of mouse squamous epithelia. EMBO J. 2007 Mar 7;26(5):1234-44.

27

Research & Activities

Division of Cellular and Molecular Biology

Department of Signal Transduction
Research Group

Professor
Research Associate
Postdoctoral Fellow
Postdoctoral Fellow
Postdoctoral Fellow
Postdoctoral Fellow

Nobuyuki Takakura, M.D., Ph.D.
Hiroyasu Kidoya, Ph.D.
Yumi Kinugasa, Ph.D.
Hisamichi Naito, M.D., Ph.D.
Tomomi Mohri, Ph.D.
Yinglu Han, Ph.D.

It is well known that the development of normal tissues and organs requires the generation of tissue-speciﬁccells from stem cells. The maintenance of this stem cell system also requires the generation of an appropriate
microenvironment. Blood vessels are the most essential structures in tissues and organs, as without blood vessel
formation, almost all tissues cannot develop (there are some exceptions). In our research group, we are
analyzing the molecular mechanisms by which blood vessels form in physiological and pathological conditions,
including in cancers and inﬂammation. We are also elucidating the mechanisms that cause stem cells to
associate closely with blood vessels. Ultimately, we wish to employ our results to establish strategies that will
inhibit the malignant progression of various diseases. Our speciﬁc research projects are as follows:
I. Analysis of the molecular mechanism of blood vessel formation
1) Molecular analysis of sprouting angiogenesis from preexisting vessels, with a particular focus on the Tie2
receptor.
2) Identiﬁcation and characterization of adult endothelial stem cells (the CD31-positive side population cells).
3) Molecular characterization of arterio-venous patterning, with a particular focus on the apelin/APJ system.
4) Development of a system that delivers drugs into blood vessels.
II. Molecular analysis of self-renewal in normal and cancer stem cells
1) Mechanism of stem cell reprogramming.
2) Analysis of cell cycle regulation in stem cells, with a special focus on Galectin-3 and the GINS complex.
3) Bioimaging of the niches that are inhabited by living cancer and normal stem cells.
4) Establishment of a strategy that can inhibit the formation of the vascular niche inhabited by cancer stem
cells.

Figure 1. Vascular niche of cancer
stem cells.
After treatment of a tumor with
angiogenesis inhibitors, the mature
blood vessels at the tumor edge
persist (left panels). Cancer stem cells
marked by PSF1 (a member of the
GINS DNA replication factor family
that is expressed by the self-renewing
normal stem cell population in avariety of tissues and organ) arepresent and proliferate in the vascularniche represented by these mature
blood vessels (right panels). Molecularanalysis of vascular niche formation
may be highly useful for thedevelopment of a new therapy forcancer stem cells.

28Research & Activities

Figure 2. Maturation of blood vessels at thetumor edge.
Hematopoietic stem cells (HSCs) migrate into
hypoxic tissues, produce angiopoietin-1 (Ang1),
and stimulate Tie2 on endothelial cells (ECs),
which results in EC migration andproliferation (upper panel). Many HSCs
accumulate at the edge of the tumor during
the early stage of tumorigenesis and a portionof HSCs may diﬀerentiate into mural cells(MCs) and stabilize blood vessels. Ang1 from
HSCs and MCs promotes the production of
apelin by ECs, thereby stimulating the apelin
receptor APJ on ECs. The activation of APJ
induces the proliferation and assembly of ECs,
resulting in blood vessel enlargement.

Figure 3. Disruption of the vascular niche in
the tumor limb.
Our goal is to completely inhibit tumor
growth by destroying mature blood vessels in
the tumor environment. For this purpose, weneed a drug delivery system that will deliverdrugs that speciﬁcally block the blood vessel
maturation functions of ECs in the tumor.

Recent publications
１．Kidoya H, Naito H, Takakura N. Apelin induces enlarged and non-leaky blood vessels for functional recovery fromischemia. Blood in press
２．Nagahama Y, Ueno M, Miyamoto S, Morii E, Minami T, Mochizuki N, Saya H, Takakura N. (2010). PSF1, a DNA
replication factor expressed widely in stem and progenitor cells, drives tumorigenic and metastatic properties.
Cancer Res. 2010 Feb 1;70(3):1215-24.
３．Ueno M, Itoh M, Sugihara K, Asano M, and Takakura N. (2009). Both Alleles of PSF1 are Required for
Maintenance of Pool Size of Immature Hematopoietic Cells and Acute Bone Marrow Regeneration. Blood. 2009 Jan
15;113(3):555-62.
４．Kidoya H, Ueno M, Yamada Y, Mochizuki N, Nakata M, Yano T, Fujii R, Takakura N. (2008). Spatial and temporalrole of the apelin/APJ system in the caliber size regulation of blood vessels during angiogenesis. EMBO J. 2008
Feb 6;27(3):522-34.
５．Yamada, Y., and Takakura, N. (2006). Physiological pathway of diﬀerentiation of hematopoietic stem cell population
into mural cells. J Exp Med. 2006 Apr 17;203(4):1055-65.
29

Research & Activities

Research Center for Infectious Disease Control

Department of Molecular Protozoology
Research Group

Professor
Assistant Professor
Assistant Professor
Postdoctoral Fellow
Postdoctoral Fellow

Toshihiro Horii, Ph.D.
Nobuko Arisue, Ph.D.
Takahiro Tougan, Ph.D.
Masanori Yagi, Ph.D
Kohhei Tetsutani, M.D., Ph.D.

Malaria is a serious threat to global human health. More than 40% of the world s population lives inmalaria-endemic areas and two million people succumb to the disease annually (Fig. 1). Controlling malaria has
become more challenging since the emergence of drug-resistant malaria parasites. This has intensiﬁed the needfor novel drug target strategies and an eﬀective malaria vaccine. Our department is focused on thedevelopment of both anti-malarial vaccines and drugs. We are also seeking to understand the mechanisms thatthe malaria parasite uses to survive in the host.
(1) Development of a recombinant vaccine based on the malaria protein SERA.
We are developing a malaria vaccine that is based on SE36, which is a recombinant protein that spans an aminoacid sequence in the serine repeat antigen (SERA) of malaria parasites (Fig. 2). We and co-researchers inmalaria-endemic areas have demonstrated that naturally acquired immunity against malaria correlates
exclusively with the development of anti-SERA IgG3 antibodies. We have also shown that, after vaccination
with SE36, many types of animals, including chimpanzees, develop antibodies that inhibit the growth of malaria
parasites (Fig. 3). Together with the Kanonji Institute of the Research Foundation of Osaka University, we haveconstructed a system by which the SE36 malaria vaccine can be mass-produced (Fig. 4). In 2005, we conducteda phase I clinical trial in Japan with SE36 to assess its safety and immunogenicity. All vaccine-administeredvolunteers were sero-converted and showed no serious adverse events. We are currently in the process ofconducting additional phase Ib clinical trials in an endemic region in Uganda. This project is under taken incollaboration with the Research Foundation for Microbial Diseases of Osaka University.
We are also studying the function of the SERA molecule in the parasite and characterizing the host immune
response against SERA. In addition, in collaboration with colleagues in Uganda, Thailand, Indonesia and theSolomon Islands, we have started a new research project that aims to develop a Plasmodium vivax vaccine.

Fig. 1: Patients waiting at the Out-patient
Department of Apac Hospital in Northern
Uganda: major victims of malaria arechildren under the age of 5 years.

30

Fig. 2: Processed fragments of P. falciparum SERA
and the structure of the recombinant SE36.

Research & Activities

Fig. 3: Model that explains how anti-SERA IgG inhibits
erythrocytic P. falciparum growth.

Fig. 4: The SE36 malaria vaccine forclinical trials is produced under Good
Manufacturing Practices (GMP) at
the Kanonji Institute of the Research
Foundation for Microbial Diseases ofOsaka University.

(2) Identiﬁcation of SERA genes from several Plasmodium species.
The Plasmodium SERA gene family consists of several gene members. To trace the evolution of the SERA
genes, we identiﬁed the SERA genes of several Plasmodium species and constructed the SERA gene family tree
(Fig. 5). Transcription and polymorphic analyses are being used to search for functional or vaccine target
molecules in the Plasmodium SERA genes.

Fig. 5:

An overview of the Plasmodium SERA gene family.

Recent publications
１．Horii T, Shirai H, Jie L, Ishii JK, Palacpac Q N, Tougan T, Hato M, Ohta N, Bobogare A, Arakaki N, Matsumoto Y,
Namazue J, Ishikawa T, Ueda S, Takahashi M. Evidences of protection against blood-stage infection of Plasmodium
falciparum by the novel protein vaccine SE36. Parasitol Int. in press.
２．Kimata-Ariga Y, Saitoh T, Ikegami T, Horii T, Hase T. Molecular interaction of ferredoxin and ferredoxin-NADP+
reductase from human malaria parasite. J Biochem. 2007 Dec;142(6):715-20.
３．Arisue N, Hirai M, Arai M, Matsuoka H, Horii T. Phylogeny and evolution of the SERA multigene family in thegenus Plasmodium. J Mol Evol. 2007 Jul;65(1):82-91.
４．Palacpac NM, Leung BW, Arisue N, Tanabe K, Sattabongkot J, Tsuboi T, Torii M, Udomsangpetch R, Horii T.
Plasmodium vivax serine repeat antigen (SERA) multigene family exhibits similar expression patterns in
independent infections. Mol Biochem Parasitol. 2006 Dec;150(2):353-8.
５．Krungkrai SR, Tokuoka K, Kusakari Y, Inoue T, Adachi H, Matsumura H, Takano K, Murakami S, Mori Y, Kai Y,
Krungkrai J, Horii T. Crystallization and preliminary crystallographic analysis of orotidine 5'-monophosphate
decarboxylase from the human malaria parasite Plasmodium falciparum. Acta Crystallogr Sect F Struct Biol Cryst
Commun. 2006 Jun 1;62(Pt 6):542-5.
31Research & Activities

Research Center for Infectious Disease Control

Department of Virology
Research Group

Professor
Associate Professor
Assistant Professor
Postdoctoral Fellow
Postdoctoral Fellow
Postdoctoral Fellow
Postdoctoral Fellow
Postdoctoral Fellow

Kazuyoshi Ikuta, Ph.D.
Keizo Tomonaga, D.V.M., Ph.D.
Takeshi Kurosu, D.V.M., Ph.D.
Yohei Watanabe, D.V.M., Ph.D.
Yuji Inoue, Ph.D.
Masahiro Sasaki, Ph.D.
Mayo Yasugi-Ueda, D.V.M., Ph.D.
Juan Fernando Arias, MD, Ph.D.

The research in this department focuses on several viruses and prions that target the immune, respiratory, andcentral nervous systems, with the aim of understanding the mechanisms by which they replicate and induce
disease. Our ultimate objective is to devise ways to control these agents, remove them from blood products, anddiagnose them rapidly.
(1) Infections of the immune system
We are working on the mechanism(s) by which HIV establishes a persistent/latent infection of the immune
system. In particular, we are currently characterizing Thai and Indian patient-derived HIV subtypes, which
are the most prevalent subtypes in the world. In addition, we are characterizing the mechanism by which
dengue viruses derived from South Asian countries induce hemorrhagic fever.
(2) Infections of the respiratory system
We recently succeeded in preparing human neutralizing monoclonal antibodies against an inﬂuenza virus thatinduces typical acute infectious disease in the respiratory region. Since the epitope that is recognized by the
monoclonal antibodies is highly conserved and has a conformational structure, we are working in collaboration
with several companies to develop a new type of vaccine bearing this conformational epitope.
(3) Infection of the nervous system
Borna disease virus (BDV) has several unique infection features, namely it exhibits highly neurotropic,
noncytopathic replication and a long-lasting persistent infection. Epidemiological studies have shown that awide variety of vertebrate species can be naturally infected with BDV. Interestingly, BDV has been suggested
to be associated with human neuropsychiatric disorders. Our focus regarding this virus involves: 1) its
epidemiology, 2) its replication, and 3) the mechanisms that mediate its persistence and neuropathogenesis.
(4) Blood-borne infections
In collaboration with a company, we are working on methods to remove infectious agents such as parvovirus
B19, SARS-corona virus, hepatitis E virus, and prions from blood products.
(5) Rapid diagnosis kits
There are many techniques that are used to diagnose virus infections, namely immunoﬂuorescence, ELISA,
Western blot, and PCR assays. We are currently working in collaboration with several companies to develop
rapid diagnosis kits against several infectious diseases.

32

Research & Activities

Human monoclonal antibodies (MAbs)
that show neutralizing activity against
a broad range of strains within the
H3N2 subtype recognize a highly
conserved conformational epitope (redand green) within the HA protein.
Since similar corresponding regions
are also detected in other subtypes,
we are currently working on thedevelopment of a vaccine that can
induce neutralizing antibodies against
inﬂuenza virus.

We found bornavirus N gene-relatedendogenous elements in the genomesof several mammals, including humans.
Such elements, known as EBLN, may
encode functional proteins in someprimate species.

Recent publications
１．Horie M, Honda T, Suzuki Y, Kobayashi Y, Daito T, Oshida T, Ikuta K, Jem P, Gojobori T, Coﬃn JM, Tomonaga
K. Endogenous non-retroviral RNA virus elements in mammalian genomes. Nature. 2010 Jan 7;463(7277):84-7.
２．Yamashita A, Kawashita N, Kubota-Koketsu R, Inoue Y, Watanabe Y, Ibrahim MS, Ideno S, Yunoki M, Okuno Y,
Takagi T, Yasunaga T, Ikuta K. Highly conserved sequences for human neutralization epitope on hemagglutininof inﬂuenza A viruses H3N2, H1N1 and H5N1: Implication for human monoclonal antibody recognition. Biochem
Biophys Res Commun. 2010 Mar 19;393(4):614-8.
３．Watanabe Y, Ohtaki N, Hayashi Y, Ikuta K, Tomonaga K. Autogenous translational regulation of the Borna
disease virus negative control factor X from polycistronic mRNA using host RNA helicases. PLoS Pathog. 2009
Nov;5(11):e1000654
４．Yunoki M, Kubota-Koketsu R, Urayama T, Sasaki T, Du A, Konoshima Y, Ideno S, Fukunaga Y, Morikawa S, Hiroi
S, Takahashi K, Okuno Y, Hagiwara K, Ikuta K. Signiﬁcant neutralizing activity of human immunoglobulin
preparations against pandemic 2009 H1N1. Br J Haematol. 2009 Nov 24;143:953-955.
５．Kubota-Koketsu R, Mizuta H, Oshita M, Ideno S, Yunoki M, Kuhara M, Yamamoto N, Okuno Y, Ikuta K. Broadneutralizing human monoclonal antibodies against inﬂuenza virus from vaccinated healthy donors. Biochem
Biophys Res Commun. 2009 Sep 11;387(1):180-5.
33

Research & Activities

Genome Information Research Center

Department of Experimental Genome Research
Research Group

Professor（SUP）
Masaru OKABE, Ph.D.
Associate Professor
Takeshi MIWA, Ph.D.
Associate Professor（SUP）
Masahito IKAWA, Ph.D.
Assistant Professor
Hidetoshi HASUWA, Ph.D.
Assistant Professor
Naokazu INOUE, Ph.D.
Assistant Professor（SUP）
Ayako ISOTANI, Ph.D.
SA Assistant Professor（SUP） Yuhkoh SATOUH, Ph.D.

In the past, naturally-mutated animals were used to elucidate themechanisms of various diseases. In the post-genome project era,
however, genetically manipulated animals that can serve as animalmodels for human diseases play a key role in such investigations. Ourlaboratory assists other research facilities in generating suchgenetically manipulated animals, as shown by our web page(http://kumikae01.gen-info.osaka-u.ac.jp/EGR/index.cfm).
Thisobjective is undertaken in collaboration with the Animal Resource
Center for Infectious Diseases.
Research Projects
We were the ﬁrst in the world to produce a genetically altered green
mouse that glows in the dark. These mice are highly useful for many
types of research, including stem cell transplantation andregeneration. By utilizing these animals, we showed that the sex ofmurine embryos can be determined at the preimplantation stage.
These mice have been used to study the fertilization process (Fig. 1)(3) and the sex determination mechanism in germ cells.

Fig. 1. A transgenic mouse line whose
sperm express green ﬂuorescentprotein (GFP) in their acrosome andred ﬂuorescent protein (RFP) in theirmitochondria.
Thisdevelopment
makes it possible to obtain live imagesof sperm in vivo (3).

We are also interested in the fertilization process in terms ofself-nonself recognition. By utilizing homologous recombination
technology, we showed protein IZUMO1 as the ﬁrst sperm factor thatplays an essential role in the fusion of sperm with eggs; we also foundrecently that the sperm protein SPESP1 is needed for the productionof fully fusion-competent sperm (Figs. 2 and 3) (1, 2).

Fig. 2. Izumo1 KO sperm accumulate in
the perivitelline space of the egg because
they cannot fuse with the egg (1).

Fig. 3. The membraneof the entire equatorial
segment area isdetached in almost all
acrosome-reacted
Spesp1-deﬁcient sperm(right) (2).

34

In addition to our studies on the sperm-egg interaction, we are studying the roles the placenta plays infeto-maternal immune tolerance. Since we believe gene functions are best observed in live animals, we sought amethod that would permit genetic manipulation of the placenta. We were eventually successful in developing a
Lentiviral vector-based method that mediates the genetic manipulation of the placenta without aﬀecting theembryos (Fig. 4) (4).

Research & Activities

Fig. 4. Placenta-speciﬁc gene manipulation.
GFP-transgene expression at E14.5 aftergene manipulation. Shown are embryos inmice with (left) an untransduced placenta,(middle) a placenta that had been
subjected to the normal transgenic
procedure, and (right) a placenta that had
been altered by our newly developed
method for placenta-speciﬁc genetic
manipulation (4).

We are also interested in understanding the biological function of non-coding RNA such as miRNA. The miRNAknock-out technique is being used to identify the roles miRNA plays in live animals.
Miwa s group is using genetically manipulated animals to investigate the molecular biological mechanisms thatare involved in human diseases, especially cardiovascular diseases. To understand the cellular and molecular
aspects of vascular smooth muscle (SM) cell growth in atherosclerotic plaques, we characterized thetranscriptional mechanisms of one SM-speciﬁc gene, the SM alpha-actin (SmaA) gene. Since SmaA is also
expressed in many tissues during acute inﬂammation, we are currently analyzing its gene expression system
and its functional roles (Fig. 5) (5). We are also analyzing the molecular pathogenic mechanisms of diastolic heart
failure by using a Dahl salt-sensitive rat model. Speciﬁcally, we are currently investigating how the endothelin
and renin-angiotensin systems participate in the excessive hypertrophy and ﬁbrosis that eventually leads todiastolic heart failure.

Fig. 5. The human SM alpha-actin
promoter (left) expresses in theembryonic aorta. However, point
mutations in the enhancer regionof this promoter, including the-1M (center) and 4M (right)
mutations, eliminate this speciﬁc
expression pattern (5).

Recent publications
1. Inoue N, Kasahara T, Ikawa M, Okabe M. Identiﬁcation and disruption of sperm-speciﬁc angiotensin converting
enzyme-3 (ACE3) in mouse. PLoS ONE. 2010. 5, e10301.
2. Fujihara Y, Murakami M, Inoue N, Satouh Y, Kaseda K, Ikawa M, Okabe M. Sperm equatorial segment protein 1,
SPESP1, is required for fully fertile sperm in mouse. J Cell Sci. 2010. 123, 1531-1536.
3. Hasuwa H, Muro Y, Ikawa M, Kato N, Tsujimoto Y, Okabe M. Transgenic mouse sperm that have green acrosome
and red mitochondria allow visualization of sperm and their acrosome reaction in vivo. Exp Anim. 2010.
59,105-107.
4. Okada Y, Ueshin Y, Isotani A, Saito-Fujita T, Nakashima H, Kimura K, Mizoguchi A, Oh-hora M, Mori Y, Oagata
M, Oshima RG, Okabe M, Ikawa M. Complementation of placental defects and embryonic lethality bytrophoblast-speciﬁc lentiviral gene transfer. Nat Biotechnol. 2007. 25, 233-237.
5. Takeji M, Moriyama T, Oseto S, Kawada N, Hori M, Imai E, Miwa T. Smooth muscle a-actin deﬁciency in
myoﬁbroblasts leads to enhanced renal tissue ﬁbrosis. J Biol. Chem. 2006. 281,40193-40200.

35Research & Activities

Genome Information Research Center

Department of Genome Informatics
Research Group

Professor
Professor (SUP)
Assistant Professor
Assistant Professor (SUP)SA Researcher

Teruo Yasunaga, Ph.D.
Tatsuya Takagi, Ph.D.
Naohisa Goto, Ph.D.
Norihito Kawashita, Ph.D.
U. Chandimal de Silva, M.Sc.

We use high performance computers to study the genome information of various organisms in an eﬀort toidentify new biological phenomena and to understand how organisms evolve. In addition, we develop softwaretools for bioinformatics and molecular biology. We also operate a computer system that can be used to analyze
genome sequence data. This system is available to researchers in our university and we hold training courses
in genome analysis at least once every year.
(1) Large-scale analysis of genomes
Today, the complete genome sequences of more than 1,000 organisms are available. We are currently analyzing
this enormous body of genome data by using bioinformatics and molecular evolution techniques. We are also
developing software and algorithms that facilitate large-scale genome analysis. These include CONSERV, a
conserved sequence ﬁnder. When this algorithm was used to analyze the complete genome sequences of 266
organisms, it identiﬁed invariant sequences that may have been present in the last common ancestor of all
extant life forms (Goto et al, 2007; Fig. 1). In addition, we are striving to understand the evolutionary pathwaysof the inﬂuenza virus by comprehensively analyzing its genome (Fig. 2).

Figure 1. Large-scale genome analysis of 266
organisms revealed a sequence that isconserved in almost all known genomes.

36

Figure 2. Comprehensive analysis of the inﬂuenza
virus genome.

Figure 3. Next generation sequencing
enables sequencing of an entire
genome in one run.

Research & Activities(2) Next generation sequencer data analysis
The recently developed next generation sequencing technology has made it possible to sequence the entire
genome of any microorganism in one sequencer run. Thus, each run produces a massive amount of nucleotide
sequence data. We are developing software that can handle this data and have set up an analysis system that isused for microorganism sequencing projects that are performed in collaboration with other laboratories (Fig. 3).

Figure 4. Genome Information ResearchCenter Computer System.

(3) Operation of a computer system that permits the analysis of genome information at Osaka University.
We provide computer resources for researchers in our university. We also provide mirrored access to major
nucleotide, protein, and genome databases through our servers (Fig. 4), which are fully synchronized with themother servers and kept up to date at all times.

Recent publications
１．Yamashita A, Kawashita N, Kubota-Koketsu R, Inoue Y, Watanabe Y, Ibrahim MS, Ideno S, Yunoki M, Okuno Y,
Takagi T, Yasunaga T, Ikuta K. Highly conserved sequences for human neutralization epitope on hemagglutininof inﬂuenza A viruses H3N2, H1N1 and H5N1: Implication for human monoclonal antibody recognition. Biochem
Biophys Res Commun. 2010 Mar 19;393(4):614-8.
２．Nakamura S, Yang CS, Sakon N, Ueda M, Tougan T, Yamashita A, Goto N, Takahashi K, Yasunaga T, Ikuta K,
Mizutani T, Okamoto Y, Tagami M, Morita R, Maeda N, Kawai J, Hayashizaki Y, Nagai Y, Horii T, Iida T, Nakaya
T. Direct metagenomic detection of viral pathogens in nasal and fecal specimens using an unbiased
high-throughput sequencing approach. PLoS One. 2009;4(1):e4219.
３．Yamashita A, Goto N, Nishiguchi S, Shimada K, Yamanishi H, Yasunaga T. Computational search forover-represented 8-mers within the 5'-regulatory regions of 634 mouse testis-speciﬁc genes. Gene. 2008 Dec
31;427(1-2):93-8.
４．Yoshida M, Yamashita A, Idoji Y, Nishiguchi S, Shimada K, Yasunaga T, Yamanishi H. In silico study of a novel
gene evolved from an ancestral SVIP gene and highly expressed in the adult mouse testes. Int J Mol Med. 2008
Aug;22(2):143-8.
５．Goto N, Kurokawa K, Yasunaga T. Analysis of invariant sequences in 266 complete genomes. Gene. 2007 Oct
15;401(1-2):172-80.

37

Research & Activities

Genome Information Research Center

Department of Infection Metagenomics
Research Group

Professor（SUP）
Professor（SUP）
SA Professor（SUP）
SA Associate Professor（SUP）
Assistant Professor（SUP）
SA Assistant Professor

Toshihiro Horii, Ph.D.
Teruo Yasunaga, Ph.D.
Tetsuya Iida, Ph.D.
Takaaki Nakaya, Ph.D.
Naohisa Goto, Ph.D.
Shota Nakamura, Ph.D.

1. RAPID (Robotics Assisted Pathogen IDentiﬁcation)
Under the aegis of the Program of Research Centers for Emerging and Re-emerging Infectious Diseases of
MEXT, Japan, we are constructing, in collaboration with the Omics Science Center, RIKEN, a framework called
RAPID that will facilitate the emergency diagnosis of infectious diseases. We are also cooperating with theresearch centers of eight countries in Asia and Africa in an eﬀort to identify the causative agents in naturally
occurring outbreaks.
2. Metagenomic Diagnosis of Infectious Diseases
Metagenomic analysis allows us to diagnose many of the major human infectious diseases (including respiratory
tract infections, enteric infections, and blood-borne infections) by using a single common protocol. In addition, topre-empt zoonotic disease outbreaks, we are seeking to identify new pathogenic microorganisms inanimal-derived samples that may have zoonotic potential.
3. Metagenomic Analysis of the Intestinal Microbiome
The intestinal microbiome plays an important role in protecting the host from pathogen invasion. We are
currently analyzing the intestinal microbiome of patients with diarrheal diseases to elucidate how the humanhost, the intestinal microbiome, and pathogenic microorganisms interact. This will help us to understand thechanges that take place in the intestinal microbiome during the course of infection.
4. Development of Novel Methods for Pathogen Detection
To develop more eﬃcient and comprehensive methods of identifying pathogens, we are studying the eﬃcacy of
diﬀerent methods that amplify the genome of pathogenic microorganisms and subtract the host genome.

Extract DNA or RNA
Next-generation sequencer

DATA analysis

Fig. 1. Metagenomic diagnosis of infectious diseases using a next-generation sequencer.

38

Research & Activities

Fig. 2. Distribution of detected organisms in viral infection cases

Fig. 3. Standard operating protocol for RAPID

Fig. 4. Next-generation sequencer,
454 GS Junior System

Recent publications
１．Nakamura S, Yang CS, Sakon N, Ueda M, Tougan T, Yamashita A, Goto N, TakahashiK, Yasunaga T, Ikuta K,
Mizutani T, Okamoto Y, Tagami M, Morita R, Maeda N, Kawai J, Hayashizaki Y, Nagai Y, Horii T, Iida T, Nakaya
T. Direct metagenomic detection of viral pathogens in nasal and fecal specimens using an unbiased
high-throughput sequencing approach. PLoS One. 2009;4(1):e4219.
２．Nakamura S, Maeda N, Miron IM, Yoh M, Izutsu K, Kataoka C, Honda T, YasunagaT, Nakaya T, Kawai J,
Hayashizaki Y, Horii T, Iida T. Metagenomic diagnosis of bacterial infections. Emerg Infect Dis. 2008
Nov;14(11):1784-6.

39

Research & Activities

International Research Center for Infectious Diseases
Department of Special Pathogens

Laboratory for Clinical Research on Infectious Diseases
Research GroupSA Professor
Assistant Professor
Postdoctoral Fellow
Postdoctoral Fellow
Research Staﬀ

Kazunori Oishi M.D., Ph.D.
Yukihiro Akeda Ph.D.
Tatsuya Nakayama Ph.D.
Zhenyu Piao Ph.D.
Yumi Hattori

The research activities in our department aim to 1) investigate the epidemiology, pathogenesis and prevention
by vaccines of pneumonia and invasive bacterial infections, 2) elucidate the mechanisms by which dengue
viruses induce disease, and 3) analyze the protein secretion systems of pathogenic bacteria. In addition, ourgroup is registered as a member of the World Health Organization (WHO)/Global Outbreak Alert & Response
Network (GOARN) and, when necessary, will join the outbreak response team in the global eﬀort to control
emerging and re-emerging infectious diseases.
1) Epidemiology, pathogenesis, and vaccine-mediated prevention of pneumonia and invasive bacterial infections
1. Study of pneumonia in Thailand
We have conducted a study project entitled "Surveillance of emerging respiratory infections and analysis ofmechanism of secondary bacterial pneumonia in Thailand". This project allowed us to investigate howvirus-host-bacteria interactions promote secondary bacterial infections in pediatric patients with pneumonia. In
2009, we examined the clinical features of 24 adult cases of pandemic H1N1 inﬂuenza-associated severe
community-acquired pneumonia at Buddachinaraj Hospital, Phitsanulok.
2. Clinical applications of the 23-valent pneumococcal polysaccharide vaccine (PPV) and the development of newpneumococcal vaccinesa) Clinical application of 23-valent PPV
We found that the combined PPV and inﬂuenza vaccine (IV) vaccination program reduced the incidence ofacute exacerbation in patients with chronic obstructive pulmonary diseases (Vaccine, 2008). An open-label,randomized study was conducted involving 786 Japanese subjects older than 65 years of age who werereceiving a routine IV. Concomitant PPV vaccination signiﬁcantly reduced the number of admissions andmedical costs for all-cause pneumonia for subjects older than 75 years. We also started a project in 2008 thatexamines the eﬀects of PPV in combination with IV on long-term‒care residents. Our goal is the nationwide androutine vaccination of the elderly in Japan.
b) Development of a nasal mucosal pneumococcal vaccine
Pneumococcal surface protein A (PspA) is known to elicit protective antibodies in animals. We havedemonstrated the eﬀects of PspA plus TLR agonist on bacterial clearance in a mouse model of pneumococcal
pneumonia (Vaccine, 2009). In addition, we have shown that the PspA nasal vaccine is eﬀective in a mousemodel of secondary pneumonia after inﬂuenza virus infection.
3. Research on Streptococcus suis infections, which are prevalent in Thailand
Streptococcus suis is an important zoonotic pathogen that causes invasive infections such as meningitis in humans
who are in close contact with infected pigs or contaminated pork-derived products. The number of such humancases is rapidly increasing in Thailand because of a tradition of consuming raw pork or blood in the north. We
showed that the clinical manifestations of serotype 2 infections are related to the genotypic proﬁles of theisolates; we also reported the clonal dissemination in humans of serotype 14, which has been a rarely occurring
serotype up until now (Figure 1). We are currently developing an epidemiological study of S. suis infections in
Phayao Province as a project of RCC-DMSc.

40

Research & Activities

2) Mechanisms by which dengue virus infections lead to thrombocytopenia
Dengue illness has become a major public health concern, particularly in tropical countries. We have conducted
a number of clinical studies in the Philippines and recently found in an ex vivo setting that patients withthrombocytopenia during acute phase secondary dengue virus infections showed increased phagocytosis ofplatelets (AJTMH, 2009). Since we found that IVIG treatment did not signiﬁcantly hasten the recovery fromthrombocytopenia in such patients (AJTMH, 2007), this suggests that the Fcγ receptor is not involved inplatelet phagocytosis by macrophages. We are currently elucidating the novel mechanism by which theplatelets are phagocytosed in this disease.
3) Protein secretion systems of pathogenic bacteria
The development of bacterial infections requires many virulence factors. Since most are proteins that are
secreted by the pathogenic bacteria, it is essential to study the mechanisms by which proteins are secreted bypathogenic bacteria during the course of infection. We are using the food-borne pathogen Vibrio parahaemolyticus
and a causative agent of pneumonia, Streptococcus pneumoniae, to study such protein secretion systems and thesecreted virulence factors.
4) Response to emerging and re-emerging infectious diseases
The objective of GOARN is to combat the international spread of infectious disease outbreaks by ensuring thatappropriate technical assistance reaches the aﬀected areas rapidly and by promoting long-term epidemic
preparedness. Our group is registered as a member of GOARN and our team will join the WHO-organizedresponse team when there is an outbreak of infectious diseases in developing countries.

Figure 1. Assumed routes of transmission and pathogenesis of S. suis infection, and the clonal
dissemination of serotype 14 infections in Thailand (J Med Microbiol, 2009).

Recent publications
１．Kerdsin A, Oishi K. Sripakdee S, Boonkerd N, Polwichai P, Nakamura S, Uchida R, Sawanpanyalert P, Dejsirilert
S. Clonal Dissemination of Streptococcus suis serotype 14 in Thailand. J Med Microbiol. 2009 Nov;58(Pt 11):1508-13.
２．Honda S, Saito M, EM Dimaano, Morales PA, Alonzo MTG, Suarez LC, Koike N, Inoue S, Kumatori A, Matias RR,
Natividad FF, Oishi K. Increased platelet phagocytosis from patients with secondary dengue virus Infection byhuman macrophages. Am J Trop Med Hyg. 2009 May;80(5):841-5.
３．Akeda Y, Okayama K, Kimura T, Dryselius R, Kodama T, Oishi K, Iida T, Honda T. Identification andcharacterization of a type III secretion-associated chaperone in the type III secretion 1 of Vibrio parahaemolyticus.
FEMS Microbiol Lett. 2009 Jul;296(1):18-25.
４．Oma K, Zhao J, Ezoe H, Akeda Y, Koyama S, Ishii KJ. Kataoka K, Oishi K. Intranasal immunization with amixture of PspA and a Toll-like receptor agonist induces specific antibodies and enhances bacterial clearance inthe airways of mice. Vaccine. 2009 May 21;27(24):3181-8.
５．Chen M, Ssali F, Mulungi M, Awio P, Yoshimine H, Kuroki R, Furumoto A. Tanimura S, Kityo C, Nagatake T,
Mugyenyi P, Oishi K. Induction of opsonophagocytic killing activity with pneumococcal conjugate vaccine inhuman immunodeficiency virus-infected Ugandan adults. Vaccine. 2008 Sep 8;26(38):4962-8.
６．Furumoto A, Ohkusa Y, Chen M, Kawakami K, Masaki H, Sueyasu Y, Iwanaga T, Aizawa H, Nagatake T, Oishi K.
Additive effect of pneumococcal vaccine and influenza vaccine on acute exacerbation in patients with chroniclung disease. Vaccine. 2008 Aug 5;26(33):4284-9.

41

Research & Activities

International Research Center for Infectious Diseases
Department of Special Pathogens

Laboratory for Infection Cell Biology
Research GroupSA Associate Professor
SA Researcher
Postdoctoral Fellow

Yukako Fujinaga, Ph.D.
Yo Sugawara, Ph.D.
Takuhiro Matsumura, Ph.D.

Many bacterial toxins are able to severely damage the host, even at very low concentrations. Most are enzymes
that act catalytically and with high speciﬁcity on functional host cell molecules, thereby markedly modulating
host homeostasis. The toxins are also often highly eﬃcient in accessing their target molecule in the host. The
ingenious transport systems involved often exploit the fundamental membrane traﬃcking machinery and thefunctions of intracellular organelles. Therefore, studies seeking to elucidate toxin traﬃcking could provide us
with valuable information about basic cellular function, as well as aiding our understanding of the pathology
induced by these toxins and helping us to develop eﬀective therapeutic strategies against them. We are
currently engaged in studying the transport mechanisms of the botulinum neurotoxin complex, which must
pass down the digestive tract and cross the epithelial barrier lining the intestine to cause food-borne botulism.

Figure (a) Schematic depiction of thebotulinum neurotoxin complex (16S
and 12S toxins). Orally ingested
neurotoxin complexes cross theintestinal epithelial barrier to cause
food-borne botulism. (b) 16S toxin(green) penetrates the intestinal
epithelium. (c) Interaction of botulinum
neurotoxin complexes with theintestinal epithelial barrier. The HA ofthe botulinum neurotoxin complex
binds
E-cadherin
anddisrupts
E-cadherin‒mediatedcell-to-celladhesion, thereby disrupting theepithelial paracellular barrier.

Recent publications
１．Sugawara Y, Matsumura T, Takegahara Y, Jin Y, Tsukasaki Y, Takeichi M, Fujinaga Y. Botulinum HA disrupts
the intercellular epithelial barrier by directly binding E-cadherin. J Cell Biol 2010., (in press)
２．Jin Y1, Takegahara Y1, Sugawara Y, Matsumura T, Fujinaga Y. Disruption of the epithelial barrier by botulinum
hemagglutinin (HA) proteins - Diﬀerences in cell tropism and the mechanism of action between HA proteins oftypes A or B, and HA proteins of type C. Microbiology. 2009 Jan; 155(Pt 1): 35-45. 1 These authors are contributed
equally.
３．Matsumura T, Jin Y, Kabumoto Y, Takegahara Y, Oguma K, Lencer WI, Fujinaga Y. The HA proteins ofbotulinum toxin disrupt intestinal epithelial intercellular junctions to increase toxin absorption. Cell Microbiol. 2008
Feb; 10(2): 355-364.
４．Matsumura T1, Fujinaga Y1, Jin Y, Kabumoto Y, Oguma K. Human milk SIgA binds to botulinum type B 16S toxin
and limits toxin adherence on T84 cells. Biochem Biophys Res Commun. 2007 Jan 26; 352(4), 867-872.
５．Fujinaga Y. Transport of bacterial toxins into target cells: Pathways followed by cholera toxin and botulinum
progenitor toxin. J Biochem. 2006 Aug; 140(2), 155-160.
42

Research & Activities

International Research Center for Infectious Diseases
Department of Special Pathogens

Research GroupSA Associate Professor Takaaki Nakaya, Ph.D.
Postdoctoral Fellow
Tomo Daidoji, D.V.M., Ph.D.

Research & Activities

Laboratory for Viral Pathogenesis and Immunity◆ Molecular mechanism of H5N1 avian inﬂuenza virus pathogenesis
In recent years, the highly pathogenic avian inﬂuenza virus (AIV) H5N1 emerged from southeast Asia andraised serious worldwide concern about the risk of an inﬂuenza pandemic. However, how H5N1 induces diseaseremains poorly understood. We are using in vitro and animal experiments to study the role the viral
glycoprotein hemagglutinin (HA) plays in viral growth and cell toxicity. Recent achievements are listed below:
By genetically changing H5 AIV by recombinant DNA techniques, H5N1-HA has been shown to be one of the
major viral factors that determine lethality in mice. We conﬁrmed that the pathogenicity of HA depends on its
cleavage sequence, which is consistent with previous observations. However, our further experiments suggestthat other region(s) and amino acids of H5N1-HA may also participate in the pathogenicity of H5N1.
That H5N1-HA can induce signiﬁcant cellular toxicity was demonstrated by in vitro experiments. We showedthat H5N1-HA‒speciﬁc cell toxicity (apoptosis) was observed in porcine as well as human primary airway
epithelial cells. Similar results were also observed in primary cells from a water fowl known to be a natural AIV
host. In contrast, HA proteins from previously isolated AIVs, including the H5 subtype, did not induce thissevere cell toxicity. Thus, the genotype of HA may be critical for the pathogenicity and/or cellular toxicity of
H5N1 AIVs.
Metagenomic analysis(human specimens)

Pathogenesis
in vitro infection experiments(primary cells form human airways)of FluAnimal experiments(recombinant inﬂuenza viruses)◆ Metagenomic analysis of viral pathogens in humans: Development of Pathogen Identiﬁcation System byusing a high-throughput Next-Generation DNA sequencer (RAPID system; Department of Infection
Metagenomics)
We are establishing a protocol to rapidly obtain the whole genome information of viral pathogens. This is
expected to signiﬁcantly accelerate the speed with which pathogens can be identiﬁed. Using this protocol, we
have successfully demonstrated the presence of pathogenic microbes in clinical human samples without
resorting to conventional selective procedures for speciﬁc pathogens.
Recent publications
１．Daidoji T, Kaihatsu K, Nakaya T. The role of apoptosis in inﬂuenza virus pathogenesis and the mechanisms
involved in anti-inﬂuenza therapies. Curr Chem Biol [Review, in press]
２．Ueda M, Daidoji T, Du A, Yang C-S, Ibrahim M-S, Ikuta K, Nakaya T. Highly pathogenic H5N1 avian inﬂuenza
virus induces extracellular Ca2+ inﬂux, leading to apoptosis in avian cells. J Virol. 2010 84(6):3068-78.
３．Nakamura S, Yang C-S, Sakon N, Ueda M, Tougan T, Yamashita A, Goto N, Takahashi K, Yasunaga T, Ikuta K,
Mizutani T, Okamoto Y, Tagami M, Morita R, Maeda N, Kawai J, Hayashizaki Y, Nagai Y, Horii T, Iida T,
Nakaya T. Direct metagenomic detection of viral pathogens in nasal and fecal specimens using an unbiased
high-throughput sequencing approach. PLoS ONE. 2009 4(1):e4219.
４．Du A, Daidoji T, Koma T, Ibrahim M-S, Nakamura S, de Silva U-C, Ueda M, Yang C-S, Yasunaga T, Ikuta K,
Nakaya T. Detection of circulating Asian H5N1 viruses by a newly established monoclonal antibody. Biochem
Biophys Res Commun. 2009 378(2):197-202.
５．Daidoji T, Koma T, Du A, Yang C-S, Ueda M, Ikuta K, Nakaya T. H5N1 avian inﬂuenza virus induces apoptotic
cell death in mammalian airway epithelial cells. J Virol. 2008 82(22): 11294-307.

43

Research & Activities

International Research Center for Infectious Diseases
Department of Infectious Disease Control

Laboratory of Genomic Research on Pathogenic Bacteria
Research GroupSA Professor
Postdoctoral Fellow
Postdoctoral Fellow
Postdoctoral Fellow
Postdoctoral Fellow

Tetsuya Iida, Ph.D.
Kaori Izutsu, Ph.D.
Shigeaki Matsuda, Ph.D.
Natsumi Okada, Ph.D.
Hirotaka Hiyoshi, Ph.D.

This research group is studying pathogenic bacteria from the genomic point of view.
Our main research targets are as follows:
1. Characterization of the mechanism(s) used by bacterial pathogens to infect host organisms by identifying
infection-related changes in pathogen genome expression: To understand the molecular mechanisms by which
bacterial pathogens infect host organisms, we are using DNA microarrays and other molecular methods toinvestigate the changes in the genome expression pattern of various bacterial pathogens that occur during theirinteraction with their target host.

Figure 1. Whole genome sequence of Vibrio parahaemolyticus

2. Analysis of the mechanism(s) that lead to the emergence of new infectious diseases: The unique features ofvarious newly emerged bacterial pathogens are being studied by analyzing their genomes and comparing themwith those of other bacterial strains.

44Research & Activities

3. Investigation of the life cycles of bacterial pathogens in their natural environment: Based on what is currently
understood about various bacterial pathogens, we are seeking to characterize their life cycles in their natural
habitats.

Figure 2. Characterization and comparison of the genomes of pathogenic
bacteria by using DNA microarrays

4. Development of new methods for the rapid identiﬁcation of bacterial pathogens based on genomic
information: To rapidly diagnose bacterial infections, a novel system for identifying bacterial pathogens bylarge-scale DNA sequencing is being developed.

Recent publications
１．Kodama T, Gotoh K, Hiyoshi H, Morita M, Izutsu K, Akeda Y, Park KS, Cantarelli VV, Dryselius R, Iida T, Honda
T. Two regulators of Vibrio parahaemolyticus play important roles in enterotoxicity by controlling the expression ofgenes in the Vp-PAI region. PLoS One. 2010 Jan 13;5(1):e8678.
２．Okada N, Iida T, Park KS, Goto N, Yasunaga T, Hiyoshi H, Matsuda S, Kodama T, Honda T. Identiﬁcation andcharacterization of a novel type III secretion system in trh-positive Vibrio parahaemolyticus strain TH3996 reveal
genetic lineage and diversity of pathogenic machinery beyond the species level. Infect Immun. 2009
Feb;77(2):904-13.
３．Dryselius R, Izutsu K, Honda T, Iida T. Diﬀerential replication dynamics for large and small Vibrio chromosomes
aﬀect gene dosage, expression and location. BMC Genomics. 2008 Nov 26;9:559.
４．Nakamura S, Maeda N, Miron IM, Yoh M, Izutsu K, Kataoka C, Honda T, Yasunaga T, Nakaya T, Kawai J,
Hayashizaki Y, Horii T, Iida T. Metagenomic diagnosis of bacterial infections. Emerg Infect Dis. 2008
Nov;14(11):1784-6.
５．Kodama T, Rokuda M, Park KS, Cantarelli VV, Matsuda S, Iida T, Honda T. Identiﬁcation and characterization of
VopT, a novel ADP-ribosyltransferase eﬀector protein secreted via the Vibrio parahaemolyticus type III secretion
system 2. Cell Microbiol. 2007 Nov;9(11):2598-609.

45

Research & Activities

International Research Center for Infectious Diseases
Department of Infectious Disease Control

Laboratory of Malariology
Research Group

Invited Professor
Postdoctoral Fellow
Postdoctoral Fellow
JSPS PD Fellow

Kazuyuki Tanabe, D. Sc., D. Med. Sc.
Kenji Hikosaka, D. Agr. Si.
Shin-ichiro Tachibana, D. Agr. Si.
Hajime Honma, D. Agr. Si.

Malaria imposes a huge burden on human health. We are studying the genetic diversity of malaria parasites inorder to elucidate their history and adaptive evolution mechanisms.
(1) Population genetics of malaria parasites
The human malaria parasite Plasmodium falciparum infects only humans. Consequently, the evolution of thisparasite is presumed to be intimately associated with humans. To examine this association, we are investigating
the genetic diversity of P. falciparum populations from diverse geographic areas. We have observed a clear
negative correlation between the within-population genetic diversity and the geographic distance from East
Africa to Asia and Oceania, which mirrors the isolation-by-distance of modern humans. Age estimates support
the notion that modern humans carried the parasite along during their colonization of the world.

Fig. 1. Negative correlation between the within-population
genetic diversity of P. falciparum and the geographic distancefrom East Africa to Asia and Oceania. This suggests that theparasite spread together with modern humans during theirout-of-Africa colonization.

(2) The evolution of antigenic polymorphism in malaria parasites
One way malaria parasites evade the host s immune system is through antigenic polymorphism. We areinvestigating human parasite populations from diverse geographic areas, as well as monkey malaria parasites, toreveal the evolutionary origins and population genetic mechanisms that drive antigenic polymorphism inimmune-target genes.
(3) Co-evolution between malaria parasites and their hosts
While malaria parasites infect all classes of terrestrial vertebrates (mammals, birds, and reptiles), each parasite
species infects only one speciﬁc host. Such wide host range and high host speciﬁcity indicate that malaria
parasites have a close relationship with their hosts. To examine this parasite-host co-evolution, we compared theevolutionary histories of malaria parasites and their hosts. We found that all of the extant lineages of malaria
parasites originated during a period of rapid diversiﬁcation that involved host switches.

46

Research & Activities

Fig. 2. Malaria big bang in theevolution of extant malaria
parasites. The diversiﬁcation(beige bar) occurred muchlater than the emergence of
host animals, which suggeststhat host switching led to thediversiﬁcation of the parasite
lineage.

(4) Genome sequencing of a P. vivax-related monkey malaria parasite, P. cynomolgi
The human malaria parasite P. vivax became a parasite of humans by host switching from a monkey. To clarify
the genetic changes that are speciﬁc to P. vivax, we are genome sequencing a P. vivax-related monkey malaria
parasite, P. cynomolgi. The sequencing is currently at the gap closure and annotation step, and a draft genome
will be complete soon. Comparative genomics has already revealed gene gains/losses that are speciﬁc to P. vivax.

Fig. 3. Genome sequencing of P. cynomolgi,
a monkey malaria parasite that is closely
related to P. vivax, by using the new
sequencing system (in collaboration with
Dr. S. Kawai at Dokkyo Med. Univ.).

Recent publications
１．Tanabe K, Mita T, Jombart T, Eriksson A, Horibe S, Palacpac N, Randord-Cartwright L, Sawai H, Sakiyama N,
Ohmae H, Nakamura M, Ferreira MU, Escalante AA, Prugnolle F, Björkman A, Färnert A, Kaneko A, Horii T,
Manica A, Kishino H, Balloux F. Plasmodium falciparum accompanied the human expansion out of Africa. Curr. Biol.
2010 (in press).
２．Hikosaka K, Watanabe Y, Tsuji N, Kita K, Kishine H, Arisue N, Palacpac N M Q, Kawazu S, Sawai H, Horii T,
Igarashi I, Tanabe K. Divergence of mitochondrial genome structure in the apicomplexan parasites, Babesia and
Theileria. Mol Biol Evol. 2010 May; 27 (5): 1107-16.
３．Sawai H, Otani H, Arisue N, Palacpac N, de Oliveira Martins L, Pathirana S, Handunnetti S, Kawai S, Kishino H,
Horii T, Tanabe K. Lineage-speciﬁc positive selection at the merozoite surface protein 1 (msp1) locus of Plasmodium
vivax and related simian malaria parasites. BMC Evol Biol. 2010 Feb 19; 10: 52.
４．Culleton R, Ndounga M, Zeyrek F Y, Coban C, Casimiro P N, Takeo S, Tsuboi T, Yadava A, Carter R, Tanabe K.
Evidence for the transmission of Plasmodium vivax in the Republic of Congo, West Central Africa. J Infect Dis. 2009
Nov 1; 200 (9): 1465-9.
５．Hayakawa T, Culleton R, Otani H, Horii T, Tanabe K. Big bang in the evolution of extant malaria parasites. Mol
Biol Evol. 2008 Oct; 25 (10): 2233-9.

47

Research & Activities

International Research Center for Infectious Diseases
Department of Infectious Disease Control

Laboratory of Combined Research on Microbiology and Immunology
SA Associate Professor

Research Group

Hiroki Nagai, Ph.D.

Protein secretion is a process of fundamental importance for bacterial pathogenesis. Whether they deliver
toxins or directly inject eﬀector proteins into the cytoplasm of host cells, bacterial protein secretion systems
play a central role in modulating eukaryotic cell functions. Legionella pneumophila are Gram-negative bacteriathat are found ubiquitously in soil and freshwater environments. Once inhaled by humans, Legionella infections
can result in a severe form of pneumonia known as Legionnaires' disease. Legionella use a type IV secretion
system to deliver eﬀector proteins, and this mediates the establishment of a replicative niche in host cells. The
goal of our research is to understand at the molecular level how Legionella subvert host cellular functions toaccomplish their successful intracellular replication. To this end, the following research projects are currently
in progress.
(1) Analysis of the structure and function of the type IV secretion apparatus.
There is essentially nothing known about the substrate transfer across eukaryotic and bacterial membranes
that occurs via type IV secretion systems. Furthermore, the macromolecular structure of the type IV secretion
apparatus is largely unknown. To address these questions, we are working towards the structural andfunctional analysis of the type IV secretion apparatus from Legionella.
(2) Analysis of eﬀector proteins that translocate from Legionella to host cells.
We previously demonstrated that RalF is translocated by the type IV secretion system of Legionella into hostcells and is required for the recruitment of host ARF proteins to Legionella-containing vacuoles. We also
recently demonstrated that the eﬀector LubX acts as an E3 ligase and targets another eﬀector for proteasomal
degradation within host cells. LubX is the ﬁrst eﬀector protein that has been shown to target and regulate
another eﬀector within host cells.

Fig. 1 Type IV apparatus localizes tobacterial poles (Green).

Fig. 2 Discovery of metaeﬀector.

Recent publications
１．Kubori, T., Hyakutake, A. and Nagai, H. Legionella translocates an E3 ubiquitin ligase that has multiple U-boxes
with distinct functions. Mol. Microbiol. 2008;67(6),1307-1319.
２．Nagai, H., Cambronne, E.D., Kagan, J.C., Amor, J.C., Kahn, R.A. and Roy, C.R.. A C-terminal translocation signal
required for Dot/Icm-dependent delivery of the Legionella RalF protein to host cells. Proc. Natl. Acad. Sci. USA.
2005;102,826-831.
３．Amor,J.C., Swails, J., Roy, C.R., Nagai, H., Ingmundson, A., Cheng, X., and Kahn, R.A. The structure of RalF, an
ARF guanine nucleotide exchange factor from Legionella pneumophila, reveals the presence of a cap over theactive site. J. Biol. Chem, 2005;280, 1392-1400.

48

Research & Activities

Oﬃce of Combined Program on Microbiology and
Immunology

Research Group

Research promotion group
Education promotion group

Associate Professor
Associate Professor

Yoshiko Murakami, M.D., Ph.D.
Hodaka Fujii, M.D., Ph.D.

Research promotion
To directly promote cross-disciplinary microbiology and immunology research, we are implementing thefollowing plans.
1. Organization of the Awaji international forum on infection and immunology, which is held in September
annually.
2. Organization of the research progress report, which is produced every two months by our institute.
3. Organization of the symposium and the research presentation, which are held annually.
4. Organization of interactive projects that involve the Institut Pasteur in France, Chonnam University in Korea,
and the Research Collaboration Center on Emerging and Re-emerging Infections in Thailand.
These activities aim to facilitate cooperative research on microbiology and immunology by promoting researchcollaboration, information exchange, personal exchange between laboratories, and preparing the research
environment.

Research & Activities

Oﬃce activities
Our institute and the Immunology Frontier Research Center are world premier institutes in the ﬁelds ofmicrobiology and immunology, respectively. These institutes are located next to each other. To take maximum
advantage of this situation, our oﬃce works to encourage cross-disciplinary microbiology and immunology
research as follows:

Research & Activities

Oﬃce of Combined Program on Microbiology and Immunology

Education promotion
To facilitate seamless cross-disciplinary research on microbiology and immunology, we also direct a
multidisciplinary graduate program on microbiology and immunology. This task includes designing thecurriculum and its contents. We also organize an Open House of the institute and guide new students.

Research Group

Associate Professor

Yoshiko Murakami, MD. Ph.D

I have an additional appointment in the Department of Immunoregulation, where I serve as the leader of the
PNH group. This group is performing the following studies (see details on the Department page):
1. Investigation of the pathogenesis of paroxysmal nocturnal hemoglobinuria (PNH), an acquired
glycosylphosphatidylinositol (GPI) deﬁciency.
2. Investigation of the pathogenesis of inherited GPI deﬁciency.
3. Investigation of the functional signiﬁcance of GPI-anchored proteins by using the Pgap3 KO mouse in which
GPI-anchored proteins fail to localize within the raft due to defective GPI anchor remodeling.
Recent publications
1. Sena CB, Fukuda T, Miyanagi K, Matsumoto S, Kobayashi K, Murakami Y, Maeda Y, Kinoshita T, Morita YS.
Controlled expression of branch-forming mannosyltransferase is critical for mycobacterial lipoarabinomannan
biosynthesis. J Biol Chem. 2010 Mar 9. doi: 10.1074/jbc.M109.077297
2. Kanzawa N, Maeda Y, Ogiso H, Murakami Y, Taguchi R, Kinoshita T. Peroxisome dependency of alkyl-containing
GPI-anchor biosynthesis in the endoplasmic reticulum. Proc Natl Acad Sci U S A. 2009 Oct 20;106(42):17711-6.
3. Almeida AM*, Murakami Y*, Baker A, Maeda Y, Roberts IA, Kinoshita T, Layton DM, Karadimitris A. Targeted
therapy for inherited GPI deficiency. N Engl J Med. 2007 Apr 19;356(16):1641-7(* equally contributed).
4. Almeida AM*, Murakami Y*, Layton M, Hillmen P, Sellick G, Maeda Y, Richards S, Patterson S, Kotsianidis I, Mollica
L, Crawford D, Baker A, Ferguson M, Roberts I, Houlston R, Kinoshita T, Karadimitris A. Hypomorphic promoter
mutation in the mannosyltransferase-encoding PIG-M gene causes inherited glycosylphosphatidylinositol
deficiency. Nat. Med., 12:846-851. 2006(* equally contributed).
5. Inoue N, Izui-Sarumaru T, Murakami Y, Endo Y, Nishimura J, Kurokawa K, Kuwayama M, Shime H, Machii T,
Kanakura Y, Meyers G, Wittwer C, Chen Z, Babcock W, Frei-Lahr D, Parker C, Kinoshita T. Molecular basis ofclonal expansion of hematopoiesis in two patients with paroxysmal nocturnal hemoglobinuria (PNH). Blood 2006
108:4232-4236.

49Research & Activities

Oﬃce of Combined Program on Microbiology and
Immunology

Fujii Group
Research Group

Associate Professor Hodaka Fujii, M.D., Ph.D.
Assistant Professor Toshitsugu Fujita, Ph.D.

We are developing novel technologies to address important questions
in biology. In addition, we are analyzing mechanisms that regulatetheimmunesystembyusingstate-of-the-art
transgenic/knock-out/knock-in technologies.
I. Development of novel technologies to elucidate fundamental
principles of the immune system(1) We developed the inducible translocation trap (ITT) system toidentify the signal-induced nuclear translocation of signaling proteins(Fig. 1). ITT is the ﬁrst non-protein‒speciﬁc technology that canidentify nuclear-translocating proteins; it also enables the analysis ofthe "translocatome", namely the entire set of proteins that translocate
to the nucleus in response to a deﬁned extracellular stimulus. We willuse ITT to (i) identify and characterize signal-induced
nuclear-translocating proteins, (ii) perform high-throughput screeningof small compounds that aﬀect the nuclear translocation of particularsignaling proteins, and (iii) screen an RNAi library to identify proteinsthat regulate the nuclear translocation of signaling proteins.
(2) We are developing the insertional chromatin immunoprecipitation(iChIP) system to isolate speciﬁc genomic regions that retain their invivo conformation. This system will enable us to perform unbiased
molecular biological and biochemical analyses of the chromatin
structure of speciﬁc genomic regions and to identify the molecules(proteins, DNA, RNA, and others) that interact with these regions.
iChIP will also help us to elucidate molecular transcriptional
regulation, cell diﬀerentiation, and lineage commitment mechanisms,
especially those involved in lymphocyte development.
II. Analysis of immune regulation mechanisms and development oftherapies for autoimmune diseases(1) We identiﬁed a novel phosphorylated nuclear protein, Cyclon,
whose expression is induced in T cells when they are activated. We
found that Cyclon regulates the activation-induced cell death of T
cells by modulating the expression levels of Fas (Fig. 2). We are
currently using transgenic and gene-deﬁcient mice to analyze the invivo functions of Cyclon and the molecular mechanisms by which it
regulates Fas expression. The knowledge generated by these studies
will be used to develop eﬀective therapies of autoimmune diseases.
(2) We identiﬁed GARP, an activated T-regulatory cell (T-reg)-speciﬁccell surface molecule, and showed that it plays an important role in
the immune suppression that is mediated by T-regs. We are
currently elucidating the in vivo function of GARP by usingtransgenic and gene-deﬁcient mice.

Figure 1. The inducible translocation
trap system.

Figure 2. Normalization of the splenomegaly ininterleukin-2 receptor α-chain‒deﬁcient miceby the transgenic expression of Cyclon.

Recent publications

50

１．Hoshino A, Fujii H. Insertional chromatin immunoprecipitation: a method for isolating specific genomic regions. J
Biosci Bioeng. 2009 Nov;108(5):446-9.
２．Saint Fleur S, Hoshino A, Kondo K, Egawa T, Fujii H. Regulation of Fas-mediated immune homeostasis by anactivation-induced protein, Cyclon. Blood. 2009 Aug 13;114(7):1355-65.
３．Wang R, Kozhaya L, Khaitan A, Fujii H, Unutmaz D. Expression of GARP selectively identifies activated human
Foxp3+ regulatory T cells. Proc Natl Acad Sci U S A. 2009 Aug 11;106(32):13439-44.
４．Singh AP, Buscaglia CA, Wang Q, Levay A, Nussenzweig DR, Walker J, Winzeler EA, Fujii H, Fonoura BMA,
Nussenzweig, V. Plasmodium circumsporozoite protein promotes the development of the liver stages of theparasite. Cell. 2007 Nov 2;131(3):492-504.
５．Hoshino A, Hirst JA, Fujii H. Regulation of cell proliferation by interleukin-3-induced nuclear translocation of
pyruvate kinase. J Biol Chem. 2007 Jun 15;282(24):17706-11.

Research Collaboration Center in Overseas
Research & Activities

Thailand - Japan
Research Collaboration Center on
Emerging and Re-emerging Infections
SA Professor

Shigeyuki Hamada, D.D.S., Ph.D.

It was believed until recently that infectious diseases could be conquered through the development ofchemotherapies and vaccines. However, the recent worldwide emergence of new infectious diseases andreemergence of infectious diseases that were once considered to be controlled has seriously challenged thisnotion. Under these circumstances, intensive research that closely monitors and rapidly analyzes emerging andre-emerging infections is urgently required. Since a variety of infectious diseases can spread rapidly acrossnational borders, it is obvious that these diseases cannot be controlled by the independent eﬀorts of individual
nations.

Research Collaboration Center in Overseas

Director

To this end, Osaka University founded the Research Collaboration
Center on Emerging and Re-emerging Infections (RCC-ERI) in theThai National Institute of Health (NIH), Department of Medical
Sciences, Ministry of Public Health of Thailand in 2005.
The facility consists of P2 and P3 biohazard containment
laboratories and various other equipment and facilities on 600-m2 of
ﬂoor space. Previously, most of the research projects conductedabroad were short-term, with the researchers only staying for a
few months to complete their experiments. Due to the installationof the RCC-ERI, researchers are now able to stay for longer
periods of time. The RCC-ERI aims to carry out research projects
on both emerging and re-emerging infections in close collaboration
with the researchers at the NIH, while at the same timedeveloping the talents of young scientists from Japan andSoutheast Asian countries in the ﬁeld of infection.
To conduct basic and applied research and to develop humanresources, the RCC-ERI consists of two sections that are devoted
to bacterial and viral infection research. In addition, we aim toestablish an eﬀective system that would (i) provide informationthat would help to prevent the emergence of emerging andre-emerging infections, and (ii) promptly activate a variety ofcountermeasures for such a disease emerged, including developing
therapeutics or vaccines. Finally, we wish to begin collaborations
with laboratories from the nations that neighbor Thailand so that
we can be at the frontline with the capacity to quickly respond toany globally spreading infectious disease.

P2-level laboratory

P3-level laboratory
51

Research Collaboration Center in Overseas
Section of Bacterial Infections
Research GroupSA Professor
SA Lecturer
SA Researcher
SA Researcher
Research Fellow
Research Fellow

Shigeyuki Hamada, D.D.S., Ph.D.
Yumi Kumagai, Ph.D.
Kazuhisa Okada, Ph.D.
Natsuko Kishishita, Ph.D.
Amonrattana Roobthaisong, M.Sc.
Chetsada Boonthimat, M.Sc.

The Section of Bacterial Infections pays special attention to emerging and reemerging bacterial diseases thatare prevalent or are broken out in Asian countries. We study the molecular epidemiology of enteric or systemic
bacterial infections. Moreover, in collaboration with the National Institute of Health, Department of Medical
Sciences, Ministry of Public Health of Thailand, we develop detection and identiﬁcation techniques for the
diagnosis of bacterial diseases.
It has been reported that pneumonia, tuberculosis, and acute diarrheal diseases are associated with highmorbidity and mortality rates in Thailand. Consequently, in the Program of Promotion of Research Network for
Emerging and Reemerging Infectious Diseases during the 2010‒2014 ﬁscal years, we prioritize research on
enteric infectious diseases in Thailand that are caused by Vibirio cholerae and diarrheagenic E. coli.
In addition, Streptococcus suis, which is generally pathogenic and frequently isolated from diseased pigs, has beenfound to cause several systemic (zoonotic) infectious diseases in humans, namely meningitis, infective
endocarditis, and toxic shock-like syndrome. This has mainly been observed in Asian countries, including
Northern Thailand. We survey this emerging zoonotic infection closely, and elucidate the molecularpathogenesis of S. suis infections.

Cholera surveillance in the border of Thailand and Myanmar

Recent publications
１．Okada K, Chantaroj S, Roobthaisong A, Hamada S, Sawanpanyalert P. A Cholera Outbreak of the Vibrio cholerae
O1 El Tor Variant Carrying Classical CtxB in Northeastern Thailand in 2007. Am J Trop Med Hyg, 2010. May;
82(5):875-8.
２．Puiprom O, Chantaroj S, Gangnonngiw W, Okada K, Honda T, Taniguchi T, Sawanpanyalert P. Identiﬁcation ofcolonization factors of enterotoxigenic Escherichia coli with PCR-based technique. Epidemiol Infect2010.Apr;138(4):519-24.
３．Okada K, Chantaroj S, Taniguchi T, Suzuki Y, Roobthaisong A, Puiprom O, Honda T, Sawanpanyalert P. A rapid,
simple, and sensitive loop-mediated isothermal ampliﬁcation method to detect toxigenic Vibrio cholerae in rectal
swab samples. Diagn Microbiol Infect Dis 2010. Feb;66(2):135-9.
４．Maruyama F, Kobata M, Kurokawa K, Nishida K, Sakurai A, Nakano K, Nomura R, Kawabata S, Ooshima T,
Nakai K, Hattori M, Hamada S, Nakagawa I. Comparative genomic analyses of Streptococcus mutans provide
insights into chromosomal shuﬄing and species-speciﬁc content. BMC Genomics 2009. Aug 5;10:358.
５．Kasai S, Okada K, Hoshino A, Iida T, Honda T. Lateral transfer of the lux gene cluster. J Biochem 2007.
Feb;141(2):231-7.
52

Research Collaboration Center in Overseas

Research GroupSA Professor
SA Professor
SA Researcher
Postdoctoral Fellow
Postdoctoral Fellow
Research Fellow
Research Fellow
Research Fellow
Research Fellow

Naokazu Takeda, Ph.D.
Masanori Kameoka, Ph.D.
Yong-Gang Li, Ph.D.
Uamporn Siripanyaphinyo, Ph.D.
Sompong Sapsutthipas, Ph.D.
Piraporn Utachee, M.Sc.
Chris Verathamjamras, M.Sc.
Samatchaya Boonchawalit, M.Sc.
Chidchanok Khamlert, M.Sc.

Research Collaboration Center in Overseas

Section of Viral Infections

Intestinal infectious diseases (enteroviral infections): It has recently become diﬃcult to isolate viruses frompatients with hand, foot and mouth diseases, and it has been suggested that enteroviruses other thanEnterovirus 71 and Coxsackie virus 16 may be involved. To grasp the prevalence of these viruses in Thailand,
molecular epideomiological studies are currently underway.
Blood-borne infectious diseases (HIV diseases/AIDS): We are performing basic studies that examine thevirological and immunological characteristics of the HIV-1 CRF01̲AE strains that are prevalent in Southeast
Asia, including Thailand. In addition, the mechanism by which HIV acquires viral drug resistance toanti-retroviral drugs is being studied.
Mosquito-borne infectious diseases (dengue fever): We are constructing infectious molecularly cloned viruses to
identify the viral genes that are involved in viral pathogenesis.

Recent publications
１．Utachee P, Nakamura S, Isarangkura-na-ayuthaya P, Tokunaga K, Sawanpanyalert P, Ikuta K, Auwanit W,
Kameoka M. Two N-linked glycosylation sites in V2 and C2 regions of human immunodeficiency virus type 1
CRF01̲AE envelope glycoprotein gp120 regulate viral neutralization susceptibility to the human monoclonal
antibody specific for CD4 binding domain. J Virol. 2010;84:4311-20.
２．Pongsuwannna Y, Guntapong R, Tacharoenmuang R, Prapanpoj M, Kameoka M, Taniguchi K. A long-term
survey on the distribution of the human G type rotavirus in Thailand. J Med Virol. 2010;82:157-63.
３．Bai GR, Chittaganpitch M, Kanai Y, Li YG, Auwanit W, Ikuta K, Sawanpanyalert P. Amantadine- andoseltamivir-resistant variants of influenza A viruses in Thailand. Biochem Biophys Res Commun. 2009;390:897-901.
４．Jinnopat P, Isarangkura-na-ayuthaya P, Utachee P, Kitagawa Y, de Silva UC, Siripanyaphinyo U, Kameoka Y,
Tokunaga K, Sawanpanyalert P, Ikuta K, Auwanit W, Kameoka M. Impact of Amino Acid Variations in Gag andProtease of Human Immunodeficiency Virus Type 1 CRF01̲AE strains on Drug Susceptibility of Virus toProtease Inhibitors. J Acquir Immune Defic Syndr 2009;52:320-8.
５．Auwanit W, Isarangkura-na-ayuthaya P, kasornpikul D, Ikuta K, Sawanpanyalert P, Kameoka M. Detection of
drug resistance-associated and background mutations in human immunodeficiency virus type 1 CRF01̲AE
protease and reverse transcriptase derived from drug treatment-naive patients residing in central Thailand.
AIDS Res Hum Retroviruses 2009;25:625-31.

53

Endowed Chair
Laboratory of Molecular Biology (Lifetechnologies Corporation-Endowed Chair)
Research Group

Professor
Fumio Imamoto, Ph. D.
Assistant Professor Takefumi Sone, D. Agr. Sc

The cells that form the multi-cellular organism control its responses to various extracellular signals by
employing dynamic inter- and intra-cellular networks of biological molecular interactions. The recent
development of ﬂuorescence imaging technologies has made it possible to investigate how these molecular
network systems function when a signal from the outer world is received. However, in most cases, researchers
use cell extracts or membrane-permeable cells to introduce the ﬂuorescence-labeled protein into theintracellular system in question. Moreover, even when the ﬂuorescent proteins are expressed in living cells,
this involves the introduction of vectors and the cell is observed under a condition where the protein isover-expressed. In contrast, in living cells, the expression level and cytological localization of each protein is
precisely regulated, which facilitates their proper functions. Thus, the observations made by studies such as
those described above may not be reﬂective of the protein in its intrinsic physiological condition. If we wish to
analyze the real dynamics of multiple target proteins by introducing their genes (cDNAs) into living cells, weneed to integrate the genes, which are regulated by their own promoter, into a deﬁnite site on the chromosome,as this is the native state of the intrinsic genes in the genome.
This department has developed further the multisite Gateway (msGW) technology, which was originally created
and developed by Invitrogen Co. We are now applying this technology to the construction of multi-gene
expression clones that permit the simultaneous introduction of the genes into culture cells. These clones aredesigned to regulate the expression of the genes at a physiological level. Our research subjects thus include:
1) Construction of msGW vectors for the high-throughput introduction of multiple genes labeled by ﬂuorescence
protein tags.
2) Development of proper promoters and IRES signals to ensure expression is at physiological levels, anddevelopment and improvement of ﬂuorescence protein tags for bio-imaging.
3) Development of technology to control the expression levels of the transgenes, and development of methods toquantify the ﬂuorescently-tagged products when they are expressed at low levels.
4) Introduction of multiple genes simultaneously and stably into deﬁnite sites on the chromosome to obtainsuperior transformed cell lines.

Fig: 1 Multisite Gateway cloning technology.

54

Fig. 2 Generation of msGW clones for multiple purposes.

Endowed Chair

Fig. 3 Introduction of an expression clone into a targeted site of the cellular chromosome.

Recent publications
１．Inoue K ., Sone T., Oneyama C., Nishiumi F., Kishine H., Sasaki Y., Andoh T, Okada M., Chesnut J.D. and Imamoto
F. A versatile nonviral vector system for tetracycline-dependent one-step conditional induction of transgene
expression. Gene Therapy 2009 Dec ; 16(12) :1383-1394
２．Nishiumi F., Sone T, Kishine H., Thyagarajan B., Kogure T., Miyawaki A., Chesnut J. D. and Imamoto F.
Simultaneous single cell stable expression of 2-4 cDNAs in HeLaS3 using φC31 integrase system. Cell Structure
and Function 2009 Mar 20 ; 34:47-59(online)
３．Sone T., Yahata K., Sasaki Y., Hotta J., Kishine H., Chesnut J.D. and Imamoto F. Multi-gene gateway clone designfor expression of multiple heterologous genes in living cells: Modular construction of multiple cDNA expression
elements using recombinant cloning. J. Biotechnol.2008 Sep 10 ; 136(3-4):113-121
４．Sasaki Y., Sone T., Yahata K., Kishine H., Hotta J., Chesnut J.D., Honda T. and Imamoto F. (2008) Multi-gene
gateway clone design for expression of multiple heterologous genes in living cells: Eukaryotic clones containing
two and three ORF multi-gene cassettes expressed from a single promoter. J. Biotechnol. 2008 Sep 10 ;136(3-4):103-112
５．Tahara K., Takagi M., Ohsugi M., Sone T., Nishiumi F., Maeshima K., Horiuchi Y., Tokai-Nishizumi, Imamoto F.,
Yamamoto T., Kose S., Imamoto N. Importin-β and the small guanosine triphosphatase Ran mediate chromosome
loading of the human chromokinesin Kid. J. Cell Biology, 2008 Feb 11 ; 180(3):493-506

55

Osaka University Global COE Program
Frontier Biomedical Science Underlying Organelle Network Biology
Research program

Our main goal is to create an interdisciplinary research center, which will coordinate work in cell biology,
microbiology/immunology, and glycobiology in order to converge on a greater knowledge of the organellar
network.
The subjects under study will range widely:• modes of communication between organelles• interactions of pathogens with the organelle network• the roles of glycosylation in determining organelle function• the eﬀects of abnormal glycosylation on disease
By combining these fundamental studies with clinical research, we will drive the creation and development of the
new ﬁeld of organelle network medicine. To this end, we will conduct research focused on achieving an integrated
understanding of human disease, and on developing technological solutions to clinical problems.
On the road to these goals, we will ask clinically relevant questions, such as:• What is the mechanism leading from invasion by a pathogen to establishment of a full-ﬂedged infection?
• How might we interfere in the interactions between pathogens and the organelle network?
• How does disruption of organelle network result in neurodegenerative diseases? To what extent does ER
quality control play a role?
• Can we exploit changes in protein and sugar chain modiﬁcations to develop novel diagnostic tools?
Diseases are not merely the consequences of single causes or single gene mutations. We recognize that diseases
are multifactorial conditions, in which many genes and environmental factors intertwine and interact in a complex
way. Based on this recognition, our Center will encourage biological and clinical research that is committed tounderstanding disease at the level of both the molecular and organelle networks.
Ultimately, our greater understanding of the organelle network, and of clinically important issues in organelle
biology, will allow us to develop novel therapeutic strategies that accelerate the medicine of the 21st century.

Education program

Young scientists will someday become the biologists and clinical researchers of the future. Therefore, we arecommitted to establishing a world-class training environment in which junior scientists are fully supported andactively trained to take global leadership roles in 21st century science.
Our training program has many aspects, all focused on training the future leaders of the new ﬁeld of organelle
network medicine:• Researcher development program - Through a series of courses taught by domestic and international
academics and industrialists, young scientists will improve their management capabilities, technical writing,English proﬁciency and grant application skills.
• Interdisciplinary graduate curriculum - Investigators will have the opportunity to study across multiple
university schools, including the Schools of Medicine, Pharmaceutical Sciences, Dental Sciences, Science andFrontier Biosciences, and the Research Institute for Microbial Disease.
• Support for interdisciplinary projects - We will support research that breaks through barriers between
departments and ﬁelds, and encourage young researchers to be creative and ﬂexible in their work.
• Meetings for international young investigators - Our young researchers will themselves organize meetings,
held in a "training camp" format, to facilitate bottom-up international exchanges.
• Construction of an international network - We will have a special staﬀ devoted to the task of expanding
cooperation with overseas centers• Securing career paths - We will provide Ph.D.-level researchers with space and funding, in order to help
move them along the road to independence. After completion of graduate training, we will cooperate with otherresearch centers to help our alumni achieve tenured positions.
• Research assistants (RAs) - RA positions will be available in order to provide ﬁnancial assistance tograduate students. Our students will also be actively encouraged and well supported to attend international
meetings.
• Overseas practical training program - In order to advance clinical training, we will create practical courses
using overseas centers.
• Graduate student exchange - Providing opportunities to study abroad.

56

We hope to create an environment where traditional barriers between departments and ﬁelds essentially don't
exist - where young scholars can quickly and eﬃciently obtain advice from researchers in diﬀerent ﬁelds.
Major changes like this don't happen by themselves - which is why our faculty will include a specially appointed
associate professor whose primary responsibility is the cultivation of our collaborative networks with otherinstitutions.

Name
Aﬃliated department, Position title, Specialized ﬁeld, Academic degree

Yoshihiro Yoneda
Graduate School of Frontier Biosciences (Department of Frontier Biosciences), Professor, Cell Biology, M.D., Ph.D.

Tatsushi Toda
Graduate School of Medicine (Division of Preventive and Environmental Medicine), Visiting Professor, Mol.Genetics & Neurology, M.D., Ph.D.

Yoshihide Tsujimoto
Graduate School of Medicine (Division of Preventive and Environmental Medicine), Professor, Medical Genetics, Ph.D.

Kiyoshi Takeda
Graduate School of Medicine (Division of Preventive and Environmental Medicine), Professor, Immunology, M.D., Ph.D.

Kazunori Tomono
Graduate School of Medicine (Division of Preventive and Environmental Medicine), Professor, Clinical Microbiology, M.D., Ph.D.

Masaya Tohyama
Graduate School of Child Development, Professor, Anatomy & Neurosci, M.D., Ph.D.

Toshikazu Nakamura
Center for Advanced Science and Innovation (Joint Research Division for Regeneartive Drug Discovery), Professor, Biochemistry, Molecular Biology, Ph.D.

Tetsuo Takehara
Graduate School of Medicine (Division of Internal Medicine), Associate Professor, Gastroenterology & Hepatology, M.D., Ph.D.

Eiji Miyoshi
Graduate School of Medicine (Division of Health Sciences), Professor, Clinical and Laboratory Medicine, M.D., Ph.D.

Yoshinao Wada
Graduate School of Medicine (Division of Internal Medicine), Visiting Professor, Mass Spectrometry, M.D., Ph.D.

Yukari Fujimoto
Graduate School of Science (Department of Chemistry), Associate Professor, Organic Chemistry, Ph.D.

Naoyuki Taniguchi
The Institute of Scientiﬁc and Industrial Research, Endowed Chair Professor, Biochemistry, M.D., Ph.D.

Tamotsu Yoshimori
Graduate School of Frontier Biosciences (Department of Frontier Biosciences), Professor, Cell Biology, Ph.D.

Hitoshi Kikutani
Research Institute for Microbial Diseases, Professor, Immunology, M.D., Ph.D.

Eisuke Mekada
Research Institute for Microbial Diseases, Professor, Cell Biology, Ph.D.

Yoshiharu Matsuura
Research Institute for Microbial Diseases, Professor, Virology, Ph.D.

Tatsuo Shioda
Research Institute for Microbial Diseases, Professor, Virology, Ph.D.

Yasuhiko Horiguchi
Research Institute for Microbial Diseases, Professor, Bacteriology, Ph.D.

Kazuyoshi Ikuta
Research Institute for Microbial Diseases, Professor, Virology, Ph.D.

Toshihiro Horii
Research Institute for Microbial Diseases, Professor, Parasitology, Ph.D.

Shizuo Akira
WPI Immunology Frontier Research Center, Professor, Immunology, M.D., Ph.D.

Taroh Kinoshita
WPI Immunology Frontier Research Center, Professor, Immunology, Ph.D.

Hisashi Arase
WPI Immunology Frontier Research Center, Professor, Immunology, M.D., Ph.D.

Atsushi Kumanogoh
WPI Immunology Frontier Research Center, Professor, Immunology, M.D., Ph.D.

Tadashi Suzuki
RIKEN (Systems Glycobiology Research Group), Team Leader, Biochemistry, D. Sc.

Yoshiki Yamaguchi
RIKEN (Syetems Glycobiology Research Group), Team Leader, Structural Biology, D. Pharm.

Division of roles

Global COE Program

Members

Coordination of a center establishment andelucidation of the organelle network
Sugar chain modiﬁcations andneurodegeneration diseases
Mechanism of cell death and organelle
Analysis of activity regulation mechanismof the natural immunity system
Analysis of antibacterial activity anddevelopment of new antimicrobial agents
Neurological function abnormalities andorganella
Treatment strategies against neurodegenerativeand renal diseases with HGF
Hepatitis onset mechanism and treatment
strategies
Development of biomarkers using sugar
chain technologies
Development of sugar chain analysis
methods
Recognition system of bacteria with its
glycoconjugate
Functional analyses of sugar
chains/proteins
Analyses of roles of membrane traﬃc in
infection/immunity
Study on the dynamics of the acquired
immunity
Analyses of factors related to toxicity
manifestation of the diphtheria toxin
Infection mechanism of Hepatitis C virus
and studies on the control methods
Studies on host factors related to HIV
infection
Analyses of functions and structures ofbacterial virulence factors
Studying emerging viral infections andtheir pathogenesis
Development of malaria vaccines andanalyses of the host-parasite interactions
Studies on innate immunity
Analysis of signiﬁcance of GPI anchor in
the host-pathogen interactions
Studies on mechanism of
immunoregulation by pathogens
Studies of immunoregulation/regulatory
molecules of cell migration
Quality controls of free sugar chains andglycoproteins
Structural analyses of glycoconjugates by
NMR

57

Research & Activities
Cancer Cell Research Group
Research Group

Associate Professor
Assistant Professor

Masuo Yutsudo, Ph.D.
Shinji Higashiyama, Ph.D.

(1) Analyses of the CapG tumor suppressor geneWe isolated a series of cell lines from a human diploid ﬁbroblast that had beentransformed in various ways; these lines included immortalized,
anchorage-independent, and tumorigenic cell lines (Figure). Analysis of their gene
expression proﬁles revealed that the tumorigenic cell line had lost CapG protein
expression. Analysis of a variety of cancer cell lines revealed that several had also
lost CapG expression. When these tumorigenic human cancer cell lines weretransfected with CapG cDNA, they all became non-tumorigenic. We also identiﬁed
a protein that interacts with CapG: this protein is an oncogene product that forms
a complex with another tumor suppressor protein. Thus, CapG may suppress
tumorigenicity by modulating the activity of a particular oncoprotein/tumor
suppressor protein complex.
(2) Cellular dediﬀerentiation involved in tumorigenesis
It is well known that cancer cells often express genes that are usually only expressed by less diﬀerentiated cells.
We found that during the malignant progression of our model cell lines, ﬁbroblast-speciﬁc gene expression was
shut oﬀ and the expression of several new genes was switched on. We are currently studying how the alterationof diﬀerentiation status relates to tumorigenesis.
(3) Post-translational regulation of tumor suppressor protein p14ARF by PANO
We isolated a novel apoptosis-inducing gene called PANO, which encodes a nucleolar protein. Our studies thenrevealed that PANO up-regulates the expression of tumor suppressor protein p14ARF by inhibiting its
degradation, and that this may induce apoptosis. We are currently investigating whether this gene participates inhuman tumorigenesis.
Recent publications
Watari, A., Takaki, K., Higashiyama, S., Li, Y., Satomi, Y., Takao, T., Tanemura, A., Yamaguchi, Y., Katayama, I., Shimakage,M., Miyashiro, I., Takami, K., Kodama, K., and Yutsudo, M. (2006). Suppression of tumorigenicity, but notanchorage-independece, of human cancer cells by new candidate tumor suppressor gene CapG. Oncogene 25, 7373-7380.

Germ Cell Group
Research Group

Associate Professor

Masami Nozaki, Ph. D.

(1) DNA methylation during spermatogenesis.
Many of the testicular germ cell-speciﬁc genes are retroposons, most ofwhich contain a CpG-rich region within their ORFs. We discovered that the
methylation of the CpG dinucleotides in the ORF represses its promoter insomatic cells and that demethylation is necessary for gene expression in
spermatogenic cells. We are analyzing the molecular basis of the epigenetic
modiﬁcations, including DNA methylation and histone methylation, which
occur in a distinct genomic region in germ cells.
(2) Unique structure of sperm chromatin.
In the mammalian sperm nucleus, the haploid genome is packaged into ahighly compact structure that contains protamines and some remaining
histones. We are analyzing the physiological importance of the somatic-like,histone-containing regions of sperm chromatin.
(3) Establishment of an in vitro germ cell diﬀerentiation system
To examine the genetic requirements that are needed for germ cell
formation and epigenetic reprogramming, we are in the process ofestablishing an in vitro developmental system based on ES cell
diﬀerentiation.
Recent publications

58

Figure legend. Generation and regulation of
testicular germ cell-speciﬁc genes. Testicularisoform genes are generated by conventional
gene duplication or retroposition. In the caseof retroposons, the inserted cDNA, which isreverse-transcribed from mRNA, cannot be
expressed in any tissue because the mRNAs
lack a promoter in the 5'-ﬂanking sequence ofthe genomic DNA.
This implies that
testicular germ cells provide an appropriate
environment for retroposon transcription andfacilitate gene expression from promoter-likesequences. This in turn suggests that therules governing gene transcription during
the later stages of spermatogenesis diﬀer
drastically from those in other cell types.

Kato Y, Kaneda M, Hata K, Kumaki K, Hisano M, Kohara Y, Okano M, Li E, Nozaki M, Sasaki H. Role of the Dnmt3
family in de novo methylation of imprinted and repetitive sequences during male germ cell development in themouse. Hum Mol Genet. 2007 Oct1; 16(19): 2272-80.

Research & Activities
Research & Activities

Laboratory of Genome Dynamics
Research Group

Associate Professor
Postdoctoral Fellow

Takashi Hishida, Ph.D.
Nami Haruta, Ph.D.

DNA damage occurs frequently in all organisms as a consequence of both endogenous metabolic processes andexogenous DNA-damaging agents. Organisms have evolved several repair and tolerance mechanisms thatremove and tolerate DNA damage and coordinate cell cycle progression. In the S phase of the cell cycle,
replication stress occurs when an active fork encounters DNA lesions or proteins that are tightly bound to theDNA. These obstacles pose a threat to the integrity of the replication fork and are thus a potential source ofgenome instability that can contribute to tumorigenesis and aging in humans. Confronted with this risk, cells
have developed fundamental DNA damage response mechanisms in order to faithfully complete DNA
replication. Our group uses Escherichia coli and budding yeast as model systems to examine the cellularresponses to DNA damage, with a special emphasis on the mechanisms that maintain genome integrity during
DNA replication.
(1) Molecular mechanisms of the post-replication repair (PRR) pathway.
The UV spectrum present in sunlight is a potent and ubiquitous carcinogen that is responsible for most of theskin cancers in humans. In the natural environment, organisms are exposed to chronic low-dose UV light(CLUV), as opposed to the acute high doses that are commonly used in laboratory experiments. Hence, to clarify
the biological signiﬁcance of speciﬁc DNA damage response pathways, understanding the cellular response to
CLUV exposure is an important approach that complements the more traditional laboratory approaches. An
experimental assay that was recently developed to analyze CLUV-induced DNA damage responses was used toshow that the PCNA polyubiquitination-dependent error-free PRR pathway plays a critical role in tolerance to
CLUV exposure. We are currently analyzing in more detail the role(s) the error-free PRR pathway plays upon
CLUV exposure. In addition, we are examining the molecular and structural foundations of PRR functions by
combining genetic, biochemical and structural approaches.

Fig. 1: DNA damage tolerance
pathway. The RAD6 pathway consistsof at least two diﬀerent Rad6-Rad18
dependent mechanisms, which include
translesion DNA synthesis andRad5-dependent template switching.

Recent publications
１．Hishida, T., Kubota, Y., Carr, A. M. and Iwasaki, H. (2009) RAD6-RAD18-RAD5 pathway-dependent tolerance tochronic low-dose UV light. Nature 457, 612-615.
２．Ohya T., Arai, H., Kubota, Y., Shinagawa, H. and Hishida, T. (2008) A SUMO-like domain protein, Esc2, is requiredfor genome integrity and sister chromatid cohesion in Saccharomyces cerevisiae. Genetics 180, 41-50
３．Hishida, T., Ohya, T., Kubota, Y., Kamada, Y. and Shinagawa, H. (2006). Functional and physical interaction ofyeast Mgs1 with PCNA: impact on RAD6-dependent DNA damage tolerance. Mol. Cell. Biol. 26, 5509-5517.
４．Hishida, T., Han, Y-W., Fujimoto, S., Iwasaki, H. and Shinagawa, H. (2004). Direct evidence that a conserved
arginine in RuvB AAA+ ATPase acts as an allosteric eﬀector for the ATPase activity of the adjacent subunit ina hexamer. Proc Natl Acad Sci USA. 101, 9573-9577.
５．Hishida, T., Han, Y-W., Shibata, T., Kubota, Y., Ishino, Y., Iwasaki , H. and Shinagawa, H. (2004). Role of the
Escherichia coli RecQ DNA helicase in SOS signaling and genome stabilization at stalled replication forks. Genes
Dev. 18, 1886-1897.

59

Research Facilities

Special Research Facilities
Common Research Facilities

Animal Resource Center for Infectious Diseases
Research Group

Head, Professor（SUP）
Masaru Okabe, Ph. D.
Associate Professor
Masahito Ikawa, Ph. D.
Assistant Professor
Ayako Isotani, Ph. D.
Assistant Professor（SUP）
Hidetoshi Hasuwa, Ph. D.
Assistant Professor（SUP）
Naokazu Inoue, Ph. D.
SA assistant Professor（SUP） Yuhkoh Satouh, Ph. D.

A key area of microbial disease research is the analysis of the interactions between the host and pathogenicorganisms. Such microbiological and immunological research is greatly facilitated by animal models,particularly since molecular biological and biotechnological methods now allow the generation of geneticallymanipulated mice that can aid our understanding of the mechanisms of infection. These experiments requirethat experimentally infected animals are managed in a suitable, safe and controlled fashion. The Animal
Resource Center for Infectious Diseases is a unique facility that was established in 1967 to meet theserequirements. The center is separated into three areas: one for animal experimentation with microbial diseasemodels (P2 and P3 level) and an SPF area. The microbial disease model animal experimentation section iscompletely air-conditioned and maintained at a negative air pressure to minimize the risk of contamination.
Each sub-area in the microbial disease model animal experimentation section has an individual pass-throughtype of autoclave to sterilize all materials before they are removed. Moreover, the exhaust air is ﬁltered toprevent the exterior dissemination of pathogenic microbes. These measures ensure that microbial diseasemodel animals are handled safely without accidental cross-contamination.
Before gaining access to this restricted facility, researchers are required to take an oﬃcial orientation tour andsubmit a research plan for committee review. The condition of the animals is inspected regularly.
The facility oﬀers services such as the generation of genetically manipulated animals, in vitro fertilization, andthe cryopreservation of mouse strains (Table １).

Figure 1: Biosafety level 3 room (Building A, 1st ﬂoor).
This is the room where disease model animal experimentation
occurs under biosafety level 3 conditions. Hemorrhagic feverwith renal syndrome-causing virus (HFRSV) was ﬁrst isolated
in this area.
In addition, animal experiments for
Creutzfeldt-Jakob Disease (CJD), severe acute respiratory
syndrome (SARS) and Acquired Immune Deﬁciency
Syndrome (AIDS) can be performed in this facility.

Table 1) No. of mouse lines produced/preserved at the facility

period1995-1997
1998-2000
2001-2003
2004-2006
2007-2009

TG mice
92
116
101
43
21

KO mice
14
23
49
76
69

TG, transgenic; KO, knock-out

60

Cryopreservation
83
178
443
331
216

Research Facilities

Special Research Facilities
Common Research Facilities

Research Group

Head, Professor（SUP）
Hiroshi Nojima, Ph. D.
Assistant Professor
Daisuke Okuzaki, Ph. D.
Assistant Professor（SUP） Norikazu Yabuta, Ph. D.

Research Facilities

DNA-chip Development Center for Infectious Diseases

Facility Management: The establishment of infectious diseases is driven by the gene expression of pathogenicorganisms within the infected host cells. To understand parasite pathogenesis and pathophysiology, and todevelop new methods to prevent and treat infectious diseases, it is necessary to identify the pathogenic genesthat are expressed in the infected host cells and to determine how they induce disease at the genetic level.
This requires the analysis of the transcriptional patterns of both the genes of the pathogenic organism and the
responsive genes of the host genome.
The DNA-chip Development Center for Infectious Diseases is a unique facility that was established in 2004 to
analyze the transcriptional dynamics and variations involved in infectious diseases. Two research approaches
are employed in this facility:(1) Transcriptome analysis using DNA-chip analyzers.
The high density DNA microarray system in this facility permits comprehensive transcriptional analysis ofgene expression in the human or mouse host, and in various pathogenic organisms. Two DNA microarraysystems, namely the Agilent-type and the Aﬀymetrix-type, are available in this center. Our real-time PCR
analysis system (ABI, PRISM7900HT-2) is also useful for more accurate quantitative analysis of thetranscriptional levels of particular genes. In addition, a novel DNA microarray system (Genopal of Mitsubishi
Rayon Co. Ltd.) is currently being used in this center. An example of its practical use is the establishment of ablood RNA-based system for the diagnosis of autoimmune diseases. This system employs a focusedmicroarray that examines the expression of ˜200 blood cell-speciﬁc and disease-related genes.
(2) Proteome analysis using mass spectrometry.
Comprehensive translational analyses are also very important in furthering our understanding of infectious
diseases. The MS/MS spectrometer installed in this facility enables the analysis of the expression, interactions
and modiﬁcations of proteins from humans, mice, and pathogenic organisms. This center is also capable ofrecent technical innovations, such as the mass spectrometric detection of pathogenic organisms that facilitates
the development of novel diagnostic systems for infectious diseases.

Fig. 1 : High density DNA microarray system.

Fig. 2 : MS/MS spectrometer.

Recent publications
１．Tougan T, Okuzaki D, Nojima H. Chum-RNA allows preparation of a high-quality cDNA library from a single-cell
quantity of mRNA without PCR ampliﬁcation.Nucleic Acids Res. 2008 Sep;36(15):e92.
２．Kobayashi S, Ito A, Okuzaki D, Onda H, Yabuta N, Nagamori I, Suzuki K, Hashimoto H, Nojima H. Expression
proﬁling of PBMC-based diagnostic gene markers isolated from vasculitis patients. DNA Res. 2008
Aug;15(4):253-65.
３．Tougan T, Onda H, Okuzaki D, Kobayashi S, Hashimoto H, Nojima H. Focused microarray analysis of peripheralmononuclear blood cells from Churg-Strauss syndrome patients. DNA Res. 2008 Apr 30;15(2):103-14.

61

Research Facilities

Special Research Facilities
Common Research Facilities

Center for genetic analysis of biological responses
Research Group<Production laboratory for genetically-manipulated animals><Laboratory for analysis of genetically-manipulated animals>Head, Professor
Masaru Okabe, Ph. D.
Professor（SUP）
Shizuo Akira, M. D., Ph. D.
Assistant Professor（SUP）
Hidetoshi Hasuwa, Ph. D.
Professor（SUP）
Taroh Kinoshita, Ph. D.
<Resource laboratory for genetically-manipulated animals>
Professor（SUP）
Atsushi Kumanogoh, M. D., Ph. D.
Visiting Professor
Kenichi Yamamura, M. D., Ph. D. Professor（SUP）
Hisashi Arase, M. D., Ph. D.
Associate Professor（SUP）
Masahito Ikawa, Ph. D.
Professor（SUP）
Hitoshi Kikutani, M. D., Ph. D.
Assistant Professor（SUP）
Ayako Isotani, Ph. D.
Professor（SUP）
Masato Okada, Ph. D.
<Laboratory for promotion of collaborative research>
Professor（SUP）
Nobuyuki Takakura, M. D., Ph. D.
Visiting Professor
Yoichiro Iwakura, Ph. D.
Professor（SUP）
Hiroshi Nojima, Ph. D.
Assistant Professor（SUP）
Naohisa Goto, Ph. D.
Assistant Professor（SUP） Naokazu Inoue, Ph. D.
SA Assistant Professor（SUP） Yuhkoh Satouh, Ph. D.
Our bodies are kept homeostatically stable by the functions of proteins produced from many genes. In otherwords, our health is essentially dependent on the balance of our gene products. Many diseases can therefore be
traced to a defect in, or malfunction of, various genes. To ﬁnd and develop new drugs or new therapies, it isimportant to identify the function of each gene in vivo. However, at present, the functions of various genes
remain poorly understood, which makes it diﬃcult to clarify their relationships with each other, or to analyze ina systematic fashion how they participate in speciﬁc diseases.
Gene-disrupted animals can be powerful tools that help us to understand the role of certain genes in vivo. Suchanimals can be highly useful as models of various human diseases as well as for screening for new drugs. Thus,
many countries are producing gene-disrupted mouse lines of entire genes as national research projects in hopesof ﬁnding cures for diseases. In this context, the Japanese eﬀort remains small and needs to produce moregene-manipulated animals in order to contribute to the search for cures to diseases. It is very important thatJapan makes a reasonable contribution to this area. Given these issues, we created a consortium composed ofthree research institutes from three universities (the Research Institute for Microbial Diseases, Osaka
University; the Institute of Medical Science, University of Tokyo; and the Center for Animal Resources andDevelopment, Kumamoto University). The
headquarters are in Kumamoto University.
This consortium allows us to share our
specialties with each other and aims to produce
many gene-manipulated animals for the studyof human diseases and their treatment. In ourcenter, we are mainly focusing on genes thatparticipate in reproduction, infection andallergy by taking advantage of an existing
disease-screening system in our university; thissystemincludesfeaturessuchas
ﬂuorescent-colored sperm and eggs (Figure 1).
We aim to use these gene-manipulated animals
to perform the translational research that will
Elucidation of intertility
eventually lead to new therapeutic systems andthe discovery of new drugs.
Figure 1. Strategy for elucidating sterility

Recent publications

62

１．Inoue N, Kasahara T, Ikawa M, Okabe M. Identification and disruption of sperm-specific angiotensin converting
enzyme-3 (ACE3) in mouse. PLoS ONE. 2010. 5, e10301.
２．Fujihara Y, Murakami M, Inoue N, Satouh Y, Kaseda K, Ikawa M, Okabe M. Sperm equatorial segment protein 1,
SPESP1, is required for fully fertile sperm in mouse. J Cell Sci. 2010. 123, 1531-1536.
３．Yamaguchi R, Muro Y, Isotani A, Tokuhiro K, Takumi K, Adham I, Ikawa M, Okabe M. Disruption of ADAM3
impairs the migration of sperm into oviduct in mouse. Bio Reprod. 2009. 81, 142-146.
４．Yamaguchi R, Yamagata K, Hasuwa H, Inano E, Ikawa M, Okabe M. Cd52, known as a major
maturation-associated sperm membrane antigen secreted from the epididymis, is not required for fertilization in
the mouse. Genes Cells. 2008. 13, 851-861.
５．Okada Y, Ueshin Y, Isotani A, Saito-Fujita T, Nakashima H, Kimura K, Mizoguchi A, Oh-hora M, Mori Y, Oagata
M, Oshima RG, Okabe M, Ikawa M. Complementation of placental defects and embryonic lethality bytrophoblast-specific lentiviral gene transfer. Nat Biotechnol. 2007. 25, 233-237.

Research Facilities

Special Research Facilities
Common Research Facilities

Head

Professor

Hiroshi Nojima, Ph.D.

Research Facilities

Biken History Museum

Biken History Museum, which will open on December 2010, will present historical materials of RIMD since its
establishment in February 1934.

Fig. 1: Perspective Drawing (inside view) of the Biken History Museum.

Fig. 2: A stereo view of the blue print.
63

Research Facilities

Special Research Facilities
Common Research Facilities

Central Instrumentation Laboratory
Head

Professor
Associate Professor
Assistant Professor

Masato Okada, Ph. D.
Masuo Yutsudo, Ph. D.
Shinji Higashiyama, Ph. D.

This laboratory was established around the late 1950 s.
Since then, it has grown to possess a variety ofhigh-performance instruments, including ultracentrifuges,
electron microscopes, a laser microdissection system, cell
sorters, automatic plasmid puriﬁcation systems, DNA
sequencers, and a mass spectrum analyzer.
Thislaboratory also provides a room installed with large liquid
nitrogen tanks for the preservation of living materials suchas cells and viruses, and a room for treating speciﬁed
injurious chemicals. Several technicians are employed tokeep the instruments in proper working condition as wellas to provide advice to beginners and ongoing support for
researchers.
In addition, they execute cell sorting,nucleotide sequencing, observation by electron microscope,and mass spectrometric analyses on samples upon request from Institute researchers. These kinds of services
will become more and more important in the future since many instruments are becoming increasingly precise
and complicated and require extensive training. Plans to accommodate such changes are currently in progress.

Radioisotope Laboratory
Head

Head, Professor
Assistant Professor

Masato Okada, Ph.D.
Shinji Higashiyama, Ph.D.

The Radioisotope (RI) Laboratory is located adjacent to
the main building of the institute and covers 403 sq.m.
It was founded in 1967, and was extended by branchlaboratories with a combined space of about 600 sq. m.
during the establishment of the North building in 1979
and of the Central Laboratory for Biological Hazardous
Microbes in 1983. In 1998, a radiation exposure room
was established on the ﬁrst basement level of the Southbuilding. The Genome Information Research Centerradioisotope laboratory joined in 2007. At present, thetotal radiation controlled area is about 1,700 square
meters.
The RI Laboratory is designed for biomedical
experiments with radioisotopes and plays an important role in the research institute. Its facilities include an RI
stockroom, a distribution room, a tissue culture room, and an area for RI- measuring equipment. Safety
requirements are met by a stringent security system that involves the use of ID cards and computerizedmanagement of radioisotopes. About 200 researchers use the RI laboratory every year.

64Research Facilities

Special Research Facilities
Common Research Facilities

Head

Professor

Tatsuo Shioda, D. Med. Sc.

Research Facilities

Central Laboratory for Biological Hazardous Microbes

This laboratory was set up in 1983 to ensure the safe handling of
biologically hazardous microbes such as the HFRS (hemorrhagic feverwith renal syndrome) virus. Since then, all experimental studies using
such microbes, including the human immunodeﬁciency virus (HIV),
have been carried out in this laboratory. The laboratory is a 3-story
building that is 550 sq. m. in area. The ﬁrst ﬂoor is reserved forexperiments using radioisotopes. The facilities are designed to protect
researchers from getting infected with pathogens and to prevent the
spread of biohazardous pathogens outside the building.
The supply of fresh air is regulated to keep room interiors at
negative pressure. Air is released from the facility through high-quality
outlet ﬁlters to minimize contamination of the outside environment.
Furthermore, each room is equipped with safety cabinets andautoclaves for the sterilization of used materials before their disposal.
The entire laboratory has been renovated from 2005 to 2007 to increase
numbers of pathogens simultaneously used in this laboratory.
In 2008 and 2009, 64 and 66 researchers, respectively, were approved by the Biosafety Committee to use thislaboratory. The microbes used included HIV, human and avian inﬂuenza viruses, SARS corona virus, andscrapie agent.

Library
HeadProfessor

Hisashi Arase, M.D., Ph.D.

The RIMD library collects academic books and journals on microbiology and immunology, as well as work on
related scientiﬁc ﬁelds such as cell biology, genetics, histology, developmental biology, biochemistry,
pharmacology pathology, microbiology and oncology. In particular, we have collected rare books on parasitologythat cannot be found at other institutes. These books are frequently accessed by visitors to the RIMD library.
Due to the construction of a new building for the Integrated Life Science Building, the RIMD library was
moved to a temporary library room set up on the 1st ﬂoor of the south building of RIMD on December 2007. At
June 2010, the RIMD library was moved to a new space located at the 1st ﬂoor of the new RIMD main building.
Since the temporary library space was quite small, we were forced to discard most of the old books andjournals. For this reason, we now only hold journals published after 1991, all of the back issues of Biken Journal,
and 13,000 books. We now purchase 70 and 20 journals published in English and Japanese, respectively. Most of
the books are kept on the bookshelves in the stock room, but textbooks and newly arrived journals arearranged on the front shelf of the bureau. Most of the materials in the RIMD library are registered on the
online index at the main Library of Osaka University, which can also be accessed by libraries throughout Japan
via the Inter Library Loan (ILL) system. Three librarians handle the RIMD library together with two
professors, two associate professors and one assistant professor who act as members of RIMD librarycommittee. These members also take care of the publication of the Annual Reports of the Research Institutefor Microbial Diseases Osaka University (online only from 2003).

65

Related Facility
World Premier International Research Center

Immunology Frontier Resarch Center●Uniqueness and Objectives
Immunology has always been a scientiﬁc strength of Japan.
In particular, Osaka University has been historicallyknown for its leading immunology researchers including
Prof. Shizuo Akira.
The Osaka University Immunology Frontier ResearchCenter (IFReC), directed by Prof. Akira, is an example ofthis reputation. IFReC was selected by the Japanese
Ministry of Education, Culture, Sports, Science andTechnology (MEXT) as one of the nation's ﬁve exclusive
World Premier International (WPI) Research Center
Initiative Programs. Established on October 1st of 2007,
the center is expected to engage in high level researchthat will make it an international leader in the ﬁeld of
immunology. Immunology investigates the mechanismsthat protect the body against microbial infection. Because
the immune system is essential for eliminating infectious
pathogens from the body, its malfunction gives rise tovarious disorders such as autoimmune diseases like AIDS,
allograft rejection during transplantation, and allergies.
The scientiﬁc aim of IFReC is to unveil a comprehensive
understanding of the dynamic immune system by employing a variety of imaging technologies andBioinformatics to immunology.
International Advisory Review Board
Director

Deputy Director
Management Committee
Board of Representatives
Laboratories
Immunology Groups
Host Defense（Shizuo Akira）

Molecular Immunology（Hitoshi Kikutani）

Immunoglycobiology（Taroh Kinoshita）

Experimental Immunology（Shimon Sakaguchi）

Immunopathology（Atsushi Kumanogoh）

Cell Signaling（Takashi Saito）

Immunochemistry（Hisashi Arase）

Lymphocyte Diﬀerentiation（Tomohiro Kurosaki）

Immune Regulation（Tadamitsu Kishimoto）

Lymphocyte Development（Fritz Melchers）

Developmental Immunology（Toshio Hirano）

Gastrointestinal Immunology（Myoung Ho Jang）

Immunodynamics（Masayuki Miyasaka）

Malaria Immunology（Cevayir Coban）

Mucosal Immunology（Kiyoshi Takeda）

Vaccine Science（Ken Ishii）

Imaging Groups
Single Molecule Imaging（Toshio Yanagida）

Biological Imaging（Masaru Ishii）

Biomedical Optics（Junji Seki）

Nuclear Medicine（Jun Hatazawa）

Biofunctional Imaging（Yoshichika Yoshioka）

Chemical Imaging Techniques（Kazuya Kikuchi）

Nano-bio Materials（Takashi Jin）

Biophotonics（Nicholas Isaac Smith）

Bioinformatics Groups
Information System（Yutaka Hata）

Systems Immunology（Daron M. Standley）

Bioinfomatics and Genomics（Diego Miranda-Saavedra）
Administrative Director

Research Planning and Management Oﬃce
General Aﬀairs Section

66

Accounting Section

To date, research in immunology has either been carried out by isolating immune cells from the body andexamining these cells in vitro or by using in vitro cultured cell lines. Although such studies have provided
many new insights on the immunology system, we still do not understand the system well enough to predict
how it will respond when a certain pathogen invades. One approach to resolving this problem is to study
immune responses in a spatiotemporal manner. Given the importance of the spatiotemporal organization of thelymphoid organs in an immune response and the importance of understanding how single immune cells behave,combining immunology techniques with imaging techniques is vital for advancing immunology to a newfrontier.
To meet our needs and expectations, new imaging techniques will need to be developed by applying aninterdisciplinary eﬀort that combines people from disparate ﬁelds like physics, computer science, andimmunology.
By integrating the immunology and imaging ﬁelds, we will be able to understand the dynamic interactions of
immune cells and their activation. This will lead to new and more eﬃcient development strategies for vaccines
and immune therapies when combating infectious diseases, cancers and autoimmunity disorders.

Related Facility●Research ﬁelds／Expected Achievements

67

Accounts
Management Expenses Grants（unit : thousand yen）
Classiﬁcation

2005

2006

2007

2008

2009

Personnel

926,795

943,574

917,415

905,437

859,673

Non-Personnel

754,260

643,140

495,488

513,073

548,947

Total

1,681,055

1,586,714

1,412,903

1,418,510

1,408,620

Other Grants（unit : thousand yen）
Classiﬁcation

2005

2006

2007

2008

2009

Contract Research

946,305

997,753

1,175,396

1,022,353

1,040,180

Donations for Research

194,528

252,863

1,215,677

187,969

343,772

Miscellaneous

6,408

7,499

4,591

3,406

2,090

1,147,241

1,258,115

2,395,664

1,213,728

1,386,042

Total

Grants-in-Aid for Scientiﬁc Research（unit : thousand yen）
Classiﬁcation

2005

2006

2007

2008

2009

MEXT Research
Grants604,530

578,559

613,870863.592

688,999

Health and Labor
Sciences Research Grants

136,220

156,049

237,575

163,278

118,789

Health and Laborother Research Grants

-

-

-

18,00013,988

21st Century COE
Program Grants

174,700

192,500

196,900-

1,048,345-

Global COE
Program Grants

-

-

-

149,599

120,037
1,149,599

915,450

927,108

1,048,345

1,194,469

941,813

Total
68

Building Area③⑦

Building Area …… 7,752㎡

⑩

④

⑤

⑨

⑧

Gross Floor Area 27,408㎡

⑬

⑫

⑥

⑩

Accounts, Building Area

Site Area …… 36,197㎡⑪①⑪ Main building（left）andIntegrated Life Science building（right）①②② South building⑦ Central Laboratory for Biological Hazardous Microbes
and⑤⑥ Animal Resource Center

Building name

Total ﬂoor numbers

Building area（㎡）Total ﬂoor area（㎡）①Main building

7

1,518

6,059

②South building

3（1basement）

1,712

4,941

DNA-chip Developmet Center and Genome Information Research Center are included③North building

3

499

1,259

④Annex

2

771

1,548

⑤Animal Resource Center A

2

640

1,293

⑥Animal Resource Center B

4

354

1,430

⑦Central Laboratory for Biological Hazardous Microbes

3

242

550

⑧Radioisotope Laborator

1

403

403

⑨Central Instrumentation Laboratory

2

378

504

⑩Depository for dangerous chemicals

1

163

163

10

1,072

9,258

3（ 1 basement）

600

2,400

⑪Integrated Life Science building⑫Animal Resource Center C（belonging to IFReC）⑬New IFReC building（tentative name）（Construction to be completed in March, 2011）

69

Map & Access
LocationJapan

To Saito Nishi

Kyoto
Tokyo
Osaka
NagoyaTo MinohKayanoOnoharaImamiyaSenri Kinran University

To
Umeda

ute

Nara

HankyuSenri Line

Senri-Chuo
Stn.

Chugo
ku

Handai Byoin Mae Stn.
MonorailNational
Museumof Ethnology
Expo Park

Expressway
Ex

Kita-Osaka
Kyuko Line

Mie

Osaka
University
Suita
CampusKita-Senri
Stn.

Ibaraki I.C.

YamadaStn.

po

Ibaraki-shi Stn.
Koen
Higashiguchi
Stn.

Suita
I.C.

Banpaku Kinen
Ou
Koen Stn.
ter
Loop
Route

To
Kawaramachi
Stn.

Meishin
Expressway

Chuo Loop
Highway

Osaka Monorail
Unobe Stn.

JR
TokaidoLine

Minami-IbarakiStn.
To Umeda
Stn.

To Awaji Stn.

To Umeda Stn.

To Kyoto Stn.

Research Institute forMicrobial Diseases,
Osaka University

su Ro

To
Osaka
(Itami)
Airport

Shiga

Osaka

Toyokawa Stn.

ki-Sett

Shin-Mido Suji

Kyoto

Shimizu

National
Cardiovascular Center

Pacific Ocean

Hyogo

Route 171oad Ibara
ctural R
Prefe

To
Ikeda

To Kyoto

YukoukaiGeneralHospital

Wakayama

Ibaraki-shi
Stn.

HankyuKyoto Line

Kinki
Expressway
To Matsubara
To
Kadoma-shi Stn.

Pacific Ocean

Transportation access
To Saito Nishi

Handai Byoin Mae Stn.

Osaka University Suita Campus

Chugoku Expressway
To Takarazuka

Kita-Senri Stn. Banpaku Kinen
Koen Stn.
Senri-Chuo Stn.

To
To
Kyoto Stn. Kyoto Stn.

Osaka (Itami) Airport

SubwayMidosuji Line

Meishin Expressway

HankyuSenri Line

Kita-Osaka
Kyuko LineTo Shin-Kobe Stn.

Yamada Stn.

JR

Shin-Osaka Stn.

To Kobe Stn.

JR Osaka
Loop Line

JR

Osaka
(Umeda)
Stn.

ine

oL

aid

k
To

sen

Sh

an
ink

To
Kadoma-shi Stn.

Kinki Expressway

Tsuruhashi Stn.

Namba Stn.

Kintetsu Line

To
Nara

JR Lines
Nankai Line

Tennoji Stn.
JR Hanwa Line

JR Shinkansen
Monorail

To Kansai International Airport and Wakayama

70●Train : 12-minute walk from Kita-Senri Station on Hankyu Senri Line.
●Monorail : 20-minute walk from Handai Byoin Mae Station on Osaka Monorail Saito Line.
●Bus : From Senri-Chuo Station :5-minute walk from Handai-Guchi Bus Stop on Hankyu Buses heading to Onohara Higashi , Toyokawa-Eki , Fujikasai .
12-minute walk from Handai Honbu Mae Bus Stop on Hankyu Buses heading to Handai Honbu Mae or Ibaraki Mihogaoka .
●Bus : From Hankyu Ibaraki-shi Station:
12-minute walk from Handai Honbu Mae Bus Stop on buses heading to Handai Honbu Mae (via JR Ibaraki Station).

Map & Access

Suita Campus
Research Institute forMicrobial Diseases

Campus Map
30

NNorth Gate

27
28

25
13

22

24

14

18

12

26

19

29
East Gate

Icho Kaikan

8

5

21
Senri Gate

15

23

West Gate

3

16
1

6

HankyuGymnasiumn

Main Gate

Expo Park

Suita Campus

i Statio

Kitasenr

92

Convention Center

4

120

20
11

17
10

119

Osaka Monorail
Handai-ByoinMae
Station7

i LineKitasenr

Expo Park

129
Expo Park West
Chu

gok

Yamada Station

uE

Os

aka

Y

am

ad

a

S

ta

xpr

ess

Mo

wa

nor

y

ail

tio

n

Open Laboratories for Advanced Bioscience and Biotechnology

1 Administration Bureau

17

2 Graduate School/School of Human Scienses

18

3 Graduate School/Faculty of Medicine

19

4 Faculty of Medicine (Dept. of Allied Health Sciences)20

5 Osaka University Hospital21

6 OSaka University Dental Hospital

22

7 Graduate School/School of Pharmaceutical Scienses

23

8 Graduate School/ School of Engineering

24

9 Graduate School of Frontier Biosciences

25

10 Graduate School of Information Science and Technology

26

11 United Graduate School of Child Development

27

12 Research Institute for Microbial Diseases

28

13 Institute for Protein Research29

14 Low Temperature Center

30

International Student Center
International Center for Biotechnology
Center for Advanced Science and Innovation
The Center for Advanced Medical Engineering and Informatics
Global Collaboration Center
Sustainability Design Center
Institute of Laser Engineering
Immunology Frontier Research Center
Institute of Scientiﬁc and Industrual ResearchInstitute of Social and Economic Reseach
Joining and Welding Research InstituteResearch Center for Ultra-high Voltage
Cybermedia Center
Research Center for Nuclear Physics

15 Radioisotope Research Center
16 Research Center for Environmental Preservation

Published by : RIMD, Osaka University／Edited By : Hiroshi Nojima, the chair of RIMD Public Relations Committee
RIMD, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan
Tel: +81-6-6877-5111, Fax: +81-6-6879-8266 URL: http://www.biken. osaka-u. ac. jp71