1Introduction“NOVARTIS Foundation’s Raison d’etre”
Akimichi Kaneko, Chairman
This year marks the 20th anniversary of the NOVARTIS Foundation (Japan) for the
Promotion of Science. Twenty years have passed since it was established as the
Ciba-Geigy Science Promotion Foundation in 1987, based on the one billion yen
donation from the Ciba-Geigy Group. We sincerely thank all people whose financial,business and spiritual supports have made our present success. I am honored to witness
this historical moment as the Chairman.
Recently I found that the 1st Annual Report describes the detailed record of how theFoundation was established. On the next page an excerpt from the part of thedescription is reprinted. I believe it is an important message for those who did notwitness the start of the foundation 20 years ago to help understanding the purpose andthe background of the establishment of the Foundation. The record shows clearly that
the Foundation’s principle has never changed since then.
Since its establishment the Foundation has aided 871 research grantees, 153 researchmeetings, 94 researchers for Europe-Japan researcher exchange program, 60 researchersfor overseas business trips and 14 researchers for other special aids. The total amount of
the aids has reached 1.38 billion yen. Many researchers who were supported at that time
are now at the forefront in each field, such as being the professors of universities or
major institutions to teach younger researchers throughout Japan.
What has the Foundation dedicated to those researchers? Even if the aided amount was
small compared to recent large-scale research funds, I imagine that they were greatly
encouraged by this, because obtaining high appreciation from this Foundation indicatesthat the granted researcher is highly evaluated. For example, researchers selected for thecompetitive Research Grant of this Foundation must first obtain the universities’
recommendation, which is already a quite high hurdle. In order to estimate grantapplicants fairly and correctly, the NOVARTIS Foundation is asking leading
researchers in the subject field for screening. We thank these researchers for kindly
sparing their precious time for us.
At this historical milestone of this Foundation, we reconfirm our resolve to continue thepromoting activities to support promising researchers, based on the unchanged
fundamental principle.

2

Background to Establishment
The NOVARTIS Foundation (Japan) for the Promotion of Science was established on September 3rd,
1987 under the authorization of the Ministry of Education by following the process below.
Started Preliminary Investigation (Aug. 1985)
It was decided that the first fundamental research laboratory in Japan shall be established as the partof the Ciba-Geigy Group. Along with the establishment, an exclusive project team was formed toinvestigate the current situation of Japanese science and what role the established organization must
play for the Japanese science by visiting prominent figures in the target fields or other company
laboratories. The original purpose of this investigation was to make a plan for the establishment.
However, as the investigation progressed, the team members realized the importance of juridical
foundations because they were greatly contributing to Japanese science. This is how the Ciba-Geigy
Group established a new juridical foundation in Japan. Since then, the team had investigated anumber of Japanese research aid foundations for about one year.
Started Preparation for Establishing Foundation (Sept. 1986)
Ciba-Geigy Limited and Nihon Ciba-Geigy K.K. decided to establish a new juridical foundation in
Japan to respond anticipated tremendous benefits from Japanese society when the new researchlaboratory would be completed. At that moment the Ciba-Geigy Group had already establishedFriedrich Miescher Institute (FMI) in Switzerland and Ciba Foundation in London, so the new
foundation would be the third one.
The project team started the preparations for establishing the new foundation and the new researchlaboratory simultaneously. First they created a charter for the new foundation. The charter was
stating three main points; the importance of creative research in Japan, the necessity of international
exchange of researchers, and the Group’s strong decision to establish a research aid foundation in
Japan as the part of the Ciba-Geigy Group. The considerably broad range of target fields including
bioscience, chemistry and polymer science was set up. Also the team members asked the selected
persons for being the founders, trustees or councilors of the new foundation.
Holding Explanatory Meeting for Establishing Foundation (Jan. 1987)
The team invited major founders to Nihon Ciba-Geigy K.K. to explain them the progress and the
purpose of the establishment.

3

Visited Scientific Research Aid Division of the Research Promotion Bureau, Ministry of
Education (Feb. 1987)
The team asked the Ministry of Education (present Ministry of Education, Culture, Sports, Scienceand Technology) in Japan for administration because the new foundation would cover such a wide
range of fields and its main purpose was to support mainly fundamental researches. Although there
were somewhat exceptional factors as it was the first foreign-financed foundation in Japan, theMinistry of Education gave the team flexible responses and detailed advices appropriately.
Receiving Donation from Ciba-Geigy Limited as Basic Fund (Mar. 1987)
Ciba-Geigy Limited proposed for donation to the new foundation; 1,000,000,000 yen as its basic
fund and 50,000,000 yen every year as its operating fund.
Holding Founders’ Meeting (June 1987)
The Founders’ Meeting was held on June 3rd. The founders were Drs. Yuichi Yamamura, Saburo
Fukui, Hitoshi Nozaki, Hiroshi Mikawa, Morio Ikehara, Yoshiro Okami and Ryo Sato, andadditionally, Messrs. Paul Dudler, Toshiaki Simizu, Peter Baumann and Max. M. Burger from
Ciba-Geigy Limited. In that meeting Dr. Saburo Fukui was elected for the representative of thefounders. Other necessary issues such as the content of the charter were also decided in this meeting.
- Quoted from the 1st issue of the Annual Report

4Contents
Part Ⅰ:
Reports from the Recipients of Novartis Research Grants(Fiscal Year 2005)
1) Biological Sciences1-1) Molecular Biology
Oncogenesis mediated by the stabilization of AU-rich element containing mRNA
Fumihiro Higashino
Department of oral pathology and biology, Division of oral pathobiological science,
Hokkaido University graduate school of dental medicine
fhigashi@den.hokudai.ac.jp

Molecular mechanism of transport of mRNA into dendritic spines
Toru Takumi
Osaka Bioscience Institutetakumi@obi.or.jp

Functional analysis of ZNRF1 and 2, a novel family of ubiquitin ligases inducedby nerve degeneration.
Toshiyuki Araki
National Institute of Neuroscience, National Center of Neurology and Psychiatry
taraki@ncnp.go.jp

Study on mechanism of genomic imprinting establishment in YAC transgenic mice
Hitomi Matsuzaki
University of Tsukuba
matsuzaki@tara.tsukuba.ac.jp

1-2) Struktural Biology
Intelligent deoxyribozyme and aptamer for the development of side effect-freedrugs
MASATO KATAHIRA
Department of Supramolecular Biology, International Graduate School of Arts andSciences, Yokohama City University,
katahira@tsurumi.yokohama-cu.ac.jp

5

NMR Structural Biology for the elucidation of the mechanisms of Parkinson's
diseaseKoichi Kato
Department of Structural Biology and Biomolecular Engineering, Graduate School of
Pharmaceutical Sciences, Nagoya City University
kkato@phar.nagoya-cu.ac.jp

1-3) Cell Biology
Analysis of molecular machinery for autophagy
SUZUKI Kuninori
National Institute for Basic Biology
kuninori@nibb.ac.jp

Cell cycle regulation by Apollon
Mikihiko Naito
Institute of Molecular and Cellular Biosciences, The university of Tokyo
mnaito@iam.u-tokyo.ac.jp

Spatio-temporal analysis of lipid second messengers with probes based on
fluorescence resonance energy transfer
Michiyuki Matsuda
Graduate School of Medicine, Kyoto University
matsudam@path1.med.kyoto-u.ac.jp

Analysis of molecular mechanisms for regeneration of retina and neural tissues bystem cells and clinical application
Noriko Gotoh
Institute of Medical Science, The University of Tokyo
ngotoh@ims.u-tokyo.ac.jp

Development of regulatory strategies for neuronal death induced by malfolded
proteins.
Imaizumi Kazunori
Division of Molecular and Cellular Biology, Department of Anatomy,
Medicine, University of Miyazaki
imaizumi@med.miyazaki-u.ac.jp

Faculty of1-4) Developmental Biology
Analysis of molecular mechanism of fusion process intermediating Izumo factor on
sperm surface.

6

Naokazu Inoue
Reserch Institute for Microbial Diseases, Osaka University
ninoue@gen-info.osaka-u.ac.jp

Periodic gene expression generates spatial pattern in mouse development
Yasumasa Bessho
Graduate School of Biological Sciences

Nara Institute of Science and Technology

ybessho@bs.naist.jp

1-6) Pharmacology
Genomic Drug Target Validation Study for Cerebrovascular Disorders.
Toshio Tanaka
Department of Molecular and Cellular Pharmacology, Pharmacogenomics andPharmacoinformatics, Division of Genomics and Regenerative Medicine, Mie University
Graduate School of Medicine
tanaka@doc.medic.mie-u.ac.jp

1-7) Plant Biology
Molecular mechanisms of photoreceptors-regulated growth of plant throughphytohormone auxin
Tatsuya Sakai
RIKEN Plant Science Centertsakai@psc.riken.jp

1-8) Animal Biology
Study on processing mechanisms of novel identified neuropeptides.
Kazuyoshi Ukena
Graduate School of Integrated Arts and Sciences, Hiroshima University
ukena@hiroshima-u.ac.jp

2) Medical Sciences2-1) Immune System
The role of cortex-to-medulla migration of developing thymocytes in establishing central
tolerance
Yousuke Takahama
Division of Experimental Immunology, Institute for Genome Research, ,University ofTokushima
takahama@genome.tokushima-u.ac.jp

72-2) Central Nervous System
Uterine sensitization-associated gene-1 (USAG-1), a novel BMP antagonist
expressed in the kidney, accelerates tubular injuries
Motoko Yanagita
Kyoto University Graduate School of Medicine
motoy@kuhp.kyoto-u.ac.jp

Studies on the calcium-dependent mechanisms regulating the activity-dependent
release of brain-derived neurotrophic factor (BDNF) at the presynaptic terminal.
Hiromu Yawo, Toru Ishizuka
Department of Developmental Biology and Neuroscience, Tohoku University Graduate
School of Life Science
yawo@mail.tains.tohoku.ac.jp

A study of mechanisms which underlie activity-dependent neural circuit formation
Nobuhiko Yamamoto
Osaka University
nobuhiko@fbs.osaka-u.ac.jp

2-3) Reproducive System
Study of epigenetic pathophysiology and effective treatment of aged infertile
oocytes
Kenichiro HATA
National Center for Child Health and Development
khata@nch.go.jp

2-5) Cardiovascular Metabolic Endocrine
Towards development of a novel therapy for atherosclerosis via the apoptosis
inhibitory factor, AIM.
Toru Miyazaki
Division of Molecular Biomedicine for Pathogenesis, Center for Disease Biology andIntegrative Medicine (CDBIM), Faculty of Medicine, The University of Tokyo
tm@m.u-tokyo.ac.jp

The role of RNA polymerase kinases on the pathogenesis of cardiac hypertrophyand heart failure
Motoaki Sano
Keio University School of Medicne

8msano@sc.itc.keio.ac.jp

Elucidation of transcriptional networks in metabolic syndrome and vasculardiseaseIchiro Manabe
The University of Tokyo Graduate School of Medicine
manabe-tky@umin.ac.jp

2-6) Gastro-intestinal
Role of IKK/NF-kappaB activation in inflammatory bowel diseases
Shin Maeda
The institute for adult diseases, Asahi life foundations-maeda@asahi-life.or.jp

2-7) Infection
Carcinogenesis and progression of gastric cancer by infection of Helicobacter
pylori in ASC-deficient mice
Shun’ichiro Taniguchi
Dept. Molecular Oncol.,Shinsu Unversity Graduate School of Medicine
stangch@sch.md.shinshu-u.ac.jp2-8) Oncology
PGE2-mediated regulation of gastric epithelial differentiation and proliferation
Masanobu Oshima
Cancer Research Institute, Kanazawa University
oshimam@kenroku.kanazawa-u.ac.jp

Role of prostaglandin E in the growth and metastasis of cancer cells.
Masaki Inada
Tokyo University of Agriculture and Technology
inada@cc.tuat.ac.jp

Functional analysis of various tumor suppressor genes in vivo.
Akira Suzuki
Dept. Molecular Biology, Akita University School of medicine
suzuki@med.akita-u.ac.jp

9Phenotypic analysis of RAD18-KO and RAD18, XPA double KO mice.
Satoshi Tateishi

Kumamoto University, Institute of Molecular Embryology and Genetics
tate@gpo.kumamoto-u.ac.jp

The role of Helicobacter pylori CagA-activated SHP-2 oncoprotein in gastriccarcinogenesis
Masanori Hatakeyama

Institute for Genetic Medicine, Hokkaido University
mhata@igm.hokudai.ac.jp

Isolation of sympathetic neural crest stem cells and neuroblastoma cancer stem
cells and the application for neuroblastoma research.
Toshihide Iwashita
Aichi Medical University
toshiiwa@aichi-med-u.ac.jp.

2-14) Others
Role of protein kinase CK2 in the treatment of glomerulonephritis.
Gozoh Tsujimoto
Department of Genomic Drug Discovery Science, Graduate School of Pharmaceutical
Sciences
Kyoto University Faculty of Pharmaceutical Sciences, Kyoto University
gtsuji@pharm.kyoto-u.ac.jp

Discovery of a protein involving in drug exclusion in the kidney; remedy for renal
failureTakaaki ABE
Tohoku University Hospitaltakaabe@mail.tains.tohoku.ac.jp

3) Chemical Sciences3-1) Organic Chemistry
Studies on the Biomimetic Cascade Synthesis of Polycyclic Terpenoids
Kazuaki Ishihara
Nagoya University
ishihara@cc.nagoya-u.ac.jp

10Development of Novel Asymmetric Reactions Based on Unprecedented
Asymmetric Amplification
Yutaka Ukaji
Kanazawa University
ukaji@kenroku.kanazawa-u.ac.jp

Efficient Synthesis of Biologically Active Nitrogen-Containing Heterocyclic
Compounds by the Carbocyanation Reactions
Yoshiaki Nakao
Kyoto University
nakao@npc05.kuic.kyoto-u.ac.jp

Studies on the Synthesis and Biological Activity of Neurotoxins Produced by
Marine Algae
Isao Kadota

Okayama University
Kadota-i@cc.okayama-u.ac.jp

Novel Synthesis of N-Containing Heterocycles via Transition Metal-Cataltyzed[2+2+2] Cocyclization
Yoshihiro Sato, PhD., Professor
Graduate School of Pharmaceutical Sciences, Hokkaido University
biyo@pharm.hokudai.ac.jp

Development of External Stimulus-triggered Auto-processing Peptides
Akira Otaka
Department of Molecular Design and Synthesis, Institute of Health Biosciences, The
University of Tokushima
aotaka@ph.tokushima-u.ac.jp

Synthesis of Native Glycoprotein by in vitro Protein Synthesis
Shino Manabe
RIKEN (The Institute of Physical and Chemical Research)
smanabe@riken.jp

3-4) Analytical Chemistry11

Design, Synthesis and Biological Application of Molecular Probes Which Convert
Cellular Biological Responses to Chemical Outpur
Kazuya Kikuchi
Osaka University, Graduate School of Engineering
kkikuchi@mls.eng.osaka-u.ac.jp

3-5) Others
Elucidation of Functional Properties of Thermostable Electron Transfer Proteins
and Their Application to Biomaterials
Yasuhiko Yamamoto
Graduate School of Pure and Applies Sciences, University of Tsukuba
yamamoto@chem.tsukuba.ac.jp

Part Ⅱ:
Reports from the Recipients of Garants for International Meeting(Fiscal Year 2005)
4th International Symposium on Autophagy (ISA):
Exploring the Frontiers of Expanding Autophagy Research
11th International Xenopus Conference
Pre-Symposium Nagoya ICOB-5 & ISCPN-25 IUPAC International Conference
on Biodiversity and Natural Products“Functional Architecture of Cortical Microcircuits” Symposium in the 29th annual
meeting of the Japan Neuroscience Society
Dynamic Organelles in Plants

12

PartⅠ
Reports from the Recipients of Novartis
Research Grants (Fiscal Year 2005)

131) Biological Sciences1-1) Molecular Biology

Oncogenesis mediated by the stabilization of AU-rich element
containing mRNA
Fumihiro Higashino
Department of oral pathology and biology, Division of oral pathobiological science, Hokkaido
University graduate school of dental medicine
fhigashi@den.hokudai.ac.jp

Introduction
E4orf6 is an oncoprotein which can cooperate with adenovirus E1A to transform primary cells.
AU-rich element (ARE) is present in a certain mRNA transcribed from the genes such as
proto-oncogene.

HuR binds to the ARE elements to protect ARE-mRNAs from the rapid

degradation. Under the condition of heat shock, HuR makes a protein complex with pp32 to export
ARE-mRNAs to the cytoplasm in a CRM1-dependent manner.
We previously identified pp32 as an E4orf6 associated protein and revealed that E4orf6 exports
ARE-mRNAs by perturbing CRM1–dependent pathway (1). To explore the purpose of this E4orf6
function, we examined the stabilization of ARE-mRNAs.

Results
We examined in detail about the effects of E4orf6 on stabilization of ARE-mRNAs, such as c-fos,c-myc and COX-2.

The stabilization was estimated by quantitative real-time RT-PCR usingtransformed baby rat kidney (BRK) cells.

Accumulated c-fos mRNA in E4orf6 expressing BRK

cells (BRK E1+E4) was about 4.0 times more abundant than that in control BRK cells (BRK E1)(Fig. 1a). BRK E1+E4 cells also expressed approximately 5.5- and 4.2-fold greater levels of c-myc
and COX-2 mRNAs, respectively, than BRK E1 cells (Fig. 1a).
For elimination of the transcriptional
activation of each gene, the cells weretreated with actinomycin D (Act. D).
After 0, 60 and 120 minutes of Act. D
treatment, the quantity of each mRNA
species in BRK cells was measured byquantitative real-time RT-PCR.

The

half-lives of c-fos, c-myc and COX-2
mRNAs in BRK E1+E4 cells were about
Fig.1

14

141, 144 and 165 minutes, respectively. These values were significantly longer than those of BRK
E1 cells (50, 50 and 73 minutes, respectively) (Fig. 1b).
Since a previous study demonstrated that c-fos mRNA was stabilized by Act. D treatment, wetherefore examined the stabilization of c-fos mRNA by using a luciferase assay system.

We

constructed pGL3-based luciferase reporter plasmids with and without the 3’-UTR of c-fos cDNA
including the AREs. In BRK E1+E4 cells, the ARE-containing reporter (pCMVGL-ARE) showed
higher luciferase activity (about 6-fold) than in BRK E1 cells (Fig. 1c; right). On the other hand, thecontrol reporter (pCMVGL) did not show such activity (data not shown). We concluded that E4orf6
has a potential to stabilize the ARE-containing mRNA.
The fact that E4orf6 can stabilize ARE-mRNAs indicates that E4orf6 should enhance theoncogenic potential mediated by ARE-mRNA.
formation assay.

To confirm this hypothesis, we carried out a colony

The expression plasmid of full-length c-myc cDNA was transfected into rat 3Y1

cells with or without the expression constructs for E4orf6 and its mutants (E4orf6 L245P, the mutant
in the oncodomain).

E4orf6 enhanced the colony formation activity of c-myc oncogene.

Furthermore, the c-myc mRNA was obviously stabilized in transformed cells expressing wild-type
E4orf6, whereas it was not observed in E4orf6 L245P-expressing cells, consistent with the results ofthe colony formation (data not shown). Therefore, the ability of E4orf6 to enhance c-myc-mediatedoncogenesis was confirmed.
To estimate the requirement of the stabilization of
ARE-mRNA for the oncogenic activity of E4orf6, we
produced HuR knockdown cells (Fig. 2a), since HuR
is the only protein that interacts with ARE-mRNA
directly.

We expected that the ARE-mRNAs are notable to be exported and stabilized in HuR knockdown
cells, even if E4orf6 is expressed in the cells.

These

cells were treated with Act.D and the amounts ofc-fos mRNA were estimated as Figure 2b.

This data

indicate that HuR is necessary for the stabilization of
ARE-mRNA mediated by E4orf6.

The export ofARE-mRNA was not observed by HuR knockdown,even if E4orf6 is expressed in the cells (data notshown).
The oncogenic activities of these cells were
estimated by soft-agar colony formation assay.
BRK cells expressing E1 and E4orf6 was failed tomake colony by HuR knock down (Fig. 2c).
Fig.2

15

Thus HuR is also necessary for the oncogenic activity of E4orf6.

These data suggest that HuR andthe stabilization of ARE-mRNA are necessary for the oncogenic activity of E4orf6.

Discussion & Summary
We present here that E4orf6 stabilized ARE-mRNAs such as c-fos, c-myc and COX-2 and that
E4orf6 cooperated with c-myc to transform 3Y1 cells at least in part by stabilizing c-myc mRNA.
Moreover, HuR and the stabilization of ARE-mRNA are necessary for the oncogenic activity ofE4orf6.
Since the mechanism of action of viral oncoprotein has often led to insights into actual humanoncogenesis, we believe that this behavior of mRNA plays an important role in generating humancancer.

Recently, a systematic comparison between HuR expression in a variety of cancers andtheir normal tissue counterparts was carried out and higher HuR expression and cytoplasmicpresence in all malignancies examined was shown (2). These data indicate involvement of HuR
expression and transportation for human oncogenesis.

References(1) Higashino, F., et al., 2005 Adenovirus E4orf6 targets pp32/LANP to control the fate of
ARE-containing mRNAs by perturbing the CRM1-dependent mechanism. J. Cell Biol. 170:
15-20.
(2) de Silanes, I.L., et al., 2003. Role of the RNA-binding protein HuR in colon carcinogenesis.
Oncogene 22: 7146-7154.

161) Biological Sciences1-1) Molecular Biology

Molecular mechanism of transport of mRNA into dendritic spines
Toru Takumi
Osaka Bioscience Institutetakumi@obi.or.jp

Introduction
mRNA localization is a common mechanism operative in cells and has an essential role inlocalizing cytoplasmic determinants, controlling the direction of protein secretion and allowing the
local control of protein synthesis in neurons. In neuronal dendrites the localization and translocationof mRNA is considered as one of the molecular bases of synaptic plasticity. Recent imaging andfunctional studies revealed that several RNA-binding proteins form a large messenger
ribonucleoprotein (mRNP) complex that is involved in transport and translation of mRNA in
dendrites. However, the mechanism of mRNA translocation into dendritic spines, a site of local
synthesis of proteins, is unknown.

Results
To examine if TLS interacts with myosin-Va, we performed immunoprecipitation (IP) –immunoblotting (IB) of mouse brain lysates with specific antibodies against TLS and myosin-Va. In
the presence of EDTA and EGTA, TLS was coimmunoprecipitated with myosin-Va. However, nosignificant association of TLS with myosin-Va was observed in the presence of 100 µM CaCl2. To
see whether myosin-Va forms a complex with a specific target of TLS, Nd1-L mRNA, we performed
IP/RT-PCR. The amplified PCR products for Nd1-L mRNA migrated to the same position as thecontrol cDNA products obtained from mouse brain RNA. To test whether myosin-Va plays a role in
the transport of TLS into dendritic spines, we analyzed the localization of GFP- or red fluorescentprotein (RFP)-tagged TLS (TLS-GFP or TLS-RFP) in cultured mouse hippocampal neurons together
with that of full-length myosin-Va (BRMV) or 3 kinds of tail domains of its brain-specific isoforms,
i.e., brain short tail (BRST), brain stalk (BRSTK) and globular tail domain (GTD). Whereas
BRSTK-RFP and GTD-RFP did not affect the localization of TLS-GFP (BRSTK and GTD),
overexpression of BRST-RFP, containing both medial tail (stalk) and globular tail domains ofmyosin-Va, inhibited the translocation of TLS-GFP into spines and resulted in a granular expression
pattern within the dendrites, called “Granular” (BRST). To clarify whether the endogenous
myosin-Va plays a critical role in the transport of RNA-binding proteins into spines, we adopted

17

RNA interference (RNAi) technique. We introduced myosin-Va or control- short hairpin (sh)RNA
together with TLS-GFP by microinjection into the nucleus of primary hippocampal neurons. In
neurons expressing control-shRNA, TLS-GFP accumulated in dendritic spines (~90%,). In about
50% of the neurons challenged by 4623MyoVa-shRNA with or without RFP, TLS-GFP formed
dense clusters in dendritic shafts (p<0.0001), as observed in neurons expressing thedominant-negative myosin-Va tail domain. To investigate how TLS dynamics would be affected by
the loss-of-function of myosin-Va, we stimulated cultured hippocampal neurons expressing
TLS-GFP with 100 µM DHPG over 60 min and monitored the movement of TLS-GFP by time-lapse
confocal microscopy. The relative enrichment at the indicated time span (Ek) of TLS-GFP in spines
was significantly increased after mGluR stimulation (p<0.0005 for 40 min, p<0.0001 for 60 min).
On the contrary, in neurons expressing the dominant-negative myosin-Va tail domain (BRST-RFP),
TLS-GFP clusters were localized in dendritic shafts, and the intensity of TLS-GFP in the spines
remained low even after synaptic stimulation with DHPG. In myosin-Va-knockdown neurons
obtained by injection of 4623MyoVa-shRNA, the TLS-GFP clusters formed granules and werelocated within the dendritic shafts when stimulation with DHPG. The intensity of TLS-GFP in spines
exhibited larger fluctuations than in control neurons. Nevertheless, the translocation of TLS-GFP in
spines was impaired by knocking down myosin-Va expression. We further verified the results of theabove knockdown experiments by using neurons derived from myosin-Va-deficient dilute-lethal(dl/dl) mice. The time-lapse imaging experiments substantiated this assumption and demonstrated
that DHPG stimulation induced TLS-GFP enrichment in spines of +/+ (and/or dl/+) neurons,
whereas the induction was impaired in dl/dl neurons.

Discussion & Summary
We show that an actin-based motor, myosin-Va, plays a significant role in mRNP transport inneuronal dendrites and spines. Myosin-Va was Ca2+-dependently associated with TLS, an
RNA-binding protein, and its target RNA Nd1-L, an actin stabilizer. A dominant-negative mutant or
RNAi of myosin-Va in neurons suppressed TLS accumulation in spines and further impaired TLS
dynamics upon activation of mGluRs. The TLS translocation into spines was impeded also inneurons prepared from myosin-Va-null dilute-lethal (dl) mice, which exhibit neurological defects aswell as dilute fur color. Our results demonstrate that myosin-Va facilitates the transport/accumulationof TLS-containing mRNP complexes in spines and may function in synaptic plasticity through Ca2+
signaling.

18

Figures&Tables

References
1. Fujii, R., Okabe, S., Urushido, T., Inoue, K., Yoshimura, A., Tachibana, T., Nishikawa, T., Hicks,
G.G., and Takumi, T. (2005). The RNA binding protein TLS is translocated to dendritic spines by
mGluR5 activation and regulates spine morphology. Curr Biol 15, 587-593.
2. Fujii, R., and Takumi, T. (2005). TLS facilitates transport of mRNA encoding an actin-stabilizing
protein to dendritic spines. J Cell Sci 118, 5755-5765.

191) Biological Sciences1-1) Molecular Biology

Functional analysis of ZNRF1 and 2, a novel family of ubiquitin
ligases induced by nerve degeneration.
Toshiyuki Araki
National Institute of Neuroscience, National Center of Neurology and Psychiatry
taraki@ncnp.go.jp

Introduction
We aimed to identify proteins targeted by ZNRF (zinc finger/RING finger)1 and ZNRF2, a novel
family of ubiquitin ligases, which we previously identified to be expressed in the nervous system.
ZNRF1 was originally found to be highly induced after nerve injury in Schwann cells(1). ZNRF1
and 2 are both constitutively expressed in most neuronal cells and implicated in the synaptic vesicle
exocytosis(2). Identification of molecules targeted by ZNRF family is critically important forclarifying ZNRF-dependent protein metabolism and cellular functions regulated by ZNRF family ofE3 ligases.

Results
To characterize the biological function of ZNRF
family of E3 ubiquitin ligases, it is criticallyimportant to identify proteins which are degraded by
ZNRF-dependent proteasomal function in neurons aswell as Schwann cells.
by

To identify proteins targeted

ZNRF-dependentubiquitin-proteasomemechanism, we purified proteins which bind to
ZNRF1 or ZNRF2 from a brain protein lysate. We
found two proteins from the eluate of an affinity
column in which ZNRF proteins were cross-linked tothe column matrix, and identity of the proteins wasdetermined by mass-spectrometry.
One of the proteins was identified as glutaminesynthetase (GS)(Fig1a). GS is a metabolic enzyme
which generates glutamine using glutamate as a
substrate.

We examined whether GS can bind to20

both ZNRF1 and ZNRF2 by using recombinant GS and ZNRF proteins. We found that GS protein
binds to ZNRF1 but not to ZNRF2(Fig 1b). To confirm that GS is degraded by proteasome in a
ZNRF1-dependent manner, we examined whether the expression level of GS protein is related tothat of ZNRF1 or proteasomal function in cultured cells. We found that in neuro2a cells, forced
expression of ZNRF1 reduced the GS protein level in a dose-dependent manner(Fig 1c). TheZNRF1-dependent decrease of GS expression was not observed by introducing C184A mutation in
the ZNRF1 protein, which disrupt RING finger of the E3 ligase (Fig 1d-f).

These results suggestthat GS serves as a substrate of ZNRF1 (but not ZNRF2) E3 ubiquitin ligase.
GS is regarded as a “marker” of astrocytes and is not expressed in neurons in the central nervous
system. In the peripheral nervous system, GS is known to be expressed in satellite cells of the dorsal
root ganglia, which are regarded as non-myelinating Schwann cells, which show a phenotype similar
to Schwann cells in injured peripheral nerves. To examine whether GS is a physiological target of
ZNRF1-dependent proteasomal degradation, we first examined GS expression time course after
nerve injury in Schwann cells. We found that, in a mouse sciatic nerve transection model, ZNRF1
expression is highly induced in a segment distal to the injury site(Fig 2a-b). Side by side with thischange, we found that GS protein expression is decreased in injured nerve Schwann cell(Fig 2a).
Surprisingly, we also found that, in spite of the significant decrease of the GS protein expression, we
found that GS mRNA remains the same level before and after nerve injury in Schwann cells(Fig 2b).
This observation suggests that GS is physiological target of ZNRF1 in Schwann cells, and ZNRF1 is
responsible for regulation of GS protein expression.
Schwann cells dramatically change their phenotype
during development as well as after nerve injury.
Protein expression profile designates each phenotypeof Schwann cells, and the expression of each protein
is usually transcriptionally regulated.
Unlike most other proteins characterizing Schwann
cell phenotypes, we found that the GS expression isregulated

post-translationally

by

ZNRF1

E3

ligase-dependent ubiquitin-proteasome degradation
mechanism.
We are now determining the function of ZNRF1 and
GS during Schwann cell proliferation and peripheral
nerve myelination.
We are also examining the nature of the second
candidate which binds to ZNRF family of proteins.
We will confirm that the second protein also binds to21

ZNRF1 or 2, and characterize the function of the candidate protein as well as ZNRF in the nervous
system function.

Discussion & Summary
ZNRF1 was first discovered as a molecule up-regulated in Schwann cells of injured nerve, andZNRF2 was identified as a molecule highly similar to ZNRF1 in C-terminal zinc-RING finger
region. ZNRF1 and ZNRF2 sequences are diverse in the N-terminal area, which suggest each ZNRF
ligase has its own target molecule. Our data is compatible with this idea.
GS is now determined as a target of ZNRF1 E3 ligase. ZNRF1 is constitutively expressed in mostneurons in both central and peripheral nervous system, but GS is expressed only in glial cells. Thissuggests that ZNRF1 has substrate(s) other than GS.
To identify more candidate proteins which binds to ZNRF1 or ZNRF2, we may need to change
conditions when we screen proteins which bind to ZNRF.

References
1. Araki T. Nagarajan R. and Milbrandt J. Identification of genes induced after peripheral nerve
injury:expression profiling and novel gene discovery. J. Biol. Chem. 2001;276:34131-34141

2. Araki T, Milbrandt J. ZNRF proteins constitute a family of presynaptic E3 ubiquitin
ligases. J. Neurosci. 2003, 23: 9385-9394.

221) Biological Sciences1-1) Molecular Biology

Study on mechanism of genomic imprinting establishment in YAC
transgenic mice

Hitomi Matsuzaki
University of Tsukuba
matsuzaki@tara.tsukuba.ac.jp

Introduction
The mouse Igf2/H19 locus is subject to genomic imprinting; H19 gene is expressed only whenmaternally inherited while the neighboring Igf2 gene is paternally transcribed. This reciprocallycontrolled mono-allelic expression requires parental-specific methylation of the imprinting control
region (ICR) that is located 5' to the H19 gene.

Although it is reported that methylation imprintingof the H19 ICR is established during gametogenesis and maintained after fertilization, underlying
molecular mechanism in this process is not fully understood.

Here, we explored the mechanism of

methylation imprinting by using transgenic mouse (TgM) methodology.

Results
The differentially methylated H19 ICR functions as a landmark for the parental origin of thechromosomes, and regulates the imprinted expression of the H19 and Igf2 genes.

Thehypo-methylated maternal ICR carries an enhancer blocking insulator activity as a consequence of a
CTCF protein binding to it and prevents the distally located Igf2 gene from being activated by ashared 3’ enhancer. In contrast, a hyper-methylated paternal ICR suppresses H19 gene transcription
through promoter DNA methylation, while abolishing CTCF binding, thereby allowing Igf2 gene
expression (Figure 1).

23

To test if the ICR activity is autonomous, we introduced a 2.9-kbp ICR DNA fragment into aheterologous genomic context, i.e. 3’ end of the locus control region in the human β-globin yeast
artificial chromosome (YAC), and established single-copy YAC TgM lines.

In these mice,paternally inherited transgenic β-like globin genes were more abundantly expressed than that on thematernal allele.

In accord with this result, the paternally transmitted transgenic ICR was moreheavily methylated, and CTCF was preferentially recruited to the maternally inherited transgenic
ICR in nucleated erythrocytes.

These results demonstrated that the ICR could autonomously

recapitulate genomic imprinting within a normally non-imprinted locus.

An unexpected

observation was that hyper-methylation of the transgenic ICR was not observed in male germ cells,suggesting that methylation imprinting in the paternal transgenic ICR was established after
fertilization, and “primary mark” other than the DNA methylation may be a prerequisite for a
post-fertile, parent-of-origin specific methylation acquisition.
We then tested if the H19 ICR could establish methylation imprinting by itself.

To this end, we

injected the 2.9-kbp ICR DNA fragment into pronuclei of fertilized mouse eggs and generated six
TgM lines, each of which carried from one to five copies of transgenes inserted randomly at
non-imprinted genomic loci.

In order to distinguish transgenic from endogenous ICR sequences,

the transgenic ICR fragment was flanked by the ~0.5-kbp human β-globin sequences on both sides.
Methylation status of the transgenic ICR DNA was then analyzed by Southern blotting and bisulfite
sequencing. In nucleated erythroid cells, paternally inherited transgenic ICR was more heavily
methylated than the maternal one in five out of six TgM lines.

These results suggested that thetransgenic ICR fragment could mark its parental origin independent of its copy number and its
genomic integration site.

Discussion & Summary
In this work, we investigated how methylation imprinting of H19 ICR is established by using
TgM methodology, and showed that 2.9-kbp ICR fragment could autonomously establish
methylation imprinting at the normally non-imprinted human β-globin locus as well as other random
genomic loci.

These results suggested that the 2.9-kbp ICR fragment carried sufficient information

for marking its parental origin.

Interestingly, transgenic ICR at human β-globin locus was

hypo-methylated in germ cells, while differential, parent-of-origin specific ICR methylation wasobserved in somatic cells.

Thus, it is likely that establishment of post-fertile methylation

imprinting does not always require the differential ICR methylation in germ cells.

Distinct

molecular mechanisms may independently control establishment of methylation imprinting insomatic or germ cells.

24

References
Tanimoto K, Shimotsuma M, Matsuzaki H, Omori A, Bungert J, Engel JD, Fukamizu A.
Genomic imprinting recapitulated in the human β-globin locus.
Proc Natl Acad Sci USA. 2005. 19:10250-10255.

251) Biological Sciences1-2) Struktural Biology

Intelligent deoxyribozyme and aptamer for the development of side
effect-free drugs
MASATO KATAHIRA
Department of Supramolecular Biology, International Graduate School of Arts and Sciences,
Yokohama City University,
katahira@tsurumi.yokohama-cu.ac.jp

Introduction+

K concentration in cells is much higher than that of the outside of cells. Structure of some DNAchanges drastically when the K+ concentration increases. Guanine-rich DNA is a typical example. Its
structure is an extended single strand under low K+ conditions, while the structure becomes compact
quadruplex under high K+ conditions. We call this kind of DNA as a switching unit. Functional
nucleic acids such as deoxyribozyme and aptamer can be divided to two sub-units. Suppose these
two sub-units are joined to both the ends of guanine-rich DNA (switching unit). Under low K+
conditions (outside of cells), guanine-rich DNA is extended and two sub-units are separated.
Therefore, no deoxyribozyme or aptamer activity is exerted. Under high K+ conditions (inside ofcells), guanine-rich DNA forms a compact structure and thus two sub-units are located closely. Then,
deoxyribozyme or aptamer activity is exerted. We already demonstrated that guanine-rich DNA
composed of GGA sequences can be used for this scheme. In order to find more functional switching
unit, we have studied the structure of human telomeric DNA under physiological high K+ conditions.

Results
The structure of human telomeric DNA has been controversial: the solution structure in thepresence of Na+ has been determined to be an antiparallel quadruplex, while the crystal structure in
the presence of K+ has been reported to be a parallel quadruplex. The solution structure in thepresence of K+ has drawn intense interest, as the intracellular K+ concentration is higher than that of
Na+, but the structure remains to be elucidated. The difficulty of the structural determination is due
to conformational heterogeneity. Here, we have stabilized the major structure in a K+ solution by theunique method of proper substitutions of guanosines with 8-bromoguanosines and determined thestructure at high resolution. Then it was feasible to determine the major structure by NMR. The
root-mean-square deviations (rmsd) of the 10 final structures versus the mean structure for all heavy
atoms were 1.3±0.3 Å for a whole molecule. They were 0.8±0.1 Å for a core region. The atomic

26

coordinates have been deposited in the RCSB under Protein Data Bank accession code 2E4I. This
demonstrates that the use of 8-bromoguanosine is a powerful tool to overcome the difficulty of thestructure determination arising from conformational heterogeneity.
We have examined the possible effect of the substitution with 8-bromoguanosines on a native
structure. We substituted the guanosines which take on syn in a native structure. It was pointed outthat the 8-bromoguanosine substitution at a syn position does not cause a steric hindrance because
the Br atom is placed in the groove. Therefore, the effect of the substitution on a native structure is
expected to be rather moderate.
The obtained structure is neither the antiparallel quadruplex nor the parallel quadruplex. It is amixed-parallel/antiparallel quadruplex, in which three strands of the quadruplex are aligned inparallel, while one strand is aligned antiparallel. The positions of both the ends of DNA are closeenough. So, when two sub-units of functional nucleic acids are joined to both the ends, re-formationof the functional body is expected. Therefore, human telomeric DNA is supposed to be applicable asa switching unit.
Recently, the structure of telomeric DNA was reported by other group, in which stabilization was
brought about by mutation and resultant additional interactions. The structure of the guanine tracts
was similar between the two structures. However, difference was seen for loops connecting guanine
tracts, which may play a role in higher order arrangement of telomeres. The loop, a TTA segment,does not contain 8-bromoguanosine. Thus, the effect of the substitution on a native loop structure
should be moderate. Our lower loop is more compact than that of the other group and three residuesof the loop in our structure are located even closer to the guanine-tetrad, which contains the
8-bromoguanosine. Therefore, the steric hindrance involving 8-bromoguanosine can not be the causeof different loop structures. Similarly, the difference in the upper loop structure is not caused by thesteric hindrance involving 8-bromoguanosines. Difference was also seen for the sugar conformationof some residues. Our structure which does not contain the mutation can be utilized to design a small
molecule which stabilizes the quadruplex. This kind of molecule is supposed to inhibit a telomerase
and thus is expected to be a candidate of an anticancer drug.

Discussion & Summary
Elucidated structure of human telomeric DNA has indicated that this DNA can be used as aswitching unit as DNA composed of GGA sequences. The positions of the both ends of this DNA is
ideal to join two sub-units of functional nucleic acids.
It is found that the activity of telomerase is inhibited by the formation of the quadruplex.
Therefore, a small molecule that binds to and stabilizes the quadruplex is expected to be a candidateof an anticancer drug. Our structure determined in a K+ solution can be utilized to design anddevelop such a molecule. Precise structures of loops and sugars may be useful for such design and27development. The determination of the unique quadruplex structure must also serve as a basis tounderstand the interactions of various telomere-binding proteins with telomeres.
There are differences regarding loops and sugars between two structures by our group and the othergroup. Our structure does not include the mutation and resultant additional base pair which may be
responsible for these differences. When the structure is utilized to understand higher orderarrangement of telomeres and their interactions with telomere-binding proteins, and also to develop
an anticancer drug, these differences may be critical. Therefore, it is important to present thestructure stabilized by proper incorporation of 8-bromoguanosines, without introducing the mutation.

Figures & Tables

References(1) Matsugami, M., Xu, Y., Noguchi, Y., Sugiyama, H. and Katahira, M. (2007) FEBS J. inpress. ”Structure of human telomeric DNA under physiological ionic conditions stabilized byproper incorporation of 8-bromoguanosines, as determined by NMR”
(2) Nakagama, H., Higuchi, K., Tanaka, E., Tsuchiya, N., Nakashima, K., Katahira, M. and Fukuda,
H. (2006) Mutat. Res., 598, 120-131. “Molecular mechanism for maintenance of G-rich shorttandem repeats capable of adopting G4 DNA structures”(3) Tsuchibayashi, H., Ohyama, T., Matsugami, A., Nanjo, T., Iwai, S. and Katahira, M. (2006)
Nucleic Acids Symp. Ser. No. 50, 237-238, “Structural analysis of DNA containing (6-4)
photoproduct in complex with distamycin A by NMR”

281) Biological Sciences1-2) Struktural Biology

NMR Structural Biology for the elucidation of the mechanisms of
Parkinson's disease
Koichi Kato
Department of Structural Biology and Biomolecular Engineering,
Graduate School of Pharmaceutical Sciences, Nagoya City University
kkato@phar.nagoya-cu.ac.jp

Introduction
Recent advances in NMR structural biology have made possible the high throughput structural
determination of small simple proteins and protein domains. However, it remains as tasks with morechallenge to carry out structure biology studies of intrinsically disordered proteins, which areassociated with various neurodegenerative disorders. In the present study, we demonstrate that 920
MHz ultra-high field NMR spectroscopy enables us to cope with the difficulty in dealing with thosetroublesome proteins.

Resultsα-synuclein is a 140-amino acid protein abundantly expressed in presynaptic nerve terminals
and is studied as a model of intrinsically disordered proteins.

This protein has been identified as

the major component of the Lewy bodies (LBs) and Lewy neurites of Parkinson’s disease, dementia
with LBs and multiple system antrophy. It has been reported that α-synucleins in synucleinopathy
lesions are selectively and extensively phosphorylated at Ser129.
α-synuclein at Ser 129 promoted fibril formation in vitro.

Furthermore, phosphorylation of

However, the conformational effects ofphosphorylation of α-synuclein remain to be understood.

In the present study, we perform

ultra-high field NMR spectral analyses of α-synuclein focusing on its phosphorylation at Ser129.
We first attempted to assign 1H-15N HSQC peaks originating from the backbone amide groupsof α-synuclein by use of 920 MHz NMR spectroscopy.

While the peaks severely overlap in the

500 MHz spectrum, they were well dispersed in the 920 MHz spectrum.

The improvement of thespectral resolution enables us to achieve assignments of the backbone resonances of the α-synuclein.
We have successfully prepared a large amount of [15N]α-synuclein phosphorylated at Ser129
for detailed NMR analyses of effects of the phosphorylation.

Inspection of the 1H-15N HSQC

spectral data indicated that the C-terminal phosphorylation site interacts with the middle parts in
α-synuclein molecules.

On the basis of these data, we suggest that the phosphorylation destabilizes

29

an intramolecular interaction of α-synuclein, rendering this protein more aggregatable.
We also conducted NMR analyses of ataxin-3, a product of the causative gene ofMachado-Joseph disease. Ataxin-3 is composed of the N-terminal Josephin domain, two or threeubiquitin (Ub) interacting motifs and a polyglutamine tract.

The Josephin domain, which acts as a

deubiquitinating enzyme, assumes a papain-like fold with a cystein in the catalytic site and with aflexible helical hairpin.

To provide the structural basis for the substrate recognition by ataxin-3, we

performed NMR analyses of the interaction between the Josephin domain and Lys48-linked di-Ub(K48-Ub2), which was prepared an enzymatic reaction in vitro using Ub mutants as substrates.
NMR chemical shift perturbation data indicated that the hydrophobic surfaces of both Ub units,
which surround the isopeptide linkage, were involved in the interaction with the Josephin domain.
To determine the orientation of K48-Ub2 with respect to the Josephin domain, we made asite-specific spin labeling as a source of long-range distance information.

For this purpose, either

Lys48 of the distal Ub or the C-terminal Gly76 of the proximal Ub was substituted with cystein,which was subsequently attached to a spin label.

On the basis of the paramagnetic relaxation

enhancement date, we identified the amino acids residues of the Josephin domain in close spatial
proximity of the unpaired electron in the complex. Upon complex formation, the HSQC peakoriginating from Leu93 exhibited line broadening due to the effect of the spin label attached to the
distal Ub, whereas the spin label at the C-terminal of the proximal Ub induced line broadening of the
HSQC peaks from the catalytic residues of the Josephin domain.
On the basis of these NMR data, we propose a hypothetical model of the interaction between
ataxin-3 and Lys48-linked polyubiquitin chain, which provides insights into the deubiquitinating
function of ataxin-3.
We also provided structural basis of molecular recognition by Ub and a variety of Ub-likeproteins, including the proteasome-binding domain of parkin, on the basis of NMR spectral data.

30

Discussion & Summary
In summary, we successfully applied ultra-high field NMR spectroscopic techniques forstructural analyses of proteins as causative gene products of neurodegenerative disorders.

This

spectroscopic approach allows us to discuss molecular properties of phosphorylated α-synucleinfrom the views of structural biology. Based on the results obtained in the present study, we arefurther carrying out NMR analyses focusing on interactions between small molecule inhibitors andisotopically labeled α-synuclein.

We also succeeded in determination of the mode of interaction

between ubiquitin chain and deubiquitinating enzyme using the Josephin domain of ataxin-3 as amodel enzyme.
The NMR spectral data will provide structural basis of the pathogenic mechanisms ofneurodegenerative caused by protein dysfunction and/or aggregations.

This will open up a new

avenue for developing design and discovery of drugs for therapy and diagnosis of neurodegenerative
disorders.

311) Biological Sciences
1-3) Cell Biology

Analysis of molecular machinery for autophagy
SUZUKI Kuninori
National Institute for Basic Biology
kuninori@nibb.ac.jp

Introduction
Autophagy is a bulk degradation process that is conserved in eukaryotic cells and functions in theturnover of cytoplasmic materials and organelles. When eukaryotic cells face nutrient starvation, theautophagosome, a double-membraned organelle, is generated from the pre-autophagosomal structure(PAS). In the yeast Saccharomyces cerevisiae, 16 ATG (autophagy-related) genes are essential for
autophagosome formation. Most of the Atg proteins are involved in the PAS, leading toautophagosome production. However, the mechanism of PAS organization remains to be elucidated.
Here, we performed a systematic and quantitative analysis by fluorescence microscopy to develop ahierarchy map of Atg proteins involved in PAS organization.

Results
Comprehensive analysis of Atg protein localization
Our systematic analysis aims to elucidate the mechanisms of PAS organization. First, we examined
whether each Atg protein localizes to the PAS. Second, we investigated whether PAS localization ofeach Atg protein is perturbed by disruption of another ATG gene using quantitative fluorescencemicroscopy.
We constructed 16 yeast strains expressing GFP-fused Atg proteins from their native loci andexamined these Atg-GFP strains using fluorescence microscopy and found that 11 Atg proteins
exhibited perivacuolar punctuate structures that colocalized with Ape1p (a PAS marker), showingthat these Atg proteins were indeed localized to the PAS (Fig. 1). To determine which specific Atg
proteins were required for the PAS localization of the various Atg proteins, all of the ATG genes
were individually disrupted in each Atg-GFP strain, for a total of 165 (11 x 15) disruptants. Thesecells were treated with rapamycin for five hours to induce autophagy and analyzed by fluorescencemicroscopy. The PAS targeting index (PTI) was introduced to quantify the level of Atg protein
recruitment to the PAS.

32

Atg11p recruits Atg proteins for the Cvt pathway
under autophagy-inducing conditions
The PTI analysis showed that localization of most Atg
proteins was impaired in the absence of Atg17p. This
fact led us to hypothesize that Atg17p is a scaffold
protein for PAS organization. If this is true, no Atg
proteins should localize to the PAS in the absence ofAtg17p. However, the PTI of Atg8p in ∆atg17 cells
was at the similar level as in wild-type cells.
Interestingly,

GFP-Atg8p

dots

disappearedin∆atg11∆atg17 cells. We concluded that Atg11p is a
scaffold protein for the Cvt pathway, a kind of
selective autophagy that transports Ape1p to thevacuole. In combination with this prior study, our datasuggest that Atg17p plays a role in the recruitment ofAtg proteins for the autophagic pathway and that
Atg11p plays a similar role for the Cvt pathway.
Atg17p is a scaffold protein that is responsible for
PAS organization
The PAS localization of Atg proteins is completely
absent in the ∆atg11∆atg17 mutant. If Atg17p actuallyfunctions as a scaffolding protein in PAS organization,
Atg proteins must be recruited to the site where
Atg17p localizes. We constructed Atg17p fused with aplasma membrane-targeting sequence (PM-Atg17p), then examined whether other Atg proteins
were targeted to the plasma membrane in ∆atg11∆atg17 cells expressing PM-Atg17p.
Atg9p, Atg13p, and Atg8p exhibited a cytoplasmic expression pattern in ∆atg11∆atg17 cells
carrying

an

empty

vector(Fig.

2,

Vector).

C-terminallymRFP1-fused

PM-Atg17p(PM-Atg17p-RFP) thoroughly localized to the plasma membrane (Fig. 2, left column). Intriguingly,
other Atg proteins were also recruited to the plasma membrane and essentially colocalized with
Atg17p (Fig. 2), showing that Atg17p indeed acts as a scaffold to recruit Atg proteins. Also, we
found slight differences in the localization of Atg proteins: whereas Atg13p was closely colocalizedwith PM-Atg17p (Fig. 2A), Atg9p was recruited to the plasma membrane, as visualized by thepresence of a small number of punctate fluorescence (Fig. 2B), and Atg8p formed a single dot justinside the plasma membrane (Fig. 2C).

33

Notably, PM-Atg17p-RFP did not recover the
autophagic activity of the ∆atg11∆atg17 mutant, as
determined by the failure of GFP-Atg8p delivery to
the vacuole (Fig. 2C), suggesting that these plasma
membrane-targeted Atg proteins are unable togenerate

autophagosomes.

Presumably,PASorganization at the proper site (next to the vacuole) is
required for normal autophagy. Again, this experimentclearly showed that Atg17p is a determinant of the siteof PAS organization during autophagy.

Discussion & Summary
Sixteen ATG genes are essential for autophagosome formation. Extensive analyses have elucidated
five functional groups among the Atg proteins— two ubiquitin-like systems, the PtdIns(3)-kinase
complex, Atg1 protein kinase and its regulators, and the Atg2p•Atg18p complex— but little has been
determined regarding the interrelationships between each unit in autophagosome formation. In thisstudy, we show that every functional group is organized within the PAS, which plays a pivotal role inautophagosome formation. Key to understanding Atg-mediated autophagy is elucidation of how Atg
proteins interact to organize and form the PAS. Therefore, we used fluorescence microscopy tosystematically analyze the PAS localization of Atg proteins.
From this analysis, we have developed a hierarchy diagram of Atg proteins in PAS organization.
Atg1p protein kinase and its regulators, Atg9p, and the PtdIns(3)-kinase complex I act in initial Atg
protein recruitment to the PAS. Atg8p-PE, the Atg12p-Atg5p•Atg16p complex, and the
Atg2p•Atg18p complex are recruited to the PAS with the help of the Atg proteins belonging to theabove group, and work at the PAS to generate autophagosomes. The two ubiquitin-like systems
confer autophagic activity to Atg8p and Atg12p by modifying them. This diagram suggests that
Atg17p is the most upstream component in PAS organization.

34

References
Kuninori Suzuki, Yuka Kubota, Takayuki Sekito, and Yoshinori Ohsumi
Hierarchy of Atg proteins in pre-autophagosomal structure organization. Genes Cells, 12, 209-218(2007)
Kuninori Suzuki, Yoshiaki Kamada, and Yoshinori Ohsumi
Studies of cargo delivery to the vacuole mediated by autophagosomes in Saccharomyces cerevisiae.
Dev. Cell 3, 815-824 (2002)
Kuninori Suzuki, Takayoshi Kirisako, Yoshiaki Kamada, Noboru Mizushima, Takeshi Noda, andYoshinori Ohsumi
The pre-autophagosomal structure organized by concerted functions of I genes is essential for
autophagosome formation. EMBO J. 20, 5971-5981 (2001)

351) Biological Sciences
1-3) Cell Biology

Cell cycle regulation by Apollon
Mikihiko Naito
Institute of Molecular and Cellular Biosciences, The university of Tokyo
mnaito@iam.u-tokyo.ac.jp

Introduction
Apollon is a 530K protein containing baculoviral IAP-repeat (BIR) and ubiquitin-conjugating
enzyme (UBC) domain, and regulates apoptosis by ubiquitylating caspase9, SMAC and HtrA2 forproteasomal degradation. Gene targeting study showed that apollon deficiency results in growth
retardation in some embryos, while abnormality in developmental apoptosis was not obvious,
suggesting that apollon plays a role in regulating cell proliferation. We found in this study thatapollon regulates mitosis by ubiquitylating cyclin A.

Results
When primary MEFs were cultured in vitro, the proliferation of apollon-deficient MEFs halted ataround 10 days after cultivation, while wild-type MEFs proliferated approximately 30 days (Fig. 1).
The apollon-deficient MEFs cultured in vitro for 11 days showed typical features of senescence,
flattened cell morphology and acid β-galactosidase activity (Fig. 2). These observations indicate thatapollon-deficient MEFs undergo earlier replicative senescence in culture.

From the senescent MEFs, we developed a pair of immortalized MEFs and observed their
proliferation under microscopy equipped with time-lapse video recording system and incubation
system. We found that apollon-deficient MEFs require longer period for mitosis than wild typeMEFs, while the period for whole cell cycle was almost comparable. These results suggest that36

Apollon regulates mitosis.
To study the mechanism how apollon regulates mitosis, we searched for apollon interacting proteins
with antibody array, and found that cyclin A but not cyclin B strongly interacted apollon in this array.
Co-immunoprecipitation experiment confirms apollon binds to cyclin A but not to cyclin B in cells.
Deletion analysis indicated that carboxy-terminal deletions to 1-2690 apollon fragment binds tocyclin A but further deletion seriously affected the binding. Mutaion in a conserved cystein in the
BIR domain did not affect the binding to cyclin A while it completely abolished the binding to
SMAC. These results indicate that BIR and UBC domains of apollon are not required to bind cyclin
A. They also suggest that cyclin A binds to apollon at a different site from SMAC binding.
The domain of cyclin A for binding to apollon was determined to cyclin box 1, to which CDK1 and 2
bind. Accordingly, expression of CDK1 and 2, but not CDK 4 and 6, inhibited the binding ofapollon to cyclin A. These results indicate that apollon binds to cyclin A, which is competed by
CDK1 and 2.
Since apollon contains a UBC domain, we examined if cyclin A is ubiquitylated by apollon. Whenco-expressed with FLAG-cyclin A and HA-ubiquitin, apollon, but not survivin or ubc3, enhanced
ubiquitylation of cyclin A. Unexpectedly, apollon C4638A mutant, in which a conserved cysteine
residue in the UBC domain was substituted to alanine, enhanced the ubiquitylation of cyclin A as
well as wild-type apollon, which suggests that the UBC domain of apollon is not essential toubiquitylate cyclin A in cells. They also suggest the role of apollon as an E3 ubiquitin ligase
involving other E2-UBC enzymes for cyclin A ubiquitylation.
To study the role of apollon in cyclin A degradation during mitosis, 293T cells were co-transfectedwith cyclin A-GFP, cyclin B-DsRed and siRNA against apollon. After synchronization at S-phase byaphidicolin, cells were released and observed for degradation of cyclin A-GFP by fluorescencemicroscope. During G2-phase, cyclin A-GFP and cyclin B-DsRed localizes to nuclei and cytoplasm,
respectively. At prometaphase cyclin B-DsRed translocates to nuclei, giving yellow signal in nuclei,
and then cyclin A-GFP was degraded within 20 min in control siRNA treated cells. In apollon siRNAtreated cells, on the other hand, cyclin B-DsRed translocate to nuclei at time 0, but the degradationof cyclin A-GFP was delayed to 210 min. These results indicate that apollon regulates mitosis byubiquitylating cyclin A for proteasomal degradation.

Discussion & Summary
In mammalian cell cycle, cyclin A and cyclin B are required to enter into mitosis, while theirelimination is also essential to complete mitosis. During mitosis, cyclin A and cyclin B are
ubiquitylated by anaphase promoting complex/cyclosome (APC/C) and subjected for proteasomal
degradation. However, cyclin A begins to be degraded at prometaphase when spindle checkpoint
inhibits the activation of APC/C, the mechanism of which was not elucidated. We found in this study

37that apollon binds and ubiquitylates cyclin A for proteasomal degradation. In apollon-deficient cells,
the degradation of cyclin A and progression through mitosis are delayed. These results indicate thatapollon is a ubiquitin ligase for cyclin A involved in its mitotic degradation.

References
1.Hao, Y., Sekine, K., Kawabata, A., Nakamura, H., Ishioka, T., Ohata, H., Katayama, R., Hashimoto,C., Zhang, X., Noda, T., Tsuruo, T., and Naito, M.: Apollon ubiquitinates SMAC and caspase-9,
and has an essential cytoprotection function. Nat Cell Biol, 6: 849-60 (2004)
2.Sekine, K., Hao, Y., Suzuki, Y., Takahashi, R., Tsuruo, T., and Naito, M.: HtrA2 cleaves Apollon
and induces cell death by IAP-binding motif in Apollon-deficient cells. Biochem Biophys Res
Commun, 330: 279-85 (2005)

381) Biological Sciences
1-3) Cell Biology

Spatio-temporal analysis of lipid second messengers with probes
based on fluorescence resonance energy transfer
Michiyuki Matsuda
Graduate School of Medicine, Kyoto University
matsudam@path1.med.kyoto-u.ac.jp

Introduction
Phosphoinositides (PtdIns) are a family of phospholipids containing myo-inositol as their head group.
Despite a relatively low abundance in biological membranes, PtdInss have been reported to regulatea myriad of cellular process depending on their metabolites. However, due to lack of proper probes,
their subcellular localization remains largely unknown. To visualize and quantitate the distribution ofphosphoinositides, we developed probes based on the principle of fluorescence resonance energy
transfer.

Results
FRET probes:
FRET probes for PtdIns(3,4)P2 and PtdIns(3,4,5)P3 have been reported previously. These probes
consists of a PH domain of TAPP-1 for PtdIns(3,4)P2 or GRP for PtdIns (3,4,5)P3, which is
sandwiched with yellow fluorescence protein and cyan fluorescence protein. To modify these probes
for PtdIns (4,5)P2, PtdIns(4)P, or diacylglycerol (DAG), we used PH domain of PLC-δ or FAPP1, or
C1 domain of PKCβ. In addition, to monitor the distribution of these enzymes at the plasma
membrane, we the probes were localized exclusively at the plasma membrane by using K-Ras
C-terminal region.
Distribution of PtdIns in migrating cells:
MDCK cells expressing these FRET probes were time-lapse imaged by fluorescence microscopes.
We found that all of PtdIns(4)P, PtdIns(4,5)P2, PtdIns(3,4)P2, PtdIns(3,4,5)P3, and DAG are enrichedat the leading edge of migrating cells. Inhibitors that perturb the production of any of thesemetabolites significantly impaired the cell migration. This observation strongly suggested that either
production of PtdIns (4)P or translocation PtdIns (4)P from ER to plasma membrane is accelerated.
Growth factor-induced changes in phosphoinositides:

39

Growth factor-induced changes in the level of PtdIns(4,5)P2, PtdIns(3,4)P2, and PtdIns(3,4,5)P3 were
found to be very rapid and transient. This observation suggests that the mechanism to hold thehomeostasis of phospholipids are tightly regulated even in growth-factor stimulated cells.

Discussion & Summary
We could successfully visualized the spatio-temporal regulation of PtdIns(4)P, PtdIns(4,5)P2,
PtdIns(3,4)P2, PtdIns(3,4,5)P3, and DAG in migrating cells and growth factor-stimulated cells. By
adding proper internal standards, the FRET images could be evaluated in a quantitative manner.
Such data will provide versatile information on how the metabolism of phosphoinositides isregulated in the cells. More importantly, such data can be converted into parameters that are essential
to operate kinetic simulation models. With this approach, we believe, we could understand themetabolism of and the signaling cascades mediated by phosphoinositides as a integrated system.

References
H. Yoshizaki, N. Mochizuki, Y. Gotoh, and M. Matsuda. Akt-PDK1 Complex Mediates
EGF-induced Membrane Protrusion through Ral Activation. Mol.Biol.Cell 18:119-128, 2007.

40

1) Biological Sciences
1-3) Cell Biology

Analysis of molecular mechanisms for regeneration of retina andneural tissues by stem cells and clinical application
Noriko Gotoh
Institute of Medical Science, The University of Tokyo
ngotoh@ims.u-tokyo.ac.jp

Introduction
Recently, increasing attention is paid to the regenerative medicine exploiting various stem cells
including neural or retinal stem cells (1). Neural stem cells are critical as a source of the cell
replacement therapy for the neurodegenerative disease or injured brain and spinal cord. Retinal stem
cells also have potential to be used for treatment of retinal diseases including retinal degeneration.
Therefore, it is important to clarify the molecular mechanisms underling the biology of neural stem
cells, such as the control of proliferation and differentiation. We have previously demonstrated that
FRS2alpha docking protein plays critical roles for growth and maintenance of several stem cells
including neural or retinal stem cells (2). The aim of this study is to clarify the molecularmechanisms for FRS2alpha to maintain these stem cells and to test if FRS2alpha can be used for cell
replacement therapy.

Results
Overexpression of wild type Frs2α or 8V, but not 6F, increases the size of the neurospheres
derived from E12 telencephalon.
To analyze the function of Frs2α in NSPCs, neurospheres derived from the telencephalon of E12mouse embryos were infected with retrovirus expressing wild type or mutant Frs2α, and cultured in
the presence of 2, 5, 20 ng/ml FGF2. Control was infected with empty vector, and viral infection wasmonitored by the signal from EGFP. At every dose of FGF2, overexpression of wild type Frs2α or its
constitutive active form 8V apparently increased the size of neurospheres compared with control. In
contrast, overexpression of 6F, that lacks all tyrosine phosphorylation sites, showed no apparent
effect on size of neurospheres. The distribution of diameter shows that neurospheres expressing wild
type Frs2α or 8V contains larger neurospheres than control, but not 6F. In addition, average diameterof neurospheres expressing wild type Frs2α or 8V, but not 6F, was significantly larger than control atevery dose of FGF2.

41

Overexpression of wild type Frs2α or 8V, but not 6F, increases the number of neurospheres
bFGF-specifically
FGF2 is a commonly used mitogen of NSPCs in vitro. However, it is unclear whether FGF
signaling also contributes to the self-renewal of neural stem cell. To examine whether Fgf signaling
through Frs2α plays role in the self-renewal ability of neural stem cells, we examined the number ofneurospheres formed when neurospheres were infected with retrovirus expressing wild type ormutant Frs2α.
In neurospheres derived from the telencephalon of E12 embryos, overexpression of wild type Frs2α
or 8V significantly increased the number of neurospheres, but not 6F. Similar result was obtained
when wild type or mutant Frs2α were overexpressed in neurospheres derived from the telencephalonof E14 embryos, and cultured in the presence of FGF2. In contrast, when neurospheres were cultured
in the presence of EGF, overexpression of wild type or mutant Frs2α had no effect on the number ofneurospheres.
To determine whether the effect of overexpression of Frs2α is FGF-specific, neurospheres cultured
in the presence of EGF was passed, and cultured again in the presence of EGF or FGF. Whenneurospheres were cultured again in medium containing EGF, over expression of wild type or mutant
of Frs2α did not show effect on the number of neurospheres. In contrast, when neurospheres were
cultured again in the presence of FGF2, average number of the neurospheres expressing wild typeFrs2α or 8V was significantly larger than control, but not 6F.
Activity of Erk and Akt is enhanced in neurospheres expressing wild type Frs2α or 8V but not
Frs2α-6F.
To examine the activation of the FGF signaling pathways down stream of Frs2α in theneurospheres overexpressing wild type of mutant Frs2α, components of those pathways wereanalyzed by western blotting. Control neurospheres and neurospheres overexpressing 6F showedsimilar kinetics with respect to the activation of Erk1/2 and Akt. In contrasts, neurospheres
overexpressing wild type Frs2α or 8V showed strong and long standing activation of both Erk and
Akt. In addition, activation of Shp2 and Gab1, two proteins down stream of Frs2α that are
responsible for the activation of Ras-Erk and PI3K-Akt pathway respectively, was enhanced in theneurospheres overexpressing wild type Frs2α or 8V compared with control or 6F.

Discussion & Summary
Discussion
In the present study, wild type or mutant forms of Frs2α were overexpressed in the neurosphereswith retrovirus system to analyze the function of Frs2α in NSPCs. In the presence of FGF2,
overexpression of wild type Frs2α or 8V in E12 or E14 neurospheres increased the size of42neurospheres, but overexpression of 6F did not. These results indicate that FGF2-induced tyrosine
phosphorylation of Frs2α plays an important role for the proliferation of NSPCs regardless ofdevelopmental stages examined. In contrast, overexpression of wild type or mutant forms of Frs2α
did not show effect on the proliferation of the neurospheres in the presence of EGF, suggesting thatoverexpression of Frs2α or 8V potentiates the response of NSPCs to FGF2 stimulation specifically.
Moreover, when neurospheres overexpressing wild type or mutant forms of Frs2α cultured in thepresence of EGF was passed and cultured again in the presence of FGF2 or EGF, neurospheres
expressing wild type or 8V showed efficient growth FGF2-specifically, but not 6F. These results
further confirm that the effect of overexpression of Frs2α is FGF2 specific, and indicating thatsignaling pathways down stream of Frs2α are activated strongly by the overexpression of Frs2α or
8V.
Interestingly, overexpression of Frs2α or 8V increased the number of neurospheres formed, but not
6F. This effect was also FGF2 specific, and independent of developmental stages neurospheres
derived from. This indicates that overexpression of Frs2α or 8V promotes not only the proliferationof NSPCs, but also the self renewal of neural stem cells (Figure).

Overview of Fgf signaling via Frs2
FGF

Ras-MAPK
pathway

PI3K-Aktpathway

FGFR
Grb2
bindingsites

cytosol

PTB

cell membrane

Y196
Y306
Y349
Y392

FRS2

Self renewal of neural stem cells

Y436
Y471

Shp2
bindingsites

Ras-MAPK
pathway

Proliferation of Neural progenitor
cell (Yamamoto et al., 2005 )Summary
It is important to manipulate neural stem/progenitor cells (NSPCs) in vitro in order to apply them forcell replacement therapy. Fibroblast growth factor (FGF) 2 is a commonly used mitogen for NSPCsin vitro, however, it is still unclear how FGF2 controls NSPCs. Here, we provide evidence that FGF2
controls not only proliferation but also self-renewal of NSPCs through FRS2a docking protein. We
expressed wild type FRS2α or its mutant forms in neurospheres, spherical cell clusters of NSPCs

43

using a retrovirus system. Expression of wild type FRS2α or its constitutive active form of
FRS2a-8V promoted the proliferation of NSPCs in the presence of FGF2 but not epidermal growth
factor (EGF). Interestingly, the number of FGF2-induced neurospheres, an indicator of self-renewing
ability, was also increased by expression of either protein. In contrast, expression of FRS2α-6F, thetyrosine (Y) to phenylalanine (F) mutant of all six tyrosine phosphorylation sites, did not show theseeffects on NSPCs. Moreover, we found that activation of both ERK and AKT was enhanced inneurosphere cells expressing wild type FRS2α or FRS2α-8V but not FRS2α-6F. These results
suggest that FGF2-induced tyrosine phosphorylation of FRS2α plays an important role forproliferation and self-renewal of NSPCs and raise a possibility to make use of FRS2α formanipulating NSPCs in vitro.

References
1 Okano H. Stem cell biology of the central nervous system. J. Neurosci. Res., 69, 698-707, 2002.
2 Yamamoto S., Yoshino I., Shimazaki T., Murohashi M., Hevner,RF, Lax I., Okano H., Shibuya M.,
Schlessinger J. and Gotoh N. Essential role of Shp2-binding sites on FRS2alpha for corticogenesis
and for FGF2-dependent proliferation of neural progenitor cells. Proc. Natl. Acad. Sci., USA, 102,
15983-15988, 2005.

441) Biological Sciences
1-3) Cell Biology

Development of regulatory strategies for neuronal death induced bymalfolded proteins.
Imaizumi Kazunori
Division of Molecular and Cellular Biology, Department of Anatomy,

Faculty of Medicine,

University of Miyazaki
imaizumi@med.miyazaki-u.ac.jp

Introduction
The endoplasmic reticulum (ER) stress response is a defense system for dealing with theaccumulation of unfolded proteins in the ER lumen. Recent reports have shown that ER stress isinvolved in the pathology of some neurodegenerative diseases and cerebral ischemia.

Results
In a screen for compounds that induce the ER-mediated chaperone BiP/GRP 78 (BiP), we
identified BiP inducer X (BIX). BIX induced BiP only, in a dose-dependent manner, without
induction of other molecules involved in the ER stress response. The induction of BiP mRNA by
BIX was mediated by activation of ER stress response element (ERSE) through ATF6 pathway.
Pretreatment of neuroblastoma cells with BIX reduced cell death induced by ER stress.
Intracerebroventricular pretreatment with BIX reduced the area of infarction due to focal cerebral
ischemia in mice. In the penumbra of BIX-treated mice, ER stress-induced apoptosis was suppressed,
leading to a reduction in the number of apoptotic cells.

Figures&Tables

45

Discussion & Summary
BIX induces BiP to prevent neuronal death by ER stress, suggesting that it may be a potential
therapeutic agent for cerebral diseases caused by ER stress.

References
Kudo,T., Kanemoto,S., Hara, H., et al. A molecular chaperone inducer protects neurons from ER
stress. Cell Death and Differentiation, in revision.

461) Biological Sciencese1-4) Developmental Biology

Analysis of molecular mechanism of fusion process intermediating
Izumo factor on sperm surface.
Naokazu Inoue
Reserch Institute for Microbial Diseases, Osaka University
ninoue@gen-info.osaka-u.ac.jp

Introduction
It is said that one tenth of human couples are suffering from infertility.

In fact, in our country,

at least one out of hundred new born babies are treated with ART (Assisted Reproductive
Technology).

However, the molecular basis for fertilization is poorly understood, especially in the

final sperm-egg fusion step.

During the long history of research in fertilization, many factors

involved in sperm-egg fusion have been reported.
assessed by utilizing gene targeting.

Until now, almost of these factors have been

Representative of factor, CD9, which belongs totetraspan-membrane protein family on the egg membrane was found to be essential for fusion, butsperm-related fusion factors remain unknown.

Using a fusion-inhibiting monoclonal antibody, we

identified a mouse sperm fusion-related antigen, Izumo.
incapable of fusing with eggs.

Izumo -/- mice were healthy but wereHere we show that we identified novel Izumo-binding factor byusing sperm originated from Izumo- 6 x His-transgenic mice line.

Results
A sperm and an egg meet, recognize each other, and fuse to create a new generation of life. The
factors involved in this important membrane fusion event, fertilization, have been sought for a longtime.

Recently, CD9 on the egg membrane was found to be essential for fusion (1), butsperm-related fusion factors remain unknown.

Using a fusion-inhibiting monoclonal antibody, we

identified a mouse sperm fusion-related antigen, cloned the gene and discovered that the antigen was
a novel immunoglobulin superfamily protein.
gene-disrupted mouse line.

We termed the gene Izumo and produced a

Izumo -/- mice were healthy but males were sterile.

They produced

normal looking sperm that bind to and penetrate the zona pellucida, but were incapable of fusingwith eggs. Human sperm also contain Izumo and addition of the anti-human Izumo antibody left
the sperm unable to fuse with zona-free hamster eggs (2).
To examine whether if the phenotype was directly derived from the lack of Izumo on sperm, we
performed the rescue experiment by crossing Izumo -/- mice with transgenic mouse lines generated

47

to express Izumo- 6 x His-tag using the testis-specific calmegin promoter.

The sterile phenotype

was rescued with the transgenically expressed Izumo on mouse sperm.
To identify the factor (s), which interact to Izumo on sperm, we employed His-tag pull down assay.
We prepared sperm lysate containing 1% Brij 97 detergent in Izumo knockout background mouse
Izumo transgenic mice with 6 x His-tag obtaining from intercrossing beforehand. Then, magnetic
beads conjugated with anti-His-tag antibody were added to sperm lysate.

The protein (s) coupledwith anti-His-tag were passed through the magnetic column to attach the Izumo and Izumo binding
protein, the factor was identified by separation of the extracts by SDS-PAGE, sequentially, subject it
to liquid chromatography tandem mass spectrometry (LC-MS/MS).

Consequentially, these 9

peptides which were 100% identical to a part of the sequence listed in RIKEN full length databasewere found.
The novel protein was 80 kDa and highly similar to testicular ACE (angiotensin-converting
enzyme), which is involved in sperm-zona interaction owing to glycosylphosphatidylinositol (GPI)-releasing activity (3), but, this protein was partially conserved important zinc binding motif
regulating center of active site.

Besides, this gene showed testis specific expression pattern inmRNA level, therefore we named it testicular ACE 2 (tACE2).
polyclonal antibody using recombinant tACE2.

We promptly produced anti-tACE2

After that, we could obtain the data that confirm

the evidence of directly binding tACE2 to Izumo each other by performing immunoprecipitation
experiment.
Taken together, these data suggest that there is possibility that both Izumo and tACE2 are incooperation with each other to maintain membrane order attributing to trigger the membrane fusion,and are essential factors to accomplish sperm-egg fusion on the sperm side.

Discussion & Summary
So far, it has been reported that testis ACE functions not only a key regulator of blood pressure
together with dipeptidyl carboxypeptidase, but also sperm-zona pellucida binding utilizing a
glycosylphosphatidylinositol (GPI)-anchored protein releasing activity (3).

Thus, taken together

with our data suggests that ACE seems to be multi-functional gene rather than only act as anangiotensin-converting.

If tACE2 also involves in fertilization, in particular, sperm-egg fusion

process, although we need further gene targeting experiment as proved in Izumo knock out mouse,
the study of ACE would be step into new stage of expansion.

Hopefully, these finding not only

provides insight into the enigmatic fusion mechanism but also promises future benefits for theclinical treatment of infertility and the potential development of novel contraceptive strategies.

48

References
1. Miyado K, et al.: Requirement of CD9 on the egg plasma membrane for fertilization. Science.
287: 321-324, 2000.
2. Inoue N, et al.: The immunoglobulin superfamily protein Izumo is required for sperm to fuse witheggs. Nature. 434: 234-238, 2005.
3. Kondoh G, et al.: Angiotensin-converting enzyme is a GPI-anchored protein releasing factor
crucial for fertilization. Nat Med. 11: 160-166, 2005.

49

1) Biological Sciences1-4) Developmental Biology

Periodic gene expression generates spatial pattern in mouse
development
Yasumasa Bessho
Graduate School of Biological Sciences

Nara Institute of Science and Technology

ybessho@bs.naist.jp

Introduction
In the mouse development, the most prominent repetitive structure is somite, which gives rise torepetitive pattern of mouse body such as vertebrae, ribs and muscles. A bilateral pair of somites buds
off from the anteriormost end of the presomitic mesoderm (PSM). A new somite is formed every 2 hin the mouse, and this periodic event is govern by a molecular clock. Previously, we revealed themechanism of the molecular clock, which includes a negative feedback loop of gene expressions. In
this project, we aimed at elucidating how the molecular clock generates the regular interval patternof somotes.

Results
In the mouse PSM cells, the expression of several genes, including basic helix-loop-helix gene Hes7,
oscillates synchronously with somite formation[1,2]. Notch signaling induces the transcription ofHes7, and induced Hes7 inhibits its own promoter to repress the transcription of Hes7. Notch
signaling also activates Lunatic fringe (Lfng), which encodes a Notch modulator, and Hes7 inhibits
the expression of Lfng periodically [3]. Additionally, gain- or loss-of function of Hes7 disrupts bothoscillatory gene expression and periodic segmentation of somites as well as the regular interval
pattern of somites[2](Bessho, unpublished data). Thus, this negative feedback loop of Hes7 serves as
the central mechanism of the gene oscillation, which controls periodic segmentation as a biologicalclock.
Fgf8 is expressed in the posterior end of the PSM and displays a gradient to anterior PSM.
Because excess FGF signaling pushes the segmentation border to anterior, the gradient of FGF
signaling serves as spatial information to determine the position of the segmentation[4,5]. In the tailbud region, the posterior end of the embryo, mesenchymal cells proliferate extensively, andconsequently the embryo elongates posteriorly. At the same time the gradient of FGF signaling
gradually sifts posteriorly and decides the new segmentation border every 2 h, which thesegmentation clock counts, to generate even grained somites. Thus, gene oscillation and FGF

50

gradient determine the timing and the position of segmentation, respectively.
To elucidate how gene oscillation and FGF gradient are combined and how their information isconverted to precise morphological pattern, we assumed putative mediates, which should modulate
the FGF signaling periodically in a 2 h cycle depending on the gene oscillation. FGF signaling has
several feedback inhibitors. We investigated the expression pattern of those genes in the mouse PSM.
Among them, we found that Sprouty4 is expressed prominently in the PSM and the pattern is similar
to that of Fgf8. But the expression pattern of Sprouty4 is various in different embryos. Someembryos have strong signal in the posterior PSM, some show in the middle, the others display strong
staining in the anterior PSM (Fig. 1).

Fig. 1 Sprouty4 shows different expression pattern in each embryo.
E10.5 embryos show variable expression pattern of Sprouty4. Uncx4.1 is used asa marker for formed somites. Among 104 embryos, 42 show strong Sprouty4
expression in the posterior PSM, 39 show in the middle and 23 show in theanterior PSM respectively

Next we compared the expression of Sprouty4 with that of that of Lfng, which oscillates in the same
phase as Hes7[2]. All embryos Sprouty4 and Lfng are co-expressed in the same region of the PSM(Fig. 2), indicating that Sprouty4 oscillates in the same phase as Hes7 and Lfng. In Hes7 KO
embryos, in which the expression of Hes7 does not oscillate[2], we saw uniform expression of
Sprouty4 in the PSM (data not shown). Thus, Hes7 is essential for the oscillation of Sprouty4,
suggesting Hes7 may periodically inhibit the transcription of Sprouty4. Taken all together, Sprouty4
could be a candidate of a mediator that combines the temporal information of gene oscillation andspatial information of FGF gradient in the mechanism of periodic somite formation.

Fig. 2 Sprouty4 oscillates in phase with Lfng.
The posterior embryos including PSM were cut into half sagittally, andSprouty4 and Lfng were detected separately. In each phase, both signals are
similar except the anterior most signal of Lfng.

51

Discussion & Summary
Sprouty4 protein is implicated as an inhibitor that blocks the FGF/MAPK cascade, although theprecise molecular mechanism remains elusive[6]. In this study, we showed that the expression of
Sprouty4 mRNA oscillates in the mouse PSM, synchronously with somite segmentation under thecontrol by Hes7. It strongly suggests that the expression of Sprouty4 protein also oscillates in thesame cycle, and that Sprouty4 protein inhibits the FGF signaling periodically in the PSM. Thus next
steps in this project are examining the expression pattern of Sprouty4 protein and measurement of
the FGF activity spatiotemporally in the PSM. At the same time, we need to examine whether theoscillation of Sprouty4 is essential for the somite formation by gain- and loss-of function experimentof Sprouty4.
In this project, we identified a candidate molecule that combines temporal and spatial information,and it might be a critical mechanism of formation of repetitive pattern in mouse development.

References
1. Bessho Y, Miyoshi G, Sakata R, Kageyama R: Hes7: a bHLH-type repressor gene regulated by
Notch and expressed in the presomitic mesoderm. Genes Cells 2001, 6:175-185.
2. Bessho Y, Sakata R, Komatsu S, Shiota K, Yamada S, Kageyama R: Dynamic expression andessential functions of Hes7 in somite segmentation. Genes Dev 2001, 15:2642-2647.
3. Bessho Y, Hirata H, Masamizu Y, Kageyama R: Periodic repression by the bHLH factor Hes7 isan essential mechanism for the somite segmentation clock. Genes Dev 2003, 17:1451-1456.
4. Dubrulle J, McGrew MJ, Pourquie O: FGF signaling controls somite boundary position andregulates segmentation clock control of spatiotemporal Hox gene activation. Cell 2001,
106:219-232.
5. Sawada A, Shinya M, Jiang YJ, Kawakami A, Kuroiwa A, Takeda H: Fgf/MAPK signalling is a
crucial positional cue in somite boundary formation. Development 2001, 128:4873-4880.
6. Sasaki A, Taketomi T, Kato R, Saeki K, Nonami A, Sasaki M, Kuriyama M, Saito N, Shibuya M,
Yoshimura A: Mammalian Sprouty4 suppresses Ras-independent ERK activation by binding
to Raf1. Nat Cell Biol 2003, 5:427-432.

521) Biological Sciences
1-6) Pharmacology

Genomic Drug Target Validation Study for Cerebrovascular Disorders.

Toshio Tanaka
Department of Molecular and Cellular Pharmacology, Pharmacogenomics andPharmacoinformatics, Division of Genomics and Regenerative Medicine, Mie University
Graduate School of Medicine
tanaka@doc.medic.mie-u.ac.jp

Introduction
Delayed cerebral vasospasm often develops 3 to 9 days after aneurysmal SAH. In 27% to 38% of
patients with SAH, a delayed, neurological ischemic deficit occurs. Despite successful surgicaltreatment for intracranial aneurysm and postoperative management, 26% to 38% of such patients
suffer sequelae or die of symptomatic severe vasospasm. Although extensive research has been
carried out into the mechanisms underlying delayed cerebral vasospasm and many advanced
therapies have been attempted, we still have no successful treatment for this condition.

Results
The hydroxyl radical scavenger (±)-N, N’-propylenedinicotinamide is an agent that is especiallybeneficial

indelayedvasospasm.

We

found

that

intravenous

injectionof(±)-N,

N’-propylenedinicotinamide exhibit spasmolytic effects with induction of Heme Oxigenase-1(HO-1) in basilar arteries in rat model of SAH, but not in normal rat. Furthermore, (±)-N,
N’-propylenedinicotinamide promotes cGMP accumulation as a downstream of HO-1 in basilar
arteries.

Discussion & Summary
The supposed pathogenetic role of oxygen free radicals in SAH and other type of neuronal injuries
has stimulated interest in antioxidants and free radical scavengers as potential therapeutic agents. [±]-N, N’-propylenedinicotinamide; nicaraven, a water- and oil-soluble derivative of nicotinamide
demonstrates a radical scavenging action putatively specific to the hydroxyl radical. In regard to
SAH pathophysiology, AVS pharmacological profile can be summarized as follows: it scavenges
hydroxyl radicals; exhibits anticontractile activity of 15-hydroperoxyarachidonic acid; suppresses
arterial spasm following SAH in dogs; improves neurological deficits after SAH in rats by decreased

53

blood-brain barrier permeability change. Furthermore, in a multicenter placebo-controlled
double-blind clinical trial, AVS significantly ameliorated the delayed ischemic neurologic deficits
following aneurysmal SAH, with a marked improvement in the Glasgow Outcome Scale (GOS)
scores at 1 month, and a marked decreased in the overall mortality.
The present study used intracisternal injection of antisense HO-1 ODN to identify the protective
effects of HO-1 in cerebral vasospasm in rat model of SAH. In conclusion, the therapeuticmechanism of (±)-N, N’-propylenedinicotinamide is owed its enhancement of intrinsic HO-1
induction in pathophysiological state, subsequently to increased cGMP-induced vascular smooth cellrelaxation.

Figures & Tables

541) Biological Sciences
1-7) Plant Biology

Molecular mechanisms of photoreceptors-regulated growth of plant
through phytohormone auxin
Tatsuya Sakai
RIKEN Plant Science Centertsakai@psc.riken.jp

Introduction
The phototropic response of Arabidopsis thaliana seedlings is initiated by the blue-light (BL)photoreceptors, phototropin (phot) 1 and phot2, and by their signal transducers, NPH3 and RPT2.
Although previous studies have indicated that phot1 contains BL-activated autophosphorylation
activity and binds to NPH3 and RPT2, its biochemical function during the induction of thephototropic response remains unclear. In addition, photoreceptors, phytochromes and cryptochromes
regulate hypocotyl growth under specific conditions by suppressing negative-gravitropism,
modulating phototropism and inhibiting elongation. Although those effects seem to be partiallycaused via the regulation of the phytohormone auxin, the molecular mechanisms underlying thisprocess are still poorly understood.

Results
In our current study, we show that phot1 regulates the functional expression of NPH3 and RPT2 in adifferent manner. Our results indicate that the NPH3 protein is phosphorylated in seedlings underdark or red-light conditions and is dephosphorylated under BL. Phot1 is necessary for thisBL-dependent dephosphorylation of NPH3. Our immunoprecipitation analysis further demonstrates
that NPH3 dephosphorylation facilitates the effective association of this protein with RPT2 in vivo.
We determine the phosphorylation domain of NPH3 and reveal the negative effect of NPH3 on thegravitropic response. In addition, phot1 induces the expression of RPT2 in seedlings under BL
conditions through post-transcriptional regulation. In contrast, we show that phot2 is not involved inthese regulatory pathways for NPH3 and RPT2.
On the functional analyses of phytochrome and cryptochrome on the tropic responses, wedemonstrate that the flabby mutation enhances the phytochrome-inducible hypocotyl bending in
Arabidopsis. The FLABBY gene encodes the ABC-type auxin transporter, PGP19, and its expression
is suppressed by the activation of phytochromes and cryptochromes. These results therefore revealthat phytochromes and cryptochromes have at least two effects upon the tropic responses of the

55

hypocotyls in Arabidopsis; one is the enhancement of hypocotyl bending through the suppression of
PGP19, and another is a hypocotyl bending-inducing mechanism, which is independent of PGP19.
The auxin polar transport assay and the DR5::GUS expression analysis suggest that phytochromes
inhibit the basipetal auxin transport and induce the asymmetric distribution of auxin in hypocotyls.
Further, activations of phytochromes and cryptochromes decrease auxin content in the aerial portionof the seedlings.

Discussion & Summary
Our results thus suggest the possibility that the post-transcriptional events of NPH3 and RPT2 are
the underlying mechanisms that trigger the phot1-induced phototropic response. In addition, thecontrol of auxin transport and accumulation by phytochromes and cryptochromes may be a criticalcomponent of the regulation of hypocotyl growth in response to light.

Figures & Tables

561) Biological Sciences
1-8) Animal Biology

Study on processing mechanisms of novel identified neuropeptides.

Kazuyoshi Ukena
Graduate School of Integrated Arts and Sciences, Hiroshima University
ukena@hiroshima-u.ac.jp

Introduction
Recently, we identified a novel hypothalamic neuropeptide with a C-terminal LPLRFamide
sequence in the quail brain (1). This avian neuropeptide was shown to inhibit gonadotropin releasefrom the cultured anterior pituitary. This peptide is the first hypothalamic peptide that inhibited
gonadotropin release reported in vertebrates. We, therefore, termed it gonadotropin-inhibitory
hormone (GnIH). After this finding, we found that GnIH-related peptides were present in the brainsof other vertebrates, such as mammals, birds, amphibians, and fish (2). These GnIH-related peptides
possessed a LPXRFamide (X=L or Q) motif at their C-termini in all investigated animals.

Results
We further characterized the GnIH cDNA from the quail brain and found that the deduced GnIH
precursor polypeptide encoded one GnIH and two gene-related peptides (GnIH-RP-1 and
GnIH-RP-2). These peptides possessed a LPXRFamide (X=L or Q) motif at their C-termini.
Subsequently, GnIH-RP-1 and GnIH-RP-2 were identified as mature endogenous peptides usingmass spectrometric analyses. In addition, N-terminally extended forms of GnIH and GnIH-RP-2
were found to be present in the quail hypothalamus and termed as GnIH-18 and GnIH-RP-2-18,
respectively.
In the present study, I investigated that GnIH and GnIH-RP-2 were processed from theirN-terminally extended forms, GnIH-18 and GnIH-RP-2-18, in the hypothalamus by biochemical andimmunohistochemical analyses. Firstly it was found that GnIH-RP-2 was produced by the incubation
with GnIH-RP-2-18 and hypothalamic extracts using high performance liquid chromatography(HPLC) and MALDI-TOF mass spectrometric analysis. This result showed that the quail
hypothalamus contained a novel processing enzyme which recognize between arginine (R) andserine (S) residues. Secondly I raised specific antiserum against N-terminal region of GnIH-18 andcomparedthelocalizations

of

GnIH

and

GnIH-18

using

the

fluorescencedoubleimmunohistochemical analysis. This result showed that GnIH-18 was mainly localized in the soma

57

and GnIH was present in the nerve fibers throughout the brain, suggesting that the mature peptide
GnIH was localized in the synapse vesicle of the nerve terminal (Figure 1).

Discussion & Summary
Before this study, N-terminally extended forms of GnIH and GnIH-RP-2 (GnIH-18 and
GnIH-RP-2-18, respectively) have been thought as variants of GnIH and GnIH-RP-2.

In this study

it was found that GnIH and GnIH-RP-2 were processed from their N-terminally extended forms,
GnIH-18 and GnIH-RP-2-18, in the quail hypothalamus by biochemical and immunohistochemical
analyses. This result suggests that the unique N-terminal processing mechanism including Kex2-likeprocessing enzyme, carboxypeptidase and peptidylglicine α-amidating monooxygenase may bepresent in the precursor polypeptide of GnIH in the avian brain (Figure 2). In addition, this novel
processing enzyme may recognize between arginine (R) and serine (S) residues.

References
1) Tsutsui et al. (2000). Biochem. Biophys. Res. Commun., 275, 661-667.
2) Ukena & Tsutsui (2005). Mass Spectrom. Rev., 24, 469-486.

582) Medical Sciences2-1) Immune System

The role of cortex-to-medulla migration of developing thymocytes inestablishing central tolerance
Yousuke Takahama
Division of Experimental Immunology, Institute for Genome Research, ,University ofTokushima
takahama@genome.tokushima-u.ac.jp

Introduction
Immature CD4+CD8+ thymocytes, which are generated in the thymic cortex, are induced upon
positive selection to differentiate into mature T lymphocytes and relocate to the thymic medulla. It
was recently shown that a chemokine signal via CCR7 is essential for the cortex-to-medulla
migration of positively selected thymocytes in the thymus.

However, the role of thecortex-to-medulla migration in T-cell development and selection has remained unclear.

Using micethat are deficient in CCR7 or its ligands (CCR7L), we addressed whether T cells may be exported
from the thymic cortex without accumulation in the medulla and whether central tolerance toorgan-specific self-antigens may be affected by the absence of medulla migration.

Results
Our results showed that the pharmacological inhibition of S1P-mediated thymocyte egress in
CCR7- or CCR7L-deficient mice results in the accumulation of mature thymocytes in the cortex,
suggesting that mature thymocytes may be exported via the S1P-dependent mechanism from thecortex in the absence of CCR7 signals. We also showed that in the absence of CCR7-dependent
medulla migration, mature thymocytes are incapable of acquiring tolerance to lacrimal and salivary
glands and are potent in inducing autoimmune exocrinopathy similar to Sjögren’s syndrome.

Discussion & Summary
The present study showed that the developmental kinetics and the thymic export of mature
thymocytes were undisturbed in mice lacking CCR7 or CCR7L. However, the thymocytes thatwere generated in the absence of CCR7 or CCR7L were potent in causing autoimmune
dacryoadenitis and sialadenitis in mice, and were thus incapable of establishing central tolerance toorgan-specific antigens.

These results indicate that CCR7-mediated cortex-to-medulla migration ofthymocytes is essential for establishing central tolerance rather than for supporting the maturation or

59

export of thymocytes.

Figures & Tables

References
Nitta T, Takahama Y. The lymphocyte guard-IANs: regulation of lymphocyte survival by
IAN/GIMAP family proteins. Trends Immunol. 28:58-65 (2007)
Liu C, Saito F, Liu Z, Lei Y, Uehara S, Love PE, Lipp M, Kondo S, Manley NR, Takahama Y.
Coordination between CCR7- and CCR9-mediated chemokine signals in pre-vascular fetal thymus
colonization. Blood. 108:2531-2539 (2006)
Nitta T, Nasreen M, Seike T, Goji A, Ohigashi I, Miyazaki T, Ohta T, Kanno M, Takahama Y. IAN
family critically regulates survival and development of T lymphocytes. PLoS Biology. 4(4):e103(2006)
Kurobe H, Liu C, Ueno T, Saito F, Ohigashi I, Seach N, Arakaki R, Hayashi Y, Kitagawa T, Lipp M,
Boyd RL, Takahama Y. CCR7-dependent cortex-to-medulla migration of positively selected
thymocytes is essential for establishing central tolerance. Immunity. 24:165-177 (2006)
Takahama Y. Journey through the thymus: stromal guides for T-cell development and selection.
Nature Rev Immunol. 6:127-135 (2006)
Hollander G, Gill J, Zuklys S, Iwanami N, Liu C, Takahama Y. Cellular and molecular events during
early thymus development. Immunol Rev. 209:28-46 (2006)

602) Medical Sciences2-2) Central Nervous System

Uterine sensitization-associated gene-1 (USAG-1), a novel BMP
antagonist expressed in the kidney, accelerates tubular injuries
Motoko Yanagita
Kyoto University Graduate School of Medicine
motoy@kuhp.kyoto-u.ac.jp

Introduction
Dialysis dependency is one of the leading causes of morbidity and mortality in the world, andonce end-stage renal disease develops, it cannot be reversed by currently available therapy. Although
the administration of large dose of bone morphogenetic protein-7 (BMP-7) has been shown to repair
established renal injuries and improves renal function, regulatory mechanism of endogenous BMP-7
remains elusive.

Results
Here we show that uterine sensitization-associated gene-1 (USAG-1), novel BMP antagonist
abundantly expressed in the kidney, is the central negative regulator of BMP function in the kidney,
and that USAG-1 deficient mice (USAG-1-/- mice) are resistant to kidney injuries. USAG-1-/- mice
exhibited prolonged survival and preserved renal function in acute and chronic renal injuries. Renal
BMP signaling, assessed by phosphorylation of Smad proteins, is significantly enhanced in
USAG-1-/- mice during renal injury, and the administration of neutralizing antibody against BMP-7
abolished reno-protection in USAG-1-/- mice, indicating that renoprotection in USAG-1-/- mice was atleast in part attributed to the enhancement of BMP-7 function. Furthermore, expression analysis of
BMP antagonists in the kidney revealed that USAG-1 was the most abundant BMP antagonist in thekidney, and its expression overlapped with that of BMP-7. From these findings, we conclude that
USAG-1 plays a critical role in the modulation of reno-protective action of BMP.

Discussion & Summary
The results in the study justify the therapy targeted towards USAG-1: drugs or neutralizing
antibodies that inhibits binding between USAG-1 and BMP, or gene-silencing therapy for USAG-1
would enhance the activities of endogenous BMP, and might be a promising way to develop novel
therapeutic methods for severe renal diseases. Because the expression of USAG-1 is confined to thekidney in adult mice and humans, it would be a better target for therapeutic trials than the

61administration of BMP-7 itself, whose target cells are widely distributed throughout the body.

Figures & Tables

References
1Motoko Yanagita, Shuichiro Endo, Katsu Takahashi, Tomohiko Okuda, Fumihiro Sugiyama,Satoshi
Kunita, Satoru Takahashi, Atsushi Fukatsu, Toru Kita, Takeshi Sakurai
USAG-1, a novel BMP antagonist abundantly expressed in the kidney, accelerates kidney injuries.
J Clin Invest. 116: 70-9, 2006.
2 Motoko Yanagita
Modulator of bone morphogenetic protein activity in the progression of kidney diseases.
Kidney Int. Sep;70(6):989-93, 2006
3 Motoko Yanagita
Uterine Sensitization Associated Gene-1 (USAG-1): A Bone Morphogenetic Protein Antagonist in
the Kidney. (book chapter)“TRANSFORMING GROWTH FACTOR-β IN CANCER THERAPY”, edited by Sonia B.
Jakowlew, PhD, Humana Press, in press

622) Medical Sciences2-2) Central Nervous System

Studies on the calcium-dependent mechanisms regulating theactivity-dependent release of brain-derived neurotrophic factor(BDNF) at the presynaptic terminal.
Hiromu Yawo, Toru Ishizuka
Department of Developmental Biology and Neuroscience, Tohoku University Graduate Schoolof Life Science
yawo@mail.tains.tohoku.ac.jp

Introduction
The brain-derived neurotrophic factor (BDNF), one of neurotrophins in the centranl nervous system,has been known to be involved in the hippocampus-dependent mechanisms of learning and memory.
Here we investigated the mechanisms regulating BDNF release at the large mossy fiber presynaptic
terminal of hippocampus. Our optical methods have to be improved in signal/noise ratio for further
investigation.

Results
Two Sindbis virus vectors were conventionally made; one with a construct of a fusion protein of
preproBDNF and Venus, one of green fluorescent protein derivatives,; the other with a Ds-Red
derivative, mCherry (a generous gift from Dr. R. W. Tsien). A mixture of BDNF-Venus vector andmCherry vector were inoculated stereotaxically in the dentate gyrus of mouse hippocampus (P18-25)
under ketamine-xylazine anesthesia. After 2-3 days, BDNF-Venus was distributed at mossy fiber
terminals which was morphologically detected by mCherry in the acute slice under confocal
microscopy. Fluorescent intensity of BDNF-Venus (515 nm) and mCherry (630 nm) was measured
simultaneously from a single mossy fiber terminal. By a electrical stimulation of mossy fiber, thefluorescence at 515 nm was reduced. But that at 630 nm was not. So the reduction of 515 nm
fluorescence reflects BDNF release from the terminal. To remove synchronous signals derived fromslice movement, 515/630 ratio was used as the BDNF release-specific signal. This response
depended on extracellular calcium, and disappeared by either removing extracellular calcium ortreating with N-methylemaleimide (NEM). We made a transgene plasmid which consists of CAG
promoter sequence, floxed polyA-Neo and preproBDNF-Venus and obtained six mouse lines whichhave this transgene in their genomic DNA. The genetically recombinant mice were made withgamma1-Cre knockin mice which express cre recombinase specifically in the hippocampal CA3

63pyramidal cells. However, none of six lines were not expressing BDNF-Venus phenotypically. It issuggested that the insertion of transgender may be inadequate for recombination.

Discussion & Summary
These observations would provide first and direct evidences that BDNF is actually released from thegiant mossy fiber boutons through activity-dependent exocytosis. It is suggested that BDNF isreleased from the MF terminals during induction of long-term potentiation (LTP). This study
developed a optical methods to investigate directly the release of BDNF from individual mossy fiber
presynaptic terminals of hippocampus. However, the methods have to be improved to reveal themechanisms regulating BDNF release. Since the transgenic system appear to be ideal for improving
the signal/noise ratio, several trials of foundation would be necessary to establish the line expressing
BDNF-Venous stably. We are planning to generate the transgenic system which would conditionallyexpress BDNF-Venous and mCherry.

Figures & Tables

References
Suyama, S., Ishizuka, T., Yawo, H. (2006) Activity pattern-dependency of BDNF release from mossy
fiber terminals of hippocampus. J. Physiol. Sci. 57 (Suppl.), S169.
Suyama, S., Hikima, T., Araki, R., Ishizuka, T., Yawo, H. (2007) Exocytosis dynamics in a single
mossy fiber terminal of hippocampus. J. Physiol. Sci. 58 (Suppl.), S154.

642) Medical Sciences2-2) Central Nervous System

A study of mechanisms which underlie activity-dependent neuralcircuit formation
Nobuhiko Yamamoto
Osaka University
nobuhiko@fbs.osaka-u.ac.jp

Introduction
During development neural connections are formed by not only developmentally regulated guidance
molecules but also neural activity such as firing and synaptic activity. Thalamocortical (TC) axon
branching in the mammalian neocortex is a well-characterized system in which to investigate theactivity-dependent mechanisms. The aim of this study is to reveal how TC axon branching is
modified by neural activity by using organotipic cocultures of the thalamus and cortex.

Results
TC axon branching in cocultures of the thalamus and cortex was examined by introducing enhanced
yellow fluorescent protein (EYFP)-encoding plasmid into thalamic cells. Most of the individually
distinguishable EYFP-labeled axons formed elaborate branches primarily in the target layer, layer 4of cortical explants after 2 weeks in vitro. This indicates that TC axon branches are formed in vitro
with laminar specificity resembling that found in vivo, in accordance with previous findings. To
investigate whether electrical activity was generated in thalamic and cortical cells in vitro, firing
activity was recorded by the multi-electrodes dishes that were implanted into culture dishes. Indeed,
spontaneous firing was observed as single units and field potentials in both thalamic and corticalexplants (Fig. 1), and was prominent at the later stages, when axonal branching were formed
abundantly. This finding raises the possibility that spontaneous activity may affect axonal branching.
To test this possibility, cocultures
were subjected to pharmacologicaltreatment with blockers of sodium
channels or glutamatergic synaptictransmission during the second
week in vitro. As a result, TC axon
branching was decreased in thesetreatedcultures.

Quantitative

65

analysis showed that in the presence of TTX, APV or DNQX, the number of branch points wasdecreased dramatically. Laminar specificity of axonal branching was also reduced under thesetreatments. To further examine what aspects of axonal branching were affected by thepharmacological treatments, the dynamics of TC axon branching was studied by time-lapse imaging
in the presence or absence of these blockers. Over the observation period, TC axon arbors became
larger and more complex. The average number of branch points and the total axon length increased
gradually as development progressed, but the analysis of individual axonal branches revealed thatTC axonal branches did not increase monotonically but remodeled actively. More than 80% of
branches showed changes in length. Most added and elongating branches were enriched in the upper
layers including layer 4. In contrast, the distribution of lost and retracting branches was more
uniform over all cortical depths. In drug treated slices, the total number of branches was
substantially decreased compared with the untreated group, but approximately 90% of the branches
were dynamic exhibiting both growth and loss. The laminar distribution of growing and eliminating
branches in the treated slices was nearly constant across all cortical layers. As development of axon
branching can be attributed to the accumulated difference between the number of added or
elongating branches and the number of retracting or lost branches, the differences per day were
calculated for the target and non-target layers. In untreated cultures the change in the number ofbranch points was much higher in layer 4 than the deep layers. The difference in layer 4 wasdramatically reduced as a result of drug treatment. On the other hand, we did not find that stablebranches were more prevalent in the target layer under any conditions. Furthermore, growth motility
was not different between the treated and untreated slices. Thus, our results demonstrate that neuralactivity is required for lamina-specific TC axon branching.

Discussion & Summary
Our present results demonstrated that lamina-specific TC axon branching was formed whenspontaneous firing activity increased in both thalamic and cortical neurons. The results further
showed that TC axon branching was generated dynamically by branch addition and elimination witha preference toward branch accumulation in the upper layers. This is consistent with the previous
finding that a branch-promoting molecule is expressed in the target layer, whereas abranch-inhibiting molecule is expressed in all cortical layers. Moreover, the pharmacological study
clearly showed that lamina-specific branching with the remodeling process was reduced by blockadeof firing or synaptic activity. These findings indicate that neural activity including synaptictransmission could play a role in regulating the proper expression of these branch-regulating
molecules. Alternatively, activity might modify the expression and responsiveness of the receptors
on growing TC axons or affect downstream signaling mechanisms in TC axons.

66

References
Uesaka N, Hirai S, Maruyama T, Ruthazer ES, Yamamoto N (2005) Activity dependence of corticalaxon branch formation: a morphological and electrophysiological study using organotypicslice cultures. J Neurosci 25:1-9
Uesaka N, Yamamoto N (2006) Activity-dependent regulation of cortical axon branching.
Neuroscientist 12, 102-106.
Uesaka N, Hayano Y, Yamada A, Yamamoto N (2007) Interplay between laminar specificity andactivity-dependent mechanisms of thalamocortical axon branching. J Neurosci 27, 5215-23.
Yamamoto N, Higashi S, Toyama K (1997) Stop and branch behaviors of geniculocortical axons: atime-lapse study in organotypic cocultures. J Neurosci 17:3653-3663.
Yamamoto N, Yamada K, Kurotani T, Toyama K (1992) Laminar specificity of extrinsic corticalconnections studied in coculture preparations. Neuron 9:217-228.
Yamamoto N, Inui K, Matsuyama Y, Harada A, Hanamura K, Murakami F, Ruthazer ES, Rutishauser
U, Seki T (2000) Inhibitory mechanism by polysialic acid for lamina-specific branchformation of thalamocortical axons. J Neurosci 20:9145-9151.

672) Medical Sciences2-3) Reproducive System

Study of epigenetic pathophysiology and effective treatment of aged
infertile oocytes
Kenichiro HATA
National Center for Child Health and Development
khata@nch.go.jp

Introduction
Oocytes are one of the most sensitive reproductive systems for aging. To clarify molecularmechanism(s) of aging of oocytes, we are trying to estimate epigenetic modifications in detail. Ouraims of this study is, 1. Study of molecular mechanisms for epigenetic modification in
gametogenesis, and 2. Epigenetic estimation of aged oocytes.

Results
1. Study of molecular mechanisms for epigenetic modification in gametogenesis.
Dnmt3L (DNA methyltransferase 3 like, a putative regulator of DNA methylation) is an essential
molecule for region specific DNA methylation in gametogenesis. We screened candidates molecules
which can interact with Dnmt3L by yeast two-hybrid method. Finally we got two genes and now
biochemical assay and genetic assay with these candidate genes are proceeding.
2. Epigenetic estimation of aged oocytes.
To perform DNA methylation assay with aged oocytes, we have been collecting aged oocytes fromold femal mice. Since only a few dozens of oocytes are collected from aged mice we still need timeto prepare enough number of oocytes. Additionally, we could not get high quality DNA and RNA
from small number of oocytes. This could be a constitutional problem of methods and we are trying
to modify common methods..

Discussion & Summary
Aged oocytes can be rescued by transfusion of cytoplasm of donated young oocytes (Figure 1.).
Clone organisms have been produced by nuclear transfer of somatic cells. These evidences clearlyshow that nuclei of aged oocytes or somatic cells can be rejuvenated by unknown factors incytoplasm of young oocytes. Epigenetic factors such as Dnmt3L, are decreased in aged oocytes andknown as essential factors for early mouse development. These evidences strongly suggest that

68

Dnmt3L regulate region specific DNA methylation in oocytes. On the other hand, aged oocytes lose
such epigenetic modification system(s) and aberrant DNA methylation may cause loss of early
embryos. We continue to study epigenetic pathophysiology of aged oocytes.

References
Yokomine T, Hata K*, Tsudzuki M, Sasaki H: Evolution of the vertebrate DNMT3 gene family: apossible link between existence of DNMT3L and genomic imprinting. Cytogenet Genome Res 113:
75-80 (2006).
Hata K*, Kusumi M, Yokomine T, Li E, Sasaki H: Meiotic and epigenetic aberrations in
Dnmt3L-deficient male germ cells. Mol Reprod Dev 73: 116-122 (2006).

692) Medical Sciences2-5) Cardiovascular Metabolic Endocrine

Towards development of a novel therapy for atherosclerosis via theapoptosis inhibitory factor, AIM.
Toru Miyazaki
Division of Molecular Biomedicine for Pathogenesis, Center for Disease Biology andIntegrative Medicine (CDBIM), Faculty of Medicine, The University of Tokyo
tm@m.u-tokyo.ac.jp

Introduction
Atherosclerosis, one of the most representative diseases for the Metabolic Syndrome, reveals themost frequent cause of humans’ death in advanced nations (refs. 1, 2). So far, this disease has mainly
and almost solely been regulated by controlling cholesterol levels in the body. Due to the recent
drastic change of our life style, however, prevention of the increase of cholesterol in the body is
becoming more and more difficult. Therefore, it is apparently needed to develop a novel therapy foratherosclerosis independent of control of cholesterol levels. Recently, we have shown that AIM, anapoptosis inhibitory protein, is restrictedly produced in lesional macrophages upon uptake ofoxidized LDL (oxLDL), via activation of nuclear receptors LXR/RXR. This response supports
macrophage survival and accumulation within the lesion, leading to overt atherosclerosis (Fig. 1). In
AIM-deficient mice, lesional macrophages are far more susceptible to apoptosis induced by oxLDL,
and inconsequence, the lesion size is dramatically reduced compared to wild-type mice, after achallenge with high-fat diet (ref. 3).
Based on these results, our aim is toestablish a therapeutic applicationofAIM-suppressionforatherosclerosis in human. For thisrationale, our specific aims are: (1)Development
anti-AIM
Establishmentoffunctional

antibodies;of(2)
inducible

AIM-deficient animals; and (3)Identification of AIM-receptor.

70

Results(1) Development of functional anti-AIM antibodies:
We have generated recombinant mouse AIM protein (HA-tagged) of up to 2mg, through anantibody (anti-HA) column purification. By immunizing mice with the protein, we have established
25 independent anti-AIM monoclonal antibodies that capture native AIM protein. 15 out of 25
recognize both human and mouse AIM. By using these antibody clones, we first created an Elisa
system for serum AIM detection (Fig. 2). Our system sensors AIM in human serum in a very
sensitive fashion. We are now analyzing a large
図２：ヒト血清AIMの検出

numbers of sera from patients who harborvarious cardiovascular diseases. Secondly, we
possible therapeutic effect of these antibodies foratherosclerosis in mouse models. We will assess

Western Blot

ELISANormalized O.D.

are now preparing mouse experiments to test

if multiple injections of antibodies could preventthe disease in LDLR-deficient animals with a

0.8
0.6
0.4

A

0.2

patient

B

C

D

高脂血症 正常 心血管系疾患

A

B

C

D

Fig.2

high-fat diet.
(2) Establishment of inducible AIM-deficient animals:

A targeting vector was designed to generate a conditional AIM-deficient mouse line. By using thevector, we have obtained several clones of ES cells in which the AIM gene was successfullytargeted via homologous recombination. The ES clones were transmitted into germ line of mice, andnow the resulting offspring is cross bred with inducible Cre-transgenic mice and LDLR-deficientmice. After the breeding is completed, we will assess whether the shut-off of AIM gene istherapeutically efficient in mice that harbor different levels of atherosclerosis.
(3) Identification of AIM-receptor:
We have established two types of cDNA expression libraries from mouse macrophage cell lines. By
using the recombinant AIM protein described above, we will soon start screening these libraries for
AIM-binding (AIM-receptor) protein(s).

Discussion & Summary
During the grant year, we have achieved a reasonable progress in the proposed researches. Further
efforts to complete the whole project will contribute in developing a new therapy for atherosclerosis
via functional modulation of AIM, which will definitively be required in a near future. In addition,
our Elisa system for serum AIM levels will be a useful diagnostic method for progression ofatherosclerosis including early stages.

71

References
1. Glass, C. K. & Witztum, J. L. Atherosclerosis. the road ahead. Cell 104: 503-516, 2001.
2. Lusis, A. J. Atherosclerosis. Nature 407: 233-241, 2000.
3. Arai, S., Shelton, J.M., Chen, M., Bradley, M.N., Castrillo, A, Bookout, A.L., Mak, P.A.,
Edwards. P.A., Mangelsdorf, D.J., Tontonoz, P. & Miyazaki, T.

A role of the apoptosis

inhibitory factor AIM/Spa/Api6 in atherosclerosis development. Cell Metab. 1: 201-213, 2005.

722) Medical Sciences2-5) Cardiovascular Metabolic Endocrine

The role of RNA polymerase kinases on the pathogenesis of cardiac
hypertrophy and heart failure
Motoaki Sano
Keio University School of Medicne
msano@sc.itc.keio.ac.jp

Introduction
The Cdk7/cyclin H/menage-a-trois 1 (MAT1) heterotrimer has proposed functions in transcription as
the kinase component of basal transcription factor TFIIH and is activated in adult hearts by Gq-,
calcineurin-, and biomechanical stress-dependent pathways for hypertrophic growth. This study isdesigned to ivesigate the role of Cdk7/cyclin H/MAT-1 in cardiac growth and metabolism.

Results
Using cardiac-specific Cre, we have ablated MAT1 in myocardium. Despite reduced Cdk7 activity,
MAT1-deficient hearts grew normally, but fatal heart failure ensued at 6–8 weeks.
By microarray profiling, quantitative RT-PCR, and western blotting at 4 weeks, genes for energy
metabolism were found to be suppressed selectively, including targets of peroxisome
proliferator-activated receptor γ coactivator 1 (PGC-1).
Cardiac metabolic defects were substantiated in isolated perfused hearts and isolated mitochondria.
Consistent with the impairments in gene expression, we confirmed reduced enzyme activities oftricarboxylic acid cycle and respiratory chain complexes. In Langendorff-perfused hearts, oleate andglucose oxidation were reduced by at least 60% and 50%, respectively. Likewise, state III respirationand the respiratory control ratio were reduced in mitochondria from MAT1-deficient hearts, withlittle change in ADP/O
In culture, deleting MAT1 with Cre disrupted PGC-1 function: PGC-1α failed to activate
PGC-1-responsive promoters and nuclear receptors, GAL4-PGC-1α was functionally defective, and
PGC-1β was likewise deficient. PGC-1 bound to both MAT1 and Cdk7 in coprecipitation assays.

73

Discussion & Summary
By cardiac-specific deletion of MAT1, we found that loss of MAT1 and the ensuing loss of
TFIIH-associated Cdk7 activity result in a late-onset metabolic cardiomyopathy. We identified ablock to the functional activity of PGC-1α and PGC-1β in MAT1-deficient cells and showed that
PGC-1 interacts physically with MAT1 and Cdk7. This block to metabolic gene expression via
defective operation of the PGC-1 family implicates MAT1 in the control of cardiac metabolism, with
deficient coactivation by PGC-1 the responsible molecular event.

Figures & Tables

References
Menage-a-trois 1 is critical for the transcriptional function of PPARgamma coactivator 1.Sano M,
Izumi Y, Helenius K, Asakura M, Rossi DJ, Xie M, Taffet G, Hu L, Pautler RG, Wilson CR, Boudina
S, Abel ED, Taegtmeyer H, Scaglia F, Graham BH, Kralli A, Shimizu N, Tanaka H, Makela TP,
Schneider MD. Cell Metabolism. 2007 Feb;5(2):129-42

742) Medical Sciences2-5) Cardiovascular Metabolic Endocrine

Elucidation of transcriptional networks in metabolic syndrome andvascular disease
Ichiro Manabe
The University of Tokyo Graduate School of Medicine
manabe-tky@umin.ac.jp

Introduction
Metabolic syndrome is an important risk factor for cardiovascular disease.

However, exact

mechanisms by which metabolic syndrome leads to atherogenesis are not clear.

In the present

study we analyzed the role played by the transcription factor KLF5, which we have previously
shown to play an important role in tissue remodeling in the cardiovascular system, in metabolic
stress response.

We also analyzed the structural changes in adipose tissue obesity.

Results
Previously we have shown that KLF5 heterozygous knockout (KLF5+/) mice showed much reduced
neointima formation in vascular disease models.

The mice also exhibited reduced cardiac

hypertrophy and fibrosis in the angiotensin II-induced hypertrophy model.

Interestingly, KLF5 isalso expressed in metabolic tissues, such as adipose tissue, skeletal muscle and pancreatic islets.

In

adipose tissue, we have shown that KLF5 is an essential component of the transcription factor
network that controls adipocyte differentiation.

KLF5 is induced by the transcription factors

C/EBPβ and C/EBPδ, and once expressed it PPARγ2 transactivates PPARγ2 in concert with C/EBPβ
and C/EBPδ, thus mediating the early and late differentiation programs.
analyze KLF5’s role in metabolic tissues in adult animals, we fed KLF5+/-

In the present study, tomice with high-fat diet.

KLF5+/- mice showed resistance to diet induced obesity and insulin resistance although they ate
more food than wild-type mice, suggesting that KLF5+/- mice is protected from metabolic syndrome.
The O2 consumption was elevated in skeletal muscle of KLF5+/- mice, suggesting that enhancedenergy expenditure partly explain their phenotypes. We also found that the structural integrity ofpancreatic islets was maintained in KLF5+/- fed with high-fat diet, and the mice showed better
insulin secretion, suggesting that KLF5 plays a role in stress response in pancreatic islets.
results demonstrate the KLF5 is important for response to metabolic stress.

These

Given that KLF5 plays

an essential role in pathogenesis of cardiovascular diseases, the results suggest that there arecommon molecular mechanisms that control response to various stresses in the cardiovascular and75

metabolic systems.

To address this we developed a novel living tissue imaging method andanalyzed obesity of adipose tissue.

We found that obesity of adipose tissue involves adipogenesis,

angiogenesis, adipocyte death, and macrophage infiltration.

We further addressed the role ofangiogenesis in obesity by administering anti-VEGF antibody to db/db mice.

The anti-VEGF

antibody treatment inhibited not only angiogenesis but also adipogenesis, indicating that
adipogenesis is closely coupled with angiogenesis in obesity of adipose tissue.

Becauseadipogenesis takes place in the cell clusters and is coupled with angiogenesis, we designated theseadipogenic cell clusters as adipo-/angiogenic cell clusters.

The adipo-/angiogenic cell clusters

consisted of differentiating adipocytes, blood vessels and lectin+ CD68+ small stromal cells.
Adipo-/angiogenic cell clusters were most often found in 8-week-old db/db mice, and in elder mice,
another type of cell clusters called crown-like structure were found.

Crown-like structurescontained macrophages engulfing dead adipocytes. As such, our live imaging demonstrated thatobesity of adipose tissue involves dynamic interplays between adipocytes, endothelial cells, andstromal cells including macrophages and lectin+ CD68+ cells.
tissue obesity can also be found in atherogenesis.

The changes taking place in adipose

Results of the present study strongly suggest that

there are the common molecular mechanisms operating both in the cardiovascular and metabolic
systems in obesity, metabolic syndrome, and atherosclerosis.

Discussion & Summary
Our living tissue imaging demonstrated the dynamic structural changes in obesity of adipose tissue.
The changes are similar to those found in atherosclerosis and can be considered as chronic
inflammation (Fig. 2). These findings and the findings that KLF5 plays roles in stress responses inboth cardiovascular and metabolic systems suggest common molecular mechanisms that operate inobesity and adipogenesis.

KLF5 appears to be such a transcription factor that is involved indevelopment of metabolic syndrome as well as atherosclerosis.

Future studies on the common

molecular mechanisms would elucidate how metabolic dysfunction in metabolic syndrome lead toatherosclerosis.

76

Fig.1Adipo-/angiogenic cell clusters andcrown-like structures
White arrow, adipo-/angiogenic cell c l u s t e r s .
R e d a r r o w, c r o w n - l i k e s t r u c t u r e s .

Fig.2 Obesity of adipose
tissue and atherosclerosis

772) Medical Sciences2-6) Gastro-intestinal

Role of IKK/NF-kappaB activation in inflammatory bowel diseases
Shin Maeda
The institute for adult diseases, Asahi life foundations-maeda@asahi-life.or.jp

Introduction
Inflammatory bowel diseases (IBD) are associated with the infiltration into the intestinal lamina
propria of mononuclear cells, such as macrophages or lymphocytes, which overexpress the productsof nuclear factor-kappa B (NF-κB) target genes, including the proinflammatory cytokines tumor
necrosis factor α (TNFα) and IL-6.
The present study was conducted to investigate the effect of specific inhibition of IKKβ-mediated
NF-κB activation on murine colitis using the well-characterized NEMO-binding domain (NBD)
peptide (1) and IKKβ knockout mice.

Results
Inhibition of IKK blocks LPS-induced NF-κB activity and proinflammatory cytokine expression
Initially, we examined the effect of NBD peptide on NF-κB activation by LPS in J774.1 murine
macrophages, which is the major cell type that accumulates in murine colitis. IκBα phosphorylationand degradation were observed after 15-30 min of LPS treatment, indicating that the NF-κB
signaling pathway had been activated. The NBD peptide (10 µM) inhibited the phosphorylation anddegradation of IκBα by LPS treatment. NF-kB binding activity was also inhibited by NBD in the
EMSA analysis (Figure 1). To explore the effect of NBD on LPS-mediated inflammatory cytokine
expression in macrophages, we treated J774.1 cells with LPS with or without NBD peptide,collected the supernatant, and determined the IL-6, IL-1β, and TNFα concentrations by ELISA. The
IL-6, IL-1β, and TNFα levels were markedly
increased, and the wtNBD peptide reduced theconcentrations

of

IL-6,

IL-1β,

and

TNFα

in

dose-dependent manners. Almost same results wereobtained in IKKβ knockout macrophages isolated fromthe IkkβF/F mice crossed with Mx1-Cre transgenic
mice.

78

Effect of IKK blockade on DSS-induced colitis
To determine the function of IKKβ/NF-κB in colitis, mice were given 3% DSS in their drinking
water for 5 days with NBD peptide, and were monitored for weight loss, which is a characteristic ofsevere intestinal inflammation. After 5 days, the body weights of mice without NBD peptide started
to decrease, until day 10 when they were sacrificed and analyzed; a 14% total decrease in bodyweight was recorded on Day 10. In contrast, the body weights of mice with the NBD peptide showed
only a 5% total decrease on Day 8 and thereafter, they started to recover. Histologic analyses
revealed that the severity and extent of inflammatory lesions in the colons of NBD peptide-treatedmice were significantly (p < 0.05) lower than in untreated mice, and that they had smaller areas ofulceration. Numerous macrophages had infiltrated in the DSS-mediated colitis mice, as determinedby F4/80 immunostaining (Figure 2). After DSS exposure, IKKβ/NF-κB activity determined byphospho-IκBα and nuclear p65 immunostaining was found to have decreased in the colons of NBD
peptide-treated mice compared to untreated mice. The amounts of cyclooxygenase-2 (Cox-2) andIL-6 protein, which are expressed in colitis, were significantly lower in the colons of NBD
peptide-treated mice relative to their non-treatedcounterparts. Phospho-IκBα, nuclear p65, Cox-2, andIL-6 were predominantly expressed in F4/80-positive
macrophages within inflammatory lesions. Micetreated with wtNBD peptide expressed lower amounts
of mRNAs for the pro-inflammatory cytokines IL-6,
IL-1β, and TNFα in their colons relative to untreatedmice. Almost same results were obtained in IKKβ
knockout mice generated by crossing IkkβF/F mice with
Mx1-Cre transgenic mice.
Treatment of TNBS-induced colitis by administration of NBD peptide
We next examined the effects of the NBD peptides in another murine colitis model that involves
the administration of TNBS in ethanol into the mouse rectum. Initially, we monitored the survival ofmice and found that mice treated with the NBD peptide had a significantly increased survival rate(80%) relative to untreated mice (40%). Treatment with ethanol, which used as the TNBS vehicle,
did not result in mortality. Whereas mice that received TNBS exhibited progressive weight loss,
which is a characteristic of severe intestinal inflammation, those treated with the NBD peptide
showed significantly less weight loss and started to recover on Day 4 after TNBS administration.
Histologic analyses revealed that the severity and extent of inflammatory lesions in the colons of
NBD peptide-treated mice were significantly (p < 0.05) lower than in untreated mice, and thatulcerated areas were smaller than those seen in the DSS model. Mice treated with NBD peptide
expressed lower levels of mRNAs for pro-inflammatory cytokines in their colons relative to Et-OH

79

treated mice. These results indicate that IKKβ/NF-κB activation is one of the critical regulators in
TNBS colitis, and that blockade by NBD peptide may offer a new therapeutic approach for human
IBD.

Discussion & Summary
In the present study, we demonstrate that NBD peptide, which is a well-characterized IKK
inhibitor, ameliorates colitis-related disease in two mouse models of IBD. Among the common
cellular pathways activated in IBD, NF-κB is thought to be the most common pathway central tocell activation and the production of various inflammatory mediators, including a variety ofcytokines. Thus, inhibition of NF-κB activity is a promising strategy for blocking inflammation. To
date, several investigators have reported that the use of an NF-κB decoy with or without viraldelivery is very effective for ameliorating murine IBD. Compared with other NF-κB inhibitors, the
NBD peptide has the important advantage that it works upstream of the master kinase IKK in thecytoplasm. Since NF-κB is a dimer that consists of several NF-κB family members, inhibitors must
be designed for several activation domains. In contrast, the NBD peptide blocks IKK activity,
which results in the blockade of most NF-κB activity.
Many anti-inflammatory drugs for the treatment of IBD inhibit NF-κB activity, at least partially.
Of these, glucocorticoids and 5-aminosalicylic acid are reported to be strong inhibitors of NF-κB
activity, which is thought to be one of the reasons behind their efficacy. We have demonstrated that
the NBD peptide, which can potently inhibit NF-κB activity, ameliorates murine colitis bypreventing the expression of proinflammatory cytokines and may represent a novel therapeutic
approach for IBD.

References
1. May, M. J., F. D'Acquisto, L. A. Madge, J. Glockner, J. S. Pober, and S. Ghosh.
2000. Selective inhibition of NF-κB activation by a peptide that blocks theinteraction of NEMO with the IκB kinase complex. Science 289:1550-1554.

802) Medical Sciences2-7) Infection

Carcinogenesis and progression of gastric cancer by infection of
Helicobacter pylori in ASC-deficient mice
Shun’ichiro Taniguchi
Dept. Molecular Oncol.,Shinsu Unversity Graduate School of Medicine
stangch@sch.md.shinshu-u.ac.jp

Introduction
ASC is an adaptor molecule originally found in apoptotic cells, and activates Caspase-1
responsible for the maturation of the pro-inflammatory cytokines IL-1β and IL-18. Thus, ASC plays
an important role in innate immunity. Helicobacter pylori is now known to cause persistent gastritis
and is linked to ulcer disease and gastric cancer. Cytokines are important in the host defense against
microbial pathogens, including H. pylori. And H. pylori are known to induce IL-1β. Further more, IL-1β
gene cluster polymorphisms is suspected of enhancing production of IL-1β, and is associated withincreased risk of gastric cancer.
In this study we’ll determine the role of ASC in the host defense against gastric colonization andcarcinogenesis by Helicobacter pylori using ASC deficient mice.

Results
Specific-pathogen-free 5 to 8 week-old male mice ASC.KO were inoculated intragastrically withmouse-adapted H. pylori isolate (Sydney Strain 1), and assessed for the degree of gastritis andbacterial load at 2 and 10 weeks after the last inoculation (n=6~10). Results were compared withthose obtained from H. pylori-infected C57BL/6 mice.
Mice were orally inoculated with 0.5ml of H. pylori SS1 108 CFU/ml concentration 3 times at theintervals of 24 hours. Infection status in inoculated mice was assessed using the gastric biopsy urease
test, microbial culture, and by immunohistology. Mice were classified as H. pylori-positive if they
were positive by histology, culture or biopsy urease test.
1)Colonization of H. pylori in wild type and ASC-/- mice
The number of colonized bacteria in the stomach was determined at 2 and 10 weeks after the lastchallenge.

The number of H. pylori colonized in the stomach of ASC-/- mice tended to be higher

than that of Wild-type mice, at 2 and 10 weeks after infection. Repeat of this experiment is under
way to confirm statistical difference in colonization levels between ASC-/- and Wild type mice. This
result suggests that ASC may play a protective role against H. pylori infection.

812) Histopathological examination of Wild type and ASC-/- Mice after infection.
To investigate the role of ASC in gastric inflammation induced by H. pylori infection, the histology
of stomachs samples of Wild type and ASC.KO mice was examined after infection with H. pylori
SS1.

Two weeks after infection,

more number of mononuclear and polynuclear neutrophils cells

in the submucosa and the lamina propria was observed in ASC.KO mice than in wild-type mice,probably reflecting the nature resistant to programmed cell death in ASC-/- mononuclear andpolynuclear neutrophils as compared with wild type cells. Ten weeks after infection(morechronically infected-state), strong infiltration of neutrophils and lymphoid follicles appeared in the
submucosa and the lamina propria of wild-type mice,; in contrast in ASC.KO mice; only mild
infiltration of inflammatory cells recognized as lymphocytes was observed. The existence of
H.pylori was observed more frequently in ASC-/- mice than in wild type mice.
３）Cell viability under stressful conditions
When cells were placed under various stressful conditions, such as staravation, wound, LPS
treatment in vitro as well as in vivo, we observed that ASC-/- cells(smooth muscle cell, neural cell,cornea cells etc.) were resistant to cell death in association with enhanced autophagy(data notshown).

Discussion & Summary
Our results of the current studies focused on H. pylori-ASC interactions showed that ASC deficiency
significantly attenuates chronic inflammation, and increased colonization density, suggesting that
ASC may contribute to increase severity of H. pylori-induced gastric inflammation, while also
contributing to reduce bacterial colonization.
The results are consistent with the findings of higher gastric colonization by H. pylori

but minimal

gastritis in IFN-γ-/- mice than in wild-type mice. The lowered IFN-γ expression is observed in
ASC.KO mice, because of the lack IL-18, a strong inducer of IFN-γ . The hallmark of the gastricinflammatory response to H. pylori is its capacity to persist for decades, with increased risk ofcorpus atrophy, which is considered as a pre-malignant gastric lesion. It still remains to be clarified,
however, whether loss of ASC protects H. Pylori-infected ASC.KO mice against carcinogenesis, due
to the lower inflammation, or the loss of ASC promotes carcinogenesis, due to the lack ofprogrammed cell death. To answer the above questions, further investigations, such as experiments
using ras-transgenic ASC-/- mice, are under way.

82

Figures & Tables
Pathology and immunohistological analysis ofgastric mucosa of ASC-/- and Wild-type mice
infected with SS1 strain of H. pylori.
FIG-1: Photomicrographs of H&E-stained andimmuno-stained, by 2 weeks after infection. A
massive infiltration of inflammatory cells (mostly
neutrophils) in the submucosa and lamina propria of
ASC.KO mice was observed, whereas only a mild
inflammation was seen in Wild-type mice. Uniform
moderate bacterial colonization was observed in all
experimental groups by immunosa of ostaining; asmall decline in bacterial colonization was observedin Wild-type mice as compared with ASC.KO
mice(FIG-3).

FIG-2: By 10 weeks after infection, the bacterialload was significantly reduced in Wild-type mice.
The reduced H. pylori colonization in Wild-typemice was histomorphologically associated with astrong infiltration of neutrophils and lymphocytes ascompared with H. pylori-infected ASC.KO mice.

FIG-3: Higher bacterial load in the gastric mucosa
of ASC.KO mice compared to wild-type C57/BL6
mice. Colony counts were determined from gastricbiopsy material encompassing the entire length of
the gastric mucosa. At 2 weeks, n=6 ASC.KO miceand n=10 C57BL/6 mice. At 10 weeks, n=6
ASC.KO mice and n=10 C57BL/6 mice. Data areexpressed as mean± SD for each experimentalgroup. Statistical studies between groups wasdetermined by student’s t-test.

References
1. Naoki S. et al. Role of IFN-y in H. pylori-induced gastric inflammatory responses in mousemodel. Infection and Immunity. Jan. 1999.
2. Shun’ichiro Taniguchi , Junji Sagara
Regulatory molecules involved in inflammasome formation with special reference to a key
mediator protein, ASC. Seminars in Immunopathology”Inflammasomes” Vol 29, 2007
Guest editor: Michael McDermott(in press)

832) Medical Sciences2-8) Oncology

PGE2-mediated regulation of gastric epithelial differentiation andproliferation
Masanobu Oshima
Cancer Research Institute, Kanazawa University
oshimam@kenroku.kanazawa-u.ac.jp

Introduction
In gastric and intestinal cancer tissues, expression of COX-2 and mPGES-1 is induced, which
results in production of PGE2.

Epidemiological and experimental analyses have indicated that

inhibition of COX-2 and PGE2 pathways by treatment with inhibitors suppressed gastrointestinal
carcinogenesis, indicating that PGE2 plays a key role in tumor development. To investigate the roleof PGE2 in gastric epithelial differentiation and proliferation, we have constructed transgenic mice
(K19-C2mE mice) expressing both COX-2 and mPGES-1 in gastric mucosa simultaneously.

Here,

we demonstrate that increased PGE2 signaling impaired normal differentiation in the gastric mucosa
of K19-C2mE mice.

Results
We have generated K19-C2mE mice using keratin 19 (K19) gene promoter that is transcriptionallyactive in gastric epithelial cells including undifferentiated cells.

In this mouse model, hyperplastic

tumorous lesions were developed in the proximal glandular stomach, which was suppressed bytreatment of the mice with a COX-2 selective inhibitor, NS-398.

Histologically, tumorous lesions

were consisting of well-differentiated epithelial cells with increased level of BrdU incorporations,indicating that these lesions were hyperplasia.

Major components of such lesions were Alcian-blue

positive mucous cells that were not found in the normal gastric mucosa. Moreover, inflammatory
cell infiltration was found in submucosa of the hyperplasia and metaplasia lesions.

These results

indicate that increased PGE2 signaling causes inflammation-associated hyperplasia and mucous
metaplasia in the stomach.
In the glandular stomach of K19-C2mE mice, expression of proinflammatory cytokines, TNF-α,
IL-1β and IL-6, is elevated significantly. Among these cytokines, it has been reported that TNF-α
and IL-1β play important roles in carcinogen-induced tumorigenesis in the skin and hepaticmelanoma metastasis, respectively. To investigate the roles of TNF-α and IL-1β in PGE2-induced
inflammtion and metaplastic hyperplasia, we crossed knockout mice of TNF-α (Tnf) and IL-1

84

receptor α chain (Il1r1) genes with K19-C2mE mice to construct respective compound mutant mice,and examined gastric phenotypes at 20 weeks of age.

In the control K19-C2mE mice, metaplastic

hyperplasia was found in the proximal glandular stomach with inflammatory infiltration.

However,

hyperplastic growth was suppressed significantly in the Tnf (−/−) K19-C2mE mice with reduced
mucosal thickness to the wild-type level (Fig. 1).

Histologically, inflammatory cell infiltration was

not found in the submucosa of the Tnf (−/−) K19-C2mE stomach.
metaplasia was not detected in the compound mutant mice.

On the other hand, IL-1β inhibition did

not affect gastric phenotypes of K19-C2mE mice (Fig. 1).
TNF-α-associatedConsistently, mucous cell

Accordingly, it is possible that

inflammatory

response is required for PGE2-induced
hyperplasia and mucous cell metaplasia
in the stomach.
It

has

been

demonstrated

that

TFF2/spasmolytic polypeptide-expressing mucous metaplasia (SPEM) is associated withintestinal-type gastric cancer in human and Helicobacter-infected mice, suggesting that SPEM is apreneoplastic metaplastic lesion of gastric cancer.

Because mucous cell metaplasia was also foundin the K19-C2mE mouse stomach, we examined expression of TFF2 by RT-PCR and in situ
hybridization. The mRNA level of TFF2 in the K19-C2mE gastric lesion was increased significantly
compared with that of wild-type mice. Consistently, the number of TFF2-expressing cells wasincreased in the tumor tissues, while TFF2 positive cells were found only in neck region of wild-type(Fig. 2).

Importantly, expression of TFF2 wasfound only in the gland neck of the Tnf (−/−)
K19-C2mE mice with reduced level of TFF2 mRNA,suggesting that TNF-α-associated inflammation
causes SPEM formation with TFF2 positive
epithelial cells (Fig. 2).

Recently, it has beenreported that MUC6 is also expressed in the TFF2
positivemucousmetaplastic

cells.

Inthe

K19-C2mE gastric lesions, MUC6 expression was
also found in the TFF2 positive cells, which was
suppressed in the Tnf (−/−) K19-C2mE mice.

Only

mucous neck cells in the stomach are positive forboth TFF2 and MUC6.

Accordingly, our results

suggest that TFF2+ MUC6+ double positive SPEM
cells are derived from mucous neck cells.
These results, taken together, indicate that increased PGE2 signaling causes TNF-α-associated85

inflammation, which impairs differentiation of gastric epithelial cells, resulting in development ofpreneoplastic SPEM lesions.

Because SPEM cells are frequently found in the H. pylori-infected

mice, and H. pylori infection induces PGE2 production through induction of COX-2 and mPGES-1,
the present results suggest that H. pylori infection plays a causal role for SPEM formation by
induction of PGE2 pathway.

Discussion & Summary
We showed here that transgenic mice expressing both COX-2 and mPGES-1 (K19-C2mE mice)develop hyperplasia and mucous metaplasia associated with inflammatory responses.

To further

investigate possible roles of inflammatory cytokines in gastric tumorigenesis, we introduced
knockout mutations for tumor necrosis factor (TNF)-α (Tnf) and interleukin (IL)-1 receptor α chain(Il1r1) into K19-C2mE mice.

Tnf (−/−) K19-C2mE mice showed significant suppression of

hyperplasia and metaplasia, while gastric lesions remained unaffected in compound mutants of
K19-C2mE with Il1r1.

Importantly, spasmolytic polypeptide/trefoil factor 2-expressing metaplasia(SPEM) in the K19-C2mE stomach was also suppressed in the Tnf (−/−) K19-C2mE mice.

These

results indicate that TNF-α-dependent inflammation is responsible for PGE2-induced developmentof preneoplastic SPEM.

Therefore, it is possible that inhibition of TNF-α-dependent inflammation

together with eradication of Helicobacter is an effective prevention strategy for gastric cancer.

References
1. Oshima M, Oshima H, Matsunaga A, and Taketo MM. Hyperplastic gastric tumors withspasmolytic polypeptide-expressing metaplasia (SPEM) caused by TNF-α-dependent
inflammation in COX-2/mPGES-1transgenic mice. Cancer Res 65: 9147-9151, 2005.
2. Oshima H, Taketo MM, and Oshima M. Destruction of pancreatic β-cells by transgenic inductionof prostaglandin E2 in the islets. J Biol Chem 281: 29330-29336, 2006.
3.Oshima H, Matsunaga A, Fujimura T, Tsukamoto T, Taketo MM, and Oshima M. Carcinogenesis
in mouse stomach by simultaneous activation of the Wnt signaling and prostaglandin E2 pathway.
Gastroenterology 131: 1086-1095, 2006.

862) Medical Sciences2-8) Oncology

Role of prostaglandin E in the growth and metastasis of cancer cells.
Masaki Inada
Tokyo University of Agriculture and Technology
inada@cc.tuat.ac.jp

Introduction
Prostaglandin E2 (PGE2) has been implicated in tumorigenesis and tumor progression in several
human cancers.

Membrane-bound PGE synthase-1 (mPGES-1) is an inducible enzyme that

regulates the PGE biosynthesis.
not clear.

However, the role of mPGES-1 in cancer growth and metastasis is

Recently, we have reported that lipopolysaccharide-induced inflammatory bone loss is

significantly attenuated in mPGES-1-deficient mice (mPges1-/-) [1].

In this study, we examined the-/-role of PGE2 in cancer growth and metastasis, using mPges1 .

Results
We first examined the role of PGE2 in cancer metastasis to lung and bone, using a cancer
metastasis model. By the intravenous injection of mouse malignant melanoma B16 cells (B16)
into wild-type mice (WT), a number of metastatic foci were detected as black in lung on day 18
after the injection.
(Fig. 1).

In mPges1-/-, lung metastasis was significantly attenuated compared with WT

By the intravenous injection of B16 into WT, metastatic regions were detected as black inthe distal femurs on day 18 after the injection.

In mPges1-/-, the metastasis of B16 could be

detected, but the metastasis area, shown as black, wasmarkedly reduced compared with WT (Fig. 1).

To

examine the role of the PGE2 in osteolysis due tocancer metastasis, we measured the femoral bonemineral density (BMD) by dual energy X-ray
absorptiometry.

The

BMD

was

significantly

decreased by B16 metastasis in the femurs of the WT,
but the femoral BMD did not decrease in mPges1-/after the B16 injection.

The levels of PGE2 in bonemarrow supernatants collected from WT were
elevated by the metastasis of B16.

However, the87

PGE2 level in bone marrow supernatant was low and not elevated by the metastasis of B16 inmPges1-/-.

These results suggested that the mPGES-1-dependent PGE2 production by host cells is

critical for lung and bone metastasis and the osteolysis ofcancer cells.
We next examined the role of PGE2 in tumorigenesis,
using a subcutaneous tumor formation model.

By thedorsal subcutaneous injection of B16 into WT, B16 tumor
was formed and grown rapidly from day 6 to 12 (Fig. 2).
In mPges1-/-, B16 tumor could form, but the growth rate of
B16 tumor was significantly attenuated compared with WTfrom day 6 to 12 (Fig. 2).

PGE2 did not influence thegrowth of B16 cells in vitro.

Furthermore, we detected

mPGES-1 and cyclooxygenase-2 (COX-2) mRNA in B16
tumor in vivo, but not cultured B16 cells in vitro.

These

results suggested that autocrine action of PGE2 in host cells
is essential for the growth of B16 tumor in vivo.

Discussion & Summary
In the present study, we clearly showed the attenuation of cancer growth and metastasis inmPges1-/-.

Therefore, mPGES-1-mediated PGE2 production has the essential roles in cancer

growth, metastasis and cancer-induced osteolysis in bone. Recently we have reported thatprostaglandin E receptor EP4 antagonist suppresses osteolysis due to cancer metastasis [2].
Therefore, mPGES-1-mediated PGE2 production by host osteoblasts and autocrine action of PGE2
via EP4 on host osteoblasts play an essential role in osteolysis due to cancer metastasis.

Our study

suggested that the inhibition of PGE synthesis and its signal pathways might provide a possible
therapeutic approach to cancer metastasis of various tissues including bone.

References[1] Inada, M., Matsumoto, C., Uematsu, S., Akira, S. and Miyaura, C. (2006) Membrane-boundPGE synthase-1-mediated PGE2 production by osteoblast plays a critical role inlipopolysaccharide-induced bone loss associated with inflammation. J. Immunol., 177:
1879-1885.
[2] Takita, M., Inada, M. Maruyama, T. and Miyaura, C. (2007) Prostaglandin E receptor EP4
antagonist suppresses osteolysis due to bone metastasis of mouse malignant melanoma cells.
FEBS lett., 581: 565-571.

882) Medical Sciences2-8) Oncology

Functional analysis of various tumor suppressor genes in vivo.
Akira Suzuki
Dept. Molecular Biology, Akita University School of medicine
suzuki@med.akita-u.ac.jp

Introduction
Suppression of cancer suppressor genes expression is frequently involved not only in various
cancers but also in other non-cancerous diseases. We will try to analyze the functions of cancer
suppressor genes such as PTEN, ING1, and cancer suppressor candidate PICT1, which stabilizes
PTEN, by reverse genetics methods.

Results
First of all, we had analyzed the functions of PTEN in bronchioalveolar epithelial cells. Because
the PTEN-/- mice show early embryonic lethality (~E9.0), we turned to generated a bronchioalveolar
epithelium-specific null mutation of Pten in mice

[SOPtenflox/flox mice] that was under the control

of doxycycline. Ninety percent of SOPtenflox/flox mice that received doxycycline in utero[SOPtenflox/flox (E10-16)] died of hypoxia soon after birth. Most of the surviving SOPtenflox/flox(E10-16) mice, as well as mice that received doxycycline postnatally [SOPtenflox/flox (P21-27)],
developed spontaneous lung adenocarcinomas. Urethane treatment accelerated the number and sizeof lung tumors that developed in SOPtenflox/flox mice of both ages. Histological and biochemical
examination of the lungs of SOPtenflox/flox (E10-16) mice revealed hyperplasia of bronchioalveolar
epithelial cells and mesenchymal cells such as myofibroblasts, enlarged alveolar epithelial cells, andimpaired production of surfactant proteins. Thus, abnormal thickness of the blood-air barrier andreduction in the production of surfactant proteins caused by impaired alveolar epithelial celldifferentiation may contribute to the respiratory failure of SOPtenflox/flox (E10-16) embryos. In
addition, numbers of bronchioalveolar stem cells (BASCs), putative initiators of lungadenocarcinomas, were increased. Lungs of both SOPtenflox/flox (E10-16) and (P21-27) mice showed
increased expression of Spry2, which inhibits the maturation of alveolar epithelial cells. Levels of
Akt, c-Myc, Bcl-2 and Shh, which are shown to positively regulate stem cell self-renewal, were also
elevated. Furthermore, K-ras was frequently mutated in adenocarcinomas observed in SOPtenflox/flox(P21-27) lungs. The combined effects of Pten deficiency, K-ras mutation and perhaps othertumorigenic events may further increase the proliferation of BASCs such that they eventually initiate

89lung adenocarcinoma formation. These results indicate that Pten is essential for both normal lungmorphogenesis and the prevention of lung carcinogenesis, possibly because this tumor suppressor isrequired for BASC homeostasis (J.Clin. Invest. in revision).
PICT1 stabilizes PTEN protein and its expression is markedly reduced in many brain tumors.
Suppression of PICT1 expression causes the instabilization of PTEN protein, and eventually causes
the activation of Akt. Therefore PICT1 is thought to be a cancer suppressor candidate. We therefore
generated null mutation of PICT1 in mice. PICT1-deficient ES cells show decreased expression of
PTEN. However, they showed enhanced apoptosis and reduced cell growth. PICT1+/- mutant miceshow resistant to carcinogen-induced tumors, and PICT1-/- mice show early embryonic lethality.
These results suggest that PICT has another target besides PTEN and PICT1 is mainly involved inenhanced cell growth.
Finally, we had generated null mutation of ING1, a cancer suppressor genes which controls theexpression of p53 and the chromatin remodeling, in mice. About one third of the ING-/- neonates areembryonic lethal but surviving mice began to develop malignant lymphomas at about one-year old
age.

Discussion & Summary
We had analyzed the function of cancer suppressor genes (PTEN and ING1) and cancer
suppressor candidate gene (PICT1) in vivo by generating the null mutation of these genes in mice.
Especially using the bronchioalveolar epithelium-specific Pten mutant mice, we havedemonstrated that an intact PI3K/Pten/Akt pathway in bronchioalveolar cells isessential for normal lung morphogenesis and BASC homeostasis as well as for theprevention of lung tumorigenesis. Our results suggest that the PI3K/PTEN/Aktpathway, particularly in BASCs and/or cancer stem cells, may be an attractive
therapeutic target for the treatment of lung adenocarcinomas in humans.

References
Hamada et al. Genes and Dev 19(17), 2054-65, 2005
Horie et al. J. Clin. Invest 113(12), 1774-83, 2004
Stambolic et al. Cell 95(1), 29-39, 1998
Okahara F et al. Mol. Biol. Cell 17(11), 4888-95, 2006
Okahara F et al. J. Biol Chem 279(44), 45300-3, 2004

90

Figures & Tables

912) Medical Sciences2-8) Oncology

Phenotypic analysis of RAD18-KO and RAD18, XPA double KO
mice.
Satoshi Tateishi
Kumamoto University, Institute of Molecular Embryology and Genetics
tate@gpo.kumamoto-u.ac.jp

Introduction
Cellular DNA is continually damaged by intrinsic sources; reactive oxygen species and extrinsic
sources; UV light irradiation. If these DNA damages are not repaired, accumulation of the DNA
damages cause cellular death or carcinogenesis. Most DNA lesions are removed prior to DNA
replication by the nucleotide excision, in which XPA gene is involved. On the other hand, unrepaired
lesions encountered by the DNA replication machinery during S-phase cause replication fork stalling.
RAD18 gene is involved in the resumption process, operationally defined as post-replication repair(PRR), and is characterized by re-initiation of DNA replication without removal of the lesion on atemplate strand

Results
Ⅰ．UV sensitivity and skin cancer formation of mice
We have prepared RAD18-Knockout mice. At first, we examined MED (minimal erythema dose) of
wild-type mice, XPA-KO mice and RAD18KO mice. MED

of wild-type mice, XPA-KO mice and2

RAD18KO mice are 2500, 400 and 2000 J/m , respectively.
XPA-KO mice is known to form skin cancer by irradiation of 400-2000 J/m2 of UV/day

for 20weeks. We examined whether RAD18-KO mice forrm skin cancer by the UV-irradiation. After 20
week UV-irradiation to the mice, no skin cancer was observed (Fig. 1). We next prepared
XPA-RAD18 double knockout mice by mating. These mice appeared to grow normally. We
expected that the formation of skin cancer due to XPA deficiency will be rescued by RAD18
deficiency. However, XPA-RAD18 double knockout mice showed almost same MED as that of
XPA-KO mice and form sever ulcer even at low
dose of UV (400J/m2). These results suggestthat formation of skin cancer due to XPA
deficiency
deficiency.

was

not

rescued

by

RAD18

92

Ⅱ．Progressive degeneration of testicular stem cell of RAD18-KO mice by aging.
We have shown that testicular germ cells in RAD18-KO mice degenerate by aging (Fig. 2). By
aging, the weight of testis became lesser andsperm number dramatically decreased at 1 year
age. To explore role of RAD18 in testicular stem
cell, we examined localization of RAD18 intestis.

RAD18

specifically

localizedatspermatogonial cells.

Discussion & Summary
RAD18 have pivotal role in post-replication repair. In yeast, RAD18 regulated various translesion
polymerase, such as polymerase η, Ι and ζ. Polymerase η, and ζ. are thought to be involved inerro-free and error-prone DNA synthesis, respectively. RAD18 knockout ES cells showed lower
induced mutation frequency as compared to that of wild-type (ref.) Thus, we expected that skincancer formation in XPA-KO mice were suppressed by deficiency of RAD18. However,
RAD18-XPA double knockout mice showed low MED and severe ulcer at low dose of UV
irradiation. The mechanism of formation of skin cancer was dominant as compared to role of
RAD18 in post-replication repair. Actually, aged testicular stem cells appeared to be removed fromtestis in RAD18KO mice, thus we speculated that RAD18 has a role to maintain genome integrity
especially in stem cells.

References
1.

Tateishi, S., Niwa, H., Miyazaki, J., Fujimoto, S., Inoue, H. andYamaizumi, M.

Enhanced Genomic Instability and Defective Postreplication Repair in RAD18 Knockout
Mouse Embryonic Stem Cells.
Mol. Cell. Biol. Vol. 23, No. 2 : 474-481, (2003)

932) Medical Sciences2-8) Oncology

The role of Helicobacter pylori CagA-activated SHP-2 oncoprotein ingastric carcinogenesis
Masanori Hatakeyama
Institute for Genetic Medicine, Hokkaido University
mhata@igm.hokudai.ac.jp

Introduction
Helicobacter pylori virulence factor CagA is directly injected from H. pylori into H. pylori-attachedgastric epithelial cells via the bacterial type IV secretion system (TFSS) (1). H. pylori-injected CagA
then undergoes tyrosine phosphorylation by Src family kinases and specifically binds andderegulates SHP-2 tyrosine phosphatase (2), gain-of-function mutations of which have been reported
in a variety of human malignancies (3). The purpose of this study was to identify in vivo substrates
of CagA-activated SHP-2, which contribute to gastric carcinogenesis.

Results
CagA-deregulated SHP-2 induces cell-morphological transformation showing an extremely
elongated cell-shape termed the hummingbird phenotype in gastric epithelial cells. Notably,
hummingbird cells easily detach from the culture plate, indicating that CagA impairs the celladhesion system. We thus decided to investigate the effect of CagA on focal adhesion kinase (FAK),
which regulates cell adhesion by controlling the formation of focal adhesion spots. Expression of
CagA in AGS human gastric epithelial cells resulted in reduced levels of FAK tyrosine
phosphorylation. Decreased FAK phosphorylation was dependent on CagA tyrosine phosphorylation
and required a CagA region, which mediates CagA-SHP-2 interaction, indicating a role of
CagA-activated SHP-2 in the reduced levels of FAK tyrosine phosphorylation. Consistently, ectopic
expression of a constitutively active SHP-2 in gastric epithelial cells resulted in the decrease in FAK
phosphorylation levels. A substrate-trapping mutant of SHP-2 was capable of forming a stablecomplex with CagA. Furthermore, SHP-2 specifically dephosphorylated FAK in an in vitro
phosphatase assay. From these results, we concluded that FAK is an in vivo substrate of
CagA-activated SHP-2 phosphatase.
There are six potential tyrosine phosphorylation sites in FAK. To identify tyrosine residues thatare dephosphorylated by SHP-2, we generated a series of the FAK mutants that lack various
combinations of the potential tyrosine phosphorylation sites. Ectopic expression of these FAK

94mutants revealed that FAK is phosphorylated on tyrosine-397, -576, -577 in gastric epithelial cells
and CagA-activated SHP-2 dephosphorylated all of these tyrosine residues. Notably,
phosphorylation of tyrosine-576 and -577, both of which are located on the activation loop of thekinase catalytic domain, is required for full activation of FAK kinase activity. This in turn indicatedthat FAK is inhibited when CagA-activated SHP-2 dephosphorylates it. Consistent with this idea,
expression of CagA potently downregulated FAK kinase activity in gastric epithelial cells.
The above observation raised the possibility that inhibition of FAK is responsible at least in partfor the induction of the hummingbird phenotype. To address this possibility, we inhibited
endogenous FAK kinase activity by expressing two distinct dominant-negative FAK mutants. As
expected, expression of these mutants in AGS cells induced elongated cell-shape, which was highly
reminiscent of the hummingbird cells induced by CagA. Conversely, expression of a constitutively
active FAK mutant abolished the activity of CagA to induce the hummingbird phenotype. Theseobservations collectively indicated that inhibition of the FAK kinase activity by CagA-activatedSHP-2 plays an important role in the morphogenetic activity of CagA.
To consolidate inhibition of focal adhesions by CagA, CagA-expressing cells were stained with ananti-phospho-FAK antibody, which specifically recognizes the active form of FAK and found that
focal adhesion spots containing active FAK were strongly reduced in cells expressing CagA.
Intriguingly, there still remained a small amount of active FAK molecules, which are specificallyenriched at the tips of the membrane protrusions, in CagA-expressing cells with the hummingbird
phenotype. This observation indicated that a specific compartmentalization of active FAK, which has
escaped from CagA-stimulated SHP-2, may promote assembly of new focal adhesion complexes thatgenerate precursor sites for membrane protrusions. Such a polarized localization of active FAK may
also be important for a single cell to move from one place to another with a small number of focal
adhesions (Figure 1).

Discussion & Summary
In this work, we identified FAK as an in vivo substrate of CagA-activated SHP-2 phosphatase.
FAK is a legitimate downstream target of CagA because it plays pivotal roles in cell adhesion and95cell morphology as well as cell motility. Two lines of evidence support the idea that reduced FAK
activity plays a role in the morphogenetic activity of CagA. First, a constitutively active mutant of
FAK, which exhibits phosphorylation-independent enhanced kinase activity, abolished induction of
the hummingbird phenotype by CagA. Second, ectopic expression of dominant-negative FAK wascapable of inducing hummingbird-like phenotype. Although many studies have implicated FAK as apositive regulator of cell motility in response to integrin signaling, recent studies have also
demonstrated that downregulation of FAK activity plays an important role in growth factor-inducedchanges in cell morphology and cell movement. Impaired cell adhesion and increased motility by
CagA may be involved in promotion of gastric carcinogenesis caused by cagA-positive H. pylori
infection.

References
1. Segal, E. D., J. Cha, J. Lo, S. Falkow, and L. S. Tompkins. 1999. Altered states: involvement ofphosphorylated CagA in the induction of host cellular growth changes by Helicobacter pylori.
Proc. Natl. Acad. Sci. USA 96: 14559-14564 (1999)
2. Higashi, H., R. Tsutsumi, S. Muto, T. Sugiyama, T. Azuma, M. Asaka, and M. Hatakeyama. 2002.
SHP-2 tyrosine phosphatase as an intracellular target of Helicobacter pylori CagA protein.
Science 295: 683-686 (2002)
3. Mohi, M. G. and Neel, B. G. The role of Shp2 (PTPN11) in cancer, Curr. Opin. Genet. Dev. 17:
23-30 (2007)

962) Medical Sciences2-8) Oncology

Isolation of sympathetic neural crest stem cells and neuroblastoma
cancer stem cells and the application for neuroblastoma research.
Toshihide Iwashita
Aichi Medical University
toshiiwa@aichi-med-u.ac.jp.

Introduction
Neuroblastoma, one of the common solid cancers in child, arises from sympathetic nerve derived
from trunk neural crest. Recently, the neural crest stem cells have been identified in sciatic nerve andenteric nerve in rat embryo whose cell surface immunophenotype were p75+/p0- and p75+/alpha 4integrin+, respectively. But, it has been still unknown the relationship between neuroblastoma andneural crest stem cells. We first try to identify the neural crest stem cells in the sympathetic nerve
tissue to investigate whether or not, the neural crest stem cells can be the sauce of neuroblastoma.

Results
Adrenal gland that contains the largest sympathetic ganglion of rat postnatal day 0 was digested incollagenase 4 and trypsin-EDTA solution in order to disaggregate into single cell. The single cellsuspension was incubated with the monoclonal antibody against p75 (low affinity nerve growth
factor receptor) and followed by incubation with FITC conjugated anti-mouse antibody. One
hundred P75 positive cells per each well were isolated by FACS (fluorescence activated cell sorting)and grown in the self-renewal media which contained rich chicken embryo extract, insulin-growth
factor 1 and basic fibroblast growth factor on the fibronectin coated dish under the hypoxic condition(6% CO2 and 1% O2) for seven days. They were incubated in the differentiation media containing
lower concentration of growth factor and no chicken embryo extract for another five days to getthem differentiated. Plating efficiency was very low and around 3%. The colonies were fixed by 4%paraformaldehyde and immunocytochemically stained using the antibodies against peripherin which
is a specific marker for peripheral neurons and GFAP (glial fibrillary acidic protein) which is a
specific marker for glia cells. We found that around one-third of colonies contained both peripherin
positive neuron and GFAP positive glial cells. Two-third colonies contained only glial cells. Around
1% of p75 positive cells was sympathetic neural crest stem cells. In addition to adherent culture, we
performed floating neurosphere culture to make sure the existence of neural crest stem cells in
sympathetic nerve tissue. Ten thousand P75 positive cells per each well were grown in the

97self-renewal media for floating culture for ten days. The number of neurosphere was around five
(The efficiency was around 0.05%). The floating neurospheres were put on the fibronectin coated foradherent culture in the differentiation media for five days and followed by immunocytochemical
analysis using both anti-peripherin and anti-GFAP antibodies. We found the differentiated primaryneurospheres contained both neuron and glia. A couple of floating primary neurospheres weredigested by trypsin-EDTA to disaggregate and incubated in the self-renewal media for ten days andin the differentiation media for another five days and followed by immunocytochemical analysis
using both anti-peripherin and anti-GFAP antibodies. We revealed secondary neurospheres gave rise
to both neuron and glia as same as primary neurospheres.

Discussion & Summary
We concluded that some p75 positive cells in the sympathetic nerve were neural crest stem cells.
Since the plating efficiency of the adherent culture for sympathetic neural crest stem cells was very
low compared to those of neural crest stem cells derived from sciatic nerve and enteric nerve, wehave been trying to increase the purity of sympathetic neural crest stem cells using various
antibodies, such as CD9, CD81, c-ret and HNK-1.

References
Iwashita T, Kruger GM, Pardal R, Kiel MJ, and Morrison SJ.
Hirschsprung disease is linked to defects in neural crest stem cell function.
Science. 2003 301:972-976

982) Medical Sciences2-14) Others

Role of protein kinase CK2 in the treatment of glomerulonephritis.
Gozoh

TsujimotoDepartment of Genomic Drug Discovery Science, Graduate School of Pharmaceutical Sciences
Kyoto University Faculty of Pharmaceutical Sciences, Kyoto University
gtsuji@pharm.kyoto-u.ac.jp

Introduction
Glomerulonephritis (GN) is a progressive inflammation that may be caused by a variety ofunderlying disorders. It is the primary cause of chronic renal failure and end-stage renal disease,which require dialysis and transplantation worldwide. Immunosuppressive therapy has been used totreat GN clinically, but this treatment has had insufficient therapeutic effects. We show that protein
kinase CK2 is a key molecule in the progression of GN. cDNA microarray analysis identified
CK2alpha, the catalytic subunit of CK2, as a GN-related, differentially expressed gene. Our results
show that CK2 plays a critical role in the progression of immunogenic renal injury, and therefore,
CK2 is a potential target for GN therapy.

Results
Expression profiling was carried out by using mRNA from the renal cortex of anti-GBM GN orcontrol rats on day 28 and cDNA microarrays enriched for clones representing rat kidney genes. We
selected 43 of 3,000 cDNAs that were examined, in which the expression levels differed by >2-foldintensity from controls. The expression of 29 genes, including CK2α, TGFβ1, osteopontin, andcollagen IVα1 were up-regulated, whereas the expression of 14 genes, including pendrin andorganic anion transporter 1, were down-regulated. Expression profiling performed in the renal cortex
of prednisolone-treated anti-GBM GN rats showed that 18 up-regulated and 7 down-regulated
GN-related genes, respectively, were repressed by prednisolone treatment. TGFβ1 , osteopontin,
collagen IVα1, pendrin, and organic anion transporter 1 were previously reported as genes forwhich expression levels change during the development of renal disease. Real-time RT-PCR analysis
on these genes further verified that the microarray data accurately represented gene expression inanti-GBM GN rats. Among the differentially expressed genes, we focused on one gene, CK2α, thatwas overexpressed in the anti-GBM GN rats. Furthermore, the histologic evaluation was conductedon human renal biopsy specimens obtained from untreated lupus nephritis and IgA nephropathy
patients. In all specimens examined, CK2α was overexpressed in the glomeruli, and in some99

cases, in the peritubular interstitium. Hence, overexpression of CK2α appeared to be closely
associated with glomerular injury not only in the GN animal models but also in GN patients. We
further found that an antisenseDNA against CK2αwas therapeutically effective in anti-GBM GN
rats. To provide mechanistic insight into the role of CK2 in GN, we examined in vivo the effect of
CK2 inhibition on apoptosis, proliferation, inflammation, and fibrosis, all processes that are relevant
to resolution and/or progression of GN. The results showed that the protective effects of CK2
inhibition in GN may result from its suppression of ERK-mediated cell proliferation, and its
suppression of inflammatory, as well as fibrotic processes that are enhanced in GN; however, CK2
inhibition apparently does not result in increased apoptotic activity. Administration of either
antisense oligodeoxynucleotide against CK2alpha or low molecular weight CK2-specific inhibitors
effectively prevented the progression of renal pathology in the rat GN models. The resolution of GN
by CK2 inhibition may result from its suppression of extracellular signal-regulated kinase-mediatedcell proliferation, and its suppression of inflammatory and fibrotic processes that are enhanced inGN.

Discussion & Summary
We have isolated a GN-related gene, CK2, by microarray analysis performed on kidney cDNA fromexperimental GN model rats, and demonstrated that in vivo inhibition of the kinase ameliorates therenal dysfunction and histological progression. Because diverse insults can induce similar
clinicopathologic presentations in GN, a marked overlap among downstream molecular and cellularresponses has been suggested. Hence, pharmacologic agents that inhibit common underlying cellularmechanisms are expected to prove effective in treating glomerular diseases of diverse etiologies. Ourpresent study indicates that CK2 could be an ideal therapeutic target for treating immunogenic GN.

Figures & Tables

100

References
Yamada M, Katsuma S, Adachi T, Hirasawa A, Shiojima S, Kadowaki T, Okuno Y, Koshimizu TA,
Fujii S, Sekiya Y, Miyamoto Y, Tamura M, Yumura W, Nihei H, Kobayashi M, Tsujimoto G.
Inhibition of protein kinase CK2 prevents the progression of glomerulonephritis.
Proc Natl Acad Sci U S A. 102(21):7736-41, 2005.

1012) Medical Sciences2-14) Others

Discovery of a protein involving in drug exclusion in the kidney;
remedy for renal failure
Takaaki ABE
Tohoku University Hospitaltakaabe@mail.tains.tohoku.ac.jp

Introduction
It was found that the newly discovered OATP-R is a gene that expresses only in human kidney, andthat the protein produced by OATP-R is a membrane protein transporting various drugs from blood
into the kidney.
The transporting protein has a function to take in drugs and substances of renal failure into the cell
membrane or discharge them to the outside of the cell in the kidney.

OATP-R was also confirmed

as a transporting protein for digoxin, which is a drug used for the therapy of heart failure andarrhythmia.

Digoxin is discharged from the kidney, and its discharge route was previously

considered to be controlled mainly by multi-drug resistant MDR genes.

OATP-R plays animportant role in the uptake of digoxin from the blood to the kidney and controls its concentration in
the blood, which shows the possibility of its application to therapy for kidney diseases.

The aim of

this study is to clarify the mechanism of OATP-R and to develop a new therapy for renal failure.

Results
Tubular secretion of digoxin appears to be a major route of eliminating into urine.

In the past,glomerular filtration was considered as a main elimination pathway, because digoxin excretion iscorrelated with glomerular filtration rate (GFR). However, co-administration of several drugs withdigoxin (i.e. verapamil, quinidine, amiodarone etc.) decreases the renal excretion of digoxin without
affecting GFR.

Because digoxin secretion system is reported to be different from OAT and OCT

systems, these data further suggest that other type(s) of molecule(s) should be involved in thebasolateral transport of digoxin.

We have recently isolated kidney specific organic aniontransporter OATP-R and established MDCK cell line to characterize the pharmacological function.
The establishment of OATP-R expressing cells leads to the characterization and development of adrug for renal failure.
In addition, we focused on the manipulation of genes in the proximal tubules.

The proximal tubule

works for maintaining body fluid homeostasis and drug metabolism. Although there are many

102

different channels or transporters in proximal tubule, which work for this aim, it remains unclear
how they work in vivo on account of difficulty of separation and gene manipulation of intact
proximal tubule. To resolve this problem, we made a new genetically modified mouse which
express GFP and rtTA in proximal tubule specifically by mating ROSA-rtTA-GFP-loxP mouse andSglt2-Cre mouse which has specific expression of Cre protein in proximal tubule. Thus, we will beable to use tet-on system to express any target gene in proximal tubule specifically under thepresence of doxycycline by mating this new mouse and the other mouse which is inserted the target
gene into the downstream area of tet-o gene. We would clarify the mechanism of OATP-R at
proximal tubule in vivo .

Discussion & Summary
The expression level of rat oatp-R is decreased in the renal failure model. This reduction might give
one of the reasons for reduction of digoxin and renal failure substances excretion in the renal failurestate.
The manipulation of OATP-R might play a key role to eliminate and be a clue to develop a new
remedy for renal failure patients.

Figures & Tables

103

References
Mikkaichi T. et al.

Isolation and characterization of novel kidney-specificProc. Natl. Acad. Sci. USA 101: 3569-3574, 2004

digoxin transporters.

1043) Chemical Sciences3-1) Organic Chemistry

Studies on the Biomimetic Cascade Synthesis of Polycyclic
Terpenoids
Kazuaki Ishihara
Nagoya University
ishihara@cc.nagoya-u.ac.jp

Introduction
Biomimetic polyene cyclization is an important key step in the concise total synthesis ofpolycyclic natural products.

In particular, the Lewis acid-promoted diastereoselective cyclizationof polyenic aldehyde acetals to 4β(axial)-alkoxypolycycles has been established by Johnson et al.
However, excess SnCl4 is often required as Lewis acid and there are no methods available for thesynthesis of 4α(equatorial)-alkoxypolycycles.

We report here the SnCl4 (10 mol%)-catalyzed

polycyclization of homo(polyprenyl)arene analogues bearing terminal siloxyvinyl groups, whichwere much more reactive than other initiators such as acetals, aldehydes, and ketones

Results
The α（equatorial)/β(axial) selectivity of 4-siloxy group at polycycles could be controlled by the
nucleophilicity of pro-C(9) and the steric effect of a silyl group.
The proposed mechanism is shown in Figure 1. 4α-Selective cyclization of polyenic silyl enol
ethers would concertedly proceed through antiperiplanar (chair–chair-like) transition state (TS) 1.
On the other hand, 4β-selective cyclization of polyenic silyl enol ethers would proceed stepwise
through synclinal TS-2 or 3 stabilized by Coulomb attractive interaction (minimalization of charge
separation) between O and pro-C(10).

A strong nucleophilicity of pro-C(9), a (6E)-geometry and abulky silyl group would effectively favor TS-1, while a weak nucleophilicity of pro-C(9), a(6Z)-geometry and less steric hindrance of a silyl group would favor TS-2 or 3.
Based on the experimental results, two natural diterpenoids, 18-norabieta-8,11,13-trien-4-ol (6),which has antibacterial activity, and its epimer 11 were synthesized from 4 and 7 with >99% 4α and>99% 4β, respectively (Scheme 1).

The anti-herpes active diterpenoid 9, a synthetic intermediateof 11, was also synthesized with >99% ds.

105

Figure 1. Proposed tansition-state assemblies 19-21

Scheme 1. Total Syntheses of Epimers 6and 11

Discussion & Summary
Although it was difficult to directly generate silyloxocarbenium ion intermediates fromaldehydes and ketones with silyl Lewis acids, we succeeded in their catalytic generation with SnCl4
from silyl enol ethers instead of carbonyl compounds.

The present results demonstrate thesynthetic advantages of using polyprenoid analogues bearing a terminal siloxyvinyl group as
substrates of polyene cyclization with respect to both the reactivity and 4α/4β-diastereocontrol.

References
Catalytic diastereoselective polycyclization of homo(polyprenyl)arene analogues bearing terminalsiloxyvinyl groups
Muhammet Uyanik, Kazuaki Ishihara, Hisashi Yamamoto
Org. Lett. 2006, 8(24), 5649–5652.

1063) Chemical Sciences3-1) Organic Chemistry

Development of Novel Asymmetric Reactions Based on
Unprecedented Asymmetric Amplification
Yutaka Ukaji
Kanazawa University
ukaji@kenroku.kanazawa-u.ac.jp

Introduction
The development of practical and efficient methods for construction of chiral molecules is an
essential part of programs to explore new medicinal and agrochemical agents. We have used a
strategy to design novel chiral system derived from metal salts of tartaric acid ester, in which two
substrates bound to the metal centers might be ideally oriented and/or activated for subsequent
reaction.

Based on this proposal, we studied a nucleophilic addition of alkynylzinc reagents toacyclic nitrones, and unprecedented asymmetric amplification was realized.

Results
The asymmetric addition of alkynylzinc reagents, prepared in situ from dimethylzinc and 1-alkynes,
to acyclic nitrones was investigated by utilizing di(t-butyl) (R,R)-tartrate as a chiral auxiliary toafford the corresponding optically active (R)-N-(propargylic)hydroxylamines. In order to confirm
how the reaction proceeded, the time course of the reaction was observed and the enhancement ofenatioselectivity was found along the progress of the reaction.

Base on this observation,

unprecedented enhancement of enantioselectivity was realized by the addition of methylzinc salt of
product-like hydroxylamine to afford the (R)-N-(propargylic)hydroxylamines up to 95% ee.
The catalytic version of the present reaction of alkynylzinc reagents was also achieved. The
influence of the stereochemistry of the product-like additive was investigated.

When themethylzinc salt of (R)- or (S)-product-like hydroxylamine was used as the additive instead of that ofracemic product-like hydroxylamine, the enantioselectivity was surprisingly decreased.

By theaddition of methylzinc salt of the product-like racemic hydroxylamine, enantioselectivity of theN-(propargylic)hydroxylamines was again enhanced up to 96% ee.
To the contrary, the asymmetric addition reaction of diphenylzinc to C-alkynyl nitrone was also
investigated by utilizing di(t-butyl) (R,R)-tartrate as a chiral auxiliary.
corresponding (S)-N-(propargylic)hydroxylamines was obtained.

In this case, theBy the addition of the same

methylzinc salt of product-like hydroxylamine again realized enantiomeric enhancement.

107

Furthermore, in the catalytic version of this phenylation of C-alkynyl nitrone was examined.
Although chiral induction was very poor without the product-like additive, the presence of the
product-like additive dramatically improved the enantioselecitivy.

Discussion & Summary
In conclusion, by the addition of a product-like racemic substrate, the asymmetric amplification wasobserved, and the corresponding N-(propargylic)hydroxylamines were obtained with excellent
enantioselectivities.
enanchiomeric

So far, the precise mechanism and the reason why such unprecedented

enhancement

wasobserved

are

not

yet

clear.

The

products

N-(propargylic)hydroxylamines were found to be readily crystallized in racemic form rather than inenantiomerically

pure

form.

In

1

H

NMR,

chemical

shiftsofN-(propargylic)hydroxylamines and enantiomerically pure ones are not same each other.

racemic
These

observation suggested that even the methylzinc salts of N-(propargylic)hydroxylamines readily
dimerize in solution, which induced the formation of active chiral environment containing
methylzinc salt of di(t-butyl) (R,R)-tartrate.

Figures & Tables

Entry
1
2
3
4
5
6
7
8
9
10

R2
Ph

BrC6H4BrC6H4
Ph

Ph
Ph
SiMe3

p

R1
Ph

o

A
racemic
racemic
RS
racemic
racemic
racemic
racemic
racemic
racemic

n
0.0
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3
0.3

solvent
CH2Cl2CH2Cl2CH2Cl2CH2Cl2
Et2O
toluene
EtPh
EtPh
EtPh
tolueneYield/%
87
90
74
86
89
92
84
73
77
57

ee/%
68
83
51
73
83
91
95
90
96
88

108

References(1) Catalytic Asymmetric Addition of Alkynylzinc Reagents to Nitrones
Konishi, A.; Wei, W.-L.; Kobayashi, M.; Fujinami, S.; Ukaji, Y.; Inomata, K. Chem. Lett. 2007,
36, 44-45.
(2) Preparation of Novel N-Sulfonylated (S,S)-2,3-Diaminosuccinate-Type Chiral Auxiliaries andApplication to an Asymmetric 1,3-Dipolar Cycloaddition Reaction of Nitrile Oxides to Allyl
Alcohol
Serizawa, M.; Ukaji, Y.; Inomata, K. Tetrahedron: Asymmetry 2006, 17, 3075-3083.
(3) Enantioselective Diels-Alder Reaction of o-Quinodimethanes by Utilizing Tartaric Acid Ester asa Chiral Auxiliary
Takinami, M.; Ukaji, Y.; Inomata, K. Tetrahedron: Asymmetry 2006, 17, 1554-1560.
(4) Asymmetric 1,3-Dipolar Cycloaddition of Nitrones with an Electron-Withdrawing Group to
Allylic Alcohols Utilizing Diisopropyl Tartrate as a Chiral Auxiliary
Ding, X.; Taniguchi, K.; Hamamoto, Y.; Sada, K.; Fujinami, S.; Ukaji, Y.; Inomata, K. Bull.
Chem. Soc. Jpn. 2006, 79, 1069-1083.
(5) Asymmetric Addition of Alkynylzinc Reagents to Nitrones Utilizing Tartaric Acid Ester as a
Chiral Auxiliary
Wei, W.-L.; Kobayashi, M.; Ukaji, Y.; Inomata, K. Chem. Lett. 2006, 35, 176-177.

1093) Chemical Sciences3-1) Organic Chemistry

Efficient Synthesis of Biologically Active Nitrogen-Containing
Heterocyclic Compounds by the Carbocyanation Reactions

Yoshiaki Nakao
Kyoto University
nakao@npc05.kuic.kyoto-u.ac.jp

Introduction
Direct cleavage of a R–CN bond followed by reconnection of both R and CN with two carbons in
unsaturated carbon–carbon bonds, namely carbocyanation reaction,1 provides ready access to highly
functionalized nitriles from simple ones with perfect atom economy and, thus, should be of great
synthetic value. The purpose of this research is to develop and apply the carbocyanation reaction toconcise synthesis of nitrogen-containing heterocyclic compounds.

Results
We have extensively studied the carbocyanation reaction in the last three years, and found for the
first time that aryl cyanides add across alkynes in the presence of Ni/PMe3 catalyst.1 To expand thescope of nitriles applicable to the carbocyanation reaction, we first examined the addition of allyl
cyanides across alkynes. After screening of several combinations of a metal catalyst and a ligand, we
have found that the combination of Ni(cod)2 and P(4-CF3–C6H4)3 is optimum to achieve theallylcyanation reaction in highly stereo-, regio-, and chemoselective manners.2 The addition of
α-siloxyallyl cyanides across alkynes proceeded at the γ-carbon exclusively to give aldehyde
products after acidic hydrolysis (Scheme 1). It is worth noting that functional groups such as a C–Cl
bond, which readily undergoes the oxidative addition to nickel(0), are tolerated under the present
reaction conditions. We then turned our attention to the carbocyanation reaction across otherunsaturated compounds, and found that 1,2-dienes undergo the addition of cyanoformate esters(Scheme 2).3 For example, the addition of ethyl cyanoformate across 3-methyl-1,2-butadiene took
place exclusively at the internal double bond in the presence of Ni/PMe2Ph catalyst to afford ethyl
2-(2-cyanoprop-2-yl)acrylate in 51% yield. The adduct underwent 1,4-addition reaction withbutylcopper/BF3•OEt2 to give the corresponding β-cyanoester, which further afforded a substituted
γ-lactam, a versatile synthetic intermediate for five-memebered nitrogen-containing heterocyclic
compounds, upon treatment with NaBH4 in the presence of CoCl2. Whereas the carbocyanation

110

reaction of simple 1-alkenes has been elusive due to competitive β-hydrogen elimination to giveHeck-type adducts, norbornene and norbornadiene undergo the aryl- and allylcyanation reaction in a
cis-fasion from the exo-face (Scheme 3).4 On the other hand, intramolecular arylcyanation of alkenes
works well, giving oxyindole product having a quaternary carbon (Eq. 1). The use of a catalytic
amount of AlMe2Cl, which would act as a Lewis acid cocatalyst to coordinate to the cyano groupand promote the oxidative addition step,5 is crucial for this particular reaction. This intramolecular
reaction would be useful for the synthesis of alkaloids having a hexahydropyrrolo[2,3-b]indole
structure such as (–)-physostigmine.

111

Discussion & Summary
In summary, we have demonstrated several types of carbocyanation reactions of unsaturated
compounds. The transformations described above have no piece of precedents and, thus, haveintroduced an important innovation in retro-synthetic analysis of target molecules. The reaction
should find many applications to production of not only nitrogen-containing heterocycles but alsofine chemicals, synthetic intermediates for pharmaceuticals, and monomers for functional polymers.
Further efforts to expand the scope of the chemistry to other nitriles and unsaturated compounds
including enantioselective version are ongoing subjects in our laboratories.

References
１．(a) Nakao, Y.; Oda, S.; Hiyama, T. J. Am. Chem. Soc. 2004, 126, 13904–13905. (b) Nakao, Y.;
Oda, S.; Yada, A.; Hiyama, T. Tetrahedron 2006, 62, 7567–7576.
２．Nakao, Y.; Yukawa, T.; Hirata, Y.; Oda, S.; Satoh, J.; Hiyama, T. J. Am. Chem. Soc. 2006, 128,
7116–7117.
３．Nakao, Y.; Hirata, Y.; Hiyama, T. J. Am. Chem. Soc. 2006, 128, 7420–7421.
４．Nakao, Y.; Yada, A.; Satoh, J.; Ebata, S.; Oda, S.; Hiyama, T. Chem. Lett. 2006, 790–791.
５．Nakao, Y.; Yada, A.; Ebata, S.; Hiyama, T. J. Am. Chem. Soc. 2007, 129, 2428–2429.

1123) Chemical Sciences3-1) Organic Chemistry

Studies on the Synthesis and Biological Activity of Neurotoxins
Produced by Marine Algae
Isao Kadota
Okayama University
Kadota-i@cc.okayama-u.ac.jp

Introduction
Polycyclic ethers, produced by marine algae, show potent neurotoxicity by binding to the ionchannels and cause massive fish kills and human food poisoning.

Since further studies arehampered by the limited availability from nature, chemical synthesis has been the sole realistic way
to obtain sufficient amounts of the natural products. Moreover, the unusual structures of thesecompounds are particularly attractive targets for synthetic chemists.

Results
Yessotoxin is a disulfated polycyclic ether isolated from the digestive glands of scallops,
Patinopecten yessoensis.1 Due to its novel structural features and biological activities, yessotoxin
has attracted the attention of synthetic chemists. As a part of total synthetic study of yessotoxin,
we investigated the stereocontrolled synthesis of the IJK ring segment.

A cyclization precursor,

having an aldehyde and acrylate moiteties, was synthesized from 2-deoxyribose.

SmI2 mediatedcyclization of the substrate gave the IJ ring system with high stereoselectivity in good yield.
Further modification on the side chain was carried out to give an epoxy alcohol, which was
subjected to the acid catalyzed cyclization to afford the IJK ring segment in a highly region- andstereocontrolled manner.2
Next we investigated the formal total synthesis of hemibrevetoxin B.

This compound was

isolated from the cultured cells of the red tide organism Gymnodinium breve by Shimizu in 1989.3
The unique structural features have attracted the attention of synthetic chemists, and a number ofstrategies have been investigated. The synthesis was started from triacetyl glucal as achioral source,
and the AB ring segment was prepared by known procedures including an acid catalyzed
epoxy-alcohol cyclization.

The C ring was constructed by the intramolecular allylation of

α-acetoxy ether prepared from the corresponding ester derivative.

The D ring moiety was

successfully prepared by the ring-closing olefin metathesis mediated by the second generation
Grubbs catalyst.

Finally, hydrogenation of the D ring olefin and removal of the protective group on

113

the side chain was performed with H2/Pd-C to give the known synthetic intermediate.4

Discussion & Summary
In conclusion, we have achieved the synthesis of the IJK ring segment of yessotoxin via SmI2
mediated cyclization and acid catalyzed cyclization as key ring-closure processes in highly
stereocontrolled manner.

The present work is the first example of the synthesis of the IJK ring

segment of yessotoxin.
Furthermore, a formal total synthesis of hemibrevetoxin B was performed by the intramolecularallylation of an α-chloroacetoxy ether and ring-closing metathesis as key reactions.
convergent strategy employed makes the synthesis highly efficient.

The

Further application of thesestrategy to the synthesis of other marine natural products is in progress.

Figures & Tables

References（１） Murata, M.; Kumagai, M.; Lee, J. S.; Yasumoto, T. Tetrahedron Lett. 1987, 28, 5869-5872.

（２） Kadota, I.; Abe, T.; Sato, Y.; Kabuto, C.; Yamamoto, Y. Tetrahedron Lett. 2006, 47, 6545-6548.
（３） Prasad, A. V. K.; Shimizu, Y. J. Am. Chem. Soc. 1989, 111, 6476-6477.
（４） Kadota, I.; Abe, T.; Ishitsuka, Y.; Touchy, A. S.; Nagata, R.; Yamamoto, Y. Tetrahedron Lett.
2007, 48, 219-221.

1143) Chemical Sciences3-1) Organic Chemistry

Novel Synthesis of N-Containing Heterocycles via Transition
Metal-Cataltyzed [2+2+2] Cocyclization

Yoshihiro Sato, PhD., Professor
Graduate School of Pharmaceutical Sciences, Hokkaido University
biyo@pharm.hokudai.ac.jp

Introduction
A [2+2+2] cocyclization is one of the promising methodology for the synthesis of various
aromatic compounds.

Recently, we have reported a Pd-catalyzed [2+2+2] cocyclization ofα,ω-diynes and arynes and its application to the synthesis of natural aryl-naphthalene lignans, suchas taiwanins C, E, and dehydrodesoxypodophyllotoxin.1

This project mainly focused on thedevelopment of a transition metal-catalyzed [2+2+2] cocyclization of α,ω-diynes and heteroarynes.

Results
It is known that a heteroaryne such as 2,3-pyridine 2 or 3,4-pyridyne 4 can be produced in situ bytreatment of the corresponding precursors 1 or 3 with CsF, respectively (Scheme 1).2

The pyridines

are homologues of the arynes such as a benzyne, and it is expected to have the unique property.
However, there is only a few examples of Diels-Alder type reactions of 1,3-dienes and pyridines in
the literature, and a [2+2+2] cocyclization of the pyridines has not been reported so far.

First, weinvestigated a [2+2+2] cocyclization of α,ω-diynes 5 and 3,4-pyridynes 4 generated the precursors 3
under the various conditions.

As the results, it was found that a nickel complex could promote the[2+2+2] cocyclization of diynes and 3,4-pyridynes affording isoquinoline derivatives in good yields(Table 1).

In this reaction, an electron-donating substituent on the pyridine ring of the 3,4-pyridyne

precursor was tolerated, and the reaction of diyne 5c and 3b or 3c gave the corresponding substituted
isoquinoline 6cb or 6cc in good yields, respectively.

On the other hand, it was found that anelectron-withdrawing substituent on the pyridine ring of the precursor retarded the [2+2+2]
cocyclization.

Thus, the reaction of 5c and 3d gave isoquinoline 6cd in a moderate yield.

Discussion & Summary
In summary, we succeeded in developing the novel nickel-catalyzed [2+2+2] cocyclization ofα,ω-diynes and 3,4-pyridynes for the first time, affording isoquinoline derivatives in good yields.3

115

The present convergent strategy should pave the way for the novel synthesis of various isoquinoline
derivatives by virtue of a variety of combinations of diynes and 3,4-pyridyne precursors.

Figures & Tables

References
1. Sato, Y.; Tamura, T.; Kinbara, A.; Mori, M. Adv. Synth. Catl. 2007, 349, 647-661.
2. (a) Walters, M. A.; Shay, J. J. Synth. Commun. 1997, 27, 3573.
(b) Tsukazaki, M.; Snieckus, V. Heterocycles 1992, 33, 533.
3. Iwayama, T.; Amagasa, K.; Sato, Y., manuscript in preparation.

1163) Chemical Sciences3-1) Organic Chemistry

Development of External Stimulus-triggered Auto-processing Peptides
Akira Otaka
Department of Molecular Design and Synthesis, Institute of Health Biosciences, The Universityof Tokushima
aotaka@ph.tokushima-u.ac.jp

Introduction
Control of the function of peptides by external stimuli has been receiving increasing attention due
to its potential utility in chemical biology. An external stimulus-induced peptide bond cleavage
seems to allow peptides to be converted to their corresponding active (or inactive) forms. Therefore,
we attempted to synthesize an auto-processing peptide which can be changed to other form by an
external stimulus.

Results
1,2

Inspired by the trimethyl lock system,

we designed stimulus-responsive peptide 1, which canrelease a functional peptide after a stimulus-induced removal of a phenolic protective group (PG)
and subsequent processing reaction (Scheme 1). Designed peptide 1 features nucleophilic
involvement of a regenerated phenolic hydroxyl group to an adjacent peptide bond to release
functional peptides. In this paper, we chose a photo-removable o-nitrobenzyl (o-NB) andphosphatase-removable phosphate group for phenolic protection to afford stimulus-responsive model
peptides.

Photo-responsive model peptide 8 was synthesized as shown in Scheme 2. Aldehyde 2[2] was
α-aminated with di-tert-butyl azodicarboxylate in the presence of pyrrolidine. After reduction of thealdehyde group with sodium borohydride, the resulting alcohol was protected with a TBS group to
give silyl ether 3. Trifluoroacetylation of the terminal nitrogen in 3 with trifluoroacetic anhydride(TFAA) and subsequent N-N bond cleavage by SmI2 in the presence of HMPA and tert-BuOH gave

117

amino alcohol 4. The benzyl group on 4 was removed by hydrogenolysis and the generated phenolichydroxyl group was protected with an o-nitrobenzyl group to afford o-NB ether 5. After removal of
the TBS group of 5 under acidic condition, a two-step oxidation was performed to give carboxylic
acid 6. The Boc group on 6 was removed with hydrogen chloride in ethyl acetate to yield an amine,
which was reprotected with an Fmoc group to give Fmoc protected photo-responsive processing
device 7. The total yield of Fmoc derivative 7 amounted to 11% over 12 steps starting from aldehyde
2. Finally, incorporating amino acid 7 into the peptide by standard Fmoc solid phase peptide
synthesis (SPPS) afforded photo-responsive model peptide 8 as a diastereomeric mixture.
To examine the photo-reactivity of model peptide 8, we performed the photo-processing reaction
outlined in Scheme 2. Peptide 8 in 20% MeCN/phosphate buffer (pH 7.6) was irradiated by UV light(>365 nm) for 3 min and then incubated at 37 °C. The reaction progress was monitored by HPLC
and the peptides were characterized by ESI-MS. The
o-nitrobenzyl group of peptide 8 was completely
removed with 3 min of UV irradiation to generate
deprotected intermediate 9. Afterward, intermediate 9
was converted to corresponding processing products
10 and 11 via incubation for 2 h. Monitoring theprocessing

reactionclarifiedthat

half-lifeofintermediate 9 was approximately 34 min.
To demonstrate the applicability of this processing
systemtootherstimuli,we

designed

phosphatase-responsive model peptide 12 in which
the phenolic hydroxyl group was protected withphosphate (Scheme 2). Peptide 12 was synthesized by
Boc SPPS. A phosphatase-responsive processing
reaction of peptide 12 was examined in the presenceof alkaline phosphatase derived from calf intestine.
Model peptide 12 was incubated with alkaline
phosphatase in Tris-HCl buffer (pH 7.9) at 37 °C.
After 24 h, HPLC and ESI-MS analyses clarified that
substrate 12 completely disappeared and processing
products 10 and 13 were obtained in good purity. In
the absence of an alkaline phosphatase, hydrolysis of
the phosphate moiety was not detected under these
conditions.

118

Discussion & Summary
In conclusion, we developed photo- and phosphatase-responsive self-processing peptide. The
processing reaction was triggered by the deprotection of the protective group on the phenolichydroxyl moiety and processing products were obtained in good purity. Reaction monitoring
revealed that the reaction rate depends on the amino acid next to the stimulus-responsive residue.
Application of the stimulus-responsive processing device-containing functional peptides in chemical
biology field is in progress.

References
1. K. L. Amsberry, R. T. Borchardt, J. Org. Chem. 1990, 55, 5867.

2.

M. Caswell, G. L. Schmir, J. Am. Chem. Soc. 1980, 102, 4815; S. Milstien, L. A. Cohen, J. Am. Chem. Soc. 1972, 94, 9158; R.
T. Borchardt, L. A. Cohen, J. Am. Chem. Soc. 1972, 94, 9166.

1193) Chemical Sciences3-1) Organic Chemistry

Synthesis of Native Glycoprotein by in vitro Protein Synthesis

Shino Manabe
RIKEN (The Institute of Physical and Chemical Research)
smanabe@riken.jp

Introduction
For investigation of oligosaccharide modification of protein, homogenous structure glycoprotein
is strongly required. By pure organic synthetic methodology, the limitation of preparation of large
molecule glycoprotein exists. In this project, the synthetic approach will be investigated by
combining the synthetic organic chemistry and in vitro protein synthesis to overcome the problem.

Results
We chose the C-mannosylated tryptophan (C-Man-Trp) and O-linked N-acetyl glucosamine(O-GlcNAc) as target molecules. The target sugar modifications are related to diseases and thought
to be important molecules from biological point of view.
Firstly, the amino group protection group of C-Man-Trp was investigated. After synthesis of
C-Man-Trp by our own strategy, the amino group of amino acid moiety was protected either Boc or
Fmoc group. The aminoacylation reaction of the protected C-Man-Trp to the suppressor t-RNA wasachieved by use of ribozyme developed by Suga in high yields. After aminoacylation reaction,
removal of protecting group was found to be difficult. In the case of Boc group, the instability of
C-Man-Trp under acidic conditions is the problem, while in the case of Fmoc group, the ester groupbetween RNA and amino acid was not stable during deprotection of Fmoc under basic conditions.
Then, we turned out the target to the O-GlcNAc. After preparation of O-GlcNAc in an efficientmanner, the amino group was protected by Boc group. After aminoacylation to suppressor RNA, theBoc group was removed. The synthesis of RNA linked sugar amino acid was achieved. By using
above RNA-linked sugar amino acid, the preparation of protein was attempted. However, in vitro
protein synthesis is difficult at this stage, probably because of the steric bulkiness and hydrophilicityof the sugar moiety.

Discussion & Summary
We have achieved the efficient synthesis of glyco-amino acid, Fmoc C-Man-Trp, Boc-C-Man-Trp,

120

Boc-O-GlcNAc-Thr. By using the ribozyme developed by Suga, the amino acylation reaction of thesugar-amino acids to the suppressor RNA was achieved in a efficient manner and in high yields.
The limitation of in vitro protein synthesis was also found. Probably because of the steric bulkinessor the hydrophilic nature of the sugar part, the protein formation. The protein synthesis is currently
under investigation.

Figures & Tables

1213) Chemical Sciences3-4) Analytical Chemistry

Design, Synthesis and Biological Application of Molecular Probes
Which Convert Cellular Biological Responses to Chemical Outpur
Kazuya Kikuchi
Osaka University, Graduate School of Engineering
kkikuchi@mls.eng.osaka-u.ac.jp

Introduction
Phosphorylation and dephosphorylation are general biochemical modifications seen on a wide
variety of biomolecules.

Phosphorylation is catalyzed by kinases, and dephosphorylation byvarious phosphatases, for instance, alkaline phosphatase, acid phosphatase, serine/threonine
phosphatases, and tyrosine phosphatases, etc.

For studying about dephosphorylation of

biomolecules, fluorometric assays that can be applicable to bioimaging experiments are paid more
attention by many researchers.

Results
Development of fluorescent probes detecting phosphatase activity is important, because
phosphorylation and dephosphorylation regulate a lot of metabolic pathways in living organism.
For clarifying the biological role of each phosphatase by fluorescence measurement, it is desirable
that the fluorescent probe has the specificity toward the target enzyme.

However, widely-usedfluorescent probes such as DiFMUP have almost no enzyme specificity among phosphatases. This
is in part because all phosphatase probes are aryl phosphate monoesters, and the aryl
monoester-based structure does not possess flexibility to modify the basic structures for having an
enzyme specificity.

Here, we report a novel design strategy for fluorescent phosphatase probes

which have an alkyl phosphate monoester.

There were no fluorescent probes based on alkyl

monoester because dephosphorylation does not change its electronic characters much as aryl
monoester. The design strategy is based on a phenomenon that an anionic group adjacent to
7-hydroxyl group of 7-hydroxycoumarin increases the pKa of the hydroxyl group.

Using thisstrategy, we developed novel fluorescent probes for phosphatases and investigated theirphotophysical and enzymological properties.

They showed ratiometric changes of the excitation

spectra by addition of acid phosphatase (ACP).

They showed no spectral changes by any protein

tyrosine phosphatase or serine/threonine phosphatase and very small changes by alkaline
phosphatase (ALP).

In conclusion, we developed fluorescent probes detecting phosphatase activity

122

using a novel design strategy.
various phosphatases.

This strategy would lead to development of specific probes for

The pKa modulation approach is widely applicable strategy for designing

fluorescent probes targeted for other enzymes.

Discussion & Summary
A novel design strategy for development of fluorescent probes detecting phosphatase activities wasintroduced.

This design principle is based on that the pKa change of 7-hydroxyl group of

7-hydroxycoumarin derivatives are induced by neighboring anionic groups such as a phosphatemonoester group, and that the pKa change induces the excitation spectral change.

We applied thisprinciple to the probe design and developed fluorescent phosphatase probes that have an alkyl
phosphate monoester as the enzyme-recognition group.

Our synthesized probes were

dephosphorylated by acid phosphatase (ACP) efficiently and showed their excitation spectral shift.
The result indicates that the modification of the linker can change the affinity to enzyme
dramatically.

Figures & Tables
R

O
H

R

O
Pi

O

phosphatase

R
O

O

HO

H+ -H+O

H+ -H+
R

Pi
O

O

X

protonated form
λex = 330 nm

O

X

HO

O
X

O

deprotonated form
λex = 380 nm

Fluorescence Intensity

Pi

HO

X

330
380
Wavelength (nm)Figure 1.

Hypothesized scheme of the excitation spectral change of 7-hydroxycoumarinderivatives by phosphatases.

R: alkyl group.

X: linker.

123

(a)
Fluorescence Intensity (a. u.)

200
150
360 min180 min60 min30 min
0min

100
50
0

280

320
360
400
Wavelength / nm

440

(b)
Fluorescence Intensity (a. u.)

200
150

50
0

Fluorescence Intensity (a. u.)

(c)

280

320
360
400
Wavelength / nm

440

200
150
120 min60 min30 min10 min
0 min

100
50
0

Figure 2.

240 min180 min
120 min30 min
0 min

100

280

320

360

400

440

Excitation spectra of (a) 3a, (b) 4a, and (c) 5a before and after addition of acidphosphatase (ACP) in 100 mM HEPES buffer solution (pH 7.4). Emission wavelength was 470
nm.

References
1. Komatsu, T., Kikuchi, K., Takakusa, H., Hanaoka, K., Ueno, T., Kamiya, M., Urano, Y., Nagano, T.
Design and Synthesis of an Enzyme Activity-Based Labeling Molecule with Fluorescence
Spectral Change. (2006) J. Am. Chem. Soc., 128, 15946-15947.
2. Terai, T., Kikuchi, K., Iwasawa, S., Kawabe, T., Hirata, Y., Urano, Y., Nagano, T.

Modulation of

Luminescence Intensity of Lanthanide Complexes by Photoinduced Electron Transfer and Its
Application to a Long-Lived Protease Probe. (2006) J. Am. Chem. Soc., 128, 6938-6946.

1243) Chemical Sciences3-5) Others

Elucidation of Functional Properties of Thermostable ElectronTransfer Proteins and Their Application to Biomaterials
Yasuhiko Yamamoto
Graduate School of Pure and Applies Sciences, University of Tsukuba
yamamoto@chem.tsukuba.ac.jp

Introduction
Monoheme Class I cytochromes c (cyts c), in which heme Fe is coordinated to His N and Met S
atoms as axial ligands at the redox center, are some of the best characterized redox active proteins.
Homologous Class I cyts c, thermophilic Hydrogenobacter thermophilus cytochrome c552 (HT) andmesophilic Pseudomonas aeruginosa cytochrome c551 (PA), exhibit a unique thermodynamic
property, i.e., despite their structural similarity together with their 56 % sequence identity, theoxidized form of HT is significantly more stable than that of PA, as reflected by the large difference
in denaturation temperature (Tm).

Site-directed mutants of PA, for which amino acid substitutions

were selected with reference to the corresponding residues in HT, exhibited thermostabilities
between those of PA and HT.

In this study, we elucidated the molecular mechanisms responsible

for the thermostability and redox regulation of these proteins.

Results
In order to determine the relationship between the redox property and overall protein stability, we
first carried out electrochemical measurements at 25 ºC using cyclic voltammetry (CV).

The

observed redox potentials (E˚') were in the range of +0.2 - +0.3 V, i.e. similar to those reported forother Class I cyts c.

The E˚' values for the PA mutants, F34Y single (sm) andF7A/V13M/F34Y/E43Y/V78I quintuple (qm), were lower than that of the wild-type, corresponding
with the increase in overall protein stability.

These results indicate that, upon mutation, theoxidized forms are more stabilized than the reduced ones are. Thus, the E˚' values of cyts c are
determined by the overall stability of the oxidized forms, which is significantly affected by themutation(s), as manifested in their Tm values.
On electrochemical measurement at 5 - 85 ºC, we observed CV responses below 70 ºC and 80 ºC
for PA and sm, respectively.

In contrast, ones for qm and HT were detected up to 85 ºC.

Plots ofE˚' against temperature (E˚'−T. plots) for the PA proteins (wild-type and the two mutants) gave a125straight line at lower temperatures and exhibited downward curvature with increasing temperature,
while those for HT exhibited a straight line over the temperature range examined. The temperature
at which the plots started to show curvature correlated with the overall protein stability. The
curvature observed for the PA proteins reflects that the oxidized form becomes thermodynamicallymore favorable than the reduced one does as the thermal denaturation proceeds.

This is because theoxidized heme becomes more favorable with removal of the hydrophobic heme environment on
denaturation, as demonstrated by model studies.
From the E˚'−T. plots, we estimated the enthalpic (∆H˚') and entropic (∆S˚') contributions to theE˚' value.

For the PA proteins, the ∆H˚' values increased consistently with increasing protein

stability, whereas the ∆S˚' values remained essentially unchanged. These results suggest that theeffect of the mutations in the PA protein on the E˚' value is mainly an enthalpic contribution. In
addition, the least negative ∆H˚' value observed for HT was consistent with its highest stability
among the oxidized forms of the proteins.

Furthermore, the least negative ∆S˚' value of HT

indicates that enthalpy-entropy compensation occurs in the redox thermodynamics as found in theother electron transfer proteins such as cupredoxins and rubredoxins.
Next, we directly compared the stability of the heme active site where the electron transfer
reaction occurs in cyts c. On 1H NMR spectroscopy, signals due to Fe-coordinated Met side chain
protons are often resolved in the spectra of both the oxidized and reduced forms of cyts c. The
temperature-dependent appearance of these signals facilitates direct comparison of the stability of
the heme active site.

Such Met signals were observed up to 95 ºC in the spectra of the reduced

forms of all the cyts c examined, indicating their high structure stability around the heme active site
under the conditions used for the electrochemical analysis.

In contrast to the reduced form, Metsignals were barely observed in the NMR spectrum of the oxidized form of PA at 80 ºC, suggestingthat its heme active site structure was no longer retained above this temperature. Met signals could
be observed up to 91 ºC for sm, demonstrating that the denaturation temperature for the heme active
site in sm increased by >10 ºC due to the mutation, F34 to Y.

The stability of the heme active site

in the oxidized form of PA was further enhanced by quintuple mutations, Met signals being exhibitedup to 95 ºC, although the signals were not as sharp as observed for the reduced form at the same
temperature.

These NMR results led to two conclusions:(1) In individual proteins, the heme

active site of the reduced form is more stable than that of the oxidized one.

(2) The mutations thatstabilize the overall protein structure of a PA protein are also responsible for the stability of the heme
active site in the oxidized form.

Furthermore, as previously demonstrated, the stability of the heme

active site in the oxidized forms of the proteins, as determined by NMR, positively correlates withthe stability of the Fe-S bond, which was inferred from the dissociation temperature obtained
through variable temperature measurement of the 695-nm absorption band characteristic of the Fe-S
bond in the oxidized proteins.

126

Discussion & Summary
The findings described above, together with those described in our previous study, demonstrated
that the overall protein stability, Fe-S bond stability, and redox property of cyt c are mutually relatedwith each other.

The protein conformational change induced by the mutations, which increases theoverall stability of the oxidized form, happens to determine the Fe-S bond stability.

Since theoverall protein stability of the reduced form is less affected by the mutations than that of theoxidized one, the E˚' values of the mutants decrease with respect to that of the wild-type PA in an
enthalpic manner.

Generally, oxidized heme is less favorable in the hydrophobic environment of

the heme protein pocket compared with reduced heme.

In the mutants, the oxidized heme isstabilized to some extent through the stronger Fe-S bond, leading to the regulation of redox property
in the proteins.

This is how the protein structure influences functionality of the cyts c.

Figures & Tables

127

References
Takahashi, Y., Sasaki, H., Takayama, S. J., Mikami, S., Kawano, S., Mita, H., Sambongi, Y., andYamamoto, Y., Further enhancement of the thermostability of Hydrogenobacter thermophilus
cytochrome c552, Biochemistry, 45, 11005-11011 (2006).
Mita, H., Ohyama, T., Tanaka, Y., and Yamamoto, Y., Formation of a complex of
5,10,15,20-tetrakis(N-methylpyridinium-4-yl)-21H,23H-porphyrin with G-quadruplex DNA,
Biochemistry, 45, 6765-6772 (2006).
Iwanami, K., Mita, H.,Yamamoto, Y., Fujise, Y., Yamada, T., and Suzuki, T., cDNA-derived aminoacid sequences of myoglobins from nine species of whales and dolphins, Comp. Biochem. Physiol.
B145, 249-256 (2006).
Nagao, S., Hirai, Y., Kawano, S., Imai, K., Suzuki, A., and Yamamoto, Y., Heme orientational
disorder in human adult hemoglobin reconstituted with a ring fluorinated heme and its functional
consequences, Biochem. Biophys. Res. Commun. 354, 681-685 (2007).
Suematsu, N. J., Ogawa, Y., Yamamoto, Y., and Yamaguchi, T., Dewetting self-assembly of
nanoparticles into hexagonal array of nanorings, J. Colloid Interface Sci., 310, 648-652 (2007).
Takayama, S. J., Takahashi, Y., Mikami, S., Irie, K., Kawano, S.,Yamamoto, Y., Hemmi, H.,
Kitahara, R., Yokoyama, S., and Akasaka, K., Local Conformational Transition of Hydrogenobacter
thermophilus Cytochrome c552 Relevant to Its Redox Potential, Biochemistry, 46, in press (2007).
Characterization of N-terminal amino group-heme ligation emerging upon guanidine hydrochloric
acid-induced unfolding of Hydrogenobacter thermophilus ferricytochrome c552, Tai, H., Kawano, S.,
and Yamamoto, Y., J. Biol. Inorg. Chem, submitted for the publication.

128Part Ⅱ
Reports from the Recipients of Garants
for International Meetings(Fiscal Year 2005)

129

4th International Symposium on Autophagy (ISA):
Exploring the Frontiers of Expanding Autophagy Research
1. Representative
Eiki Kominami, the organizer of the 4th International Symposium on Autophagy
Professor of Biochemistry, Juntendo University School of Medicine, Tokyo, Japan
2. Opening period and Place
October 1-5, 2006; Toray Conference Center, Mishima-shi, Shizuoka-ken, Japan
3. Number of participants
145 persons(52 persons from foreign countries)

4. The total cost
15,300,000 yen
5. Main use of the subsidy
Support for travel expenses of invited speakers and attendees from foreigncountries

6. Results and Impression
Autophagy is the major catabolic system responsible for the turnover of bulky cellularconstituents including cell organelles, such as mitochondria, endoplasmic reticulum, andperoxisomes. Autophagy is induced under nutrient-starved conditions and positively or
negatively controlled by hormones and growth factors. In starvation-induced autophagy, anisolation membrane appears in the cytosol, where it gradually elongates to enwrap
cytoplasmic constituents. Subsequently, the edges of the membrane fuse together to form
double-membrane vesicles termed autophagosomes. Autophagosomes rapidly fuse withlysosomes, and their lumenal contents together with the inner membrane are degraded bylysosomal hydrolases. More than 20 ATG genes that are essential to autophagosome
formation have been identified. Many of the ATG genes are involved in autophagy-specificubiquitylation-like modification (ATG conjugation) systems, in which two modifiers,
Atg12 and Atg8, are activated by a common E1-like enzyme Atg7 and transferred to two
E2-likeenzymes

Atg10

and

Atg3,

respectively,

to

form

Atg12-Atg5

and

Atg8-phosphatidylethanolamine (PE).
The most important role of autophagy is the supply of amino acids under nutrient-poor
conditions or fasting in eukayotic cells from yeasts to mammals. Autophagy substantially130

contributes to house keeping of the cells and cellular remodeling during differentiation anddevelopment of multicellular organisms, such as fly, worm, and slime mold. Impairment of
autophagy has been implicated in the progress of diseases such as cancer andneuromuscular diseases. Recently, autophagy has also attracted increasing attention in the
study of cell death mechanism. It has been also demonstrated that autophagy significantly
contributes as a cell defense system against invading bacteria.
The 4th ISA, chaired by Eiki Kominami, was held at the Toray Conference Hall in
Mishima-shi, Shizuoka-ken from October 1 through October 5, 2006. The symposium
consists of 5 oral sessions (44 talks) and two poster sessions (53 titles).
Session 1: Molecular mechanism of autophagy
The session dealt with topics concerning the mechanism of ATG conjugation reactions,
autophagosome formation, and autophagic proteolysis. There is substantial progress in
structure-function relationship of Atg8; essential amino acid residues for its function(Ohsumi Y., Elazar Z.), structural comparison between Atg8 and Atg8-PE (Inagaki, F.), a
new role of Atg8-PE as a phospholipid-tethering machinery (Nakatogawa H.), and so on.
These results owed much to what crystal structure analyses have revealed.
Autophagy is essentially a nonselective catabolic system, but, under some particular
circumstances, it is thought to function as selective degradative system. One such example
is degradation of peroxisomes via autophagy. In yeast, Pichia pastoris, many new genes
have been identified and shown to be involved in selective degradation of the organelle(Subramani, S., Sakai Y., Dunn W., Fujiki Y.). It should be also emphasized that there are
growing lines of evidence for the importance of autophagy that occurs constitutively evenin nutrient-rich conditions. Some presentations clearly showed that constitutive autophagy
significantly contribute to maintenance of cellular homeostasis as a house keeping
mechanism in neuronal tissues (Komatsu M., Bjorkoy G.).
Session 2: Autophagy in infection and immunity
It has been long believed that bacteria invading a cell mimic phagocytic substrates andare incorporated into phagosomes to enter host cytoplasm. Some bacteria (group A
Streptococcus, Streptococcus pyogenes, Mycobacterium tuberculosis, etc.) in the lumen of
phagosome (endosome) break through the endosomal membrane into cytoplasm andproliferate. More recently, it has been demonstrated that these bacteria in the hostcytoplasm are frequently incorporated in autophagosomes and subsequently transported tolysosomes to be degraded (Yoshimori T., Deretic, V., Colombo M., Nakagawa I.). Thus,
autophagy functions as a cellular innate immune system. Although autophagosomes

131

enwrapping bacteria are formed dependent on ATG genes, their size is extremely larger
than that of ordinary autophagosome formed under starvation conditions. Formation ofthese larger autophagosomes may be controlled by distinct signaling pathway (Yoshimori
T.). It is also noted that some bacteria in the cytoplasm have a mechanism to escape thesurveillance of host autophagic machinery by inhibiting ATG conjugation reactions(Nakagawa I.).
Session 3: Regulation of autophagy
This session mainly focused on cell signaling pathways controlling autophagy.
Historically, two signaling pathways have been proposed as the major regulatory system for
autophagy. One is insulin-dependent signaling cascade leading to mTor activation(Nobukuni T., Neufeld T., Meijer A.) and the other is the cascade responding to amino acidstarvation (Kadowaki, M.). Researchers have paid more attention on mTor interacting
proteins, such as TSC1/2-Rheb (Nobukuni T.), on one hand, and attempted to unveil thelink between mTor signaling pathway and autophagy. Atg1 as an essential component
connecting the two has been proposed (Neufeld T.). Also, new approach to elucidate how
the cell recognizes the decrease in extracellular amino acid levels has been conducted(Mizushima, N.).
Session 4: Autophagy and cell death“Autophagy and cell death” is a hot subject of recent publications in this field. More andmore controversies have arisen about the relationship between autophagy and cell death,such as apoptosis and necrosis. Notably, there are growing numbers of articles reporting
autophagic cell death or type II cell death. Autophagy has been essentially considered as aprotective mechanism necessary for cell survival, i.e., the supply of energy source andbuilding blocks under nutrient austerity and the removal of denatured or misfolded
proteins unfavorable for cell activities. However, in some particular cases, in which
apoptosis is inactivated, it has been reported that cells filled with numerous autophagic
vacuoles died (Uchiyama Y.). This type of cell death is called autophagic cell death.
Studies using various cancer cells indicated that whether autophagy operates as aprotective mechanism for cell survival or it promotes cell death is dependent on cell typeand conditions used (Kondo S.). Meanwhile, inactivation of autophagy using siRNA orgene targeting techniques in the cell caused apoptosis (Codogno P.).
Session 5: Autophagy in disease and aging
In many neurodegenerative diseases, whether they are familial or sporadic, there is a

132

common mechanism of disease occurrence. Conformation disorder of a certain proteincauses misfolding of the protein, which results in the formation of insoluble protein
aggregates. These aggregates cause inactivation of ubiquitin-proteasome. As a result, theaggregates stimulate autophagic responses and enhance autophagosome formation.
However, sequestered protein aggregates cannot be degraded after maturation of
autophagosme to autolysosome.

Thus, impairment of autophagy is a common hallmark

of many neurodegenerative diseases. Some ATG gene products essential for autophagy
accumulate along with misfolded protein aggregates, such as Huntington with expanded
poly glutamine tracts (Kopito R.). In order to prevent the outbreak of the diseases,
administration of drugs, which enhance autophagy, to human may be a potential
therapeutic procedure (Rubinsztein D.).
7.

Additional description

7.1

Number of participating countries:
18 countries

7.2

Approvals:
The International Symposium on Autophagy (ISA) was originally planned to present
and discuss about unpublished data to unveil the mechanism of macroautophagy,
microautophagy, and pexophagy of yeasts and mammalian cells. The symposium has also
focused on newly discovered functions of autophagy in diverse fields of biology andpathobiology. The 1st ISA chaired by Yosinori Ohsumi was held in 1997 at Okazaki, Japan,
which was succeeded three years later by the 2nd ISA organized by Patrice Codogno at
Aix-les-Bains, France. Then, the 3rd ISA was organized by Yasuo Uchiyama and held in
2002 at Osaka University Medical School. The 4th ISA chaired by Eiki Kominami washeld at Toray Conference Center at Mishima, Shizuoka-ken in

October 2006.

133

11th International Xenopus Conference
1. Representative
Makoto Asashima, the chairman of organizing committee
2. Opening period and Place
September 12-16, 2006
3. Number of participants
314 persons(214 persons from foreign countries)

4. The total cost
5,591,681 Yen
5. Main use of the subsidy
Support for travel expenses of the special plenary lecturer (Dr. Yoshio Masui)
6. Result and Impression
The International Xenopus Conferences have been held biannually andalternately in the United States or Europe since April 1986 to discuss aboutcell and developmental biology reseach using an amphibian species Xenopus.
Xenopushas

been

contributing

toclarifymolecular

mechanisms

ofdevelopment and to establish common principles to understand how animal
including human is developed. For the first time in 20 years, however, the 11th
Xenopus Meeting was held in Kisarazu City, Japan, between September 12thand 16th 2006. We Japanese organizing committee considers that this is basedon Japanese Xenopus researches’ contribution to the community and the fieldis highly appreciated and thus this an extreme honor. The Xenopus Meeting forthis year 2006 was held in the midst of tremendous progress in such
epoch-making topics as genomic interpretation, genetic expression control,
regeneration, transformation, organogenesis, germ cells, transgenic methods,
proteomics and bioinformatics. Excellent presentations related to these themes
were delivered by oral as well as poster presentations, including excellent
plenary lectures “The cytoskelton of the early embryo” by Dr. Chris Wylie(Cincinnati Children’s Hospital Medical Center, USA) who contributed to the
Xenopus community not only scientifically but also by acting as the former

134

chief editor of a distinguished journal in developmental biology field“Development” and “Genome-wide and gene-specific analyses in Xenopus” by
Dr. Jim Smith (Gurdon Institute, Cambridge, UK) who is one of the pioneers of
the research on “embryonic inductions”. In addition, as the special plenary
lecturer, Dr. Yoshio Masui who won the Lasker Prize in 1998 for his innovative
contributions in understanding cell division through the discovery of
maturation promoting factor, a protein that controls cell division in fertilized
eggs, gave an excellent speech before banquet.
Scientific sessions were Session 1: Cell Fate Specification, Session 2: Axis
determination and Pattern Formation, Session 3: Signal Transduction, Session
4: Cell Polarity and Cell Migration, Session 5: Gene Expression, Session 6: Cell
Cycle, Session 7: Organogenesis and Remodeling, Session 8: Xenomics andcovered most of essential topics in the field.
At the end of the conference, we all recognized that in the last few years,
outstanding achievements have been made in the study of not only Xenopus
laevis but also on X. tropicalis. We learned that in the next few years, a drastic
and revolutionary change would happen in the filed by introducing genetics
with Xenopus tropicalis.

We believe that the role of studies using Xenopus

has been truly remarkable in the life sciences, and it was indeed meaningful at
this time that the researchers who use Xenopus in their studies gathered fromall over the world and introduced their findings and discussed their results.
Beside US (83) and Japan (100), Canada (5), Asian countries include Korea (28),Singapore (3), China (1), and European countries are Germany (14), France (7),Belgium (7), Czech (2), Italy (2), Switzerland (1), Netherlands (1), Spain (1),and Israel (1). Mid- and South America are Ecuador (1), Brazil (1), and Chile(1). Especially, participation of young researchers including posdoc andgraduate students from many countries, which is partly supported by thetravel fellowship from the organizing committee, proved that Xenopus is andwill continue to be an important model animal in life sciences.
Location of the conference was also splendid. Kazusa Akademia Park issituated in a quiet location not far from the Narita/Tokyo and Haneda
International Airports. This somewhat isolated location enhanced intensive
discussions among participants before and after meeting each day and thischoice of the conference site was highly appreciated by all attendees.
Finally, we all organizing committee members would like to express our sincere
thanks to all foundations including Novartis Foundation for the Promotion of

135

Science for their generous support and industries that had product exhibitions
during the conference for their donations that made this conference possible.
7. Additional description
7.1 Number of participating countries:
32 countries and 1 area
7.2 Approvals:
The International Xenopus Conrefence was first organized by Drs. John Gurdon, Igor
Dawid, and Jim Maller 20 years ago and the series of meeting have been held every 2
years in either US or in Europe by the local organizing committee. This time, 11th IXC wasapproved by the Japanese Society of Developmental Biologists.
7.3 Satellite symposium:
Prior to the main conference, on September12, a satellite conference on Xenopus genomics“Xenomics, the next leap forward” organized by Rob Grainger and Richard Harland washeld at the same conference site. Approximately 120 people attended and contributed toactive discussions.

136

Pre-Symposium Nagoya
ICOB-5 & ISCPN-25 IUPAC International Conference
on Biodiversity and Natural Products
１．Representative
Prof. Youji Sakagami, the chairman of organizing committee
２．Opening period and Place
July 21−23, 2006
Hamanako Royal Hotel (4396-1 Yamazaki, Yuto-cho, Hamamatsu, Shizuoka
431-0101)
３．Number of participants
60 persons(10 persons from foreign countries)

４．The total cost
2,632,356 yen
５．Main use of the subsidy
Support for travel expenses of invited speakers from foreign countries
６．Result and Impression
This pre-symposium was held as one of six satellite symposiums of the main conference,
ICOB-5 & ISCPN-25 IUPAC International Conference on Biodiversity and Natural
Products (at Kyoto).
The big main conference is held in alternate year under auspices of IUPAC and relevantorganizations of the host country. Its purpose is that researches all around the world gather
to exchange and discuss the newest information and problems on biodiversity and natural
products chemistry regarding isolation, chemical structural elucidation, biosynthesis,
chemical biology, etc. The conference is a large-scale one, which included 17 plenary and
76 invited lectures, and approximately 600 oral and poster presentations as the scientificprogram, as well as 1,350 participants from all over the world.
On the other hand, unlike the main conference, this pre-symposium held at a scenic
region near Nagoya is rather small so as to provide an relaxed circumstance of theacademic researches and cultural exchanges not only for theinvitedforeign

researchers but also Japanese researchers and students around

Nagoya.

Therefore

137

the participants (approximately 60) seemed to deepen exchanges each other. The scientificprogram of the pre-symposium, entitled ‘Bioorganic Chemistry and Chemical Biology,’
included 12 invited lectures in the fields of discovery of novel natural products, chemical
biology, biosynthesis, and chemical syntheses. The titles of lectures are follows:“Chemical studies on the red sweat of the hippopotamus” (by Associate Prof. Kimiko
Hashimoto at Kyoto pharmaceutical Univ.), “Bioorganic chemistry of the action plants:
circadian rhythmic movement and memory in higher plants” (by Prof. Minoru Ueda at
Tohoku Univ.), “Development of marine kabiramides as the new fluorescent probes for
actin assay” (by Prof. Khanit Suwanborirux at Chulalongkorn Univ., Thailand), “A
cyanobacterial origin of diverse biologically active Sponge and Tunicate natural products”(by Prof. William Gerwick at UC San Diego), “Chemical biology in the interaction
between proteins and small molecules” (by Dr. Hiroyuki Osada at RIKEN), “Chemical
genomics with natural products” (by Prof. Ho Jeong Kwon at Yonsei Univ., Korea),“Application of chiroptical spectroscopy (electronic CD and optical rotation) in natural
products research” (by Prof. Nina Berova at Columbia Univ., US), “The biosynthetic
potential of the myxobacterium Chondromyces crocatus – molecular biology andbiochemistry” (by Prof. Rolf Müller at Saaland Univ., Germany), “Using synthetic organic
chemistry to prove biological mechanisms” (by Prof. Dang Yang at Univ. Hong Kong, P. R.
China), “Formal total synthesis of enantiopure ingenol” (by Prof. Chin-Kang Sha at
National Tsing Hua Univ., Taiwan), “Synthetic studies on heterocyclic natural products”(by Prof. Marco Ciufolini at Univ. British Columbia, Canada), and “Synthesis of rare
anticancer agents isolated from marine sponges” (by Prof. Ian Paterson at Cambridge
Univ., UK). In addition, 18 poster presentations with a 5-minute oral presentation were
performed by young researchers and graduate students. The foreign invited speakers
enjoyed an excursion visiting a natural cave in the third day morning. Most participants,
especially the foreign invited speakers looked satisfied with the pre-symposium. Since
Nagoya is well known as an active place in the natural products chemistry field, thesymposium was successful and productive.
７．Additional description
7.1 Number of participating countries:
9 countries
7.2 Approvals:
The international Union of Pure and Applied Chemistry (IUPAC) is a worldwide

138

scientific organization founded in 1919 to objectively address global issues in the chemical
sciences. The co-organizers were the International Union of Pure and Applied Chemistry(IUPAC), the Chemical Society of Japan, the Pharmaceutical Society of Japan, Japan
Society of Bioscience, Biotechnology, and Agrochemistry, Science Council of Japan, andthe organizing committee
7.3 Satellite symposium:This symposium itself is one of the satellite symposiums of the Kyoto main
conference.
Follows are a list of the satellite symposiums:
Pre-symposiums were in Nagoya (July 21-23) and Tokushima (July 21-23).
Post-symposiums were in Sapporo (July 29-30), Sendai (July 30), Tokyo (July31), and Fukuoka (July 30).

139

“Functional Architecture of Cortical Microcircuits” Symposium in the
29th annual meeting of the Japan Neuroscience Society
1. Organizer
Yumiko Yoshimura, Nagoya University
Yoshiyuki Kubota, The National Institute for Physiological Sciences
2. DateJuly 19-21, 2006
Kyoto International Conference Hall
Takaragaike, Sakyo-ku, Kyoto, Japan 606-0001
3. Number of participants at the symposium
350 persons including 50 persons from foreign countries
4. The total cost
1,800,000 yen
5. Main use of the subsidy
Support for hotel expenses of invited speakers
6. Result and Impression
The aim of this symposium was to promote the spread of knowledge in regard to recent
advances in cortical microcircuit studies. We invited four distinguished speakers, Dr.
Gabor Tamas from the University of Szeged in Hungary, Dr. Jackie Schiller from theTechnion Medical School in Israel, Dr. Edward M. Callaway from the Salk Institute in the
U.S.A., and Dr. Sacha B. Nelson from Brandeis University in the U.S.A. At the
symposium these experts introduced recent data from cutting-edge studies on the synaptic
connectivity of cortical neurons using multiple electrode and optical recording, andelectron microscopic observations. A key focus of the meeting was the fundamental
characteristics of neuron subtypes defined by genetic expression patterns, physiologicalcharacteristics, and in vivo functional architecture. All who attended the symposium
enjoyed this wonderful, informative opportunity, and there was lively give and takebetween the expert guest speakers and the audience. I would like to express my sincere
thanks to the Novartis Foundation for the Promotion of Science for the support of this
symposium.

140

7. Additional description
A satellite symposium was held on July 24-26 in Okazaki.

141

Dynamic Organelles in Plants
１．Representative
Tetsuro Mimura, the organizer

2．Opening period and Place
June 14 – June 17, 2006
Okazaki conference center at Aichi, Japan

3．Number of participants
206 persons(15 persons from foreign countries)

4．The total costc.a. 6,900,000 yen
5．Main use of the subsidy
Support for travel expenses of invited two foreign speakers
6．Result and Impression
Since plants spread their roots in the ground, they must survive in a given environment.
To adapt the environment, they positively utilize the environmental changes in the life
cycle as important signals that are necessary for their survival. Recent studies have shownthat plant cells could induce, degenerate and differentiate their organelles to adapt theenvironmental changes. The flexibility of the plant organelles is the basis of the strategy
for the environmental adaptation in plants.
Thus, studies on organelles dynamics are newly developing in the field of modern plantbiology on the basis of molecular techniques.
This symposium intended to provide an excellent opportunity to review the recent
advances on the plant organelle biology.
Many international conferences are being held on the plant organelles.
ofHowever, mostthose meetings are focusing on the individual organelle, such as, chloroplasts,

mitochondria, golgi apparatus etc.

Our conference is exceptional for focusing on the allkinds of plant organelles and their dynamics dependent on the environmental conditions.
The aim of this scientific research is to clarify the molecular mechanisms underlying
induction, differentiation, and interaction of organelles, and to understand integratedfunction of individual plant through the organelle dynamics.

We also expect that this142

field will provide perspective for increase of food production, production of functional
foods and phytoremediation in the future.
For these subjects, the following sessios were held; (1) Differentiation and Degradation,(2) Biogenesis and Protein transport, (3) Post-Genome approach, (4) Metabolic Regulationand Signal Transduction, (5) Integrated Functions.
Throughourt 4 days, more than 200 persons took part in the conference.
graduate students and younger staffs were also included in participants.

Many

In total, 33 oral

lectures and 89 posters were presented.
In the evening session of the first day (Jun., 14), the plenary lecture by Prof. Asada who
is a world-famous scientist on the photosynthesis was held.

We enjoyed his interestingtalk and exciting data about his main subject, “Photoreduction and Photoexcitation of
Oxygen and Relaxation of Photon Excess Stress in Chloroplasts”.

Not only that, Prof.

Asada prepared many beautiful photographs of Japanese scenery and flowers for foreign
guests.
Above 5 sessions cover from molecular structure of proteins which compose organelles
to physiological roles of organelles in integrated functions of a whole plant.

In oralpresentations, we enjoyed researches of international top scientists. If I should point out
a matter on this symposium, we could not prepare enough discussion time, and English
discussion was still inactive among Japanese participants.

Recent younger Japanese aremuch better at speaking English than the elder generation.

But they were very quiet

during oral presentations.
Instead of that, their poster presentations were very active.

On the first day, after oralpresentations, poster sessions were held.

Each poster presenter was given 1 minutes for

explaining data in front of all participants.

We call this presentation the poster marathon.

All presenters from famous professors to master course students have equally shown theirdata for 1 minutes. Although it took longer time (two times of one hour running), it was
highly reputed by foreign participants as the exciting attempt.
In the evening of the second night on Jun. 15, the banquet was held.

There, allparticipants enjoyed in talking each other.
Throughout the conference, I was impressed that research level of organelle biology inJapan is quite high.
very well.

Furthermore, the younger generation for this field are growing up

However, the research competition in the world is severe.

make a constant challenge to progress organelle biology in plants.

7．Additional description
7．1

Number of participating countries : 6 countries including JapanWe also have to