Annual Report (2009)

No.21
The NOVARTIS Foundation (Japan)
for the Promotion of Science

平成 21年度

財団年報 第 21号

Contents
I.

Introduction

はじめに .......................................................................... 7

Akimichi Kaneko, MD, PhD
Chairman of the Board of Trustees

II. Reports from the Recipients of Novartis Research Grants
研究報告 ............................................................................................. 9
1. Analysis of new zebrafish mutants that show the defects
in gut epithelium formation ................................................................................... 10
Yutaka Kikuchi
Hiroshima University

2. Regulation of endothelial cell-cell adhesion
by lysophosphatidic acid and its molecular mechanism ........................................ 13
Junken Aoki
Tohoku University

3. The roles of cardiac chromatin remodeling factors, SWI/SNF complexes ............... 16
Jun K. Takeuchi
Global Edge Institute, Tokyo Institute of Technology

4. Molecular biological identification of stemness ....................................................... 19
Shinji Masui
Research Institute, International Medical Center of Japan

5. How dose the deficit of histone deacetylase activity
cause anxiety and depression? ............................................................................... 22
Yoshiharu Kawaguchi
Institute for Developmental Research, Aichi Human Service Center

6. Regulation of EGF receptor/PI3K/Akt signal transduction pathway
by a novel PDK1-interacting protein Aki1 ......................................................................... 25
Naoya Fujita
Japanese Foundation for Cancer Research

7. The role of transcription factor FoxO1 in pancreatic beta cell regeneration ................ 28
Tadahiro Kitamura
Metabolic Signal Research Center, Institute for Molecular and Cellular Regulation,
Gunma University

8. Studies on post-translational modifications of proteinsby endogenous nitrated nucleic acids ................................................................................... 30
Hirokazu Arimoto
Tohoku University- 1 -9. Concomitant inhibition of Mdm2–p53 interaction and Aurora kinases
or PI3K/Akt/mTOR synergistically induces p53-mediatedmitochondrial apoptosis ........................................................................................................... 32
Kensuke Kojima
Wakayama Medical University

10. The role of TRPC channel/NCX1 transporter coupling in vasospasm ........................... 34
Takahiro Iwamoto
Department of Pharmacology, Fuculty of Medicine, Fukuoka University

11. Neuropharmacological research on the role of environmental factors
during development in psychological abnormalities ....................................................... 37
Yukio Ago
Laboratory of Medicinal Pharmacology, Graduate
School of Pharmaceutical Sciences, Osaka University

12. Development of anti infectious-disease drugs based on natural products ..................... 40
Kenichi Akaji
Kyoto Prefectural University of Medicine,
Graduate School of Medical Science

13. Study on molecular mechanism of infectious particle production ofhepatitis C virus .......................................................................................................................... 43
Makoto Hijikata
Institute for Virus Research Kyoto University

14. Regulation of genome and cellular functions
by the SUMO modification system. ..................................................................................... 46
Hisato Saitoh
Department of Biological Sciences, Graduate School of Science and Technology,
Kumamoto University.

15. Analysis of barrier function at heterochromatin region ...................................................... 51
Masaya Oki
University of Fukui

16. Analysis of regulatory mechanism of extracellular signals
by receptor-mediated endocytosis ......................................................................................... 54
Jiro Toshima
Tokyo University of Science

17. Multifunctional rare earth metal-catalyzed asymmetric synthesis of
optically active small cyclic compounds and application tobiologically active compounds synthesis ............................................................................ 57
Shigeki Matsunaga
The University of Tokyo

18. Synthetic studies on maitotoxin, a marine polycyclic ether .............................................. 60
Tadashi Nakata
Tokyo University of Science- 2 -19. Molecular analysis of temperature adaptation of Plasmodium parasite ........................ 63
Hirotaka Kanuka
National Research Center of Protozoan Diseases,
Obihiro University of Agriculture and Veterinary Medicine

20. Genetic mutations associated with DOK7 myasthenia andabnormal Dok-7/MuSK signaling ......................................................................................... 66
Yuji Yamanashi
Institute of Medical Science, University of Tokyo

21. Development of screening methods for synthetic ligands
based on specific fluorescent labeling of target proteins ............................................... 69
Hirokazu Tamamura
Tokyo Medical and Dental University,
Institute of Biomaterials and Bioengineering

22. Reconsideration of the concept of the unitary psychosis
using new animal models ......................................................................................................... 73
Kohichi Tanaka
Tokyo Medical and Dental University

23. Application of ADLib system to rational designof humanized antibodies against tough antigens. ............................................................. 76
Kunihiro Ohta (with Lin Waka, Tohru Terada)
Graduate School of Arts & Sciences, Dept. of Life Sciences,
The University of Tokyo

24. The role of microRNA in endochondral ossification ........................................................... 78
Hiroshi Asahara
National Research Institute for Child Health and Development

25. Role of Gi signaling in vascular endothelial cells ................................................................ 80
Hiroshi Kataoka, Akiyoshi Uemura
Center for Developmental Biology RIKEN Kobe

26. Origin and mechanism of differentially methylated regions (DMR)
in mammals .................................................................................................................................. 82
Tomoko Kaneko-Ishino
Tokai University School of Health Sciences

27. Contribution of calumin to mouse ontogeny
through regulation of NFAT activity .................................................................................... 85
Tetsuo Yamazaki
Dept. of Biol. Chem., Grad. Sch. of Pharm. Sci., Kyoto Univ.

28. Analysis on the mechanism for generation of leukemic stem cells ................................ 88
Tetsuya Nosaka
Mie University Graduate School of Medicine- 3 -29. Synaptic plasticity linking chronic pain and enhanced emotional responses ............ 90
Fusao Kato
Department of Neuroscience,
The Jikei University School of Medicine

30. Clinical application on metabolic syndrome via exhaustive analysis ofbile acid function. ....................................................................................................................... 93
Mitsuhiro Watanabe
School of Medicine, Keio University

31. Mechanisms of aggregation and segregation of cortical neurons .................................... 96
Kazunori Nakajima
Keio University School of Medicine

32. Regulation of mRNA stability by heat shock cognate protein 70
under the control of cytokines ................................................................................................ 99
Hirotaka Matsui
Research Institute for Radiation Biology and Medicine,
Hiroshima University

33. Replenishment of innate immune system via Toll-like receptor signals ..................... 101
Yoshinori Nagai
Immunobiology and Pharmacological Genetics,
Graduate School of Medicine and Pharmaceutical Science for Research.
University of Toyama

34. Analysis of transcriptional regulatory network of vascular differentiation ................ 105
Kyoko Ohashi-Ito
The University of Tokyo

35. Quantity and quality control of antibody response
by a degradation system of Bach2 ...................................................................................... 108
Kazuhiko Igarashi
Tohoku University Graduate School of Medicine

36. Regulation of soma/germ-line distinction by small RNAs. ............................................. 110
Kunio Inoue
Department of Biology, Graduate School of Science,
Kobe University

37. Comprehensive understanding of molecular mechanismsthat can control all sequentially happens in the ocular dominance columns
from developmental stage to adult. ..................................................................................... 112
Koichi Tomita
Center for Genetic Analysis of Behavior,
National Institute for Physiological Sciences

38. Efficient Synthesis of Biologically Active Nitrogen-Containing
Heterocyclic Compounds by the Carboamination Reactions ..................................... 115
Takuya Kurahashi
Department of Material Chemistry, Graduate School of Engineering,
Kyoto University- 4 -39. Studies on cytokinetic block in cellular senescence ........................................................... 117
Eiji Hara
The Cancer Institute of Japanese Foundation for Cancer Research

40. Physiological functions of D-serine in mammals and development of
its enzymatic assay system .................................................................................................... 120
Tohru Yoshimura
Graduate School of Bioagricultural Sciences, Nagoya University

41. Functional Role of Heme-Protein Interaction
in Monooxygenase Cytochrome P450 ............................................................................... 123
Takashi Hayashi
Graduate School of Engineering, Osaka University

42. Basic research for the olfactory disease occurrence
and its degenerative therapy .................................................................................................. 126
Akio Tsuboi
Laboratory for Molecular Biology of Neural Systems
Research Institute for Frontier Medicine, Nara Medical University

43. Pre-mRNA splicing mechanism of human Dystrophin gene .......................................... 128
Akila Mayeda
Institute for Comprehensive Medical Science (ICMS),
Fujita Health University

44. Small molecules that induce the cell adhesion .................................................................... 131
Motonari Uesugi
Institute for Integrated Cell-Material Sciences, Kyoto University

45. Analysis of the signaling system for a novel lung cancer oncogene,
EML4-ALK, and its clinical application ............................................................................ 135
Hiroyuki Mano
Jichi Medical University

46. Dysfunction in cardiovascular system using micethat lost a novel membrane-associated estrogen receptor ............................................ 139
Yoichi Mizukami
Center for Gene Res. Yamaguchi Univ.

47. Analyses of DMRT1 gene knockout medaka, which reverse sex
in genetic male ........................................................................................................................... 142
Masaru Matsuda
Utsunomiya University

48. Development of novel approach for liver failureusing genetically modified hepatocytes ............................................................................. 145
Kazuo Ohashi
Institute of Advanced Biomedical Engineering and Science,
Tokyo Women's Medical University- 5 -III. Reports from the Recipients of Grants for International Meetings
研究集会報告 ................................................................................... 148
International Symposium on Biologically Active Peptides:
Peptide Diversity ........................................................................................................................... 149
Kazuhiro Takahashi
Tohoku University

The CMDS 2008 Conference Report ............................................................................................ 151
Yoshitaka Tanimura
Kyoto University

The 38th Annual Meeting of the Japanese Society for Immunology(JSI) .......................... 154
Kayo Inaba
Kyoto University

28th Sapporo Cancer Seminar: International Symposium“TGF-β signaling and cancer” .................................................................................................. 156
Kohei Miyazono
University of Tokyo

8th International Conference on Protein Phosphatases
Protein Phosphatases : From Genome to Disease ............................................................... 158
Takashi Matozaki
Gunma University

16th International Coference on Biomagnetism(BIOMAG2008) ......................................... 160
Shinya Kuriki
Hokkaido University

The 51st Annual Meeting of the Japanese Society for Neurochemistry ............................ 163
Masatoshi Takeda
Osaka University

IV.

The 22nd (fiscal year 2008) Promotion Report
第 22 期（2008 年度）助成事業報告 .................................................... 165

V.

The 22nd (fiscal year 2008) Financial Report
第 22 期（2008 年度）財務報告 ........................................................... 170

VI.

List of the Board Members
役員名簿 ........................................................................................ 171

VII. Information from the Secretariat
事務局便り ...................................................................................... 173- 6 -Introduction“The Novartis Foundation (Japan) for the Promotion of Science:
The past six years and future”

Akimichi Kaneko, MD, PhD
Chairman of the Board of Trustees
Six years have passed since I was appointed as the Chairman of the Board ofTrustees, Novartis Foundation (Japan) for the Promotion of Science, in June 2003. Thisis my 6th INTRODUCTION for the Annual Report. I sincerely appreciate the assistanceand warm encouragement extended by the members of the Board of Trustees, theauditors, the Board of Councilors and the Selection Committee. The Foundation has met
a financial crisis in the past few years, but the powerful support by the Novartis Pharma
KK enabled us to sustain our activity without interruption. Sustainability is one of our
responsibilities to the scientists’ community as an organization recognized as a Public
Benefits Promoting Foundation.
In the past 6 years we celebrated the 20th anniversary of our establishment. Now weare facing at the reorganization of the foundation under the new law to reform the
Public Interest Corporation System in Japan. As I mentioned in the 2007 and 2008
Annual Reports, the aim of the foundation remains unchanged after the reorganization.
It is to contribute the promotion of science by helping and supporting the creative
researches of the science in Japan.
This annual report includes research and meeting reports written by the 2008
grantees. From these reports, you can vividly feel their passion and seriousness inpursuing research activities. Their passion is the source of energy to promote theirresearch activity. I strongly believe that the aim of the Foundation is to help keeping
their passion and I would like to do our best toward this goal.

- 7 -

はじめに

「これまでの財団とこれからの財団」

理事長

金子章道

平成 15 年 6 月にノバルティス科学振興財団の理事長をお引き受けしてから早くも満 6 年を経過しました。この財団年報の巻頭言を書くのも 6 回目になります。この間、当財団の事業活動を継続して支援してくださった理事、監事、評議員、選考委員の皆様に深く感謝いたします。金融危機に伴う財政の悪化にも見舞われましたが、ノバルティスファーマ社の継続した強力な経済的支援によって事業を継続することが出来ました。ここに御礼申し上げたいと思います。事業の継続が特定公益増進法人に認可されている当財団の社会的責任であります。
この 6 年間の財団の活動を振り返ってみますと創立 20 周年を迎えたこと、新法人組織への移行準備があります。一昨年、昨年の年報巻頭言にも書きましたが、新組織になりましても財団の目的とする基本的な考え方は変りません。我が国の生命科学とこれに関連する研究領域における研究振興を果たしていくこと、これが本財団の基本的な方針です。
本年度もこの財団年報に 2008 年度助成金を受けられた方々の報告書を収録いたしました。受賞者の皆様の研究に対する熱い想い、真摯な取り組み態度が感じられる
素晴らしい報告書集です。このような研究に対する熱い想いが研究を推進していく上で非常に大事なエネルギーの源です。この熱意を冷まさないよう多少なりとお力添えをすることが研究助成金の目的と考え、努力を続けて行きたいと思っております。

- 8 -

II.
Reports from the Recipients ofNovartis Research Grants- 9 -Analysis of new zebraﬁsh mutants that show the defects
in gut epithelium formation.
Yutaka Kikuchi
Hiroshima University
yutaka@hiroshima-u.ac.jp

Abstract
The digestive system plays an essential role in vertebrate physiology. However, the molecularmechanisms underlying the differentiation and maintenance of digestive organs are not wellunderstood. To analyze the molecular mechanisms, we have performed a genetic screening using
zebrafish and have isolated morendo (mor) and legato (leg) mutants, which exhibit hypoplastic
digestive organs. The phenotypic analyses revealed that mor is required for the endoderm-intestinetransition. In addition, we identiﬁed a responsible gene for mor by genetic mapping. We are now
analyzing the function of Mor in the formation of endodermal organs.
Keywords: zebraﬁsh, mutant, gut, organogenesis

Introduction
The digestive system plays an essential role in vertebrate physiology. In vertebrate, after
specification of endoderm, the endodermal cells move to the most ventral layer of the embryoand form gut tube. The endodermal organs, such as liver, pancreas and lung, are induced anddifferentiated from the gut tube through the interaction between endodermal cells and mesenchymal
cells. Although several factors have been identiﬁed for the morphogenesis of digestive organs, themolecular mechanisms underlying the differentiation and maintenance of these organs are not wellunderstood. To identify genes required for the formation of digestive organs, we performed a genetic
screen using zebrafish. We have isolated morendo (mor) and legato (leg) mutants, which exhibit
hypoplastic digestive organs. In this study, we focus on the phenotypic analyses of mor mutant andthe identiﬁcation of responsible gene for this mutant.

Results
To determine the developmental step and the spatial region at which mor gene is required, weanalyzed the gene expression for endoderm and digestive organs. Expression pattern of early
endodermal marker foxA3 was indistinguishable between wild-type (WT) and mor embryos at 2.5
day postfertilization (dpf) (Fig. 1 A, B). However, by 5 dpf, the expression of the enterocyte speciﬁc
gene, intestine fatty binding protein (ifabp), liver marker, fatty acid binding protein 10 (fabp10), andexocrine pancreas marker, trypsin were signiﬁcantly reduced in mor mutants (Fig. 1 E-L). On theother hand, the expression of the endocrine pancreas marker, insulin was normal in mor mutants(Fig. 1 M, N), presumably because mor may be not expressed in this tissue. And the size of liver and- 10 -

exocrine pancreas was greatly reduced in mor mutants. We further foundthat the number of intestinal epithelium cells in mor mutant embryos wasless than that in WT embryos (Fig. 2 C, D). The nucleus enlarged along the
apical-basal direction in WT, whereas some nucleus shaped circularly in
mor mutants (Fig. 2 A’, B’). The intestinal epithelium was folded in WT,
while unfolded epithelium and exfoliation of epithelial cells were observedin mor embryos (Fig. 2 C, D). In addition, the mor mutant shows themalformation of branchial arches and reduced size of swimbladder. And thesize of retina is reduced and protrusion of lens was observed in mor mutants.

To identify the responsible gene for mor mutant, we tried to genetic mapping by using positional
cloning method. It is well known that the zebraﬁsh India strain is polymorphic with respect to the
AB strain, which is a mor background. The heterozygote carriers of mor were mated to India andmapping families (map cross) were generated. We collected mor homozygote embryos from these- 11 -

map cross heterozygote ﬁsh and isolated genomic DNA from the individual homozygote embryos. In
zebraﬁsh, lots of Simple Sequence-Length Polymorphism (SSLP) markers are available for genetic
mapping. We have identified a candidate gene of mor by using these SSLP markers and found apoint mutation in coding region of this gene. Using restriction fragment length polymorphisms toanalyze linkage, we found that the point mutation segregated with the mor phenotype in 100 meiotic
events. Moreover, the candidate gene is expressed in gut tube, liver, exocrine pancreas, brain andoptic vesicles, but is not expressed in endocrine pancreas at 4 dpf. Together, the genetic data andexpression pattern of this candidate gene indicate that mor corresponds to this gene. Mor encodes
the known protein family, but the function of this protein family in the formation of endodermalorgans is not uncovered yet. We are now trying to analyze the function of Mor using molecular andbiochemical methods.

Discussion & Conclusion
Although the expression of early endodermal marker foxA3 is normal in mor mutant embryos at
2 dpf, the endodermal organ markers, such as ifabp, fabp10 and trypsin, were signiﬁcantly reduced
in mor embryos at 5 dpf. These results suggest that mor is required for the endoderm-intestinetransition. In addition, the mor mutant has reduced retina and the malformation of branchial arches,suggesting that Mor functions in formation of various organs.
It was reported that polr3b1), nil per os (npo) 2) and digestive-organ expansion factor (def)3)
mutants show hypoplastic digestive organs like mor and leg mutants. Polr3b is a RNA polymerase
III subunit. Npo has RRM domain known as RNA binding domain, and Def regulates the expressionof p53. It is not well known how these factors act cooperatively in organogenesis of the digestiveorgans. We believe that the identification of mor and leg genes and analyses of function of theseproducts will contribute to better understand the molecular mechanisms of the digestive organ
formation in vertebrate.

References
1) Yee, N., Gong, W., Huang, Y., Lorent, K., Dolan, A., Maraia, R. and Pack, M. (2007). Mutation of RNA
Pol III subunit rpc2/polr3b Leads to Deficiency of Subunit Rpc11 and disrupts zebrafish digestive
development. PLoS Biol. 5: e312.
2) Mayer, A. and Fishman, M. (2003). nil per os encodes a conserved RNA recognition motif protein
required for morphogenesis and cytodifferentiation of digestive organs in zebraﬁsh. Development 130:3917-28.
3) Chen, J., Ruan, H., Ng, S., Gao, C., Soo, H., Wu, W., Zhang, Z., Wen, Z., Lane, D. and Peng, J. (2005).
Loss of function of def selectively up-regulates Δ113p53 expression to arrest expansion growth ofdigestive organs in zebraﬁsh. Genes Dev. 19: 2900-2911.

- 12 -

リゾホスファチジン酸による内皮細胞接着の制御とそのメカニズムの解明
Regulation of endothelial cell-cell adhesion
by lysophosphatidic acid and its molecular mechanism
青木 淳賢
Junken Aoki
東北大学
Tohoku University
jaoki@mail.pharm.tohoku.ac.jp

Abstract
リゾホスファチジン酸産生酵素オートタキシンのノックアウトマウスの解析から、 血管内皮細胞の接着性を制御する新規の LPA 受容体の存在が示唆された。 本研究では、 新規 GPCR スクリー
ニング法を用い、 スクリーニングした結果、 新規の LPA 受容体を同定した。
Keywords: リゾホスファチジン酸、 オートタキシン、 血管形成、 GPCR

Introduction
本研究では生理活性脂質リゾホスファチジン酸 （LPA） の内皮細胞に対する新規機能とそのメカニズムの解明を行う。 我々は、 LPA の産生機構を解析し、 従来がん細胞の運動性促進因子として同定されていたオートタキシン （ATX） が前駆物質リゾホスファチジルコリン （LPC） から LPAを産生する酵素であることを生化学的に明らかにした。 また最近、 ATX ノックアウト （KO） マウスの解析を行い、 KO マウスは血管形成異常により胚致死であることを明らかにした。 一方、 これまで報告されている LPA 受容体の KO マウスはいずれも胎生致死とはならず、 血管内皮細胞における発現も低いため、 ATX KO マウスの表現型や内皮細胞に対する LPA 作用は既存の LPA 受
容体では説明できない。 従って、 内皮細胞に発現し血管形成に関与する新規の LPA 受容体の存在が強く示唆される。 そこで本研究では、 「ATX-LPA シグナリングの血管内皮細胞における機能を明らかにすると共に、 新規の LPA 受容体を同定する」 ことを目的とし研究を進めた。

Results
新規 LPA 受容体を得る実験手法として、 新規オーファン GPCR スクリーニング系を立ち上げた。 この方法では、 GPCR の活性化の下流で、 マトリックスメタロプロテアーゼが活性化し、 その下流で TGFa 等の EGF 受容体リガンドが活性化するいわゆる EGF 受容体の transactivation の機構を利用する。 この手法では、 TGFa はアルカリホスファターゼの融合タンパク質として発現させ、TGFa の切り出しをアルカリホスファターゼ活性で評価し （図）、 96 well plate での測定が可能であった。 約 100 個のオーファン GPCR をスクリーニングした結果、 新規 LPA 受容体 （LPA7 と命名）を見いだした新規 LPA 受容体 LPA7 は神経系の組織に比較的高い発現を示し、 また、 LPA 分
子種のうち、 不飽和脂肪酸を有する LPA で強く活性化された。

- 13 -

A Novel GPCR assay using TGFα release
TGFα shedding (%)

OD405

p-NPP
=

OD405 (sup)OD405 (sup) + OD405 (cell)

OH
O P
O OH

O2N

O2N

OH

Vehicle-treated TGFα shedding is subtracted(8-15% depending on assay condition)

LPA

ALP

ALP
TGFα

TACE

P2Y5

P

HEK293

Discussion & Conclusion
新規の LPA 受容体は、 G12/13 とカップルし、 細胞内にシグナルを伝える。 LPA による内皮細胞の接着性の制御はやはり G12/13 が関与するので、 今回、 見いだした新規 LPA 受容体が関与する可能性がある。

References
Endo T, Kano K, Motoki R, Hama K, Okudaira S, Ishida M, Ogiso H, Tanaka M, Matsuki N, Taguchi R,
Kanai M, Shibasaki M, Arai H, Aoki J.
Lysophosphatidylmethanol is a pan lysophosphatidic acid receptor agonist and is produced by
autotaxin in blood.
J. Biochem. In press
Nakamura K, Igarashi K, Ohkawa R, Okubo S, Yokota H, Kishimoto T, Ide K, Masuda A, Yamamoto T,
Saito N, Kurokawa M, Tsuji S, Okudaira S, Ikeda H, Aoki J, Yatomi Y.
Autotaxin enzyme immunoassay in human cerebrospinal ﬂuid samples.
Clin. Chim. Acta. In press
Makide K, Kano K, Kitamura H, Arima N, Aoki J.
Lysophospholipid mediators
Tanpakushitsu Kakusan Koso. 54, 29-39 (2009)
Inoue M, Ma L, Aoki J, Ueda H.
Simultaneous stimulation of spinal NK1 and NMDA receptors produces LPC which undergoes
ATX-mediated conversion to LPA, an initiator of neuropathic pain.
J. Neurochem. 107, 1556-1565 (2008)
Ohuchi H, Hamada A, Matsuda H, Takagi A, Tanaka M, Aoki J, Arai H, Noji S.
Expression patterns of the lysophospholipid receptor genes during mouse early development.
Dev Dyn. 237, 3280-3294 (2008)- 14 -

Nakasaki T, Tanaka T, Okudaira S, Hirosawa M, Umemoto E, Otani K, Jin S, Bai Z, Hayasaka H, Fukui
Y, Aozasa K, Fujita N, Tsuruo T, Ozono K, Aoki J, Miyasaka M.
Involvement of the Lysophosphatidic Acid-Generating Enzyme Autotaxin in Lymphocyte Endothelial Cell Interactions.
Am J Pathol. 173, 1566-1576 (2008)
Kano K, Arima N, Oogami M and Aoki J.
LPA and its analogs -attractive tools for elucidation of LPA biology and drug developmentCurr Med Chem. 15, 2122-2131 (2008)
Aoki J, Inoue A and Okudaira S.
Two pathways for lysophosphatidic acid production.
Biochimi. Biochem. Acta. 1781, 513-518 (2008)- 15 -

The roles of Cardiac Chromatin Remodeling Factors,
SWI/SNF Complexes
Jun K. Takeuchi
Global Edge Institute, Tokyo Institute of Technology
Takeuchi.j.ab@m.titech.ac.jp

Abstract
Generating new heart cells to repair diseased myocardium is a critical yet elusive goal. Factors
sufficient to induce cardiac differentiation have not been identified. We show that two cardiac
transcription factors, Gata4 and Tbx5, and a cardiac-speciﬁc subunit of BAF chromatin-remodelingcomplexes, Baf60c, can reprogram mouse embryonic and extraembryonic mesoderm into
beating cardiomyocytes. Gata4 with Baf60c initiated ectopic cardiac gene expression. Additionof Tbx5 allowed differentiation into contracting cardiomyocytes and repression of noncardiac
mesodermal genes. Baf60c was essential for the cardiogenic activity of Gata4 and Tbx5, partly bypermitting binding of Gata4 to cardiac genes, indicating a novel role for BAF complexes in tissuespecific regulation. The combined functions of these factors may allow reprogramming of new
cardiomyocytes for regenerative purposes.
Keywords: Chromatin remodeling complexes, Cardiogenesis, heart failure

Introduction
Heart disease is the leading cause of mortality and morbidity in the Western world. The heart has
little regenerative capacity after damage, leading to much interest in regenerative approaches using
stem cells. However, efficient generation of sufficient numbers of cardiac myocytes remains achallenge. The reprogramming of somatic cells to pluripotent stem cells, B cells to macrophages, andpancreatic exocrine cells to β-cells with limited sets of transcription factors suggests that directedinduction of a deﬁned lineage, such as heart, may be achievable.
Baf60c, a cardiac-enriched subunit of the polymorphic Swi/Snf-like BAF chromatinremodelingcomplexes, physically links DNA-binding transcription factors to BAF complexes, thereby
modulating transcription of target genes. Mouse embryos with reduced levels of Baf60c have severe
heart defects and defective cardiac differentiation(Lickert, Takeuchi et al., Nature 2004., Takeuchi etal., PNAS 2007), due to arrested differentiation and insufﬁcient expansion of cardiac progenitors.

Results
1; Three factors (Baf60c, Gata4 and Tbx5) as cardiac master genes.
In transient mice culture system with Baf60c and two cardiac transcription factors (Gata4 andTbx5), ectopic cardiomyocytes with beating were induced in lateral plate mesoderm, an amnion
layer and presomatic mesoderm. As no cardiac genes were induced by transfection of only

- 16 -

transcription factors, combination of there three factors (Baf60c, Gata4 and Tbx5) are important
for activation of cardiac gene program (cardiac hormone, cardiac contracting muscle genes andion channel genes; ﬁg.1) and haploinsufﬁcient in human heart disease.

(ﬁg.1) three factors induce cardiac gene program (from Nature 2009)
2; Reprograming mesodermal cell fate to heart, or heart lineage to other cell fate?
To understand speciﬁcation of each organ derived from mesodermal cells, tissue speciﬁc genes
expressed in pre-heart region or lateral plate mesoderm will be loss-of-functioned mating mesp1cre mouse. I utilized Mesp1, which is the best gene marker expressed in the early mesoderm layer,
and Brg1 or Sall1/4 will be unfunctioned in the mesodermal layer during embryogenesis.
2-1);Brg1f/f;mesp1-cre caused severe heart defects with no cardiac Troponin expression at e9.5.
No cardiac structure was observed in the embryo, but we have to recognize whether justcardiac cells were decreased or cell fate might be changed. To answer this question, lateralplate markers, somatic mesodermal markers will be utilized.
2-2); Sall4f/f;mesp1-cre and Sall1f/f;mesp1-cre mice caused interesting phenotypes in outflow
tract with hypomorphic RV and DA structure without islet expression, which is a cardiac
progenitor expressed in second heart ﬁeld.
To understand Sall signal directly regulate islet1 as a cardiac progenitor and unknown cardiac
progenitor, we will sort double positive cells with Sall4-lacZ;Sall1-GFP using FACS system, andthese positive cells will be differentiated in culture system.

Discussion & Conclusion
1; Our results deﬁne a combination of three factors that can execute a cardiac transcriptional programand fully reprogram noncardiac mesoderm to contractile cardiac myocytes. The induction ofectopic beating tissue in vivo in amniotes is unprecedented, and in lower vertebrates such as
Xenopus or zebrafish, exogenous transcription factors lead only to a low frequency of beatingheart tissue.
2; Unlike other contexts where DNA-binding factors were sufﬁcient to enact reprogramming, SWI/
SNF-Baf60c was required to potentiate the function of Gata4 and Tbx5, partly by allowing
binding of Gata4 to cardiac loci. These ﬁndings reveal a novel mechanism for tissue-speciﬁc gene
regulation by chromatin- remodeling complexes.

- 17 -

3; Chromatin remodeling factors may regulate the program of cell fate from mesodermal cell to
cardiac cells. The functional complexes have to be identiﬁed to understand each organ generation.

References
2009
1;Takeuchi JK.*& Bruenau BG.*
Directed transdifferentiation of mouse mesoderm to heart tissue by deﬁned factors.
Nature 459.708-711. 2009 *; equally correspondence
2; Takeuchi J.K. and Koshiba K.
Induction and differentiation of cardiomyocytes - Is there master-regulator for cardiomyocytes?
Journal of Clinical and Experimental Medicine (IGAKU NO AYUMI) (2009) 229.711-719.
3: Yoshimura K, Kitagawa H, Fujiki R, Tanabe M, Takezawa S, Takada I, Furutani Y, Yagi H, Yoshinaga
S, Masuda T, Fukuda T, Yamamoto Y, Ebihara K, Y.Li D, Matsuoka R, Takeuchi J.K, Matsumoto T.
and Kato S.
Distinct function of 2 chromatin remodeling complexes that share a common subunit, Williams
syndrome transcription factor (WSTF)
P.N.A.S. USA. (2009). In press.

2008
4: Zhu Y*, Gramolini AO, Walsh MA, Zhou Y, Slorach C, Freidberg M, Takeuchi JK, Sun H, Henkelman
M, Olson EN, Kranias E, Backx PH, Redington AN, MacLennan DH and Bruneau BG.
Tbx5-dependent pathway regulating diastolic function in congenital heart disease.
P.N.A.S. USA. (2008) 105.5519-5524.

- 18 -

Molecular Biological Identiﬁcation of Stemness
Shinji Masui
Research Institute, International Medical Center of Japanmasui@ri.imcj.go.jp

Abstract
Recent advance in the system biological approachs have been providing tremendous information onstem cell biology. However, despite many researcher’s keen interest, the common mechanism of selfrenewal in stem cells, while whether it actually exists is not clear, have not been identiﬁed. In thisstudy I postulated the existence of common mechanism of the self-renewal. I have established thesuitable materials to analyze the self-renewal mechanism more precisely.
Keywords: stem cells, differentiation, Sox2

Introduction
Stem cells are able to replicate while keeping capability of differentiation, which is called “selfrenewal”. Various types of stem cells have been described to date, while the molecular biologicalmechanisms for the self-renewal that may be commonly working among them are not known.

Results
Although they are different in terms of developmental lineages and differentiation capability, bothof neural stem cells (NSC) and trophoblast stem cells (TSC) show strong self-renewal potential andhigh level of the expression of Sox2, which have been known to be necessary for the self-renewal ofembryonic stem cells. In my/our previous work I/we have demonstrated that Sox2 is required for the
self-renewal of NSC in vivo and invitro, which are cultured as neurosphere. In addition, Sox2 is also
necessary for the self-renewal of TSC, suggesting that there could be common target genes required
for the self-renewal of the mammalian stem cell maintenance in general. In this study I sought toidentify the common mechanism of the self-renewal, by using loss-of-function of Sox2 as a probe.
Since Sox2 is known to co-operate with a different transcription factor in different types of cells,
the down-stream genes of Sox2 in NSC and TSC will be mostly different. However, a small numberof the downstream genes commonly found in both NSC and TSC will be identified as importantfactors playing pivotal role in the self-renewal. In a sense of the clinical application, the informationof genes for general self-renewal will be useful; for example, it may be possible to transduce thosegenes/gene-products to “progenitor” cells such as cardiovascular progenitor cells and pancreatic
progenitor cells, which are known to have limited capacity to replicate in normal condition, toprepare donor cells for the cellular replacement therapy for heart attack and diabetes, respectively.
NSC, cultured as neurosphere, are known to contain considerable amount of the differentiatedcells, such as neurons and astrocytes. Recently it has been reported that NSC cultured in- 19 -

2D-culture system, which is based on the directed differentiated system of ESC, show homogenous
undifferentiated NSC population, suggestingthat this system should be suitable to identify
the direct-target of Sox2 in NSC.
In my/our previous work I have establishedthe drug-inducible Sox2-knockout ESC, byusing tetracycline-regulated expression system.
In this cell line, 2TS22C, endogenous allelesof Sox2 have been deleted, and Sox2 protein
required for the self-renewal is provided by thetetracycline-regulated transgene. In the presence Fig.1of tetracycline, Sox2 protein disappeared within NS22-2 cultured 5 days in the indicated condition. For
introduction of the transgenes, I employed retrovirus

24 hours in ESC culturing condition. By using pMYs-IRES-Hygro packaged by Plat-E cells.

this cell line, I have established the drug-inducible Sox2-knockout NSC, NS22-2. In the presence of
FGF2 and EGF NS22-2 rapidly self-renew (replicating once per 24 hours), whereas in the absence of
these growth factors they differentiate neurons (TuJ1 positive) and glial (GFAP positive) cells. Uponadministration of tetracycline NS22-2 halted to self-renew in the low-density culture (Fig. 1; thisphenotype can be rescued by introduction of Sox2 transgene), whereas in the high-density culture
they continued to grow without any sign of the differentiation. Next I postulated that Sox2 protein
is receiving an unknown signal via either paracrine or cell-cell interaction, which may contributeto the protein stability of Sox2. Since it has been reported that the Notch signaling regulates Sox2
protein expression level in vivo, I tested to repress Notch signaling in high density culture condition
by using DAPT and gamma-secretase inhibitor X, both of which are known to be speciﬁc inhibitorof Notch signaling. Even in the presence of high-concentration of these inhibitor (25 µM DAPT or 2
µM inhibitor X), NS22-2 still showed similar level of growth in high density culture in the presenceof tetracycline, suggesting that Notch signaling might not be involved in this phenomenon.

Discussion & Conclusion
In this study I have established the drug-inducible Sox2 knockout NSC in 2D-culture. Upon
induction of knockout, they stopped to self-renew, indicating that this cell line is suitable to analyze
the direct down stream genes of Sox2 in NSC. Unlike previous studies by using HSC and MSC,
this study is using inﬁnitely-replicating NSC and TSC, and is focusing on the common transcription
factor Sox2 to confine the spectrum of the genes responsible for the common self-renewal
mechanism, suggesting the high probability of identification of the “stemness” genes. Sox2 isbelonging to SoxB1 family, whose members are found in all the metazoan species including sponges,
which do not have any nervous system, suggesting that the common mechanism postulated in thisstudy might be evolutionary conserved among the metazoan species.

- 20 -

References
Miyagi S, Masui S, Niwa H, Saito T, Shimazaki T, Okano H, Nishimoto M, Muramatsu M, Iwama A,
Okuda A. Consequence of the loss of Sox2 in the developing brain of the mouse. FEBS Lett.582:2811-5,
2008
Masui S, Nakatake Y, Toyooka Y, Shimosato D, Yagi R, Takahashi K, Okochi H, Okuda A, Matoba R,
Sharov AA, Ko MSH and Niwa H. Pluripotency governed by Sox2 via regulation of Oct3/4 expression inmouse embryonic stem cells. Nat Cell Biol. 9:625-635, 2007
Niche-independent symmetrical self-renewal of a mammalian tissue stem cell. Conti L, Pollard SM,
Gorba T, Reitano E, Toselli M, Biella G, Sun Y, Sanzone S, Ying QL, Cattaneo E, Smith A. PLoS Biol
3:e283, 2005

- 21 -

How dose the deﬁcit of histone deacetylase activity cause
anxiety and depression?
Yoshiharu Kawaguchi
Institute for Developmental Research, Aichi Human Service Centerkawag001@inst-hsc.jp

Abstract
Our unexpected findings that histone deacetyase 6 (HDAC6) knockout mice showed anxietyrelated behavior suggest the relevance of HDAC6 activity in mental illness. In this study, we foundthat HDAC6 is mainly expressed in monoaminergic neurons that are well involved in emotion
and mental illness. Expression of HDAC6 in brain is increased during brain development. In
neuronal cells, HDAC6 is localized to neuritis and its growth cone, where actin and tubulin areenriched. Interestingly, loss of HDAC6 causes more dendritic shape in PC12 cells. These ﬁndings
suggest that HDAC6 might play important roles in construction of neuronal network derived frommonoaminergic neurons by regulating cytoskeletal rearrangement.
Keywords: HDACs, psychiatric disease, mouse model

Introduction
Our goal of this research is to provide new therapeutics strategy and targets for mental disorders.
To approach this, we focus on an event of reversible acetylation mediated by histone deacetylase 6(HDAC6) in neurons. HDAC6 is a member of the histone deacetylase family, and is predominantly
expressed in brain tissue. Unlike other HDACs, HDAC6 is known to contribute cytoplasmic event,as HDAC6 is mainly localized in cytoplasm (ref.1-3). Interestingly, we found that HDAC6 knockout mouse show anxiety-related behavior, suggesting that HDAC6 might play an important role inemotional activity in brain.

Results
To study the role of HDAC6 in brain, we ﬁrstly examined localization of HDAC6 in mouse brain
by using immunohistochemical technique with speciﬁc antibody to HDAC6. In adult mouse brain,
HDAC6 is preferentially expressed in neurons of dorsal raphe located in midbrain (Fig1A). To
further determine to such speciﬁc expression of HDAC6 protein, we also used human postmortem
brains, and found that HDAC6 is expressed not only in neurons of dorsal raphe, but also in substantia
nigra and locus ceruleus (Fig1B and C). As those neurons in midbrain are known to be involved inemotional activity and many of mental illness, these ﬁndings strongly support our hypothesis that
HDAC6 is functionally related to emotional activity. Also, our ﬁndings provide the clue to sort out
anxiety-related behavior observed in HDAC6 KO mouse.
Next, we examined the expression of HDAC6 in mouse embryonic and post-embryonic brain- 22 -

tissue. Maximal levels of HDAC6 expression
were observed from E18.5 to 14 days after birth,suggesting that HDAC6 might also be involvedin brain development.
To determine the function of HDAC6 inneuron, we used primary culture of rat corticalneuron, and examined subcelluar localizationof HDAC6. HDAC6 was localized to soma andneurite body in primary neuron. Interestingly,
we found that HDAC6 accumulates in the tip ofeach neurite. To further verify the localizationof HDAC6, we performed co-immunostaining assay with using antibodies to functional proteinsin primary neuron. Although neither synaptic protein nor intracelluar trafficking protein was colocalized with HDAC6, HDAC6 is well co-localized with actin and tubulin in neuron. Since HDAC6
works in cytoplasmic event where remodeling of actin and tubulin is regulated to give proper cell
motility (1,4) and endocytosis (5), HDAC6 might be involved in dynamics of cytoskeleton in neurite,
which in turn, leading to neurite elongation. In accordance with this, we found morphological change
in HDAC6 knockdown PC12 cells. These cells showed dendritic shape (Fig.2) in normal condition,
and abnormal extension of dendrites induced by neuronal growth factor (NGF). These findings
demonstrate that HDAC6 might contribute neurite elongation by regulating actin and/or tubulin
remodeling in neuron.

Discussion & Conclusion
Our ﬁndings strongly demonstrate that HDAC6-mediated deacetylation event are potentially new
target to understand the molecular mechanism of mental activity in brain. As HDAC6 protein isexpressed in monoaminergic neurons in midbrain, and affects dendrite extension in PC12 cells,morphological and/or functional abnormalities in those neurons would be expected in HDAC6
KO mouse. Further analysis of HDAC6 KO mouse needs to be done. Our study for elucidation ofneuronal function of HDAC6 would provide not only new insight for the etiology of mental illness,but also contribute to form the framework of the future drug discovery effort targeting on HDAC6medeated deacetylation.

- 23 -

References
1. Hubbert, C., Guardiola, A., Shao, R., Kawaguchi, Y., Ito, A., Yoshida, M., Wang, X.F. and Yao, T.P. Identiﬁcation of HDAC6 as a microtubule-associated deacetylase. Nature, 417:455-458, 2002
2. Kawaguchi, Y., Kovacs, J.J., McLaurin, A., Vance, J.M., Ito, A., and Yao, T. -P. The deacetylase
HDAC6 regulates aggresome formation and cell viability in response to misfolded protein stress.
Cell 115: 727-738, 2003
3. Kovacs, J.J., Murphy, P.J.M., Gaillard, S., Zhao, X., Wu, J-T., Nicchitta, C.V., Yoshida, M., Toft,
D.O., Pratt, W.B., and Yao, T-P. HDAC6regurates Hsp90 acetylation and chaperone-dependent
activation of glucocorticoid receptor. Mol. Cell 18:601-607, 2005
4. Gao, Y-S., Hubbert, C.C., Lu, J., Lee, Y-S., Lee, J-Y, and Yao, T-P. Histone deacetylase 6 regulates
growth factor-induced actin remodeling and endocytosis. Mol. Cell. Bio. 27:8637-8647, 2007
5. Tran, A.D., Marmo, T.P., Salam, A.A., Che, S., Finkelstein, E., Kabarriti, R., Xenias, H.S.,
Mazitschek, R., Hubbert, C., Kawaguchi, Y., Sheetz, M.P., Yao, T-P., and Bulinski. J.C. HDAC6
deacetylation of tubulin modulates dynamics of cellular adhesions. J. Cell Science 120:14691479, 2007

- 24 -

Regulation of EGF receptor/PI3K/Akt signal transduction pathway
by a novel PDK1-interacting protein Aki1
Naoya Fujita
Japanese Foundation for Cancer Researchnaoya.fujita@jfcr.or.jp

Abstract
We identiﬁed Akt kinase-interacting protein 1 (Aki1) as a novel scaffold for the PDK1/Akt pathway.
Aki1 interacted with not only PDK1 but also Akt. Aki1 expression induced PDK1/Akt complex
formation and induced Akt activation in a concentration-dependent biphasic manner. Aki1 also
associated with epidermal growth factor receptor (EGFR) in response to EGF. Aki1 was required for
Akt activation induced by EGF but not by IGF-1. Aki1 gene silencing increased sensitivity towards
several chemotherapeutic agents, indicating that Aki1 is a novel receptor-selective scaffold for the
PDK1/Akt pathway and is a good therapeutic target to suppress EGFR-mediated cancer cell growth.
Keywords: Aki1, PDK1, Akt, EGFR, scaffold

Introduction
The PI3K/PDK1/Akt pathway regulates various cellular functions, especially cell survival and cell
cycle progression. There is increasing evidences that scaffold proteins maintain signaling speciﬁcity,
facilitate the activation of pathway components, and localize them to particular subcellular sites orto speciﬁc targets in many signaling pathways. Although many components that are involved in the
PI3K/PDK1/Akt pathway have been elucidated, little is known about how those components arecoordinated in transmitting speciﬁc signals.

Results
Molecular Identiﬁcation of Aki1 as a PDK1-Interacting Protein.
To identify novel molecules that form a complex with and regulate PDK1, a HeLa cDNA library
and a human fetal brain cDNA library were screened using an E. coli two-hybrid system. As a result,we identiﬁed a novel PDK1-binding protein. Because the protein did not have the name standing for
its function, we designated it as Aki1 [Akt kinase (PDK1) interacting protein 1].
Aki1 Activated the PDK1/Akt Signaling Pathway.
To investigate the role of endogenous Aki1 in the PDK1/Akt signaling, we designed two Aki1
siRNAs. Both Aki1 siRNAs signiﬁcantly decreased EGF-stimulated Akt phosphrylation at Thr308 (thesite is known to be phosphorylated by PDK1) and attenuated Akt kinase activity.
Aki1 was a Scaffold for PDK1/Akt Signaling.
When examining the Aki1 binding to PDK1 in mammalian cells, we found that Aki1 interacted withnot only PDK1 but also Akt. From this result, we investigated whether Aki1 functioned as a scaffold- 25 -

protein for the PDK1/Akt pathway. Aki1 expression
modulated EGF-induced Akt phosphorylation at
Thr308 in a concentration-dependent biphasic manner.
(Quantitative studies of the MAPK cascade revealed
that molecular scaffolds might either facilitate
or inhibit signal propagation depending on theirconcentration.) We also found that Aki1, PDK1, and
Akt formed a ternary complex and that Aki1 was
required for the PDK1-Akt interaction. These results
indicate that Aki1 functions as a scaffold for the
PDK1/Akt signaling (Figure 1).

Figure 1

Aki1 Formed a Multiprotein Complex Containing EGF Receptor.
To establish whether Aki1 could exhibit its ability in other growth factor signaling pathways besides
EGF, we examined the effect of Aki1 knockdown on Akt phosphorylation in response to IGF-1 or
PDGF. In contrast to EGF treatment, Aki1 knockdown did not affect the level of the phospho-Thr308Akt after treatment with IGF-1 or PDGF.
In the case of the MAPK pathway, it has been reported that scaffold proteins promote the formationof complexes containing receptor. We have shown that Aki1 interacts with PDK1 and Akt, andregulates Akt activation upon EGF signaling by functioning as a scaffold. Thus, we hypothesized that
Aki1 may also associate with EGF receptor (EGFR). Consistent with this idea, immunoprecipitation
analysis showed that Aki1 associated with EGFR and Akt in response to EGF, whereas IGF-1
stimulation did not cause the complex formation, suggesting that Aki1 selectively formed the EGFR/
Aki1/PDK1/Akt complex in response to EGF (Figure 1). Because EGF-induced formation of EGFR/
Aki1/Akt complex was interferred by a potent EGFR tyrosine kinase inhibitor AG1478, EGFR
activation might be required for the complex formation.
Aki1 Positively Regulated Cell Survival and Proliferation.
Because Aki1 positively regulated Akt kinase activity, we assumed that Aki1 might be associatedwith cell surv ival and proliferation. We performed MTT assay and revealed that Aki1 genesilencing resulted in about 40% reduction in the number of viable cells after 96 h of transfection.
Flow cytometric analysis demonstrated that Aki1 knockdown caused a 2- to 3-fold increase in
the apoptotic sub-G1 population. We next addressed whether Aki1 knockdown affects sensitivity
to chemotherapeutic drugs, such as Taxol, VP-16, and CPT. The MTT assay revealed that thesechemotherapeutic drugs, excluding Taxol, synergistically decreased cell viability in cells transfectedwith Aki1 siRNA. These results indicate that Aki1 is a critical regulator of PDK1/Akt signaling in
the maintenance of cell survival and cell proliferation.

- 26 -

Discussion & Conclusion
In this research, we identified Aki1 as a novel PDK1-interacting protein and reported its role in
EGFR/PI3K/PDK1/Akt signaling (Ref. 1). Two lines of evidence support the assumption that Aki1
is a critical regulator of EGFR/PDK1/Akt signaling. First, Aki1 is required for EGF-mediated Akt
phosphorylation and activation; second, Aki1 selectively forms an EGFR/Aki1/PDK1/Akt complex
in an EGF-dependent manner. Recently, p21-activated protein kinase 1 (PAK1) was also found to
be a scaffold for PDK1 and Akt (Ref. 2). PAK1 is reported to be required for PDGF-stimulated cell
migration. Scaffold proteins, including Aki1 and PAK1, would deﬁne the speciﬁcity of the PI3K/
PDK1/Akt pathway, respectively. We also found that tumor suppressor candidate 4 (TUSC4) wasa PDK1-interacting protein and that TUSC4 attenuated PDK1 tyrosine phosphorylation and PDK1
kinase activity (Ref. 3). Our results suggest that PDK1 is regulated by several interacting proteins
without/with affecting its kinase activity.
In addition, Aki1 knockdown increased cellular sensitivity to chemotherapeutic drugs. In manytumor types, loss of function mutation or deletion of PTEN (a negative regulator of the Akt signaling)is frequently occurred. Therefore, therapies aimed at inhibiting Aki1 functions may be useful forovercoming the chemotherapeutic drug resistance in many tumor types.

References
1. Freud-1/Aki1, a novel PDK1-interacting protein, functions as a scaffold to activate the PDK1/Akt
pathway in epidermal growth factor signaling. A Nakamura, M Naito, T Tsuruo, and N Fujita, Mol.
Cell. Biol., 28: 5996-6009, 2008.
2. Scaffolding function of PAK in the PDK1-Akt pathway. M Higuchi, K Onishi, C Kikuchi, and Y Gotoh.
Nat. Cell Biol., 10: 1356-1364, 2008.
3. TUSC4/NPRL2, a novel PDK1-interacting protein, inhibits PDK1 tyrosine phosphorylation and its
downstream signaling. A Kurata, R Katayama, T Watanabe, T Tsuruo, and N Fujita. Cancer Sci., 99:
1827-1834, 2008.

- 27 -

The role of transcription factor FoxO1
in pancreatic beta cell regeneration
Tadahiro Kitamura
Metabolic Signal Research Center,
Institute for Molecular and Cellular Regulation, Gunma University
kitamura@showa.gunma-u.ac.jp

Abstract
In this study, we generated and analyzed pancreas speciﬁc FoxO1 knockout and transgenic mice.
The results revealed that FoxO1 is an important transcription factor for the regulation of pancreatic
beta cell differentiation and neogenesis. We propose that manipulation of FoxO1 either genetically or
pharmacologically may contribute the development of new strategies to treat diabetes.
Keywords: Diabetes, pancreatic beta cell, FoxO1Introduction
We have been studying the roles of forkhead transcription factor FoxO1 in pancreatic beta cell.
Our preliminary data indicate that the expression pattern of FoxO1 during pancreas development is
identical to the pattern of Pdx1 suggesting that FoxO1 as well as Pdx1 plays an important role in beta
cell differentiation. Therefore, we tested in this study whether FoxO1 is a key transcription factor forpancreatic beta cell differentiation by using two distinct experimental strategies; (1) loss-of-function
study of FoxO1 by generating pancreas speciﬁc FoxO1 knockout mice and (2) gain-of-function study
by generating pancreas speciﬁc FoxO1 transgenic mice.

Results
By using Pdx1 promoter and FLAG tagged FoxO1 construct, we generated pancreas speciﬁc FoxO1
transgenic mice (TG). First we conﬁrmed the expression of transgene (FLAG-FoxO1) speciﬁcally inpancreas but not in the other organs by using immunohistochemistry with FLAG antibody. The mice
were born according to the mendelian rules. Blood glucose examination and histological analyses
revealed that about 20% of the Tg mice develop severe diabetes followed by the signiﬁcant reductionof beta cells. As the other 80% Tg mice showed normal blood glucose levels, we fed the mice withhigh fat high sucrose diet (HFHSD) for 8 weeks. HFHSD fed Tg mice showed severe glucose
intolerance in glucose tolerance test (GTT) and some of the mice developed overt diabetes. On theother hand, Tg mice have some other interesting phenotype. Tg mice display severe hypoplasiaof pancreatic acinar cells and marked increase in duct-like structure, which leads to the formationof pancreatic cysts at their old age. More interestingly, Tg mice exhibit islet hypervascularization,
which is associated with increased VEGF level in beta cells. By using luciferase promoter assay, weshow that FoxO1 directly regulates VEGF transcription in vitro.

- 28 -

To generate pancreas speciﬁc FoxO1 knockout mice (KO), we crossed Pdx1-Cre mice with FoxO1
ﬂox mice. We ﬁrst conﬁrmed by using real time RT-PCR and Western blotting that FoxO1 expression
level is decreased by 90% in pancreas but not in the other organs in KO mice. KO mice were bornand grew normally. Their blood glucose levels were normal. Consistent with the observations in
Tg mice, GTT examination revealed that KO mice have improved glucose tolerance compared tocontrol mice under high fat high sucrose diet condition. We found that the numbers of small islets
and more interestingly insulin-positive duct cells were increased in KO mice, indicating that beta
cell neogenesis is enhanced in these mice. However, when we crossed KO mice with db/db mice,the mice exhibited more severe glucose intolerance compared to control db/db mice, indicating thatFoxO1 deﬁciency enhanced beta cell dysfunction in the state of hyperglycemia.

Discussion & Conclusion
The results in FoxO1 Tg mice and KO mice raised the hypothesis that FoxO1’s functions inpancreatic beta cells are like double-edged sword. We conclude that FoxO1 plays important roles
in pancreatic cell differentiation and cell type speciﬁcation as well as beta cell growth and function.
Also these results suggest that FoxO1 dysfunction may account for the pancreatic beta cell failure intype 2 diabetes. We propose that manipulation of FoxO1 either genetically or pharmacologically may
contribute the development of new strategies to treat diabetes.

References
1. Kitamura, T., Y. Kitamura, M. Kobayashi, O. Kikuchi, T. Sasaki, R.A. DePinho and D. Accili.
2009. Regulation of pancreatic juxtaductal cell formation by FoxO1. . Mol. Cell. Biol. inpress.
2. Kitamura, T., Y. Kitamura, Y. Funahashi, C.L. Shawber, D.H. Castrillon, R. Kollipara, R.A.
DePinho, J. Kitajewski and D. Accili. 2007. A Foxo/Notch pathway controls myogenic
differentiation and ﬁber type speciﬁcation. . J. Clin. Invest. 117: 2477-2485.
3. Kitamura, Y., T. Kitamura, J-P. Kruse, J. Raum, R.W. Stein, W. Gu and D. Accili. 2005. FoxO1
protects against pancreatic cell failure through NeuroD and MafA induction. Cell Metab. 2: 153163.
4. Kitamura, T., Y. Kitamura, J. Nakae, C.R. Kahn, A. Efstratiadis and D. Accili. 2004. Mosaic
analysis of Insulin Receptor function. J. Clin. Invest.113: 209-219.
5. Kitamura, T., J. Nakae, Y. Kitamura, Y. Kido, W.H. Biggs III, C.V.E. Wright, M.F. White, K.C.
Arden and D. Accili. 2002. The forkhead transcription factor Foxo1 links insulin signaling toPdx1 reguration of pancreatic β-cell growth. J. Clin. Invest. 110: 1839-1847.

- 29 -

Studies on post-translational modiﬁcations of proteinsby endogenous nitrated nucleic acids
Hirokazu Arimoto
Tohoku University
arimoto@biochem.tohoku.ac.jp

Abstract
Fluorescent probes of 8-nitro-guanosine derivatives were prepared, and used for functional analysis
of nitrated nucleotides.
Keywords: 8-NO2-cGMP, ﬂuorescent probe, cytoprotective

Introduction
Nitric oxide (NO) has diverse physiological functions. Reactive nitrogen species are generated fromNO, and work as potent nitrating agents for proteins, nucleic acids, and unsaturated membrane lipids.
We, collaborated with Prof. Akaike, identiﬁed 8-nitro-cGMP, the ﬁrst endogenous cGMP derivative,in various cell lines. This compound mediates a novel post-translational modification by reactingwith proteinous thiol in cells.

Results
Our previous efforts to uncover the role of the S-guanylations, led to the identiﬁcation of Keap1,
which has been shown to be cytoprotective via HO-1 (heme oxygenase 1) upregulation. Developmentof the 8-nitroguanosine based-probes should be a promising approach to answer further biologicalquestions regarding S-guanylation at the molecular level.
Our study was initiated with exploration of an efficient synthesis of 8-nitroguanosine that
constituted a basis for chemical studies of protein S-guanylation. Use of proper protecting groups
was proven to minimize the undesired depurination of nitrated guanosines.
Having synthesized 8-nitroguanosines, we then turned our attention to the biological activities ofthese compounds. It was revealed that 8-nitroguanosine derivatives, which lack a cyclic phosphate
could also enhance the induction of HO-1 in HepG2 cells, and protected the cells from glucose
deprivation-induced cytotoxicity. This structure-activity-relationship (SAR) is useful for the designof chemical probes for the analysis of the protein S-guanylations.
An azido-containing 8-nitroguanisine probe was then prepared, and treated with rat glioma C6 celllysate. The progress of the reaction was monitored by the release of NO2- ion by the Griess method.
The S-guanylated proteins in the lysate were then labeled by a ﬂuorescent tag via a Huisgen reaction.
Fluorescent scanning of a SDS-PAGE gel conﬁrmed the presence of several labeled proteins.

- 30 -

Discussion & Conclusion
The protein S-guanilation has been attracted considerable attentions in both biological and chemicalcommunities, because 8-NO2-cGMP is the first endogenous derivative of an important second
messenger, cGMP. We have developed in this study that the efﬁcient synthetic avenue for preparationof a wide array of 8-nitroguanosines. Our SAR study would also contribute further chemical researchin this ﬁeld.
The ﬂuorescent probes developed in this study enables enzymatic digestion and sequence analysisof the labeled bands, and thus provide an insight into the possible physiological targets of protein
S-guanylation, The results will be described elsewhere soon.

References
Y. Saito, H. Taguchi, S. Fujii, T. Sawa, E. Kida, C. Kabuto , T. Akaike, H. Arimoto, Chemical
Communications, 2008, 5984-5986.
T. Sawa, M. H. Zaki, T. Okamoto, T. Akuta, Y. Tokutomi, S. K.- Mitsuyama, H. Ihara, A.
Kobayashi, M. Yamamoto, S. Fujii, H.Arimoto, T. Akaike Nature Chemical Biolology, 2007, 3, 727735.

- 31 -

Concomitant inhibition of Mdm2–p53 interaction and
Aurora kinases or PI3K/Akt/mTOR synergistically induces
p53-mediated mitochondrial apoptosis
Kensuke Kojima
Wakayama Medical University
k-koji@wakayama-med.ac.jp

Introduction
Inactivation of wild-type p53 by Mdm2 overexpression, abberant expression of Aurora kinases,
and activation of PI3K/Akt/mTOR pathway are frequent molecular events in acute myelogenous
leukemia (AML). Preclinical data for their inhibition are promising. Regarding Auroras, it remains
largely unknown if the viability of cells exposed to Aurora kinase inhibitors depends on the p53
status. We investigated the interaction of Aurora kinases and p53 pathways after their simultaneous
blockades using a small-molecule pan-Aurora kinase inhibitor, MK-0457, and a selective smallmolecule antagonist of Mdm2, Nutlin-3. We also investigated the interaction of PI3K/Akt/mTOR
and p53 pathways after their simultaneous blockade using the dual PI3K/mTOR inhibitor PI-103 andthe Mdm2 inhibitor Nutlin-3.

Results
We found that MK-0457, which itself activates p53 signaling, acts synergistically with Nutlin-3 to
induce apoptosis in wild-type p53 AML cell lines OCI-AML-3 and MOLM-13 but not in p53-null
HL-60 cells. MK-0457 and Nutlin-3 showed synergism in inducing p53, conformational change of
Bax and ∆Ψm loss, suggesting an involvement of p53-mediated mitochondrial apoptosis. Nutlin-3
constrained endoreduplication following Aurora inhibition via activation of a p53-dependent
postmitotic checkpoint and p21 induction in pseudo-G1 cells.
Regarding an interaction between PI3K/Akt/mTOR and p53 pathways, we found that PI-103, whichitself has modest apoptogenic activity, acts synergistically with Nutlin-3 to induce apoptosis in awild-type p53-dependent fashion. PI-103 synergized with Nutlin-3 to induce Bax conformationalchange and caspase-3 activation, despite its inhibitory effect on p53 induction. The PI-103/Nutlin-3
combination caused profound dephosphorylation of 4E-BP1 and decreased expression of many
proteins including Mdm2, p21, Noxa, Bcl-2 and survivin, which can affect mitochondrial stability.

Discussion & Summary
Cells treated with MK-0457 either undergo eventual apoptosis after additional cell cycles, where
the inherited polyploid genome and multiple centrosomes result in mitotic catastrophe, or apseudo-G1 arrest, primarily induced by a p53-dependent postmitotic checkpoint. Our data suggestthat p53 activation by Mdm2 inhibition efﬁciently induces apoptosis in cells with 4N and 8N DNA- 32 -

content after MK-0457 treatment. Since the enhancement of apoptosis was associated with aberrant
p21 induction and limited endoreduplication, Nutlin-3 would activate the p53-dependent postmitotic
checkpoints and cause apoptosis via potent induction of p53.
Inhibition of PI3K/Akt/mTOR signaling can augment p53-mediated apoptosis. Since inhibitors
directed at PI3K (LY294002) or mTOR (rapamycin) alone sufficiently enhanced Nutlin-induced
apoptosis, as was seen for PI-103, one of the targets for enhancing p53-mediated apoptosis appeared
to be mTOR. Inhibition of Akt is another candidate mechanism for enhancing p53-mediatedapoptosis. The dual PI3K/mTOR inhibitor PI-103 has an advantage that it shut down the negative
feedback from mTOR and to efﬁciently reduce Akt phosphorylation.
Our ﬁndings provide the molecular rationale for concomitant targeting of Aurora kinases or PI3K/
Akt/mTOR and Mdm2 in AML where TP53 mutations are rare and downstream p53 signaling ismostly intact.

References
1) Kojima K, et al. Concomitant inhibition of Mdm2-p53 interaction and Aurora kinases activates the p53dependent postmitotic checkpoints and synergistically induces p53-mediated mitochondrial apoptosis
along with reduced endoreduplication in acute myelogenous leukemia. Blood. 2008;112:2886-2895.
2) Kojima K, et al. The dual PI3 kinase/mTOR inhibitor PI-103 prevents p53 induction by Mdm2
inhibition but enhances p53-mediated mitochondrial apoptosis in p53 wild-type AML. Leukemia.
2008;22:1728-1736.

- 33 -

The role of TRPC channel/NCX1 transporter coupling in vasospasm
Takahiro Iwamoto
Department of Pharmacology, Fuculty of Medicine, Fukuoka University
tiwamoto@cis.fukuoka-u.ac.jp

Abstract
α-adrenoceptor (AR) contributes to the sympathetic regulation of various arteries. However, themolecular mechanisms underlying α1-AR-mediated vasocontraction remain obscure. We found thatphenylephrine-induced cytosolic Ca2+ elevation and contraction were greater in mesenteric arteries
from TRPC3- or NCX1-transgenic mice (Tg). In these mice, a bolus injection of norepinephrine
elicited ST elevation and AV block (coronary spasm). When we crossed TRPC3-Tg with NCX1knockout mice or NCX1.3-Tg with dominant negative TRPC3-Tg, their offspring mice did notexhibit α 1-AR-induced hypervasoreactivity. Coimmunoprecipitation experiments revealed that
NCX1 and TRPC3 are interactively enriched in caveolar raft domains of vascular myocytes. Theseﬁndings indicate that TRPC3/NCX1 coupling plays a pivotal role in regulating arterial tonus via α1AR.
Keywords: Na+/Ca2+ exchanger, TRPC channel, vasospasm

Introduction
Na+/Ca2+ exchanger (NCX) is a plasma membrane transporter expressed in various cell types(1). Membrane potential and transmembrane gradients of Na+ and Ca2+ control this bidirectional
exchanger. The mammalian NCX family comprises three isoforms (NCX1, NCX2, NCX3). NCX1
is abundant in the heart, but is also expressed in many other tissues. Extensive alternative splicingof NCX1 generates tissue-speciﬁc variants; the vascular tissue predominantly expresses NCX1.3. In
vascular smooth muscle, NCX1 is believed to exclude Ca2+ from the cytosol, but the physiologicalroles of vascular NCX1 are still unclear. Recently, we have identified the role of NCX1 in saltsensitive hypertension (2, 3). In the study, we examined the role of NCX1 in α 1-AR-mediatedvascular contraction.

Results
To evaluate the role of NCX1 in α 1-AR-mediated vasocontraction, we generated transgenic miceexpressing NCX1.3 (NCX1.3-Tg) driven by the smooth muscle a-actin promoter. Western blot
analysis showed that NCX1.3 protein was overexpressed in the aortas, but not in the hearts, ofthe transgenic mice at 6- to 8-fold the level of endogenous NCX1. Immunohistochemical staining
indicated dense localization of NCX1.3 in the media layer of aortas from NCX1.3-Tg mice. We
examined the contractile function of the mesenteric artery in response to phenylephrine (1 µM).
Phenylephrine-induced cytosolic Ca2+ elevation and contraction were greater in mesenteric arteries- 34 -

from NCX1.3-Tg mice compared with wild-type (WT) mice. Contractile responses to phenylephrine
in mesenteric arteries from both mice were suppressed by specific NCX inhibitors for Ca2+ entry
through NCX1.
To investigate the molecular mechanism of NCX1-mediated vascular responses induced by
α 1-AR agonist, we generated transgenic mice expressing WT or dominant-negative form of TRPC3(TRPC3-Tg, TRPC3DN-Tg) and TRPC6 (TRPC6-Tg, TRPC6DN-Tg). Phenylephrine-induced
cytosolic Ca2+ elevation and contraction were greater in mesenteric arteries from TRPC3-Tg and
TRPC6-Tg mice compared with WT mice. In contrast, these responses were lowered in the arteries
from TRPC3DN-Tg and TRPC6DN-Tg mice.
We injected norepinephrine intravenously into these transgenic mice to examine the pathologicalrole of enhanced vasoreactivity to α 1-AR agonist in vivo. A bolus injection of norepinephrine elicited
ST elevation and AV block in NCX1-Tg mice as well as in TRPC3-Tg mice, but not in WT mice.
When we crossed TRPC3-Tg mice with NCX1-knockout mice or NCX1.3-Tg mice with TRPC3DNTg mice, their offspring mice did not exhibit norepinephrine-induced hypervasoreactivity and theabnormal electrocardiogram. We also analyzed vasospasm using microvascular ﬁlling in NCX1.3Tg and TRPC3-Tg mice treated with norepinephrine. Microvascular ﬁlling defects (vasospasm) wereobserved in the heart of NCX1.3-Tg and TRPC3-Tg mice, but not in WT mice.
Finally, co-immunoprecipitation experiments were performed to determine whether TRPC3
physically associates with NCX1. When sepharose beads coupled to anti-NCX1 antibody wereincubated with membrane preparation of rat aortas and A7r5 cells, TRPC3 proteins were coimmunoprecipitated with NCX1.

Discussion & Conclusion
In the present study, we demonstrated that α 1-AR-mediated increase in cytosolic Ca2+ and vascular
contraction were enhanced in mesenteric arteries from NCX1.3-Tg mice. α 1-AR-mediated elevationof cytosolic Ca2+ and vasoconstriction in WT and NCX1.3-Tg mice were suppressed by specific
NCX inhibitors. These results suggest that vascular NCX1 acts as a Ca2+ entry pathway in α 1-ARmediated vascular responses. In TRPC3-Tg and TRPC6-Tg mice, vascular responses were also
augmented, while the responses were reduced in TRPC3DN-Tg and TRPC6DN-Tg mice. In
addition, the vasospasm observed in NCX1.3-Tg and TRPC3-Tg mice were improved when TRPC3
or NCX1 functions were suppressed by genetic engineering. Furthermore, co-immunoprecipitation
experiments revealed the (direct or indirect) interaction of TRPC3 and NCX1 in vascular smoothmuscle.
In conclusion, these ﬁndings indicate that TRPC3/NCX1 coupling is critical for regulating arterial
tonus via α 1-AR. This functional unit of ion channel/transporter complex may be a useful target forthe development of therapies for vasospasm.

- 35 -

References
1) Iwamoto T, Watanabe Y, Kita S, Blaustein MP. Na+/Ca2+ exchange inhibitors: a new class of calcium
regulators. Cardiovasc Hematol Disord Drug Targets 7(3):188-198, 2007.
2) Blaustein MP, Zhang J, Chen L, Song H, Raina H, Kinsey SP, Izuka M, Iwamoto T, Kotlikoff MI,
Lingrel JB, Philipson KD, Wier WG, Hamlyn JM. The pump, the exchanger, and endogenous ouabain:
signaling mechanisms that link salt retention to hypertension. Hypertension. 53(2):291-298, 2009.
3) Iwamoto T, Kita S, Zhang J, Blaustein MP, Arai Y, Yoshida S, Wakimoto K, Komuro I, Katsuragi T.
Salt-sensitive hypertension is triggered by Ca2+ entry via Na+/Ca2+ exchanger type-1 in vascular smoothmuscle. Nat Med 10(11):1193-1199, 2004.

- 36 -

Neuropharmacological research on the role of environmental factors
during development in psychological abnormalities
Yukio Ago
Laboratory of Medicinal Pharmacology, Graduate School of Pharmaceutical Sciences,
Osaka University
ago@phs.osaka-u.ac.jp

Abstract
A genetic analysis shows that genetic variants of the genes encoding pituitary adenylate cyclaseactivating polypeptide (PACAP) are associated with schizophrenia. The present study examinedthe effects of environmental factors on psychomotor abnormalities in PACAP knockout (PACAPKO) mice. Rearing in enriched conditions for 4 weeks from 4-week-old attenuated hyperlocomotion
and jumping behavior of PACAP-KO mice. This improvement was not observed in PACAP-KOmice reared in enriched conditions for 4 weeks from 8-week-old. The expression of brain-derived
neurotrophic factor (BDNF) in the hippocampus did not differ signiﬁcantly between wild-type and
PACAP-KO mice at any age and rearing in environmental enrichment increased BDNF expression
in both genotype groups in an age-independent manner. These ﬁndings suggest that the abnormal
behaviors of PACAP-KO mice may depend on environmental factors during development, especiallyadolescence, and other mechanisms rather than hippocampal BDNF may be involved in the effect ofenvironmental enrichment.
Keywords: Pituitary adenylate cyclase-activating polypeptide (PACAP), Environmental factors, Brainderived neurotrophic factor (BDNF), Neurogenesis, Gene-environment interaction

Introduction
Mice lacking the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP)
display some remarkable behavioral changes, including hyperlocomotion, jumping behavior,sensorimotor gating deﬁcits and cognitive impairment1-2). A recent genetic analysis shows genetic
variants of the genes encoding PACAP are associated with schizophrenia3). These ﬁndings suggestthat alterations in PACAP signaling may contribute to the pathogenesis of psychiatric disorders, but
the exact mechanism is not known. On the other hand, clinical studies show that not only genetic
factors but also environmental factors play a key role in pathophysiology of many psychiatricdisorders. However, there are few preclinical studies on this point. The present study investigatedthe effects of environmental factors on the behaviors, brain-derived neurotrophic factor (BDNF)
expression, neurogenesis and oxytocin contents in wild-type and PACAP knockout (PACAP-KO)
mice.

- 37 -

Results
Effects of environmental enrichment on psychomotor abnormalities in PACAP-KO mice
PACAP-KO mice show hyperlocomotion
and jumping behavior in a novel environment.
After normal nurturing, rearing in enriched
conditions for 4 weeks from 4-week-old(adolescence) attenuated hyperlocomotion
and jumping behavior in PACAP-KO mice
(Figure 1C). In contrast, rearing in enriched
conditions for 4 weeks from 8-week-old(adulthood) did not improve the abnormal
behaviors in PACAP-KO mice (Figure1D). Environmental enrichment did notalter signiﬁcantly any of the behaviors inwild-type mice. On the other hand, theeffect of environmental enrichment duringadolescence was not lasting when mice were

Figure 1
Effects of environmental enrichment duringadolescence and adulthood on the behavior of PACAP-KO mice returned to house in standard conditions for 4
The horizontal locomotor activity (left panels) and jumping behavior(right panels) of wild-type (WT) and PACAP-KO (KO) mice in the weeks from 8-week-old.
open-ﬁeld test were measured after four weeks of rearing in standard(A) or enriched (B) conditions from 4-week-old (C) and 8-week-old(D). *P < 0.05, **P < 0.01, #P < 0.05, ##P < 0.01

Effects of environmental enrichment on hippocampal BDNF expression and neurogenesis in
PACAP-KO mice
It has been shown that PACAP enhances
BDNF expression via its high-afﬁnity
receptor PAC1 in primary cultures ofcortical neurons and BDNF expression
is reduced in the hippocampus of micedeﬁcient for PAC1 receptor. Since it isreported that environmental enrichment
increases BDNF expression, the present
study analyzed the BDNF protein levelsin the hippocampus of PACAP-KO
mice. BDNF concentrations did not

Figure 2 Effects of environmental enrichment duringadolescence and adulthood on BDNF expression in the
hippocampus of mice
The BDNF protein levels of wild-type (WT) and PACAP-KO(KO) mice were quantiﬁed after four weeks of rearing in standard
or enriched conditions from 4-week-old (A) and 8-week-old (B).
**P < 0.01

differ signiﬁcantly between wild-type and PACAP-KO mice at eight and twelve weeks of age, andrearing in environmental enrichment increased BDNF expression in both genotype groups (Figure 2).

- 38 -

Intracerebroventricular infusion of PACAP promotes the proliferation of neural stem cells andneurogenesis in adult hippocampus. Thus, this study examined the effect of environmental enrichment
on cell proliferation and the survival of newborn cells in the hippocampus of PACAP-KO mice.
Injections of 5-Bromo-2’-deoxyuridine (BrdU) were used to label dividing cells, together with double
immunoﬂuorescent labeling using neuronal cell-speciﬁc marker (NeuN). BrdU incorporation in thedentate gyrus of hippocampus did not differ signiﬁcantly between wild-type and PACAP-KO mice atfour and eight weeks of age, and rearing in environmental enrichment during adolescence increased
the number of newly generated neurons, but did not affect cell proliferation in both genotype groups.
Effect of social isolation on oxytocin contents in PACAP-KO mice
We have found that isolation rearing for 2 weeks from 4-week-old enhanced hyperlocomotion
and caused aggressive behavior in PACAP-KO mice, although PACAP-KO mice reared in standard
conditions did not show aggressive behavior at any age. This may provide new insights into
understanding the interactions between genes and environment in the pathogenesis of psychiatricdisorders. It has been shown that neuropeptide oxytocin plays a pivotal role in the regulation ofsocial and aggressive behaviors and PACAP induces oxytocin release in supraoptic neurons. Thus,
this study analyzed the oxytocin contents in the brain of PACAP-KO mice. Oxytocin contents in the
hypothalamus and pituitary did not differ signiﬁcantly between wild-type and PACAP-KO mice at six
weeks of age reared in standard conditions, and social isolation rearing for 2 weeks from 4-week-old
did not alter signiﬁcantly oxytocin contents in both genotype groups.

Discussion & Conclusion
Rearing in enriched conditions for 4 weeks from 4-week-old, but not 8-week-old, attenuatedhyperlocomotion and jumping behavior in PACAP-KO mice. In addition, PACAP-KO mice rearedin social isolation for 2 weeks from 4-week-old display aggressive behavior. These ﬁndings suggestthat abnormal behaviors of PACAP-KO mice may depend on environmental factors during early
postnatal development. On the other hand, BDNF and oxytocin contents did not differ between wildtype and PACAP-KO mice and rearing in environmental enrichment increased BDNF expression
in both genotype groups in an age-independent manner. These results imply that other mechanisms
rather than hippocampal BDNF may be involved in the behaviors of PACAP-KO mice and the effectof environmental enrichment.

References
1. Hashimoto H., et al. (2001) Altered psychomotor behaviors in mice lacking pituitary adenylate cyclaseactivating polypeptide (PACAP). Proc. Natl. Acad. Sci. U. S. A. 98, 13355-13360.
2. Tanaka K., et al. (2006) Psychostimulant-induced attenuation of hyperactivity and prepulse inhibitiondeﬁcits in Adcyap1-deﬁcient mice. J. Neurosci. 26, 5091-5097.
3. Hashimoto R., et al. (2007) Pituitary adenylate cyclase-activating polypeptide is associated withschizophrenia. Mol. Psychiatry 12, 1026-1032.

- 39 -

Development of anti infectious-disease drugs
based on natural products
Kenichi Akaji
Kyoto Prefectural University of Medicine, Graduate School of Medical Scienceakaji@koto.kpu-m.ac.jp

Abstract
A novel Human T-cell leukemia virus type I (HTLV-1 [1-3]) protease inhibitor which contains
retro-inverso peptide backbone was designed. By the syntheses of all possible isomers containing
different configuration regarding hydroxyl and side-chain groups, it is supposed that the retroinverso inhibitor interact with HTLV-1 protease as the same binding formation of hydroxylethylamine inhibitors.
It is also found that the mature SARS 3CL protease [4-6] is subject to degradation at
188Arg/189Gln. Replacing Arg with Ile at position 188 rendered the protease resistant to proteolysis.
Novel peptide-aldehyde derivatives containing a side-chain-protected C-terminal Gln efficiently
inhibited the catalytic activity of the R188I mutant. The results indicate that the tetrapeptide
sequence is enough for inhibitory activities of peptide-aldehyde inhibitors.
Keywords: Protease inhibitor, HTLV-1, SARSIntroduction
Retro-inverso modification is a reverseion of original peptide sequence accompanied withreplacement of each L-amino acid with corresponding D-amino acid. Retro-inverso analog is
expected to have topological similarity of side chains and is expected to resist proteolysis since
it contains D-amino acids. However, the introduction of the retro-inverso technique in proteaseinhibitor rarely reported. In this study, HTLV-1 protease inhibitor based on retro-inverso peptide
containing hydroxylethylamine backbone was evaluated.
For the development of anti-SARS drugs, high level of expression in E. coli and the puriﬁcationof a proteolysis-resistant mutant of the SARS 3CL protease are also described. The extremely highpotency makes it possible to evaluate newly synthesized peptide-aldehyde inhibitors quantitatively
by conventional HPLC.

Results
In the studies on HTL-1 protease inhibitors, all possible isomers containing different conﬁguration
regarding hydroxyl and side-chain groups were prepared by using aldol condensation with (1S, 2R)1-Arylsulfonamido-2-indanol as a chiral auxiliary (Scheme 1). Boc-DPro-al 8 was oxidaized with
SO3.Py, and resulting aldehyde was reacted with methoxymethyltriphenylphosphonium chloride at
50°C for 3 h. The resulting product 9 was treated with PPTS to afford an aldehyde 10. (1S, 2R)-1-- 40 -

Arylsulfonamido-2-indanyl 4-methylvalerate
11 was converted to enolate and then reactedwith the aldehyde 10. W hile the aldol
condensation was conducted with chiralauxility, mixture of diastereomers wasafforded by the reaction. Each diastereomer
was separated with column chromatography
and hydrolyzed. Each par t of resulting
β-hydoxycarboxlic acids were converted tooxazolidinones to conﬁrm their conﬁguration
by homonuclear decoupling experiment.
The coupling constant of the vicinal protons Scheme 1. Synthetic scheme for retro-inverso type inhibitor 3a: (a) SO3.Py/DMSO, Et3N, 25°C, 0.5h; (b) Cl-P+Ph3CH2OMe/of oxazolidinone which derived from more tBuOK, 50°C,3 h; (c) PPTS, 25°C, 2h; (d)1M TiCl4/CH2Cl2,
DIEA, -78°C, 1.5h; (e) 30%H O , LiOH, 25°C, 18h; (f)2 2polar ester 12 was 6.3 Hz, and one from less H-DIle-DVal-DPro-DPro-NH2/WSCD/HOBt,
25°C, 24h;(g) TFA; (h) Benzylisocyanate /DIEA

polar product 13 was 2.7 Hz.

Results
From these result, it was estimated that more polar ester has syn configuration and less polar
ester has anti one. NOE spectroscopes of oxazolidinones supported our inference. Moreover,β-hydoxycarboxlic acid 14 has identical 1H-NMR spectroscopy and optical rotation to the main
product which was obtained by using Evans’ chiral auxiliary and nBu2BOTf. The β-hydoxycarboxlic
14 was coupled with tetrateptide that was synthesized on solution phase peptide synthesis using
WSCD/HOBt as coupling reagents. Boc
g roup of the resulting product 15 wasremoved with TFA and then reacted withb e n z yl iso cya n at e t o a f ford i mp rove dretro-invero type HTLV-1 inhibitor 3a.
Diastereomer3b was sy nthesized f rom
β-hydoxycarboxlic acid 15. In a similarmanner, diastereomer 3c, 3d were obtained
from (1R, 2S)-1-arylsulfonamido-2-indanyl
4-methylvalerate.
Then, as a suitable candidate for a reversible
SARS 3CL protease inhibitor, peptide- Scheme 2.
aldehydes containing the substrate sequence were synthesized. To obtain highly homogeneous
derivatives efficiently, the aldehyde group was constructed by reduction of the corresponding
Weinreb amide prepared by solution phase synthesis (Scheme 2). Starting from Fmoc-Glu-OBut,
dimethylamine was condensed to the side chain carboxyl group. Subsequent peptide chain elongation- 41 -

was conducted using a combination of Fmoc-deprotection with diethylamine and coupling with
BOP. The resulting hexapeptide amide was then reduced with LiAlH4. After deprotection with
TFA, the crude product was puriﬁed by HPLC. Peptide aldehydes containing truncated chains weresimilarly synthesized from the corresponding intermediates and puriﬁed.

Discussion & Conclusion
The inhibitory activities of synthesized compounds were examined using the mutant HTLV-1
protease [7] and R188I SARS 3CL protease. By monitoring the enzymatic reaction on analytical
HPLC, IC50 values were obtained and compared.
All diastereomers of retoro-inverso inhibitor showed inhibitory activity for HTLV-1 protease.
Among them, 3a that have R-conﬁguraion for hydroxy group and S-conﬁgulation for isobuty group
was most effective. Thus, it was supposed that the retro-inverso inhibitor 3 interact with HTLV-1
protease as a same binding formation of hydroxyl-ethylamine inhibitor.
In SARS inhibitor studies, the tetrapeptide sequence was found to be long enough for inhibitory
activity of newly synthesized peptide-aldehyde inhibitors. The activity increased in parallel withchain length; the hexapeptide aldehyde had an IC50 of 26 µM. E-64, a typical cysteine proteaseinhibitor, showed nearly the same inhibitory activity (36% inhibition at 5.6 mM) as Ac-Val-LeuGln(Me2)-CHO, which suggests that the peptide-aldehyde derivatives would be a promising
reversible SARS 3CL protease inhibitor.

References
1. Naka, H.; Teruya, K.; Bang, J. K.; Aimoto, S.; Tatsumi, T.; Konno, H.; Nosaka, K.; Akaji, K. Bioorg.
Med. Chem. Lett. 2006, 16, 3761.
2. Bang, J. K.; Naka, H.; Teruya, K.; Aimoto, S.; Konno, H.; Nosaka, K.; Tatsumi, T.; Akaji, K. J. Org.
Chem. 2005, 70, 10596.
3. Akaji, K.; Teruya, K.; Aimoto, S. J. Org. Chem. 2003, 68, 4755.
4. Thiel, V.; Ivanov, K. A.; Putics, Á.; Hertzig, T.; Schelle, B.; Bayer, S.; Weiβbrich, B.; Snijder, E. J.;
Rabenau, H.; Doerr, H. W.; Gorbalenya, A. E.; Ziebuhr, J. J. General Viol. 2003, 84, 2305-2315.
5. Fan, K.; Wei, P.; Feng, Q.; Chen, S.; Huang, C.; Ma, L.; Lai, B.; Pei, J.; Liu, Y.; Chen, J.; Lai, L. J. Biol.
Chem. 2004, 279, 1637-1642.

6. Huang, C.; Wei, P.; Fan, K.; Liu, Y.; Lai, L. Biochemistry 2004, 43, 4568-4574.
7. Teruya, K.; Kawakami, T.; Akaji, K.; Aimoto, S. Tetrahedron Lett. 2002, 43, 1487.

- 42 -

Study on molecular mechanism of infectious particle productionof hepatitis C virus
Makoto Hijikata
Institute for Virus Research Kyoto University
mhijikat@virus.kyoto-u.ac.jp

Abstract
Molecular mechanisms of infectious particle production of hepatitis C virus (HCV) was analyzed
by using recombinant HCV system. We found the core protein is included in the multimer complex
around lipid droplet in the cells as well as virions produced in the culture medium. Mutation
analyses of the core protein suggested that this multimer complex including the core would be
important for virus particle formation.
Keywords: Hepatitis C virus, particle formation, lipid droplet, core protein, multimer complex

Introduction
Hepatitis C virus (HCV) is a serious problem worldwide, with 3% of the world's population
chronically infected. The development of more effective and less toxic anti-HCV agents is desired
to eliminate the infection of this virus. To achieve this purpose, the life cycle of HCV has been
investigated using recombinant HCV culture system. Using this system, we recently found that
infectious viral particles of HCV are produced around lipid droplets (LD) in the cells(1). In thisstudy, we aimed to elicidate the precise mechanism of this viral particle formation and infectious
virus production from LD.

Results
We previously found that two types of HCV particles, showing different buoyant densities, areproduced in the culture medium in the recombinant HCV culture system. And one of the particles
with light buoyant density appeared to show the infectivity against naïve cells and to be produced
around LD in the cells, while another with heavy density did not show the infectivity and any
relationships with LD. We, therefore, ﬁrstly analyzed whether these two types of HCV particles aretransported to outside of the cells in same or different manners, by using several reagents blocking
subcellular protein transportations. Finally we found Brefeldine A (BFA), a drug known to block the
ER/Golgi apparatus protein transportation, inhibited the production of both types of HCV particles
into the culture medium. As BFA was also reported to affect on the production of LD and multiple
cellular events, we analyzed the efﬁciency of HCV genome replication, production of HCV proteins,
and the number and shape of LD in the recombinant HCV proliferating cells in the presence of BFA.
As the results, we did not see any mal effects on these events by BFA, suggesting BFA affected on
the transportation of virus particles via Golgi apparatus. Next we investigated the factors existing- 43 -

around LD and associated with HCV proteins in the cells producing infectious viral particles byproteome analysis of isolated LD fraction. Although we found several candidate proteins, knocking
down of a part of these factors in the cells producing infectious viral particles by using smallinterfering RNA did not affect the infectious particle production. Further analysis to find a realfactor(s) related with infectious virus particle production is underway. In the above experiment,
we observed that a part of HCV core protein (the core), which is an essential component of thenuclear capsid in the virion, is included in the multimer complex in the LD fraction. We found thesimilar multimer complexes with several molecular weights in the microsome fraction as well as thefraction containing HCV infectious particles in the culture medium. This suggested that the multimer
complex including the core protein plays some roles in the HCV particle formation. To identify the
responsible amino acid region of the core to form the multimer complex, we perform the deletionand the point mutational analysis in the core. We ﬁnally found a candidate amino acid responsible- 44 -

to the multimer complex formation. To examine whether the multimer complex formation isrequired for the viral particle formation, we introduced this mutation in the core encoding regionof recombinant HCV genome and examined the effect of this point mutation on the viral genome
replication, subcellular localization of the core, and infectious virus particle formation. The viralgenome replication and subcellular localization of the core on the LD were observed quite same as
those seen in the cells in which the wild type genome was replicationg. However, production of viralparticle was not detected in the culture medium of those cells.

Discussion & Conclusion
As we observed BFA inhibited the production of both infectious and uninfectious HCV particles
into the culture medium under the condition without affecting multiple cellular events except
ER/Golgi apparatus transportation, infectious virus particles produced around LD is likely to betransported to the outside the cell via ER and Golgi apparatus together with uninfectious particle.
This seem to indicate that the acquisition of infectivity was occurred, at least, during the particle
transport from around LD to ER. Although we tried to ﬁnd the factor responsible to the infectivityof the virus particle in the LD fraction from the cells producing HCV infectious particles, we did nothave a candidate molecule yet. However, we newly found that the core protein of HCV in the cells
is included in several multimer. This multimer complex may play an important role of virus particle
formation. Prevention of this multimer formation may become good target for development of novelanti-HCV strategy.

References(1) Yusuke Miyanari, Kimie Atsuzawa, Nobuteru Usuda, Koichi Watashi, Takayuki Hishiki, Margarita
Zayas, Ralf Bartenschlager, Takaji Wakita, Makoto Hijikata, Kunitada Shimotohno: The lipid droplet
is an important organelle for hepatitis C virus production., Nat. Cell Biol., 9 (9), 1089-1097, 2007

- 45 -

Regulation of genome and cellular functions
by the SUMO modiﬁcation system.
Hisato Saitoh
Department of Biological Sciences, Graduate School of Science and Technology,
Kumamoto University.
hisa@kumamoto-u.ac.jp

Abstract
Our findings suggest an expanded role for p150 as a SUMO-interacting factor and raise an
intriguing possibility that p150 plays an important role in promoting deposition of SUMO2/3 and/or
sumoylated proteins on chromatin ﬁbers during DNA synthesis in the context of DNA replication.
Keywords: SUMO, posttranslational modiﬁcation, histone chaperone, DNA replication, chromatin

Introduction
Chromatin assembly factor 1 (CAF1) is a histone chaperone that promotes chromatin assemblyin the context of DNA replication and repair. Its targeting to the sites of DNA synthesis involves aninteraction between a largest subunit, p150, and proliferating cell nuclear antigen (PCNA), a DNA
polymerase accessory factor. We report here human p150 interacts directly and preferentially withsmall ubiquitin-related modiﬁer 2/3 (SUMO2/3), for which a short polypeptide sequence 98-105 isessential and sufﬁcient.

Results
Identification of CAF1 p150 as a SUMO2/3-interacting protein. To identify a chromatinprotein(s) that interacts with SUMO, we performed a yeast two-hybrid screening using the Gal4SUMO-3G-SUMO-3G fragment as bait (see Materials and Methods) and isolated the cDNA
fragments containing a mouse homologue of the large subunit of histone chaperone CAF1 p150
(Figure 1A). As a step toward understanding the role of p150 to bind SUMO, we ﬁrst performed
an in vitro glutathione S-transferase (GST) pull-down assay to investigate whether binding afﬁnity
varied among SUMO paralogs, SUMO1, SUMO2 and SUMO3, and whether binding was direct.
When beads bound to GST-SUMO1, -SUMO2, -SUMO3, or GST-alone were incubated with a lysate
from the HEK293-p150 cells in which Flag-HA-tagged human p150 was constitutively expressed,
we found that p150 associated more efﬁciently with GST-SUMO2/3-beads than with GST-SUMO1beads (Figure 1B). p150 binding to SUMO-2/3 was direct, since bacterially expressed recombinantfull length (His)6-p150 could be retained on the GST-SUMO3 beads (Figure 1C).
Characterization of SUMO-interacting motif in p150. To determine the region responsible
for binding to SUMO2/3, we generated four kinds of N-terminal deletion mutants of human p150
(Figure 1A), and performed a GST pull-down assay. To this end, 7 amino acid residues of p150

- 46 -

Figure 1. CAF1-p150 is a SUMO-interacting protein(A) Schematic representation of human CAF1-p150, and summary of the GST pull-down assay. p150 deletion constructs
used in the experiments are represented as thin black lines. Numbers indicated on left sides of the lines correspond to aminoacid residues in p150. Table on the right summarizes interactions of each deletion mutant with (His)6-SUMO3 in the GST
pull-down assay. + indicates sufﬁcient binding, - indicates negligible binding. The previously reported domains that interact
with other proteins are indicated.
(B) p150 interacts with GST-SUMOs with a preference to GST-SUMO2/3. 500 µl of the HEK293-NFH-p150 cell lysate
was incubated with beads bound 3µg of GST-only, GST-SUMO1, GST-SUMO2 and GST-SUMO3, respectively. Following
incubation, proteins associated with the beads were analyzed by 6% SDS-PAGE followed by immunoblot assay, using theanti-Flag antibody. The total cell lysate (10% of the input) was loaded. Position of NFH-p150 is indicated by the arrowhead.
(C) Direct binding of p150 to SUMO2/3. 500 µl of the lysate from E. coli expressing recombinant full-length (His)6-p150
was incubated with beads bound to 5 µg of GST-only, GST-SUMO1, GST-SUMO2 and GST-SUMO3, respectively.
Following incubation, proteins associated with the beads were analyzed by 6% SDS-PAGE. The total bacterial lysate (10%of the input) was loaded on the left side of the gel. Proteins were visualized by immunoblot analysis using anti-(His)6
antibody. Position of (His)6-p150 is indicated by the arrowhead.
(D) GST-15098-105 binds to SUMO3. 500 µl of the lysate from E. coli expressing recombinant full-length (His)6-SUMO3
was incubated with beads bound to 5 µg of GST-only, GST-15098-105 and GST-15098-102, respectively. Following incubation,
proteins associated with the beads were analyzed by 15% SDS-PAGE. The total bacterial lysate (20% of the input) was
loaded on the left side of the gel. Proteins were visualized by immunoblot analysis using anti-(His)6 antibody. Position of(His)6-SUMO3 is indicated by the arrowhead.
(E) Sequence comparison with previously identified SUMO-interacting polypeptides. The sequences within gray region
indicate amino acid residues similar to the canonical SIM’s hydrophobic core (V/I-Χ-V/I-V/I). Black underline represents
the region similar to a negatively charged cluster of amino acids that often follows SIM’s hydrophobic core sequences.
(F) Amino acid sequence of human CAF-1 p15098-105 is highly conserved among vertebrate species. The sequences withinblack boxed region indicate amino acid residues similar to human p15098-105.

(p15098-105) were found to be sufficient for binding to GST-SUMO2/3 (Figure 1D). The results
demonstrated that substitution in the motif eliminated the interaction with SUMO3. Taken together,
these results indicate that p150 is a SUMO2/3-interacting protein and suggest that human p150965-975
is sufﬁcient and essential for interaction with SUMO2/3 in vitro.
Comparison of p15098-105 with SIMs from other species. Given that a database search detected the
peptide sequence p150965-975 is highly conserved among vertebrate species (Figure 1E), suggesting afunctionally important role of this amino acid sequences. When compared with the peptide sequencewith a recently described hydrophobic amino acid cluster of SUMO-interacting/binding motif (SIM)detected in PIAS1, PIASx, SAE2, PML IV, RanBP2/Nup358, thymine DNA glycosylase, we foundthat p150965-975 is well overlapped with the SIM, indicating that p150965-975 might represent a canonical
SIM (Figure 1F).
p150-SUMO2/3 interaction occurs in vivo. To examine whether p150 interacts with
SUMO2/3 through the interaction between p150965-975 SIM and SUMO2/3, wild-type FLAGtagged p150 and two FLAG-tagged p150 point-mutants, p150-D968A and p150-L969A,
respectively, were transiently expressed in HeLa cells (Figure 2A). In FLAG-p150 expressed- 47 -

Figure 2. SUMO2/3-p150 interaction is detectable in vivo.
(A)SUMO2/3,but not SUMO1, overlaps with p150 foci. Hela cells expressing FLAG-p150 were ﬁxed, and subjected
to indirect immunoﬂuorescence analysis using anti-FLAG antibody either with anti-SUMO1 (upper) or anti-SUMO2/3(lower) antibody. Insets on the right side of each picture show two-fold magniﬁcation of selected regions (white boxes).
Merged images are shown on the right side.
(B) SUMO2/3-p150 colocalization is impaired by a mutation in SIMp150.
Hela cells expressing FLAG-p150wt (upper lane), FLAG-p150I99A (second lane), FLAG-p150D100A (third lane) and
FLAG-p150109-938 (bottom lane) were ﬁxed, and subjected to indirect immunoﬂuorescence analysis using anti-FLAG
and anti-SUMO2/3 antibodies. Merged images are shown on the right side.

cells, we found a substantial number, but not all, of cells in which SUMO2/3 was enrichedin FLAG-p150 foci. In contrast, colocalization of SUMO2/3 with foci containing either
FLAG-p150-D968A or FLAG-p150L969A were barely observed (Figure 2B, upper panel).
Thus, these data provided evidence that MCAF1 interacted non-covalently with SUMO2/3 in vivo, and further demonstrated that p150965-975 SIM were necessary components forbinding to SUMO2/3.

Results
Identiﬁcation of CAF1 p150 as a SUMO2/3-interacting protein.
To identify a chromatin protein(s) that interacts with SUMO, we performed a yeast two-hybrid
screening using the Gal4-SUMO-3G-SUMO-3G fragment as bait (see Materials and Methods)
and isolated the cDNA fragments containing a mouse homologue of the large subunit of histone
chaperone CAF1 p150 (Figure 1A). As a step toward understanding the role of p150 to bind SUMO,
we ﬁrst performed an in vitro glutathione S-transferase (GST) pull-down assay to investigate whether
binding afﬁnity varied among SUMO paralogs, SUMO1, SUMO2 and SUMO3, and whether binding
was direct. When beads bound to GST-SUMO1, -SUMO2, -SUMO3, or GST-alone were incubatedwith a lysate from the HEK293-p150 cells in which Flag-HA-tagged human p150 was constitutively
expressed, we found that p150 associated more efficiently with GST-SUMO2/3-beads than with
GST-SUMO1-beads (Figure 1B). p150 binding to SUMO-2/3 was direct, since bacterially expressed
recombinant full length (His)6-p150 could be retained on the GST-SUMO3 beads (Figure 1C).

Characterization of SUMO-interacting motif in p150.
To determine the region responsible for binding to SUMO2/3, we generated four kinds of- 48 -

N-terminal deletion mutants of human p150 (Figure 1A), and performed a GST pull-down assay. To
this end, 7 amino acid residues of p150 (p15098-105) were found to be sufﬁcient for binding to GSTSUMO2/3 (Figure 1D). We then designed point mutations covering the motif to test their effect uponbinding to SUMO2/3 (Figure 1E). The results demonstrated that substitution in the motif eliminatedthe interaction with SUMO3. Taken together, these results indicate that p150 is a SUMO2/3interacting protein and suggest that human p150965-975 that is highly conserved among p150 orthologs
in vertebrates is sufﬁcient and essential for interaction with SUMO2/3 in vitro.
p150-SUMO2/3 interaction occurs in vivo
To examine whether p150 interacts with SUMO2/3 through the interaction between p150965-975 SIM
and SUMO2/3, wild-type FLAG-tagged p150 and two FLAG-tagged p150 point-mutants, p150D968A and p150-L969A, respectively, were transiently expressed in HeLa cells (Figure 2A). In
FLAG-p150 expressed cells, we found a substantial number, but not all, of cells in which SUMO2/3
was enriched in FLAG-p150 foci. In contrast, colocalization of SUMO2/3 with foci containing either
FLAG-p150-D968A or FLAG-p150L969A were barely observed (Figure 2B, upper panel). Thus,
these data provided evidence that MCAF1 interacted non-covalently with SUMO-2/3 in vivo, andfurther demonstrated that p150965-975 SIM were necessary components for binding to SUMO2/3.

Comparison of the interaction of SUMO3 with SIMs from p150, MCAF1 andSETDB1.
Given that a database search detected the peptide sequence p150965-975 is highly conserved amongvertebrate species (Figure 2B), suggesting a functionally important role of this amino acidsequences. When compared with the peptide sequence with a recently described hydrophobic aminoacid cluster of SUMO-interacting/binding motif (SIM) detected in PIAS1, PIASx, SAE2, PML IV,
RanBP2/Nup358, thymine DNA glycosylase, we found that p150965-975 is well overlapped with theSIM, indicating that p150965-975 might represent a canonical SIM (Figure 2A).
In previous studies, we and others identified SIM in MCAF1 and SETDB1. Given that CAF1,
MCAF1 and SETDB1 are suggested to cooperate in the context of MBD1-mediated heterochromatin
formation, it is of interest to test whether the afﬁnities of these proteins to SUMO are distinct. To
compare the mode of the interaction of SUMO3 with SIMp150, SIMMCAF1 and SIMSETDB1, we performed
an assay termed Bead Halo, which can detect molecular interactions of high and low affinity inreal time at equilibrium (REF and see Materials and Methods). As shown in Fig. 3A, GFP-SUMO3
accumulated on GST-SIMp150, GST-SIMMCAF1 and GST-SIMSETDB1 immobilized on the Sepharose
beads immobilized. By contrast, GFP protein with no SUMO-moiety, and the bead carrying GST
protein alone showed no signals, indicating that Bead Hallo assay detects specific interaction
between SUMO3 and each SIM. Furthermore, a semi-quantitative Bead Halo assay revealed thatthe afﬁnity of SUMO3-SIMMCAF1 interaction was ~5-fold higher than that of SUMO1-MCAF1-SIM
interaction, that is consistent with the previous data (REF and shirakawa personal communication).

- 49 -

Discussion & Conclusion
Using Bead Hallo method, it is demonstrated that the bona fide SUMO-interacting polypeptide
shows signiﬁcant but slightly lower afﬁnity than those of MCAF1, a previously reported SUMO2/3interacting chromatin protein involved in histone methylation and heterochromatin formation (datanot shown). Importantly the p150-SUMO2/3 interaction appeared to be evident at the replication fociwhen HA-FLAG-tagged p150 was overexpressed in a HEK293 human cell line (data not shown).
Although deficiency of p150 delocalized SUMO2/3, reduction of SUMO2/3 expression had little
effect on targeting of p150 to the foci containing PCNA. Thus, our ﬁndings suggest an expanded
role for p150 as a SUMO-interacting factor and raise an intriguing possibility that p150 plays animportant role in promoting deposition of SUMO2/3 and/or sumoylated proteins on chromatin ﬁbers
during DNA synthesis in the context of DNA replication.

References
1) Uchimura Y, Ichimura T, Uwada J, Tachibana T, Sugahara S, Nakao M, Saitoh H.
Involvement of SUMO modiﬁcaiton in MBD1- and MCAF1-mediated heterochromatin formation.
J Biol Chem. 2006 Aug 11;281(32):23180-23190.
2) Sekiyama N, Ikegami T, Yamane T, Ikeguchi M, Uchimura Y, Baba D, Ariyoshi M, Tochio H,
Saitoh H, Shirakawa M. Structure of the small ubiquitin-like modifier (SUMO)-interacting motifof MBD1-containing chromatin-associated factor 1 bound to SUMO-3. J Biol Chem. 2008 Dec
19;283(51):35966-35975.

- 50 -

Analysis of barrier function at heterochromatin region
Masaya Oki
University of Fukui
ma4sa6ya@u-fukui.ac.jp

Abstract
In this study, we focused the YCR076C and GIC1/YHR061C gene, not known of the function in theliving cell, were isolated by boundary screening. As a result, YCR076C was speciﬁcally combinedwith the constitution factor of the proteasomal 20S complex and got an experiment result to suggest
proteolysis system and participation with the heterochromatin boundary mechanism. In addition, it
developed that GIC1/YHR061C acted directly at the HMR left boundary region.
Keywords: Boundary, Heterochromatin, S.cerevisiae, Silencing, Histone

Introduction
There is the heterochromatin domain where gene expression is repressed strongly on thechromosome. Recently, when the gene should express was inserted in the heterochromatin domain
by chromosome translocation, and that various diseases were generated for having developed wasreported from a large number of laboratories. However, as for the mechanism of the molecular level
about the formation of the heterochromatin structure and the maintenance mechanism, there aremany unknown parts. We performed a systematic genomewide screen for proteins that may block the
spread of heterochromatin structure in yeast and isolated 55 proteins (1). In this study, we analyzed
YCR076C and YHR061C/GIC1, as isolated barrier proteins.

Results(1) Localization of YCR076C and GIC1/YHR061C protein in the cell.
To determine the localization of YCR076C, anti-YCR076C antibodies were prepared. By using
the anti-YCR076C antibodies, the wild-type cells were stained but there was no clear localization.
Therefore, we constructed Flag and GFP fused YCR076C and GIC1/YHR061C proteins, and stainedby anti-ﬂag antibodies and anti-GFP antibodies. However they were no clear localization.
(2) Analysis of interaction protein with YCR076C or GIC1/YHR061C.
We made a Flag fusion protein of the each Ycr076c, GIC1/YHR061C and refined interaction
protein and identiﬁed it by mass spectroscopy. The Ycr076c protein was speciﬁcally combined withthe constitution factor of the proteasomal 20S complex. It is the ﬁrst result to suggest proteolysis
system and participation with the heterochromatin domain boundary mechanism.
In addition, a lot of protein which is specifically combined with GIC1/YHR061C are
divided and I pay attention to interesting protein included in that and can go ahead
through the analysis more now.

- 51 -

(3) Analysis of heterochromatin barrier function in the living cells with ycr076c or gic1/yhr061c
deletion strain.
We constructed plasmid forgene destruction and, withhomologous recombination
technology, made ycr076c or
gic1/yhr061c gene deletionstrain. With these strains, I
confirmed it by chromatin
immunoprecipitation method(ChIP method) that I used antiSir3 antibodies for whether
a boundary function in theliving cells was lost (2). As aresult, a boundary was lost in
gic1/yhr061c gene disruption
strain in an HMR left boundary domain that was boundary one, and it developed that Sir3 protein
extended it (Figure 1). This result suggested that GIC1/YHR061C protein participated in the HMR
left boundary function directly. On the other hand, the inﬂuence was not seen in the formation of the
HMR left boundary domain in the ycr076c knockout strain.
(4) Analysis of the factor with the functional correlation.
GIC1/YHR061C includes theCdc42 binding domain thatis one of the small G protein.
There is GIC2 in budding yeast,
which has a CDC42 binding
domain equally. We constructeddouble deletion mutants,
and gic1/yhr061c∆ became synthetically lethal in combination with gic2∆ (Figure 2). This result
suggested that these proteins had correlated function.
(5) Domain analysis of GIC1/YHR061C.
We constructed boundary assay plasmid with CDC42 binding site or without it in GIC1/YHR061C.
As a result, boundary function in GIC1/YHR061C did not include a Cdc42 binding site, and an
experiment result to suggest that Cdc42 did not participate in the barrier was provided.

Discussion & Conclusion
We divided 55 genes with a function to stop the extension of the heterochromatin domain frombudding yeast all genes about 6,000.

- 52 -

In this study, I focused GIC1/YHR061C or YCR076C, which function was not yet known andanalyzed them. The together clear localization was not observed, as a result of indirect ﬂuorescencewith anti-Flag antibody and observation with EGFP in the living cell. An experiment result
to suggest that there was little protein dose in the living cell. In addition, as a result of having
speciﬁcally identiﬁed binding protein by mass spectroscopy, YCR076C was speciﬁcally interactedwith the constitution factor of the proteasomal complex which participated in proteolysis system.
A proteolysis system factor and the participation with the heterochromatin are not reported by thepresent, and it is an interesting experiment result. In addition, GIC1/YHR061C got a result to suggestthat it functioned directly in the HMR left boundary region, known as a yeast heterochromatin
domain.

References(1) Oki, M., Valenzuela, L., Chiba, T., Ito, T. & Kamakaka, RT. : Barrier proteins remodel and modify
chromatin to restrict silenced domains. Mol. Cell. Biol., 24 : 1956-1967, 2004
(2) Oki, M. & Kamakaka, RT. : Barrier function at HMR. Mol. Cell, 19 : 707-716, 2005

- 53 -

Analysis of regulatory mechanism of extracellular signals
by receptor-mediated endocytosis
Jiro Toshima
Tokyo University of Sciencejtosiscb@rs.noda.tus.ac.jp

Abstract
The yeast mating pheromone receptor, Ste2p, has been used as a model to investigate mechanismsof signal transduction, modiﬁcation, and endocytic internalization of GPCR. Using a ﬂuorescently
labeled mating pheromone derivatives (A594-a-factor), we have revealed that phosphorylation andsubsequent ubiquitination of the Ste2p facilitates α-factor recruitment to CCPs. We also demonstrated
that mono-ubiquitin binding proteins, Ent1/2p and Ede1p, mediate this recruitment.
Keywords: Endocytosis, Clathrin, Casein kinase, ubiquitin

Introduction
Many GPCRs are overexpressed in human cancers and deeply related to tumor progression. GPCRs
are currently the targets of ~30% drugs that can be used for the treatment of a wide range of humandiseases, and thus elucidating the mechanism of regulation of GPCR signaling is essential for thedevelopment of more effective and safer therapeutic agent.

Results
Using A594-α-factor (1) as a marker for ligand-bound Ste2p receptor, we found Ste2p is recruited
to CCPs in time-dependentl manner (Figure 1a and 1b). In contrast, no recruitment of the C-terminal
truncated version of Ste2p (Ste2pΔ300) to CCPs was observed (Figure 1a and 1b), indicating that thecytoplasmic region is required for Ste2 recruitment. The C-terminal region of Ste2p contains several
potential phosphorylation sites that could be phosphorylated by yeast casein kinase I homologues(Yck1p and Yck2p) (2). We identified six serine residues in the region, which correspond to thereported consensus sequences (D/E-X-X-S/T and S/T(P)-X-X-S/T ) recognized by casein kinases.
We puriﬁed GST-fused C-terminal fragment, performed in vitro kinase assay, and found that Ste2p
were efﬁciently phosphorylated by Yck2p (Figure 1c). Compared to wild-type cells, recruitment of
A594-α-factor to CCPs was much slowed at 20 min (~28.8%) in ste2-6SA (Figure 1d). Interestingly,
recruitment of Ste2p to CCPs was almost completely restored in cells expressing Ste2p-6SD,
replaced phosphorylation sites to aspartic acid to mimic phosphorylation state of Ste2p (Figure 1d).
These results clearly suggest that phosphorylation of Ste2p receptor is important for recruitment of
A594-α-factor to CCPs.
Using ste2-7KR cells, we next investigated the role of ubiquitination on Ste2p recruitment. In ste27KR cells, recruitment of A594-α-factor to CCPs was rarely observed at 20min, indicating that- 54 -

ubiquitination is dispensable
for Ste2p recruitement to
CCPs. Our next question waswhat protein mediates therecruitment of ubiquitinatedreceptor to CCPs. Previous
study reported that Ent1p,
mammalian Epsin, and Ede1p,
mammalian Eps15, containubiquitin-binding domain that
directly interacts with monoubiquitn and functions in
receptor internalization (3).
Recruitment of A594-α-factor
was significantly decreased in ent1ΔUIM ent2Δ ede1Δ strain at 20 min. This result indicates that
redundant function of Ent1, Ent2 and Ede1 is required for Ste2p recruitment to CCPs. These results
suggest that Ent1p UIM and Ede1p might work cooperatively to recruit ubiquitinated receptor to
CCPs.
Ste2-GFP has been shown to localize at endocytic compartments in the absence of ligand when
expressed in cells at steady state, indicating that Ste2p is constitutively endocytosed and transported
to the vacuole. We therefore examined how Ste2p is recruited to CCPs in ligand-independent manner,by imaging the behavior of Ste2-GFP on the plasma membrane. Ste2-GFP was recruited to CCPs in
the absence of ligand. This recruitment was much slower, compared to ligand-stimulated recruitment.
In contrast, intriguingly, Ste2p-7KR-GFP showed only ~34.5% co-localization with Sla1p at 60min.
This result indicates that ligand-independent recruitment of Ste2p also requires ubiquitination of
Ste2p.

Discussion & Conclusion
In conclusion, we revealed that yeast G protein-coupled Ste2p receptor is recruited to pre-exiting
CCPs by promoting the phosphorylation and subsequent ubiquitination. Clathrin adaptors, Ent1p,
Ent2p and Ede1p, seem to work cooperatively to recruit ubiquitinated Ste2p. Mamalian GPCRs,such as thyrotropin-releasing hormone receptor-1 (TRHR) and β1-adrenergic receptor (β1AR), havebeen shown to be recruited to CCPs in similar manner (4). Therefore, yeast and mammals appear toshare similar mechanisms for GPCR recruitment. This mechanism also could explain how GPCR
internalization is facilitated upon binding of its ligand. Low level of Ste2p ubiqutination mediates theslow constitutive receptor recruitment to CCPs, and upon ligand binding the recruitment is facilitatedby increased level of receptor ubiquitination. Which step, receptor recruitment, internalization, orboth of them, ubiquitination is required is next important question.

- 55 -

References
1. Toshima, J.Y., Toshima, J., Kaksonen, M., Martin, A.C., King, D.S., and Drubin, D.G. (2006). Spatial
dynamics of receptor-mediated endocytic trafﬁcking in budding yeast revealed by using ﬂuorescentalpha-factor derivatives. Proc Natl Acad Sci U S A 103, 5793-5798.
2. Hicke, L., Zanolari, B., and Riezman, H. (1998). Cytoplasmic tail phosphorylation of the alpha-factor
receptor is required for its ubiquitination and internalization. J Cell Biol 141, 349-358.
3. Shih, S.C., Katzmann, D.J., Schnell, J.D., Sutanto, M., Emr, S.D., and Hicke, L. (2002). Epsins andVps27p/Hrs contain ubiquitin-binding domains that function in receptor endocytosis. Nat Cell Biol 4,
389-393.
4. Puthenveedu, M.A., and von Zastrow, M. (2006). Cargo regulates clathrin-coated pit dynamics. Cell
127, 113-124.

- 56 -

Multifunctional rare earth metal-catalyzed asymmetric synthesis of
optically active small cyclic compounds
and application to biologically active compounds synthesis
Shigeki Matsunaga
The University of Tokyo
smatsuna@mol.f.u-tokyo.ac.jp

Abstract
Catalytic asymmetric synthesis of unnatural amino acids with a small strained ring was investigated.
By using the originally designed multifunctional rare earth metal catalysts, catalytic asymmetric
Mannich-type reaction of trichloromethyl ketones proceeded in high stereoselectivity. From Mannich
adducts, stereoselective reduction followed by cyclization sequence produced azetidine carboxylic
acids in good yield.
Keywords: asymmetric synthesis, asymmetric catalysis, amino acid, rare earth metal

Introduction
Optically active unnatural amino acids are important building blocks for the development of new
therapeutic agents. Therefore, the development of new efﬁcient methods for chiral unnatural aminoacid synthesis is highly demanded. The purpose of this study is to develop catalytic asymmetricmethods for the synthesis of unnatural amino acids with a small strained ring, azetidine carboxylic
acids. To realize the goal, we have designed Lewis acid/Brønsted base multifunctional rare earth
metal catalysts and trichloromethyl ketone nucleophile.

Results
For the stereoselective synthesis of azetidine carboxylic acids, we utilized trihcloromethyl ketones
as nucleophiles because trichloromethyl ketones had following two advantages, (a) the geometry
control of enolates from trichloromethyl ketones was easy due to steric bulkiness; (b) trichloromethyl
unit can be used as a key precursor for four membered ring construction.
The synthetic scheme is consisted of following three steps. Step (1): Catalytic asymmetric Mannichtype reaction of imines with trichloromethyl ketones promoted by Lewis acid/Brønsted basemultifunctional rare earth metal catalysts. Step (2): Stereoselective reduction of Mannich adducts
to produce trichloromethyl carbinol moiety. Step (3): Construction of strained four memberedazetidine ring under basic conditions. To realize the first key catalytic asymmetric Mannich-type
reaction, we designed two types of rare earth metal catalysts. For the syn-selective Mannich-type
reaction, lanthanum aryloxide-iPr-pybox + lithium aryloxide combined catalyst was the best. synMannich adducts were obtained from various aryl, heteroaryl, alkenyl, and alkyl imines in >99-72%
yield, syn/anti = >30:1-8:1, and 98-92% ee (Figure 1). For the anti-selective Mannich-type reaction,- 57 -

a lanthanum aryloxide-bidentate phosphine oxide complex was the best, giving anti-Mannich
adducts from various aryl, heteroaryl, alkenyl, and alkyl imines in 98-64% yield, anti/syn = >30:114:1 (Figure 2). Enantioselective reactions using a chiral lanthanum aryloxide-bidentate phosphineoxide complex gave products in up to 72% ee. Further improvement of enantioselectivity in antiselective reactions is under investigation. The second stereoselective reduction of Mannich adducts
proceeded with high selectivity under optimized reaction conditions for each stereoisomers, giving
products in good yields with excellent diastereoselectivity. For the reductions, DIBAL, DIBAL withtriphenylphosphine oxide additive in a ratio of 1:2, and LiAlH4 were utilized. The third cyclization
step proceeded smoothly by the treatment of products with aq NaOH at room temperature. The
cyclization proceeded through a highly reactive oxirane intermediate, and four membered ring
formed predominantly over ﬁve membered ring. After methylation with TMSCHN2, azetidine esters
were obtained in 80-71% yields.

To gain insight of the catalytic reactions in the ﬁrst step, we performed several mechanistic studies.
For the syn-selective reaction, lanthanum aryloxide-iPr-pybox + lithium aryloxide combined catalyst
was the most reactive, but the reaction also proceeded in similar enantioselectivity in the absenceof lithium aryloxide. Thus, we assumed that the lithium aryloxide is useful to accelerate the rate
determining enolate formation step, possibly by forming more Brønsted basic lanthanum/lithium atecomplex. Because the trichloromethyl moiety is very bulky, the geometry of the enolate is believed to
be ﬁxed as Z-form. Thus, the syn-adducts were obtained through cyclic transition state. For the antiselective reaction, the reaction pathway should be controlled via acyclic transition state in analogy to
Lewis base mediated anti-selective Mannich-type reactions. To realize the desired reaction, the stericbulkiness around the lanthanum metal center was important. Therefore, sterically hindered bidentate
achiral phosphine oxide additive played a key role to realize the anti-selective reactions.

- 58 -

Discussion & Conclusion
In summary, we have established efﬁcient catalytic asymmetric synthesis of unnatural amino acids
with a small strained ring, azetidine carboxylic acids. For the ﬁrst key Mannich-type reactions, twotypes of new rare earth metal catalysts were successfully developed. The present methods provide
novel stereoselective synthetic approaches for fully substituted azetidine carboxylic acids, whichwere unless otherwise difﬁcult to synthesize. With the present methods, diverse azetidine carboxylic
acids are now available, and so can be used for the design, synthesis, and screening of new
biologically active molecules. Further investigations towards the development of new therapeutic
agents using the fully substituted azetidine carboxylic acids are ongoing.

- 59 -

Synthetic studies on maitotoxin, a marine polycyclic ether
Tadashi Nakata
Tokyo University of Science
nakata@rs.kagu.tus.ac.jp

Abstract
Maitotoxin, isolated from the dinoflagelate Gambierdiscus toxicus, is the most toxic and largest
natural product. Several useful methods for construction of polycyclic ether ring system weredeveloped towards the synthesis of maitotoxin. Based on these methods, syntheses of several
segments of maitotoxin were accomplished.
Keywords: Polycyclic ether, synthesis, methyl introduction, hydroxyl inversion

Introduction
Maitotoxin, isolated from the dinoﬂagelate Gambierdiscus toxicus, is one of the most structurally
awe-inspiring and largest natural products ever isolated. The giant structure of maitotoxin contains
32 fused cyclic ether rings, 28 hydroxyl groups, 2 sulfates, 21 methyl groups, and 98 chiral centers.
The skeletal novelty, complexity, and biological activity have attracted the attention of both chemists
and biologists. We have studied new and efﬁcient methods for construction of polycyclic ether ringsystems and the synthesis of several segments of maitotoxin.

Results
We have already developed several efficient methods for the construction of cyclic ethers basedon (1) rearrangement-ring expansion, (2) endo-cyclization of styrylepoxide, (3) endo-cyclizationof methylepoxide, (4) SmI2-induced cyclization of β-alkoxyacrylate, (5) SmI2-induced cyclizationof β-alkoxyvinylsulfone, (6) SmI2-induced cyclization of β-alkoxyvinylsulfoxide. Furthermore,convergent methods for the synthesis of polycyclic ether have been developed based on (1) acetylidetriflate coupling followed by reduction of diacetal, (2) ester coupling followed by intramolecular
Barbier reaction of iodo-ester, (3) acetylide-aldehyde coupling followed by Michael cyclization ofhydroxy-ynone, and (4) ester coupling followed by intramolecular acyloin reaction.
In this project, we have newly developed efficient synthetic methods towards the synthesis ofmaitotoxin : i. e., (1) synthetic method for 2,3-trans-2-methyl-tetrahydropyran-3-ol and oxepan3-ol by unique insertion of a methyl group, (2) synthetic method for 2-methylenetetrahydropyran3-ol derivative, a key segment for the convergent synthesis of polycyclic ethers based on SuzukiMiyaura coupling, via SmI2-induced cyclization of β-alkoxysulfone or β-alkoxysulfoxide, (3) antiselective coupling by aldehyde and propenyllsulfoxide, (4) intramolecular oxy-Michael cyclizationof (2Z,4Z)-dienoic ester for construction of 2,6-anti-tetrahydropyran, and (5) inversion of hydroxyl
group.

- 60 -

Based on these methods, we have synthesized the key segments of maitotoxin. (1) Starting fromthe C-ring, which was prepared from 2-deoxy-D-ribose, the CDEF-ring was synthesized by using
consecutive SmI2-induced cyclizations of β-alkoxyacrylate and inversion of the hydroxyl group. (2)Starting from the G-ring, which was prepared from D-glucose derivative, the H- and I-rings weresynthesized by consecutive SmI2-induced cyclizations. The diol of the side chain was then introduced
by Sharpless asymmetric dihydroxylation. Thus, the synthesis of the GHI-ring system having
a 1,2-diol side chain was accomplished. (3) Starting from the K-ring, which was prepared from
D-glucose derivative, the J-ring was synthesized by Sharpless asymmetric dihydroxylation followed
by stereoselective allylation to acetal. Then, the I- and H-rings were constructed by SmI2-induced
cyclization of β-alkoxyacrylate and β-alkoxyvinylsulfoxide, respectively. The G-ring was constructedby hydroxylation of α,β-unsaturated δ-lactone and intramolecular oxy-Michael addition. Thus, the
GHIJK-ring was stereoselectively synthesized. (4) Recently, Galimore and Spencer questioned thestereochemistry of the J/K ring and proposed another possibility of syn-trans-conﬁguration. Murata
and Yasumoto originally reported anti-trans-conﬁguration as the J/K-ring. Recently, Nicolaou et al.
supported the originally assigned structure by Murata and Yasumoto through the synthetic work.
We also examined the stereochemistry of the J/K ring. We synthesized two isomers of HIJK-ringsystem, i.e., originally and newly proposed structures. Comparison of the 13C-NMR data of our
synthetic isomers with those of maitotoxin supported the original structure proposed by Murata andYasumoto. (5) Starting from the V-ring, the U- and T-rings were synthesized by 6-endo-cyclization of
methylepoxide. SmI2-induced cyclization of β-alkoxyacrylate afforded S-ring oxepane, and methylgroup at the C-2 position was inserted by newly developed method. Thus, the STUV-ring system
was synthesized. (6) The V-ring iodo-alcohol was synthesized from 2-deoxy-D-ribose, while theRS-ring carboxylic acid was constructed by SmI2-induced cyclization of β-alkoxyacrylate. The RSring carboxylic acid and the V-ring iodo-alcohol were coupled by esterification, and the product,iodo-ester, was treated by t-BuLi followed by dehydration of the resulting hemiacetal afforded
U-ring dihydropyran, which was treated by OsO4 and then EtSH to give O,S-acetal. After MCPBA
oxidation, treatment with Me3Al introduced a methyl group of the U-ring. Ring-expansion of the
S-ring with TMSCHN2 afforded S-ring oxepane, which was converted into O,S-acetal. MCPBA
oxidation followed by Me3Al treatment effected insertion of a methyl group at ST-ring junction.

- 61 -

Thus, the RSTUV-ring was synthesized. (7) The LM(NO)-ring was synthesized based on SmI2induced cyclization of β-alkoxysulfoxide. (8) The UV-WXYZ ring was synthesized based on the
SmI2-induced cyclization of β-alkoxyacrylate.

Discussion & Conclusion
In conclusion, we have developed new synthetic methods for construction of polycyclic ethers,
namely, (1) unique methyl-insertion reaction for construction of C-2 methyl cyclic ethers, (2)efficient synthesis of 2-methylene tetrahydropyran, (3) anti-selective coupling by aldehyde andpropenyllsulfoxide, (4) efﬁcient synthesis of 2,6-anti-tetrahydropyran, and (5) new inversion reactionof hydroxy-ester. These methods would be useful for synthesis of polycyclic ethers.
Based on these developed methods, we have studied syntheses of several segments of maitotoxin.
The syntheses of the CDEF-ring, the GHI-ring having 1,2-diol side chain, the HIJK-ring and its
stereoisomer, the GHIJK-ring, the LM(NO)-ring, the STUV-ring, the RSTUV-ring, and the UVWXYZ-ring were synthesized.
The synthetic segments would be useful for chemical biology study.

References(1) Synthesis of 2-methylene-tetrahydropyran-3-ol derivative, a key segment for convergent synthesis ofpolycyclic ethers based on Suzuki-Miyaura coupling.
T. Kimura, Y. Miyagawa, S. Ozawa, M. Hagiwara, T. Nakata, Heterocycles, 77, 829-835 (2009).
(2) New synthetic method for 2,3-trans-2-methyl-tetrahydropyran-3-ol and oxepan-3-ol by uniqueinsertion of a methyl group.
A. Kimishima, T. Nakata, Tetrahedeon Lett., 49, 6563-6565 (2008).
(3) Stereoselective synthesis of maitotoxin GHI-ring system having a 1,2-diol side chain.
M. Nagatomo, T. Nakata, Heterocycles, 76, 1069-1074 (2008).

- 62 -

Molecular analysis of temperature adaptation of Plasmodium parasite
Hirotaka Kanuka
National Research Center of Protozoan Diseases,
Obihiro University of Agriculture and Veterinary Medicine
kanuka@obihiro.ac.jp

Abstract
One of the most striking features of Plasmodium, the malaria parasite, is its adaptation totemperature ﬂuctuation throughout the life cycle. We identiﬁed a gene set upregulated at temperature
shift and found ADF (actin depolymerizing factor) is required for Plasmodium oocyst development
in mosquito. Rapid change of calcium concentration was observed during temperature-induced
morphogenesis of Plasmodium sporozoite. These observations suggest that intrinsic adaptation
mechanism to temperature shift between vector and host strikingly commits to Plasmodium
development.
Keywords: parasite, temperature, infection, malaria

Introduction
Mosquitoes are carriers of various pathogens. When feeding on blood to support oogenesis,
mosquitoes ingest pathogens in the circulatory system of vertebrate. In subsequent blood meals, themosquitoes transmit the pathogens to new vertebrate hosts. Anthropod-borne pathogens must adapt
dual environments for its successful infection. A genus Plasmodium, the causative agent of malaria,
has speciﬁc developmental stage in both mammalian host and vector mosquito. For its successful
development, temperature adaptation is supposed to play critical role. However, its molecularmechanism is yet to be elucidated. In this study, we particularly focused on the vector-stage of theparasite to elucidate its robustness mechanism against temperature.

Results
Previous studies suggested that temperature adaptation in Plasmodium has two aspects: switching androbustness. Plasmodium has a certain range of temperature for its successful development and changes
its range when transits between host and vector. The ﬁrst step in dissecting the robustness must be tocapture robust responses to temperature ﬂuctuation. At ﬁrst, we observed that in vitro Plasmodium
ookinete development is robust at the range from 19 to 27oC. On the other hand, Plasmodium ookinetes
failed to form oocysts at higher temperature (Figure 1). In order to observe intercellular responses toenvironmental changes, we employed calcium as a parameter and generated the transgenic Plasmodium
expressing calcium indicator protein Yellow Cameleon (YC3.60) (Nagai et al., 2004). The developmentof malaria parasite in host body starts with the transformation of sporozoites characterized by the loss of- 63 -

polarity. This transformationof Plasmodium sporozoites into
exoerythrocytic form (EEF)
occurs without host cells in vitro(Kaiser et al., 2003), suggestingthat sporozoite senses thechange of environment andstarts the transformation at thevery timing of invasion into host
body in vivo. We revealed thattemperature shift to 37oC is
sufﬁcient to induce sporozoite
transformation into early EEF.
The extracellular transformation had proceeded further to intermediate EE-like forms displaying
a more extended bulb and further retraction of the armlike remnants of the sporozoite cell body,closely resembling the progression of transformation observed during intracellular transformation.
The extended bulb showed rapid and drastic change of calcium concentration, suggesting that Ca2+dependent cytokinesis in Plasmodium transformation may be regulated by temperature shift. Next, tounderstand the molecular mechanism according to the different temperature, suppression subtractive
hybridization (SSH) was carried out. Several genes were identiﬁed by SSH comparing mRNA ofookinete cultured at 27oC versus that of 19oC. Among those candidate genes, up-regulation of ser/thr
protein kinase, hsp70 and thioredoxin-like protein was conﬁrmed by RT-PCR. We selected ADF/cofﬁlin(actin depolymerization factor), which is one of the essential proteins that accelerate turnover of actin
ﬁlaments by depolymerizing actin monomers from the pointed ends and severing ﬁlaments. The activityof ADF/coﬁlin is regulated by several mechanisms, including phosphorylation, dephosphorylation,
pH, phosphoinositides, and calcium. To address the in vivo function of ADF, we inactivated thegene in wild-type (WT) ANKA strain of Plasmodium berghei by double-cross-over recombination.
The endogenous ADF was replaced by a marker gene, DHFR. Disruption of ADF did not affectsurvival and development at both blood and sexual stages. In vector stages, we observed formation ofknock-out ookinete in mosquito midgut as well as wild-type. However, after midgut traverse, thesemutant ookinetes signiﬁcantly lostthe ability to transform into oocysts(Figure 2). These data suggest that
intrinsic adaptation mechanism totemperature shift between vector andhost directly commits to Plasmodium
development.

- 64 -

Discussion & Conclusion
The life cycle of the malaria parasite Plasmodium goes through three developmental stages(schizogony, gametogony and sporogony), each of which presents different environmental constraints
that must be met by an adaptive response in the parasite. Here we show that thermoregulation, in which
the transcription of select genes is upregulated at temperatures shift, is crucial to the developmental
transition that occurs during the transmission of Plasmodium from human to mosquito. Temperature
control is important with regard to malaria, given that vacillating ambient temperatures affect the rateof development of Plasmodium in the mosquito. Our results provide a framework for understanding
how the malaria parasite responds to its environment, as well as indicating how this process might beinvestigated further.

References
1. Kaiser K, Camargo N, Kappe SH. J Exp Med. 197(8):1045-50, 2003
2. Nagai T, Yamada S, Tominaga T, Ichikawa M, Miyawaki A. Proc Natl Acad Sci U S A. 101(29):105549, 2004

- 65 -

Genetic mutations associated with DOK7 myasthenia
and abnormal Dok-7/MuSK signaling
Yuji Yamanashi
Institute of Medical Science, University of Tokyoyyamanas@ims.u-tokyo.ac.jp

Abstract
Dok-7 is a cytoplasmic activator of muscle-specific receptor tyrosine kinase (MuSK). These
proteins are required for neuromuscular junction (NMJ) formation. Mutations in DOK7 underlie
a congenital myasthenic syndrome (DOK7 myasthenia) associated with NMJ synaptopathy. The
overwhelming majority of patients with DOK7 myasthenia have at least one allele with a mutationthat causes a truncation in the C-terminal moiety. In this study, we found that a nuclear export signal
and two SH2 target motifs in the C-terminal region play important roles in MuSK activation. Also,
we found that Dok-7 directly interacts with the cytoplasmic portion of MuSK and activates thereceptor kinase.
Keywords: myasthenia, Dok-7, protein-tyrosine kinase

Introduction
The neuromuscular junction (NMJ) formation is orchestrated by the muscle-speciﬁc receptor kinase
MuSK. The cytoplasmic adaptor-like protein Dok-7 is an essential activator of MuSK and required
for NMJ formation (1). DOK7 mutations are the cause of an NMJ disorder, DOK7 myasthenia,
which is usually associated with at least one allele with a mutation that causes a truncation in the
C-terminal moiety (2). Here, we examined roles of a nuclear export signal and two SH2 target
motifs in this region. Also, we addressed how the cytoplasmic protein Dok-7 activates receptor
kinase MuSK, which is believed to be activated by an extracellular activator Agrin.

Results
The most common mutation, 1124_1127dupTGCC, fond in the vast majority of patients with
DOK7 myasthenia eliminates two SH2 target motifs in the C-terminal moiety (2). We conﬁrmedthat the SH2 domains of the adaptor protein CrkII bind to these SH2 target motifs upon tyrosine
phosphorylation, indicating that they are functional (3). Because mutations in the SH2 target motifs
partially but significantly impaired MuSK activation, they are important for Dok-7’s ability toactivate MuSK. However, a DOK7 myasthenia-associated mutant Dok-7, which lacks the entire
C-terminal moiety, does not activate MuSK at all (3). Therefore, we sought an additional motif andfound a CRM1-dependent nuclear export signal (NES) in the C-terminal region. Indeed Dok-7 andits mutants having a mutation in the NES are localized mainly in the cytoplasm and the nucleus,
respectively. Also, Dok-7 mutant lacking the NES did not activate MuSK. Together, we concluded- 66 -

that Dok-7 requires the NES to move out from the nucleus to interact with the cell surface receptor
MuSK (3). Once Dok-7 activates MuSK and gets phosphorylated in turn, it may recruit unknown
factor(s) via the SH2 target motifs to fully activate MuSK. It should be noted that a Dok-7 mutant
harboring a large deletion in the PH domain was cytoplasmic but the PH domain fused with EGFP
accumulated in the nucleus, indicating that the PH domain of Dok-7 has the nuclear localization
activity (3).
Given that receptor tyrosine kinases are believed to transduce extracellular cues to intracellularsignaling, it is puzzling how the cytoplasmic adaptor-like protein Dok-7 could activate MuSK. We
examined if the extracellular domain and/or the transmembrane region of MuSK are required for
Dok-7-mediated activation in 293T cells, which lack endogenous Dok-7 or MuSK. The forced
expression of Dok-7 and a mutant MuSK lacking both extracellular and transmembrane portions
resulted in strong activation of MuSK to a degree comparable to that of full-length MuSK,
suggesting that Dok-7 directly interacts with the cytoplasmic region of MuSK and activates thereceptor tyrosine kinase (4). To test this notion and to exclude possible inﬂuences from other cellularcomponents, we performed in vitro phosphorylation assays with purified, bacterially expressed
cytoplasmic region of MuSK (MuSK-cyt) and Dok-7. After an extended period of phosphorylation
reaction, tyrosine phosphorylation of MuSK-cyt was faintly detectable in the absence of Dok7. However, phosphorylation of MuSK-cyt was markedly elevated in the presence of Dok-7.
Interestingly, the tyrosine phosphorylation of Dok-7 increased in parallel with that of MuSK-cyt. In
addition, a kinase inactive form of MuSK-cyt was not phosphorylated even in the presence of Dok7, indicating tyrosine phosphorylation is induced by catalytic activity of MuSK-cyt. Furthermore, as
was observed with intact MuSK in cultured myotubes, MuSK-cyt was not activated by an N-terminal
deletion mutant of Dok-7 lacking the PH domain, which is required for MuSK activation in cells.
Taken together, we concluded that Dok-7 directly interacts with the cytoplasmic portion of MuSK
and activates the receptor tyrosine kinase (4). Consistently, the forced expression of Dok-7 in theskeletal muscle of living mice induced strong activation of MuSK and elevated NMJ formation in
the central region of muscle (4).

Discussion & Conclusion
Our experiments indentiﬁed a single NES and two SH2 target motifs in the C-terminal region of
Dok-7 as key elements for MuSK activation and subsequent NMJ formation (3). Because the lossof the NES appears to be the loss-of-function mutation but the loss of the SH2 target motifs appears
to be the reduction-of-function mutation, it is predicted that there should be no patient homozygous
for the former mutation. Indeed, such a patient has not been reported whereas not a few patients arehomozygous for the latter mutation, which causes the loss of the SH2 target motifs (5, 6, 7).
It is of note that mice with a Dok-7 transgene, which overexpresses Dok-7 in the skeletal muscle,
showed no abnormality in the motor function while they have increased number of NMJs in
the correct, central region of muscle (4). Thus, overexpression of Dok-7 in the skeletal muscle- 67 -

of patients with DOK7 myasthenia, which is associated with small and simplified NMJs, may
ameliorate muscle weakness by elevated activation of MuSK from the inside of cells.

References
1. K. Okada et al., Science 312, 1802 (2006)
2. D. Beeson et al., Science 313, 1975 (2006)
3. J. Hamuro et al., J. Biol. Chem. 283, 5518 (2008)
4. A. Inoue et al., Science Signaling 2, ra2 (2009)
5. J. S. Muller et al., Brain 130, 1497 (2007)
6. J. Palace et al., Brain 130, 1507 (2007)
7. D. Selcen et al., Ann. Neurol. 64, 71 (2008)- 68 -

Development of screening methods for synthetic ligands
based on speciﬁc ﬂuorescent labeling of target proteins
Hirokazu Tamamura
Tokyo Medical and Dental University, Institute of Biomaterials and Bioengineering
tamamura.mr@tmd.ac.jp

Abstract
Development of CXCR4-speciﬁc ligands is an important issue in chemotherapy of HIV infection,
cancer metastasis, and rheumatoid arthritis and numerous potential ligands have been developed todate. However, it is difﬁcult to assess their binding mode and speciﬁcity because of uncertainties
in the structure of the CXCR4-ligand complexes. To address this problem, we have synthesized
fluorophore labeled Ac-TZ14011, which is derived from T140, a powerful CXCR4 antagonist.
Binding of Ac-TZ14011 to CXCR4 on the cell membrane was observed by ﬂuorescence microscope,and analysis of the binding data produced IC50 values of several ligands comparable to those obtainedin RI-based assays.
Keywords: CXCR4 antagonists, ﬂuorophore, imaging, high-throughput screening

Introduction
The interaction of the chemokine, CXCL12 with CXCR4 has been shown to be correlated withcancer progression and CD4+ T cell accumulation in the rheumatoid arthritis synovium. CXCR4
is also known as the second receptor of X4-type HIV-1, and numerous ligands for CXCR4 derived
from natural and synthetic compounds have been identiﬁed as inhibitors of HIV infection and cancer
metastasis. [125I]-CXCL12 has been utilized as a competitor in the assays to evaluate the CXCR4binding activity of synthetic compounds such as T140, its derivatives, KRH-1636, and AMD3100.
Experimental methods utilizing radioisotopes (RI) have advantages in the high resolution of theassays. This article describes the synthesis and use of ﬂuorophore labeled Ac-TZ14011 to analyze the
CXCR4 binding of ligands at the cell membrane and to determine the IC50 values of ligands.

Results
Ac-TZ14011, a derivative of T140 optimized for CXCR4 binding and stability in vivo by functionalgroup substitutions, was synthesized as described previously [1, 2]. The D-Lys8 was selectively
labeled with TAMRA or ﬂuorescein. A hexamethylene group was incorporated into the TAMRA or
ﬂuorescein derivative to maintain an appropriate distance between T140 residues and the ﬂuorophore.
Residues critical to the CXCR4 binding activity of TZ14011 are Arg2, Nal3, Tyr5, and Arg14, and wereassessed by screening of amino acid substitution of T140. On the basis of the previously determinedinteraction between Ac-TZ14011 and CXCR4, the ﬂuorophores labeled at D-lysine 8 were assumed
not to inhibit binding of ﬂuorescent-Ac-TZ14011 to CXCR4. To investigate if ﬂuorescent labeled- 69 -

Ac-TZ14011 maintains binding activity against CXCR4, the IC50 values of peptides were estimatedby competitive assays against [125I]-CXCL12 binding. In this assay, the IC50 of T140 was 3.7 nM.
The IC50 values for ﬂuorescein- or TAMRA-labeled Ac-TZ14011 were 14 and 11 nM, respectively.
These values indicated that ﬂuorophore labeling does not inhibit binding of Ac-TZ14011 as reported
elsewhere about binding of TAMRA-Ac-TZ14011. The binding of TAMRA-Ac-TZ14011 to a cell
membrane was observed with a laser-scanning confocal microscope to determine the speciﬁc bindingof Ac-TZ14011 to CXCR4. The CXCR4-GFP fusion protein was stably expressed in the NP-2 cellline, and TAMRA-Ac-TZ14011 binding to CXCR4 was clearly observed at the membrane in theabsence of competitors (Fig. 1 left). To assess the speciﬁc binding of ligands, excess unlabeled AcTZ14011 was added to the medium with TA MRA-Ac -TZ14011. Upon addition of Ac-TZ14011,
weak fluorescence intensity was observed on the cell membrane or cytoplasm (Fig. 1 center).
Vesicles observed in the cytoplasm show internalization of CXCR4 receptors induced by bindingof the ligands, These results indicate the speciﬁc binding of TAMRA-Ac-TZ14011 to CXCR4. To
evaluate the binding speciﬁcity for CXCR4 across the different kinds of GPCRs, HeLa cells, whichstably express CD4-CCR5, were utilized for microscopy assays.

Results
The binding of TAMRAAc- TZ14011 was observed as for the NP-2 CXCR4-GFP cell line (Fig. 1right).

Fig.1. Confocal microscopy assays of TAMRA-Ac-TZ14011 binding to CXCR4. The signals of GFP and TAMRA are
displayed in green and red, respectively. (left) Binding to NP2-GFP-CXCR4 cells. (center) Competitive binding toNP2 cells with excess amount of Ac-TZ14011. (right) Binding to HeLa-CD4-CCR5 cells.

To investigate the utility of fluorescein-labeled Ac-TZ14011, cell-based binding assays were
performed. In this binding assay, ﬂuorescein-Ac-TZ14011 was utilized as a competitor to derivativesof FC131 and the dipicolylamine-p-xylene Zn(II) complex [Zn2+-(Dpa)-p-Xyl] that were developed
as CXCR4antagonists. The binding constants of these compounds were previously estimated bycompetitive assays with [125I]-CXCL12. As a result, larger values of IC50 than those in the previous
assays were observed (Table 1). The difference of the binding constants of competitors was assumed
to be a reﬂection of the difference of IC50 values in the assays. It is especially interesting that the- 70 -

Table 1. Kd Values Determined by RI-Competition and Fluorescent Probe Competition Assays

values of IC50 as determined by ﬂuorescent- and RI-competition assays are clearly correlated. It was
clearly indicated that binding activity of compounds can be estimated by binding inhibition assays
conducted at a constant concentration of compounds.
In the application of high-throughput screening for CXCR4 ligands, it is important to be able
to rapidly determine CXCR4 binding activity. To test whether fluorescein-Ac-TZ14011 could be
useful as a ligand in high-throughput screening, binding inhibition analyses at constant compoundconcentrations were performed. Twenty-four derivatives of a cyclic pentapeptide, FC131 [3], wereprepared for the analyses as described previously. The conditions used were the same as in thebinding experiments shown in Figure 3 except that the compound concentration was kept constant at
2 µM. Nine compounds were found to induce >75% inhibition at this concentration.

Discussion & Conclusion
Structure-activity relationships of ligands for CXCR4 have been well studied, although relatively
few known ligand pharmacophores have been studied because of the difﬁculty associated with theanalysis of receptor-ligand interactions.
In the application of high-throughput screening for pharmacophores of CXCR4 ligands, it isimportant to be able to determine IC50 values rapidly. To test whether ﬂuorescein-Ac-TZ14011 could
be useful as a ligand in high-throughput screening, binding inhibition analyses at constant compoundconcentrations were performed.
Our results strongly indicate that ﬂuorescence-based ligand binding assays could be useful in the
exploration of novel pharmacophores for CXCR4 ligands and that such compounds have promise
as therapeutic agents for AIDS, breast cancer metastasis, and rheumatoid arthritis. Furthermore, thismethodology is applicable to the design of ligands for other GPCRs.

- 71 -

References
1. Tamamura, H., Omagari, A., Oishi, S., Kanamoto, T., Yamamoto, N., Peiper, S. C., Nakashima, H.,
Otaka, A., and Fujii, N. (2000) Bioorg. Med. Chem., Lett. 10, 2633–2637.
2. Tamamura, H., Hiramatsu, K., Mizumoto, M., Ueda, S., Kusano, S., Terakubo, S., Akamatsu, M.,
Yamamoto, N., Trent, J. O., Wang, Z., Peiper, S. C., Nakashima, H., Otaka, A., and Fujii, N. (2003)
Org. Biomol. Chem., 1, 3663–3669.
3. Tanaka, T., Tsutsumi, H., Nomura, W., Tanabe, Y., Ohashi, N., Esaka, A., Ochiai, C., Sato, J., Itotani,K., Murakami, T., Ohba, K., Yamamoto, N., Fujii, N., and Tmamura, H. (2008) Org. Biomol. Chem.
6(23), 4374-4377.
4. Nomura, W., Tanabe, Y., Tsutsumi, H., Tanaka, T., Ohba, K., Yamamoto, N., and Tmamura, H. (2008)
Bioconjugate Chem., 19, 1917-1920- 72 -

Reconsideration of the concept of the unitary psychosis using new
animal models
Kohichi Tanaka
Tokyo Medical and Dental University
tanaka.aud@mri.tmd.ac.jp

Abstract
Recent data suggest that excessive glutamatergic signaling in the prefrontal cortex may contributeto the pathophysiology of schizophrenia. The glial glutamate and aspartate transporter (GLAST)
regulates extracellular glutamate levels via uptake into glia, but the consequences of GLAST
dysfunction for schizophrenia are largely unknown. We showed that GLAST knockout (KO) mice
consistently showed locomotor hyperactivity to a novel environment. The locomotor hyperactivityinducing effects of the NMDAR antagonist MK-801 was exaggerated in GLAST KO relative to WT.
In addition, KO mice showed poor nesting behavior, abnormal sociability and impaired learning.
Schizophrenia-related abnormalities in GLAST KO raise the possibility that loss of GLASTmediated glutamate clearance could be a pathophysiological risk factor for the disease.
Keywrds: Glutamate, schizophrenia, GLAST, NMDA, mGluR2/3

Introduction
There is s a growing body of evidence implicating excessive glutamatergic signaling in thepathophysiology of schizophrenia. mGlu2/3 agonists, that negatively modulate glutamate
release and counteract spillover, has shown to have antipsychotic efficacy in human subjects
with schizophrenia. Glutamate receptor signaling is tightly regulated by five different glutamate
transporters which function to clear glutamate from the extracellular space. Glutamate transporter
GLAST has been shown to be mutated in patients with schizophrenia. However, the consequences of
GLAST dysfunction for schizophrenia are largely unknown. The present study assessed the role of
GLAST in schizophrenia-related behaviors via phenotypic analysis of GLAST KO mice.

Results
Glial glutamate and aspartate transporter (GLAST) KO consistently showed locomotor
hyperactivity to a novel but not familiar environment, relative to wild-type (WT) mice. The
locomotor hyperactivity-inducing effects of the NMDAR antagonist MK-801 was exaggerated in
GLAST KO relative to WT. Treatment with haloperidol or LY379268 normalized novelty-induced
locomotor hyperactivity in GLAST KO. These data demonstrated that GLAST KO mice exhibit
abnormalities on behavioral measures thought to model the positive symptoms of schizophrenia. To
further determine the role of GLAST in schizophrenia-related behaviors we tested GLAST mutant
mice on a series of behavioral paradigms associated with the negative (social withdrawal, anhedonia),- 73 -

Figure 1: GLAST KO showed locomotor
hyperactivity in a novel open field.
GLAST KO mice show increased
locomotor activity compared to WT
controls. Over the session as a whole,
GLAST KO were more active thanWT (inset). n=10-19/genotype. Data in
Figures 1-6 are Means ±SEM.

Figure 2: Decreased sociability and social novelty preference, but normal
free social interaction in GLAST KO mice. All genotypes spent more time
snifﬁng a cage containing a mouse than an empty cage, but KO spent lesstime doing so than WT (A). All genotypes spent more time snifﬁng a cage
containing a novel mouse than a familiar mouse, but KO spent less time
snifﬁng either stimulus than WT, while HET spent less time snifﬁng thenovel stimulus than WT (B). Total time spent investigating an unfamiliar
stimulus mouse in a free dyadic encounter did not differ between
genotypes (C). Time spent engaged in anogenital and non-anogenital
investigation, or aggression, did not differ between genotypes (D). n=1722/genotype. *P<.05, **P<.01 vs. WT; ##P<.01, #P<.05 vs. empty cageor familiar mouse-containing cage for same genotype.

sensorimotor gating (prepulse inhibition of startle), and executive/cognitive (discrimination
learning, extinction) symptoms of schizophrenia. GLAST knockout (KO) mice showed poor nesting
behavior and abnormal sociability, whereas KO and heterozygous (HET) both demonstrated lesser
preference for a novel social stimulus compared to wild-type littermate controls. GLAST KO, but
not HET, had a signiﬁcantly reduced acoustic startle response, but no signiﬁcant deﬁcit in prepulse
inhibition of startle. GLAST KO and HET showed normal sucrose preference. In an instrumental
visual discrimination task, KO showed impaired learning. By contrast, acquisition and extinctionof a simple instrumental response was normal. The mGlu2/3 agonist, LY379268, failed to rescue
the discrimination impairment in KO mice. These findings demonstrate that gene deletion of
GLAST produces select phenotypic abnormalities related to the negative and cognitive symptomsof schizophrenia. Altogether, gene deletion of GLAST produces select phenotypic abnormalities
related to the positive, negative and cognitive symptoms of schizophrenia. These findings raise
the possibility that loss of GLAST-mediated glutamate clearance could be a pathophysiologicalrisk factor for the disease. Our findings provide novel support for the hypothesis that glutamate
dysregulation contributes to the pathophysiology of schizophrenia and for the antipsychotic potentialof mGlu2/3 agonists.

Discussion & Conclusion
Mechanistically, the ability of NMDAR antagonists to provoke human psychosis and produce
schizophrenia-related behaviors in rodents has been linked to a loss of NMDAR-mediatedGABAergic inhibition, leading to excessive glutamate release and neuronal hyperexcitability in PFC.

- 74 -

Loss of GLAST is predicted to cause glutamate excess and extrasynaptic spillover under conditions
that provoke glutamate release, including novelty and NMDAR-mediated GABA inhibition.
Although voltametry or microdialysis measures of PFC extracellular glutamate in behaving GLAST
KO would provide direct evidence of this, indirect support for this notion is given by the abilityof LY379268 to rescue GLAST KO phenotype and is entirely consistent with the notion thatextrasynaptic mGlu2/3 receptors facilitate inhibitory control of glutamate release and mitigate theeffects of impaired clearance. In this context, our data offer novel support for the hypothesis that
excessive glutamate contributes to the pathophysiology of schizophrenia and lend further credence tothe antipsychotic potential of mGlu2/3 agonists. In summary, present data demonstrate that GLAST
KO exhibit abnormalities on behavioral measures thought to model positive and negative symptomsof schizophrenia. These disturbances were normalized by the prototypical antipsychotic haloperidol
and the mGlu2/3 receptor agonist LY379268. Present ﬁndings suggest that loss of GLAST-mediatedglutamate clearance could be a pathophysiological risk factor for schizophrenia and are particularly
intriguing given recent evidence linking genetic mutation of human GLAST (SLC1A3) withschizophrenia . More generally, our data provide novel support for the hypothesis that excessive
glutamate neurotransmission contributes to psychosis.

References
1. Karlsson, RM., Tanaka, K., Saksida, LM., Bussey, TJ., Heilig, M., Holmes, A. Assessment of glutamate
transporter GLAST (EAAT1) deficient mice for phenotypes relevant to the negative and executive/
cognitive symptoms of schizophrenia. Neuropsychopharmacology (in press)
2. Karlsson, RM., Tanaka, K., Heilig, M., Holmes, A. Loss of Glial Glutamate and Aspartate Transporter(Excitatory Amino Acid Transporter 1) Causes Locomotor Hyperactivity and Exaggerated Responses
to Psychotomimetics: Rescue by Haloperidol and Metabotropic Glutamate 2/3 Agonist. Biol Psychiatry
64. 810-814, 2008.

- 75 -

Application of ADLib system to rational design ofhumanized antibodies against tough antigens.
Kunihiro Ohta (with Lin Waka, Tohru Terada)
Graduate School of Arts & Sciences, Dept. of Life Sciences, The University of Tokyokohta@bio.c.u-tokyo.ac.jp

Abstract
We have developed new “photo-reactive magnetic beads” that can form covalent linkages withvarious chemical compounds and natural organic materials. Using the beads, combined with the
ADLib monoclonal antibody design system, we have obtained chicken B cell clones producing
antibody against tough antigens such as sphingolipids and small molecule drugs. In addition, aprocedure for “in silico-assisted rational (ISAR) CDR-grafting to convert chicken ADLib antibody(IgM) to humanized IgG has been established.
Keywords: monoclonal antibody, magnet beads

Introduction
Monoclonal antibodies are useful for the diagnosis and antibody medicines. Production of
monoclonal antibodies is normally laborious. In addition, due to the problem of immune-tolerance,some “tough antigens” such as endogenous small molecules can be hardly immunized in vivo. Using
artiﬁcially diversiﬁed chicken B cell libraries and magnetic beads, we developed a new in vitro-based
ADLib system, which enables rapid design of chicken IgM monoclonal antibodies against toughantigens. However, this system still had challenges; conjugation of small compounds to magnetic
beads and humanization of chicken IgM. We have conquered these points in this study.

Results
1) Preparation of “photo-reactive magnetic beads”:
Photoactivative linker (designed by Dr. Osada’s laboratory in RIKEN) was covalently conjugatedto M450-Epoxy beads (Dynal) via the amino group (Fig 1). We ﬁrst tried to crosslink cyclosporine
A (CsA) to the magnetic beads (PALC-magnetic beads) under the standard UV-crosslink condition
used for agarose beads (Kanoh N. et al., Angew. Chem. Int. Ed. 2005). However, due to the highoptic density of beads suspensions, it turned out that this condition is not applicable to the magnetic
beads. We ﬁnally optimized the conditions for the UV-crosslinking of the “photo-reactive magnetic
beads”, and applied to the
conjugation of various toughantigens such as small compounds(CsA, galactosylceramide, S1P)
to PALC-magnetic beads (patent- 76 -

pending). We conducted monoclonal antibody preparation by the ADLib system (Seo et al., Nature
Biotech., 2005) with such antigen-conjugated PALC-magnetic beads. Several clones producing
monoclonal antibodies were ﬁnally screened by ELISA assay.
2) Humanization of chicken ADLib antibody by in-silico assisted rational CDR grafting:We determined the DNA
sequences of an ADLib
antibody (both variable
regions of heavy and lightchains), and employed thehomology modeling using
some oth e r k n o w n I g G
3D structures (Fig 2). We
simulated a grafting of thechicken CDR1-3 segments
to human V regions, taking
account of the 3D modelingdata. We detected subtle structural alterations in the in silico simulation. Thus, a couple of amino acids
were altered to adjust the ﬁne 3D structures. Using this approach, we selected most eligible amino acidsubstitutions in silico. The in silico-desgined V-segments were incorporated into the recombinant human
IgG, and cultured cells producing the humanized ADLib antibody were constructed. We analyzed thereactivity of the humanized ADLib antibody in culture supernatants, and confirmed that the antibody
preserves afﬁnity to the original antigen.

Discussion & Conclusion
1) The method to conjugate various small compounds to PALC-magnetic beads is useful not only for
expansion of the ADLib system but also for biochemical hunting of drug binding targets. Thus,
PALC-magnetic beads has high value for life science and medical ﬁelds. The technology is now inpatent pending status.
2) In-silico assisted rational CDR grafting has succeeded in at least one ADLib antibody. This
technology will open the way to prepare humanized ADLib antibodies for therapeutic purposes.
We believe that rational design of humanized ADLib antibodies by this system will reduce thetime and costs for the monoclonal antibody design for antibody medicines in the future. Morepractical examples should be analyzed to evaluate limits and possibility of this system.

References
Kanoh N., et al., Angew. Chem. Int. Ed. 44: 3559-3562 (2005)
Seo et al., Nature Biotechnology, 23: 731-735 (2005)
Seo et al., Nature Protocols, 1: 1502-1506 (2006)- 77 -

The role of microRNA in endochondral ossiﬁcation
Hiroshi Asahara
National Research Institute for Child Health and Development
asahara@nch.go.jp

Abstract
Our study indicates that Sox9 directly promotes the cartilage-speciﬁc expression of miR140. Futurestudies will be needed to identify miR-140 target genes and the role of miR-140 in chondrocyte andcartilage function. These data may provide a new basis to understand molecular networks of cartilage
development, homeostasis and cartilage diseases.
Keywords: MicroRNAs, chondrogenesis, Sox9, arthritis

Introduction
MicroRNAs are non-coding RNAs which regulate target gene expression, however, little is known
about their regulation and function in chondrogenesis. Tuddenham et al showed cartilage speciﬁc
expression of miR-140 in mouse embryos. However, mechanisms of cartilage-specific miR-140
expression and upstream molecular signals are unknown. The present study is aimed to examine
how Sox9 regulates cartilage-speciﬁc miR-140 gene expression, identifying a new mechanism bywhich Sox9 controls chondrogenesis.

Results
We showed that Sox9 controls the cartilage speciﬁc expression of miR-140. By whole mount insitu hybridization assays primary miR-140 transcripts were detected in cartilaginous regions inmouse embryos. We next examined the expression pattern of miR-140 during chondrogenesis. The
mesenchymal cell line, C3H10T1/2 can be stimulated to undergo chondrogenic differentiation andthen shows dynamic changes in the expression of chondrocyte speciﬁc genes, including Sox9 andCol2a1. To detect pri-miR-140, RT-PCR primers were set inside in intron 12 of WWP2 to detect allabove transcripts. In this model, both pri-miR-140 and mature-miR-140 expression were increased
during chondrogenesis in parallel with Sox9 and Col2a1.
Both in Sox9ﬂox/ﬂox;Ck19–Cre and Sox9ﬂox/ﬂox;Col2a1–Cre embryos, pri-miR-140 was not detected invivo. Furthremore, in vitro deletion of Sox9 in mature chondrocytes from Sox9ﬂox/ﬂox mice by adenoCre infection decreased miR-140 expression. In the miR-140 upstream region, we identified apotential Sox9 binding site. The region between the11th and 12th exon of Wwp2 contained three Sox9
binding sites. To investigate whether this DNA sequence can physically bind to Sox9 protein, we
performed EMSA using In vitro synthesized Sox9 and 32P-labeded DNA oligo probes. In addition,
we utilized the Col2a1 enhancer sequence (48bp) which contains a well characterized Sox9 binding
sequence. In vitro synthesized Sox9 bound to the predicted Sox9 binding sites (miR-140-PSB)- 78 -

in the11th and 12th exons of Wwp2, as well as to the Col2a1 enhancer probe, and these miR-140PSB-Sox9 complexes were shifted by adding anti-Sox9 antibody. Moreover, these complexes were
diminished by adding excess amount of non-labeled miR-140-PSB and Col2a1 enhancer, suggestingthat Sox9 is binding to miR-140-PSB is speciﬁc. Two binding sites in tandem of three Sox9 bindingsites arranged in opposite orientation to each other. Sox9 bound to these tandem Sox9 binding sites(PSB1/2) and PSB3 to each other. These results support the speciﬁcity of miR-140-PSB for Sox9
binding. To test whether Sox9 is physiologically occupying this region in intact chondrocytes, we
performed chromatin immunoprecipitation (ChIP) assay using anti-Sox9 antibody. DNA obtained bychromatin immunoprecipitation from chondrocytes contained high amounts of the DNA fragment
containing three Sox9 binding sites. These results suggest that the conserved sequence nearby miR140 is occupied by Sox9 in chondrocytes.
Taken together, our findings support the idea that chondrocytes specific expression of miR-140
would be directly regulated by Sox9 during chondrogenesis and in mature chondrocytes.

Discussion & Conclusion
In this study, we demonstrated that pri-miR140 was specifically expressed in cartilage duringchondrogenesis. The potential Sox9 binding site at miR140 upstream region was identiﬁed by EMSA
and ChIP assay. Sox9 directly regulates miR140 expression as demonstrated by loss of function
studies. Taken together, this indicates that Sox9 directly promotes the cartilage-speciﬁc expressionof miR140. Future studies will be needed to identify miR-140 target genes and the role of miR-140
in chondrocyte and cartilage function. These data may provide a new basis to understand molecular
networks of cartilage development, homeostasis and cartilage diseases.

References
MicroRNA-140 is expressed in differentiated human articular chondrocytes and modulates IL-1 responses
Shigeru Miyaki, Tomoyuki Nakasa, Shuhei Otsuki, Shawn P Grogan, Reiji Higashiyama, Atsushi Inoue,
Yoshio Kato, Tempei Sato, Martin K Lotz and Hiroshi Asahara Arthritis and Rheumatism in press- 79 -

Role of Gi signaling in vascular endothelial cells
Hiroshi Kataoka, Akiyoshi Uemura
Center for Developmental Biology RIKEN Kobe
kataokah@cdb.riken.jp

Abstract
To investigate the role of Gi signaling in vivo we have generated a mouse model where Gi can beablated speciﬁcally in endothelial cells. We found that that Gi is critically required at the interface ofendocardial mesenchymal transformation. To analyze endothelial-mesenchymal transformation at thecellular level we established a culture system to see the transformation process in vitro.
Keywords: vascular endothelial cells, G protein coupled receptor(GPCR)

Introduction
GPCRs are the most promising drug targets, however their roles in real physiological context havenot been thoroughly analyzed. To systemically analyze GPCR function in vivo a mouse model was
generated which enabled us to ablate Gi signaling in a tissue specific manner（ROSA-PTX）.
We focused on the phenotypes of Gi blockade in endothelial cells. We also found RhoJ as a highly
endothelial-speciﬁc downstream effecter of G protein signaling. RhoJ knockout animals are being
generated.

Results
Generation of ROSA-PTX mice and analysis of Gi ablation in endothelial cells
A mouse model was developed enabling a Cre dependent pertussis toxin (PTX)expression
from ROSA 26 locus (ROSA-PTX mice).By crossing this line to a number of tissue-specific Cretransgenic animals we have observed phenotypes of Gi ablation in endothelial cells, endocrine,
nervous or hematopietic cells. Animals expressing PTX in endothelial cells by Tie-2 Cre(Tie-2

Fig.1

Schematic drawing of the ROSA-PTX locus
which can drive PTX expression only after Cre
excision of STOP cassette.

- 80 -

Fig.2

Tie-2 PTX animals expressing PTX in
endothelial cells die at embryonic stages
from heart failure presenting edema or
venous dilatation.

PTX) were embryonic lethal due to cardiac
failure.Tie-2 PTX embryos suffered fromhypoplastic cardiac valves and ventricularseptal defects coming from the defective
endothelial-mesenchymal transformation.
In vitro culture system for endothelialmesenchymal transformation
F2 endothelial cells were cultured withlow serum (1%FCS) condition and treatedwith TGF-beta. After 5-7 days smoothmuscle alpha actin positive cells emerged in

Fig.3

culture indicating endothelial-mesenchymal

Valvular hypoplasia and ventricular septal defects
caused cardiac failure.

transformation. Sphingosine-1-phosphate, one of the major agonists to activate Gi, seems to have arole to prevent apoptosis during mesenchymal transformation.
Screening for endothelial G protein signaling effectors
Using microarray we searched for G protein signaling molecules highly expressed in endothelial
cells. RhoJ was identiﬁed as one of such effecters.
Rho J was expressed in embryonic endothelial cells and ES derived endothelial cells, suggesting its
importance in developmental angiogenesis

Discussion & Conclusion
ROSA-PTX mice have been demonstrated to be an useful tool to probe Gi signaling in the realanimal context.We also have preliminary results thatGi blocked in platelets can be a powerfultherapeutic way to prevent thromboticdisorders by crossing ROSA-PTXmice to recently developed
platelet specific Cre mice. From the analysis of Tie-2 PTX mice we found that Gi is criticallyrequired at the interface of endocardial mesenchymal transformation. We have established an in vitro
culture system to analyze endothelial-mesenchymal transformation which will be used for microarray
analysis. We are planning to see the interactive effects of TGF-b, and sphingosine-1-phosphate.
From microarray analysis of retinal endothelial cells we found RhoJ as adownstream effecter of G
protein signalin gwhich is highly expressed in endothelial cells. Since RhoJ expression is higher inendothelial cells vs. hematopoietic cells at the diverging point between endothelial and hematopoietic
cells we speculate that RhoJ can be one of the endothelial fate determinants by inducing cellmorphology changes.

References*Regard JB#, Kataoka H#, Cano DA, Camerer E, Yin L, Zheng YW, Scanlan TS, Hebrok M, Coughlin
SR.(#equal contribution) Probing cell type-speciﬁc functions of Gi in vivo identiﬁes novel regulators of
insulin secretion. Journal of Clinical Investigation 2007 Dec;117(12):4034-43
Li G., Kataoka H., Coughlin S.R., Pleasure S.J. (2009) Identiﬁcation of a transient subpial neurogenic
zone in the developing dentate gyrus and its regulation by Cxcl12 and reelin signaling. Development
136(2):327-35.

- 81 -

Origin and mechanism of differentially methylated regions (DMR)
in mammals
Tomoko Kaneko-Ishino
Tokai University School of Health Sciences
tkanekoi@is.icc.u-tokai.ac.jp

Abstract
We have demonstrated that the mouse-specific repeat sequence (MSR) within Meg1-DMR
functioned as a silencer. The Meg1-DMR seems to function as a boundary between the maternallyexpressed upstream and paternally expressed downstream regions.
Keywords: genomic imprinting, mechanism of imprinted gene regulation, insulator, silencer/
enhancer, differentially methylated region (DMR)

Introduction
Mouse Meg1/Grb10 gene was originally identified as a maternally expressed imprinted gene inwhole body. However, in brain, it exhibits paternal expression. Interestingly, these genes have twocorresponding promoter regions. Type I transcripts from upstream promoters display maternal andbiallelic expression in almost all tissues. Type II transcripts from downstream promoters display
paternal expression in the brain.
DMR (differentially methylated region) locating on CpG island 2 (CGI 2) DMR) overlaps thebrain-speciﬁc downstream promoter; it is hypermethylated on the inactive maternal allele while nonmethylated on the active paternal allele, suggesting that DNA methyaltion directly regulates theimprinted paternal expression of the Type II transcripts.

Results
We have previously demonstrated the existence of a mouse-speciﬁc repeat sequence in the mouse
Meg1-DMR, where an insulator CTCF protein binds in a DNA-methylation-sensitive manner andhave proposed a molecular mechanism model in which the CTCF binding to the mouse-specificrepeat controls the mouse-specific maternal expression of Meg1/Grb10 Type I transcript via an
insulator function, as has been observed in the Igf2/H19 region.
In this experiment, we have addressed the mechanism of imprinted regulation by in vitro functional
assays using several vector constructs, especially the role of the mouse-speciﬁc repeat sequence
containing several CTCF binding sites within the Meg1-DMR. Unexpectedly, we found that the
MSR, by itself, could function as a silencer.
First, the insulator action was examined using two different assays: transient assay using luciferase
vectors and stable assay using neomycin resistance gene (Neo) vectors. In both vectors, the MSR
or H19 DMR insulator was positioned between the promoter and the enhancer. The MSR inserted- 82 -

in either direction significantly reduced the number of Neo-resistant colony as in the case of thecontrol H19 DMR, although that in the forward direction was weaker than the reverse direction.
These results indicate that the MSR within the Meg1-DMR had some capacity to reduce the Neo
expression. However, using these kinds of assays, it is essentially impossible to discriminate
insulator activity from silencer activity.
We then tested whether the MSR had silencer activity by using vector constructs without anenhancer. In these vectors, the luciferase gene was driven by the Meg1-Type I promoter and theMSR or the H19 DMR was placed downstream. This experiment clearly demonstrated that the MSR
in either the forward or reverse orientation could down-regulate the luciferase activity while no such
effect was observed with the H19 DMR. Importantly, its silencer function was DNA-methylationsensitive because the luciferase expression increased 2 to 3 fold when fully methylated fragments
were inserted. Human sequence corresponding to the MSR (h-MSR) has no CTCF binding sites andshowed no silencer function and DNA methylation did not affect the result. These results indicatethat the MSR containing several CTCF-biding sites within the Meg1-DMR, by itself, can play asilencer role in a DNA-methylation-dependent manner. Although it is not easy to integrate thesilencer function of the MSR with the regulatory mechanism of the Meg1/Grb10 imprinted region, it
is probable that the Meg1-DMR plays a different role from the H19 DMR.

Discussion & Conclusion
The DMR coupled with the CTCF-binding sites is very effective for the regional control of multiple
imprinted genes. The human homologous DMR region has no such CTCF-binding sequences andthe GRB10 Type I transcript exhibits biallelic expression in almost all tissues. Therefore, the functionof the CTCF-binding sequences is of considerable interest.
We previously proposed that the insulator function of the CTCF is essential for regulation of thematernally expressed Meg1/Grb10 Type I transcript in mice as in the case of the Igf2/H19 region.
Therefore, we investigated the mechanism of the imprinting regulation by Meg1-DMR using several
reporter vector constructs transfected into cultured cells, and demonstrated that the mouse-speciﬁc
repeat sequence (MSR) within Meg1-DMR, but not its corresponding human h-MSR sequence,
functioned as a silencer, although an insulator function was not excluded. The Meg1-DMR seems
to function as a boundary between the maternally expressed upstream and paternally expressed
downstream regions. Further experiments, such as identiﬁcation of CTCF-binding protein complexs
and determination of their biochemical function, will help to elucidate the precise regulation
mechanism of the Meg1/Grb10 imprinted region.

References
1. Shiura, H., Nakamura, K., Hikichi, T,. Hino, T., Oda, K., Suzuki-Migishima, R., Kohda, T., KanekoIshino, T., Ishino, F. Paternal deletion of Meg1/Grb10 DMR causes maternalization of the Meg1/
Grb10 cluster in mouse proximal Chromosome 11 leading to severe pre- and postnatal growth
retardation. Hum. Mol. Genet. 18(8), 1424-1438 (2009).

- 83 -

2. Hikichi, T., Kohda, T., Kaneko-Ishino, T. and Ishino, F. (2003) Imprinting regulation of the murine
Meg1/Grb10 and human GRB10 genes; roles of brain-speciﬁc promoters and mouse-speciﬁc CTCFbinding sites. Nucleic Acids Res., 31, 1398-1406.
3. Arnaud, P., Monk, D., Hitchins, M., Gordon, E., Dean, W., Beechey, C.V., Peters, J., Craigen, W.,
Preece, M., Stanier, P. et al. (2003) Conserved methylation imprints in the human and mouse GRB10
genes with divergent allelic expression suggests differential reading of the same mark. Hum. Mol.
Genet., 12, 1005-1019.

- 84 -

Contribution of calumin to mouse ontogeny
through regulation of NFAT activity
Tetsuo Yamazaki
Dept. of Biol. Chem., Grad. Sch. of Pharm. Sci., Kyoto Univ.
yamazakt@pharm.kyoto-u.ac.jp

Abstract
Calumin[1] is a novel endoplasmic reticulum (ER) protein and is vital for cellular Ca2+ signaling.
A majority of calumin-knockout embryos (~74%) died in utero by embryonic day (E)11.5, while
the rest were born alive but died within a day. Blood vessels in the knockout embryos and yolksacs were morphologically primitive and did not form a intricate network. Besides, the endocardial
cushion poorly developed in calumin-knockout embryos. These characteristics are observed in the
mutant mice where the NFAT family of transcription factors are genetically targeted[2], reminiscentof the functional link between calumin and NFAT. Indeed, NFATs in calumin-knockout cells were
not activated. It thus is suggested that calumin contributes to establishment of the cardiovascularsystem by mediating the activation of NFATs.
Keywords: calumin, Ca2+, NFAT, endoplasmic reticulum

Introduction
We have currently reported a novel Ca 2+-binding transmembrane protein on the ER, namely
calumin, and established its knockout mice. A majority of the knockout mice succumbed toembryonic lethality, so that we employed mouse embryonic fibroblasts as a model system todemonstrate that calumin contributes to ER Ca2+ storage, store-operated Ca2+ entry (SOCE) and ER
stress responses. In this study, we went into details about the mutant animals. On the basis of theresults obtained, we propose that calumin serves for mouse organogenesis probably by mediating theactivation of the calcineurin- NFAT signal cascade.

Results
Calumin-knockout mice die at two discrete stages of development: at mid-gestation andpostnatally
No mice homozygous for the targeted allele were detected among >300 weaned offspring produced
by intercrossing heterozygous mice. Live-born calumin-knockout mice were found at 26% of the
expected Mendelian ratio at postnatal day (P)0, but none of them at P1, indicating their death withina day of birth. To determine the stage at which a majority of the mutant mice were lost, litters fromheterozygous intercrosses were dissected out at various time points of gestation. At E9.5, ~20%of the mutant embryos were grossly normal, albeit a little smaller in size than wild-type littermates(hereafter referred to as KO-I) whereas the rest of the homozygotes, termed KO-II, displayed distinct- 85 -

growth retardation. Viability of calumin-knockout embryos steeply dropped at E10.5-11.5 andsubsequently leveled off until birth at ~26% of that predicted. It therefore is suggested that the KOII embryos (~74%) die between 9.5 and 11.5 days postcoitum, while the KO-I homozygotes survive
the rest of gestation without further loss and are born alive.
Vascular abnormalities in calumin-knockout yolk sacs and embryos
The pericardial effusion seen in the KO-II embryos at E10.5 prompted us to survey the vasculatureusing antibodies against PECAM and SMA, markers of endothelial cells and vascular smooth muscle
cells, respectively. Most of the vessels in the KO-II yolk sac and embryo proper were dilated andremained in a primitive honeycomb-like pattern, indicating a failure of primary vascular plexus toundergo remodeling, namely, angiogenesis. Differentiation into the myogenic lineage took placein the KO-II embryo, as confirmed by SMA positivity of somites. The KO-II mutants, however,exhibited a significantly reduced SMA staining not only in the yolk sac but at the dorsal aorta.
Collectively, calumin might play an indispensable role in angiogenesis by facilitating physical andfunctional interactions between endothelial and accessory cells, and the compromised vasculaturein the mutant embryos might lead to insufficient blood circulation and therefore early embryoniclethality.
Defective cardiac development in calumin-knockout embryos
At E9.5, the KO-I and -II cardiac ventricles became less trabeculated. In addition, cardiac
cushions, the future valve sites, underdeveloped in the KO-I and II hearts compared with those inwild-type controls. The cardiac cushion is formed by a complex and concerted interplay between
the endocardium and the myocardium, where a subset of endothelial cells transdifferentiate into
mesenchymal cells, a process termed endothelial-mesenchymal transformation (EMT). It is thus
suggested that calumin contributes to early heart morphogenesis by mediating EMT.
Compromised NFAT signaling in calumin-knockout embryos
Among SOCE-modulated signaling molecules, the NFAT family of transcription factors appeared to
be a likely target because abrogation of the Ca2+/calcineurin (CN)/NFAT link by genetic manipulation
results in the underdeveloped vasculature, malformed cardiac cushions and midgestational lethality,
major features of the KO-II embryos. SOCE-activated protein phosphatase CN dephosphorylates
the NFAT family members and facilitates their nuclear translocation. We then determined NFAT
phosphorylation status by immunoblotting of E9.5 whole-embryo extracts with an anti-NFATc4
antibody. NFATc4 dephosphorylation, required for NFAT to function, inefficiently takes place inthe mutant embryos. Taken together, the midgestational crisis of calumin-knockout mice could be
attributed to defective activation of NFAT family members.

Discussion & Conclusion
We have demonstrated in this study that calumin plays an indispensable role during mouse
ontogeny, the most physiological context, probably by mediating Ca2+ signals (Figure). Given that
disruption of functional coupling between calumin and NFAT is a key determinant of lethality of the- 86 -

KO-II embryos, there has been a critical question raised: What molecular mechanisms differentiate
the KO-I mice, which die neonatally, from the KO-II mice? The mice we analyzed were bred on amixed genetic background (129Sv/C57BL6). Thus, polymorphisms in modiﬁer genes could possibly
underlie the incomplete penetrance of the embryonic-lethal phenotype of calumin-knockout mice.
The most likely candidates for modiﬁer genes in this case would be those involved in the Ca2+/CN/
NFAT signaling axis. Immunoblot analysis of whole-embryo lysates argues in favor of this idea. The
presence of an extra band in the KO-I, but not the KO-II, lysates suggests that dephosphorylation of
NFAT occurs, albeit inefﬁciently. Profoundly limited but still remaining NFAT activity might render
the KO-I embryo to survive early embryonic crisis. Alternatively, modiﬁer gene effects could cause
compensatory activation of other angiogenesis-related signaling pathways implicating integrins, theTGFβ superfamily and Indian hedgehog. In either case, less severe vascular defects in the KO-I
embryos might ensure a second phenotype of neonatal lethality.

References
1. Zhang, M., et al., Calumin, a novel Ca2+-binding transmembrane protein on the endoplasmic
reticulum. Cell Calcium, 2007. 42(1): p. 83-90.
2. Graef, I.A., et al., Signals transduced by Ca(2+)/calcineurin and NFATc3/c4 pattern the developing
vasculature. Cell, 2001. 105(7): p. 863-875.

- 87 -

Analysis on the mechanism for generation of leukemic stem cells
Tetsuya Nosaka
Mie University Graduate School of Medicine
nosaka@doc.medic.mie-u.ac.jp

Abstract
MLL(Mixed Lineage Leukemia) is a homologue of Drosophila trithorax, and is involved in
majority of infant acute leukemia as MLL-fusion genes generated by chromosomal translocations. We
generated the transgenic mice inducibly expressing MLL-ENL oncogene. In vitro colony replating
assay revealed that MLL fusion gene-mediated transformation arises only from normal hematopoietic
stem cells, and not from multipotent progenitors.
Keywords: leukemic stem cell, MLL, cancer stem cell, leukemia

Introduction
MLL-related leukemia has been shown to develop from hematopoietic progenitor cells as well ashematopoietic stem cells (HSCs) by bone marrow transplantation (BMT) experiments with forced
expression of MLL fusion genes in retroviral vector. Recently Chen W et al. (1) reported that MLL
fusion gene expressed at the physiologic level transforms HSCs and common lymphoid progenitors,
but not common myeloid progenitors and granulocyte-monocyte progenitors (GMPs) by using MllAF9 knock in mice. However, non-physiologically high expression of MLL fusion gene resulted
in transformation even of GMPs. In their experiments, Mll-AF9 gene was expressed from early
embryonic stage. Here we report that induced expression of MLL fusion gene after birth at thephysiologic level results in leukemogenesis only from HSCs.

Results
Transgenic mice harboring CAG promoter-loxP-EGFP-SV40 polyA-loxP-MLL- ENL-rabbit
β-globin polyA gene (Figure) were generated in which Cre expression results in induced expressionof MLL-ENL. Mating of these transgenic mice with Mx-Cre mice resulted in development ofleukemia 2-3 months after birth without poly IC treatment, probably due to leaky expression of Cre.
Therefore, bone marrow cells from thetransgenic mice were infected with theretrovirus expressing Cre to induce MLLENL expression by excision of EGFP
cassette. Then Cre virus-infected BM
cells were subjected to colony replating
assay with cytokines supporting myeloid
cell growth. Interestingly, induced
expression of MLL-ENL on CD34 -- 88 -

KSL (c-kit+Sca1+Lineage-) cells (HSCs) led to immortalization of the cells, whereas CD34+KSL
cells (multipotent progenitor cells) did not. The difference of the results between CD34-KSL and
CD34+KSL cells in colony replating assay was very clear. Importantly, the expression levels
of MLL-ENL were comparable to those of endogenous MLL gene in both cell populations. Iffunction of MLL-fusion gene was to confer the “stemness”(or self renewal activity), the opposite
results (i.e., progenitor cells, not the HSCs, are transformed) would have been obtained. However,
our study strongly suggested that MLL leukemia is a stem cell leukemia, thereby MLL fusion proteintransforms normal stem cells into leukemic stem cells by previously unknown mechanisms.
We are currently investigating the effects of induced expression of MLL-ENL in vivo using BMT
experiments to conﬁrm that HSCs, but not progenitors, are transformed by MLL fusion gene.

Discussion & Conclusion
Our study suggested that MLL-related leukemia arises from HSCs, as in development of chronic
myeloid leukemia by Bcr-Abl. BMT experiments with retroviral transduction should be interpreted
cautiously by following reasons; first, high expression of transgene sometimes transforms nonphysiologic target cells, second, insertional upregulation of the gene near the integration site could
occur. Our observation of selective transformation of HSCs by expression of physiologic levelof MLL fusion gene will help to elucidate the molecular mechanism of generation of leukemicstem cells. Presence of MLL gene rearrangements in acute myeloid leukemia was demonstrated
to be strongly correlated with Evi-1 expression (2), which is preferentially expressed in HSCs,
supporting our working hypothesis on the origin of leukemic stem cell in MLL-related leukemia.
Recently Goyama S. et al demonstrated that Evi-1 plays an important role in MLL-ENL-mediatedleukemogenesis (3). Further study is required to identify genes essential for generation of leukemicstem cells in addition to Evi-1.

References(1) Chen W, Kumar AR, Hudson WA, Li Q, Wu B, Staggs RA, Lund EA, Sam TN, Kersey JH. Malignant
transformation initiated by Mll-AF9: gene dosage and critical target cells. Cancer Cell 13: 432-440,
2008.
(2) Barjesteh van Waalwijk van Doorn-Khosrovani S, Erpelinck C, van Putten WL, Valk PJ, van der Poel-van deLuytgaarde S, Hack R, Slater R, Smit EM, Beverloo HB, Verhoef G, Verdonck LF, Ossenkoppele GJ, Sonneveld
P, de Greef GE, Löwenberg B, Delwel R. High EVI1 expression predicts poor survival in acute myeloid leukemia:
a study of 319 de novo AML patients. Blood 101: 837-845, 2003.

(3) Goyama S, Yamamoto G, Shimabe M, Sato T, Ichikawa M, Ogawa S, Chiba S, Kurokawa M. Evi-1
is a critical regulator for hematopoietic stem cells and transformed leukemic cells. Cell Stem Cell 3:207-220, 2008.

- 89 -

Synaptic plasticity linking chronic painand enhanced emotional responses
Fusao Kato
Department of Neuroscience, The Jikei University School of Medicine
fusao@jikei.ac.jp

Abstract
In the rat models of chronic neuropathic pain, we found that adenosine and noradrenaline attenuate
the potentiated synaptic transmission between the parabrachial afferents and central amygdala
neurons to a level similar to that in non-treated or Sham-operated animals by decreasing the numberof simultaneously released glutamate-containing vesicles. We also found that, while the allodynia
response recovers after treatment of the neuropathy, this synaptic transmission remains potentiated,suggesting a morphofunctional "consolidation" of the synaptic potentiation in the central amygdalaof neuropathic rats.
Keywords: Chronic pain, amygdala, allodynia, synaptic transmission, emotion

Introduction
Acute sensation of pain is inevitably linked to negative emotion, endowing it with a “warning”
signal role. In the chronic pain, a persistent pain sensation that continues beyond the normally
expected time, this link remains aberrantly potentiated, causing clinical problems because it nolonger has “warning” roles but is only “painful”. We have already shown, in animal models ofchronic neuropathic pain, excitatory synaptic transmission to the “nociceptive amygdala”, a partof the central amygdala (CeLC) becomes potentiated (Ikeda et al., 2007). In this study, we tried toreveal the mechanism underlying this chronic pain-induced potentiation in the central amygdala.

Results
In the young Wistar rats, we made unilateral neuropathic pain models by ligating the left lumber
spinal nerve according to the method described by Chung et al. and evaluated the allodynic responses
by measuring threshold for hindpaw withdrawal responses using a series of von Frey ﬁlaments. The
neuropathic rats, but not Sham-operated or non-treated ones, showed decreased threshold within one
day. At the seventh day after operation, we made acute brain slices and recorded AMPA receptormediated postsynaptic currents (EPSCs) from neurons in the laterocapsular part of the central
amygdala (CeLC) evoked by stimulation delivered through an electrode placed on the mediolaterally
and ventrodorsally projecting afferent tract arising from the nucleus parabrachialis (PB). Biocytin
was injected to the recorded CeLC cells and morphology of these neurons was visualized withstreptavidin-conjugated AlexaFluor with a confocal microscope (Fig. 1). Analyses of releaseproperties at different extracellular Ca2+ concentrations suggested no change in the transmitter release- 90 -

properties in the potentiated synapses in the neuropathic rats. Measurement of quantal EPSC sizeby substituting Ca2+ with Sr2+ indicated that the EPSC is composed of ~20 simultaneously released
vesicles in the control left PB-CeLC synapses, whereas ~50 vesicles in the potentiated synapses in
the right CeLC when stimulated at 1 mA. The "minimal" stimulation of the PB afferents with theta
pipette electrode revealed larger-amplitude single-ﬁber EPSC in the right CeLC of neuropathic rats.
Application of adenosine and noradrenaline made appear smaller EPSCs with ~a half or ~a third
amplitude, suggesting that multiple vesicles are released synchronously in responbse to a single
action potential from the single ﬁber terminal of PB afferents in the right CeLC of neuropathic rats
(Fig. 2) and that adenosine and noradrenaline restore the monovesicular release. In those neurons
in the right CeLC showing larger-amplitude EPSC in the neuropathic models, blockade of NMDA
receptors with AP-5 failed to reduce the EPSC amplitude recorded at a holding potential of –60 mV,
indicating that this potentiation did not result from the change in voltage-dependency of the NMDA
receptor activation, in contrast to the case of acute pain (Bird et al., 2005). However, to our surprise,
the ratio of the amplitude of NMDA receptor-mediated currents recorded at +40 mV in the presenceof CNQX to that of AMPA receptor currents at –60 mV was markedly larger in the right CeLCof the neuropathic animals, pointing to a intriguing possibility that the properties of the synaptic
plasticity might be different in these potentiated neurons. These neurons were moderately spiny withextensive dendrites mostly limited within the central amygdala (Fig. 1). At the seventh day after theoperation, despite marked synaptic potentiation in the CeLC, we failed to observe speciﬁc changes
in the quantitative and qualitative properties of the astrocytes and microglial cells, as visualized with
antibodies against GFAP and Iba-1, respectively, in the both sides of CeLC, suggesting that at thislate phase of potentiation, dynamic remodeling of the synaptic process does not play the primaryrole. In another series of experiments, we tried to treat the neuropathic pain and improved theallodynia that was recuperated to a value almost similar to a pre-ligation level. In these rats, despite
almost complete absence of allodynia, the PB-CeLC transmission remained strongly potentiatedat the seventh day after operation. In contrast, the synaptic potentiation at synapses arising frombasolateral amygdala was not observed in those models with relieved allodynia.

- 91 -

Discussion & Conclusion
Taking together, these results point to a specific structural plasticity and its consolidation in the
amygdala synapses in the neuropathic pain model. Interesting features of this consolidation is that
this consolidated potentiation persists even in the disappearance of the withdrawal reﬂex at the spinal
cord level, thus indicating that nociceptive inputs are not necessary to maintain it. A novel ﬁndingis that the potentiation would be accompanied by increase in NMDA receptor components, whichmight have a strong impact to the synaptic variability in responses to associated nociceptive inputs.
These ﬁndings would provide basis for understanding the potentiated link between nociception andnegative emotion and also the clue to the potential therapeutic approaches against the drug-resistantchronic pain, which makes suffer hundreds of millions of people in the world.

References
Ikeda R, Takahashi Y, Inoue K, Kato F. NMDA receptor-independent synaptic plasticity in the central
amygdala in the rat model of neuropathic pain, Pain 127 (2007) 161–72.
Bird GC, Lash LL, Han JS, Zou X, Willis WD, Neugebauer V. Protein kinase A-dependent enhanced
NMDA receptor function in painrelated synaptic plasticity in rat amygdala neurones. J Physiol 564 (2005)
907–21.

- 92 -

Clinical application on metabolic syndrome
via exhaustive analysis of bile acid function.
Mitsuhiro Watanabe
School of Medicine, Keio University
wmitsu@sc.itc.keio.ac.jp

Abstract
We have been thinking that bile acid metabolism is important for energy metabolism 1)2)3).
In this study, we performed the analysis by metabolism products and mRNA to elucidate thebiophysiological difference between bile acid and FXR agonist.
By contraries, our result was different from previous report. Bile acid and FXR agonist did notchange adipocyte differentiation and the gene expressions such as aipocyte differentiation genemarkers (PPARγ, FAS, GLUT4, ACC1, ACC2). By the micro array analysis, a difference ofthe expression was found in a lot of gene, which participate in metabolism, inflammation, cellproliferation and apoptosis, in BAT. By the metabolome analysis, it was suggested that bile acidparticipated in amino acid metabolism in BAT.
Keywrds: FXR, Bile acid

Introduction
The farnesoid X receptor (FXR) is a member of the metabolic nuclear receptor superfamily
members of which are highly expressed in liver, intestine, kidney and adrenals. FXR plays key roles
in regulating cholesterol and bile acid homeostasis. Some reports that FXR agonist let insulin signals
increase in adipocyte were published, indicating that FXR might be good drug target for diabetes
treatment. The aim of our study was to clarify the role of FXR in metabolism by using transcriptome,
proteome and metabolome.

Results
Some reports which FXR agonist let insulin signals increase in adipocyte and the adipocyte
differentiation in 3T3-L1 cell were published. Bile acids have been shown to improve metabolic
disturbance by suppressing fat accumulation in adipose tissue and stimulating energy expenditure,according to reported our study in vivo 1)2)3). On the other hand, FXR agonist did not have any
effects against metabolic syndrome.
To investigate whether induction of differentiation medium induced differentiation by FXR agonist(GW4064) and CDCA (chenodeoxy cholic acid) was mediated by FXR activation in 3T3-L1 cells.
CDCA and GW4064 treatment did not change adipocyte differentiation and TG accumulation in
3T3-L1 cells. The gene expression of the lipogenic genes, such as PPARg, ACC1, ACC2 and FAS
mRNA, were not changed by FXR activation.

- 93 -

We evaluated the metabolic effects of either FXR agonist or bile acid administered to male
C57BL/6J mice that were fed a high fat diet. These mice received either a normal chow (C), a highfat diet (F), a normal chow with cholic acid (CA), a high fat diet with cholic acid (FA) or a high fat
diet supplemented with GW4064 for 2 weeks.
DNA microarrays were performed to elucidate biophysiological difference between bile acids and
FXR agonist in brown adipose tissue (BAT) of these mice. The DNA microarray analysis indicatedgenes involved in metabolic alteration evoked by bile acids and FXR agonist. For example, bile
acids administration was shown to induce the expression of 39 genes, and to suppress that of 34
genes. Cyclin T1, one of the important molecules in cell cycle regulation, was found in the listof genes whose expressions were altered by bile acids. In comparison with the result of bile acidadministration and that of FXR agonist, the expression of 116 genes were significantly affected.
Many interesting genes like Inhibitor of kappa B kinase gamma (IKK gamma), participating in themolecular mechanism of inﬂammation, were included in the result. In our study, genes presenting
expression changes more than 2 folds were regarded as significantly changed. Then we foundcandidates to clarify biophysiological activities of bile acids.
We measure the metabolites of C, CA, F, FA in BAT by CE-TOF/MS (capillary electrophoresis
mass spectrometry). As a result, a number of amino acid was changed. His and Cys were induced bycholic acid both in C and F. Bile acids might affect amino acid pathways in energy metabolism.

Discussion & Conclusion
Bile acids are essential constituent of bile that facilitate dietary lipid absorption and catabolism.
Bile acid also activate several signaling pathways in lipogenesis and energy metabolism. Recent
advances in FXR biology have demonstrated that FXR represents a pharmacological target as
dyslipidemia and diabetes. By contraries, our result was different from previous report. FXR agonist
did not change serum glucose, insulin sensitivity, adipocyte differentiation and the gene expressions
such as aipocyte differentiation gene markers.
We evaluated the different metabolic effects between FXR agonist and bile acid. By the micro array
analysis, we elucidated the importance of inflammation, cell proliferation and apoptosis. By themetabolome analysis, it was suggested that bile acid participated in amino acid metabolism in BAT.
The amino acid metabolism is important in energy metabolism, but there is little report about that.
Interestingly, we found that bile acid had an inﬂuence on inﬂammation and amino acid metabolism
in BAT.

- 94 -

References
1) Houten, S.M a., M. Watanabe, M a. & Auwerx, J. : Endocrine functions of bile acids. EMBO J, 25 :1419-1425, 2006. a: contributed equally
2) Watanabe, M., Houten, S. M., Mataki, C., Christoffolete, M. A., Kim, B. W., Sato, H., Messaddeq, N.,
W. Harney, J., Ezaki, O., Kodama, T., Schoonjans, K., Bianco, A. C., & Auwerx, J. : Bile acids induce
energy expenditure by promoting intracellular thyroid hormone activation. Nature, 439 : 484-489,
2006.
3) Watanabe, M., Houten, S. M., Wang, L., Moschetta, A., Mangelsdorf, D. J., Heyman, R. A., Moore, D.
D., & Auwerx, J. : Bile acids lower triglyceride levels via a pathway involving FXR, SHP, and SREBP1c. J. Clin. Invest, 100 : 1408-1418, 2004.

- 95 -

Mechanisms of aggregation and segregation of cortical neurons
Kazunori Nakajima
Keio University School of Medicine
kazunori@sc.itc.keio.ac.jp

Abstract
Migrating neurons in the developing cerebral cortex are ultimately segregated into layers beneaththe marginal zone. This may occur as a result of interplay between the fate acquisition for birthdate-dependent segregation in the ventricular side and the induction of the segregation/aggregation
properties beneath the marginal zone.
Keywords: Cerebral cortex, neuronal migration

Introduction
Cerebral cortical neurons form a six-layered structure in which their position depends on their birthdate. This developmental process requires the presence of Reelin, which is secreted by Cajal-Retzius
cells in the cortical marginal zone (MZ). However, it is still unclear whether the migration fromthe ventricular zone (VZ) to beneath the MZ is essential for the neurons to segregate into layers.
Previous transplantation studies of ferret cerebral cortical neurons suggested that their ultimate
laminar fate is, at least to some extent, determined in the VZ, but it is unknown how “laminar fate”
eventually positions cells in a speciﬁc layer.

Results
To examine the migration profile of the cortical neurons in vivo, we used the in-utero
electroporation system (Tabata & Nakajima, 2001; Tabata & Nakajima, 2002; Tabata &Nakajima, 2003; Sasaki, Tabata, Tachikawa & Nakajima, 2008; Tabata, Kanatani & Nakajima,in press). Using this system,
the pyramidal neurons
undergoing their final mitosis
in the VZ can be visualized
for a long time by transfectionof a reporter gene. As theresults, we observed that,
although the cells seemed tomigrate individually throughthe cortical plate (CP), they
accumulated beneath the
MZ when the migration was- 96 -

ﬁnally complete, suggesting that the cells probably start to form new intercellular relationships
with adjacent cells once their migration is complete. At later stages such as at postnatal day(P) 14, most of the cells that had been transfected at a certain embryonic stage (e. g. E13) werealigned around a certain layer (e. g. Layer IV). These results indicate that cortical neurons
born in embryos of the same stage preferentially localize around a speciﬁc layer in the CP after
migration.

To explore the segregation properties of mouse cortical cells that have not yet arrived beneath theMZ, embryonic day (E) 16 VZ and intermediate zone (IMZ) cells were dissociated and allowed toreaggregate for 1-4 days in vitro. The result suggested that the migrating neurons in the IMZ at E16
preferentially located near the center of the aggregates more than the proliferative cells from the
VZ. The birth-date-labeling followed by the dissociation/reaggregation culture suggested that the
segregation properties of the E16 IMZ was characteristic of the E14-born cells, which were migratingin the IMZ at E16, but they were not general properties of migrating IMZ cells. This birth-datedependent segregation mechanism was also observed in the yotari mutant cells, in which the Reelinsignal is not transduced due to a deﬁciency of the intracellular adaptor protein Dab1, suggesting thatthe birth-date-dependent cell segregation mechanism is acquired independent of Reelin signaling.
We then performed a short-term aggregation assay to investigate the adhesion properties of the
IMZ cells, because short-term aggregation assay is thought to directly reﬂect the expression andregulation of signaling molecules on the cell surface. When dissociated cells from the E16 VZ,
IMZ, and CP were cultured separately in a rotating plate for 1 hour, the IMZ cells showed astronger tendency to form large aggregates than the VZ and CP cells. These ﬁndings suggestthat the postmitotic migrating neurons in the E16 IMZ have stronger adhesion properties thanthe CP or VZ cells. To identify the adhesion mechanism that mediates the adhesive propertiesof the E16 IMZ cells, we investigated the Ca dependency of the cell adhesion. Cell adhesion- 97 -

mechanisms are classified into Ca2+-dependent and Ca2+-independent, and exposure to a low
concentration of trypsin in the presence of EDTA (“LTE-treatment”) interferes with the Ca2+dependent cell adhesion, because the Ca2+-dependent adhesion molecules are highly sensitive totrypsin in the absence of Ca2+ (Matsunami & Takeichi, 1995). In the presence of Ca2+, however,
the Ca2+-dependent molecules become resistant to trypsin, allowing selective digestion of theCa2+-independent molecules (“TC-treatment”). The preferential cell adhesion of the E16 IMZ
cells in the short-term aggregation culture was not inhibited by TC treatment but was inhibitedby LTE treatment. In addition, it was not inhibited by the RGD peptide, which suppresses
integrin-mediated cell adhesion. These results suggest that the preferential adhesion of E16 IMZ
cells is mediated by the Ca2+-dependent cell adhesion mechanism.
We then tried to ectopically express Reelin on the migratory route of the cortical neurons.
Interestingly, migrating cells that encounter the ectopic Reelin form aggregates. These aggregatesbecame bigger as development proceeded.

Discussion & Conclusion
These findings suggest that cortical neurons acquire a birth-date-dependent segregation
mechanism before their somas reach beneath the MZ. When they terminate migration beneaththe MZ, they encounter Reelin that is secreted from Cajal-Retzius neurons. There, thesegregation properties of the neurons may be induced, and, as the results, the cells may begin

to form “aggregates”, which would eventually develop into certain layers. Future studies will
be required to identify the molecular mechanisms that occur beneath the MZ in response to theReelin molecule.

References
The cortical subventricular zone-speciﬁc molecule Svet1 is part of the nuclear RNA coded by the putative
Netrin receptor gene Unc5d and is expressed in multipolar migrating cells. Shinji Sasaki, Hidenori Tabata,
Kashiko Tachikawa and Kazunori Nakajima. Mol. Cell. Neurosci., 38 (4), 474-483 (2008).
Differences of migratory behavior between direct progeny of apical progenitors and basal progenitors
in the developing cerebral cortex. Hidenori Tabata, Shigeaki Kanatani, and Kazunori Nakajima. Cereb.
Cortex, in press.

- 98 -

Regulation of mRNA stability by heat shock cognate protein 70
under the control of cytokines
Hirotaka Matsui
Research Institute for Radiation Biology and Medicine, Hiroshima University
hmatsui@hiroshima-u.ac.jp

Abstract
Cell death control of hematopoietic cells by the regulation of Bim mRNA stability
Keywords: Bim, Hsc70, mRNA stability

Introduction
We recently found a molecular mechanism for cytokine-mediated posttranscriptional regulation of
Bim mRNA by heat-shock cognate protein 70 (Hsc70), which binds to AU-rich elements (AREs) inthe 3’-untranslated region of speciﬁc mRNAs and enhances their stability. To fully understand thismechanism, we mainly analyzed the involvement of miRNAs in this mechanism.

Results
Cytokines promote cell survival of hematopoietic progenitors by negative regulation of Bim, a BH3only cell death activator in the Bcl-2 superfamily. The molecular mechanisms by which cytokines
negatively regulate Bim function differ between cell types. We found that, in IL-3 dependent Baf-3
cells, IL-3 downregulates Bim mRNA by inhibiting Hsc70-containing protein complex, which works
as an RNA stabilizing factor, from binding to Bim mRNA(1)(Figure 1).
To better insight into this mechanism, we analyzed the involvement of miRNAs in the regulationof Bim mRNA stability because miRNAs are reported to destabilize mRNAs in collaboration with
mRNA-binding proteins. By synthesizing cDNA on Bim mRNA template using miRNAs as theendogenous primer, we identiﬁed several putative cis-elements that would be bound by miRNAs.
In addition, we searched
databases such as TargetScan
and miRBase for miRNAbinding elements conserved
among human and mouse andfound that miR-181 possibly
binds to 200bp downstream ofstop codon on Bim mRNA. We
speculate that Hsc70 protein
complex would stabilize Bim
mRNA by blocking binding- 99 -

of these miRNAs to Bim mRNA. Another possibility is that Hsc70 protein may recognize miRNA/
mRNA hybrids and acts as an inhibitor of mRNA-destabilizing function of miRNAs.
We also analyzed factors in Hsc70-containing protein complex. By an immunoprecipitation
analysis, we identified poly(A)-binding protein (PABP) as a Hsc70-binding protein. As PABP
stabilizes mRNAs by mediating 5’cap and 3’poly(A) chain, it is suggested that Hsc70 stabilizes Bim
mRNA by recruiting PABP on mRNA.

Discussion & Conclusion
Our ﬁndings are unique in that cytokines control cell survival by regulating mRNA stabilities of
apoptosis-related genes. Because Bim plays a critical role in the regulation of hematopoietic cellnumber, altered regulation of Bim could be involved in leukemogenesis. For example, negative
regulation of Bim by the Bcr-Abl chimeric kinase has been shown to contribute to an increase inwhite blood cell and hematopoietic progenitor counts in the chronic phase of chronic myelogenous
leukemia(2). Thus, this mechanism could be an important pathway to identify new molecular targets
for antileukemic drugs.
To this end, further we have to analyze this mechanism in detail. We have shown that cytokines
modulate members of Hsc70 protein complex, however, how cytokines exert its function is still
unknown. In addition, the speciﬁcity of target mRNAs is unclear. We suppose that sequences around
AREs, which may be miRNA-binding cis-elements, would be critical in this regulation.

References(1) Cytokines direct the regulation of Bim mRNA stability by heat-shock cognate protein 70.
Matsui H, Asou H, Inaba T. Molecular Cell; 25(1): 99-112, 2007.
(2) Roles of Bim in apoptosis of normal and Bcr-Abl-expressing hematopoietic progenitors.
Kuribara R, Honda H, Matsui H, Shinjyo T, Inukai T, Sugita K, Nakazawa S, Hirai H, Ozawa K, Inaba
T. Mol Cell Biol; 24(14): 6172-83, 2004.

- 100 -

Replenishment of innate immune system via Toll-like receptor signals
Yoshinori Nagai
Immunobiology and Pharmacological Genetics, Graduate School of Medicine andPharmaceutical Science for Research. University of Toyama
ynagai@med.u-toyama.ac.jp

Abstract
Hematopietic stem/progenitor cells express TLRs, suggesting that bacterial/viral products may
inﬂuence blood cell formation. We now show that common lymphoid progenitors (CLPs) from micewith active HSV-1 infection are biased to dedritic cell (DC) differentiation, and the phenomenon islargely TLR9 dependent.
Keywords: Toll-like receptors (TLRs), Hematopoietic progenitors, CpG-DNA, Dendritic cells (DCs)Introduction
Hematopietic stem cells (HSCs) give rise to progenitors with potential to produce blood cell types
with remarkably stable characteristics. Although this process is tightly controlled, recent findings
suggest that hematopoiesis is dynamic and also responsive to environmental factors. We previously
found that stem/progenitor cells express functional TLR2 and 4, and TLR signals alter lymphohematopoiesis. HSCs were stimulated to enter cycle and acquire lineage markers by exposure to LPS.
LPS also caused lymphoid progenitors to produce conventional DCs (cDCs). This new mechanism
represents a potential means for pathogen products to signal the rapid generation of innate immune
cells within the bone marrow or other tissues.
We have now found that lymphoid progenitors assume other fates after HSV-1 inoculation of
normal but not TLR9-deﬁcient mice. Thus, unique differentiation pathways can be used to generate
cells of the innate immune system in response to pathogen products,

Results
1. B lymphoid progenitors in mice with acute HSV-1 infection are directed to produce dendritic cells.
A dose of 1000 pfu of the human pathogen HSV-1 was delivered to mice by corneal scariﬁcation.
Pro-B, pre-B, and B cells were all reduced in marrow of the infected mice, whereas pDC and NKlike IKDC populations were increased. The lymphoid potential of CLPs was reduced more than 95%
in cells from the infected mice, whereas formation of pDCs was strongly favored. This direction of
CLPs to DC fates was negligible in TLR9-deﬁcient mice. Thus, B lymphopoiesis is diminished, andDC production is augmented during HSV-1 infection. This phenomenon is mediated by TLR9.
2. Nature of DCs made from lymphoid progenitors primed by in vivo exposure to CpG..
Highly puriﬁed CLPs were recovered from mice 48 hours after CpG injection and placed in stromalcell-free, serum-free cultures. CLPs from treated mice gave rise to CD11c+ DC subsets, pDCs

- 101 -

and NK-like IKDCs. pDCs were the main producers of IFN-alpha in response to CpG stimulation,
whereas NK-like IKDCs secreted more IFN-gamma. Thus, CpG treatment directed the formation ofa variety of non-B lymphoid cells, including functional DCs from CLPs.
3. Lymphoid progenitors express TLR9, and their differentiation potential is altered byexposure to CpG.
RT-PCR analyses revealed that TLR9 transcripts were detectable in

HSCs. TLR9 expression

was particularly high in subsets dedicated to lymphopoiesis, including CLPs and early lymphoidprogenitors (ELPs). CLPs were stimulated with the TLR9 ligand CpG for 48 hours. B lymphopoiesis
was suppressed, and conspicuous CD19- populations emerged from the cultured cells. Further gatingon CD19- cells revealed that the new subsets included pDCs and NK-like IKDCs. Thus, a TLR9
ligand can act as a differentiation cue, deviating highly puriﬁed progenitors under deﬁned culture
conditions to new fates.

- 102 -

Discussion & Conclusion
The present study shows in vivo relevance with viral infection directing lymphopoiesis toward
production of innate effector cells. TLR9 ligands interacted with progenitors, and there was noapparent cytokine requirement for altering their fates. CLPs expose to the TLR4 ligand LPS
generated almost exclusive populations of cDCs in culture. We now show that additional types offunctional DCs emerge when the same lymphoid progenitors are ligated with CpG. IL-7 stimulation
normally causes phosphorylation of STAT5, and this response was blocked by TLR9 signaling.
One report showed that human cord blood CD34+ cells express TLR9- and CpG-elicited productionof IL-8. It will be important to determine whether lymphohematopoiesis is altered in human BM as aresult of viral infection or CpG therapy. These phenomena could pertain to other types of disease. For
example, pDCs are major mediators of autoimmunity, and chronic infections might cause excessive
DC production.
There is much to learn about differentiation pathways responsible for generating all of the many

- 103 -

specialized cells in the immune system, and we must consider how hematopoietic cells integratesignals from extrinsic pathogen products with those from normal growth and differentiation factors.
Patterns of blood cell differentiation under normal steady-state conditions might be quite different
from those used during infection.

References
R.S. Welner, R.P. Pelayo, Y. Nagai, K.P. Garrett, T.R. Wuest, D.J. Carr, L.A. Borghesi, M.A. Farrar, andP.W. Kincade. Lymphoid precursors are directed to produce dendritic cells as a result of TLR9 ligation
during herpes infection. Blood. 2008;112:3753-3761.

- 104 -

Analysis of transcriptional regulatory networkof vascular differentiation
Kyoko Ohashi-Ito
The University of Tokyokyoko@biol.s.u-tokyo.ac.jp

Abstract
The vascular system of plant is an essential to conduct water, nutrients and signal molecules throughthe whole body. To reveal the transcriptional regulatory network of plant vascular differentiation, weestablished a novel approach to analyze transcriptional regulations. We collected vectors including
coding sequences of Arabidopsis transcription factors and cloned them into estrogen inducible
plant binary vectors. We tested a new screening system using them and successfully isolated novel
regulators of VND7 that was a master regulator of protoxylem vessels. These results indicate that ourapproach could be useful to clarify transcriptional relations.
Keywords: Xylem differentiation, transcription factor

Introduction
The vascular system of plant is an essential for plants to conduct water, nutrients and signal
molecules through the whole body. The vascular bundle consists of xylem, phloem and cambium. To
understand the mechanism of vascular differentiation fully, it is important to reveal the transcriptional
regulatory network governing vascular differentiation. In this study, we established a novel screening
system as a ﬁrst step investigating the transcriptional network and found novel regulators for xylem
differentiation.

Results
In the Arabidopsis genome, about 2000 genes are predicted as transcription factors. To begin toclarify the transcriptional network of vascular differentiation, we collected about 1100 entry clones
of Arabidopsis transcription factors as gateway entry vectors (invitrogen) from Arabidopsis resource
center and made about 100 entry clones of them by ourselves. Each clone includes a coding sequenceof a transcription factor, respectively. The entry clones were mixed with a plant binary estrogen
inducible vector (destination vector, Zuo el al. 2000), and the coding sequences were transferred into
the destination vector through recombination of the gateway system. The puriﬁed estrogen inducible
vectors were mixed and used as screening pools.
VND7 is a master regulator of protoxylem differentiation (Kubo et al. 2005). Overexpression of
VND7 induced ectopic xylem differentiation in leaves and a hypocotyl. VND7 is considered to bea key transcription factor of xylem differentiation. We tried to isolate novel regulators of VND7.
We made the VND7 promoter:: YFP-NSL transgenic plants of Arabidopsis. The YFP signal was- 105 -

observed only in cells of protoxylem cell files in root tips. The vectors of screening pools weretransformed into the transgenic plants having VND7::YFP through agro bacterium. T1 seedlings
were selected and checked the expression patterns of VND7::YFP. Seedlings showed ectopic YFPsignals were selected as candidates of VND7 regulators. One seedling showed very strong YFPsignal in all cells in the root. The seedling had single transgene of LBD12/ ASL5, a member of afamily of plant speciﬁc transcription factor. We conﬁrmed that the ectopic expression was caused byoverexpression of LBD12/ASL5 through qRT-PCR (Fig. 1).
To examine whether LBD12/ASL5
binds to the VND7 promoter directly,
we performed infiltration assays using
Nicotiana benthamiana. Cotransfection
with an estrogen-inducible promoterdriven LBD12/ASL5 construct and a
GUS construct having 1kb of the VND7
promoter showed that LBD12/ASL5 wasable to activate the expression of VND7GUS gene.
Next we examined the expression patternof LBD/ASL5. Two kb of the LBD12/
ASL5 promoter-GUS construct wastransformed into Arabidopsis. The GUS
signal was observed only in quiescentcenter cells at the root tip. This result
suggests that LBD12/ASL5 may not be a true regulator of VND7 and that other members of the
LBD/ASL family may be. To examine the possibility of the regulation by other LBD/ASL genes, wechecked expression patterns of
LBD1, LBD3, LBD4, LBD11,
LBD23 and LBD24 which had
higher similarity to LBD12/ASL5. LBD1, LBD11, LBD23
and LBD24 were preferentiallyexpressed in stipules. As
a result, only LBD3 and
LBD4 showed vascularrelated expression (Fig. 2).
The GUS staining of LBD3
was observed along vascularbundles in root, cotyledons androsette leaves. The expression- 106 -

of LBD4 was seen in procambium cells in leaves and primordium of lateral roots. We conﬁrmed that
LBD3 and LBD4 were able to bind the 1kb of VND7 promoter by cotransfection assays.
These results suggest that LBD3 and/or LBD4 regulate the VND7 expression in vascular bundles
positively.

Discussion & Conclusion
To reveal the transcriptional regulatory network of plant vascular differentiation, we collected
vectors including coding sequences of Arabidopsis transcription factors and cloned them into
estrogen inducible plant binary vectors as a first step. We tested a new screening approach using
the mixture of estrogen-inducible transcription factors and successfully isolated novel regulators
of VND7 that was a master regulator of protoxylem vessels. These results indicate that our new
approach could be useful to clarify transcriptional relations although it needs to examine whether
an isolated regulator is proper. Applying our new approach could reveal a lot of transcriptional
regulatory relations between genes during vascular differentiation.

References
Zuo et al. (2000) Plant J. 24, 265-273.
Kubo et al. (2005) Genes. Dev. 19 1855-1860.

- 107 -

Quantity and quality control of antibody response
by a degradation system of Bach2
Kazuhiko Igarashi
Tohoku University Graduate School of Medicine
igarak@m.tains.tohoku.ac.jp

Abstract
By purifying the transcription factor Bach2 from B cells, we found novel ubiquitin E3 ligase
adaptor proteins that mediate polyubiquitination of Bach2. Detailed mechanistic aspects wereaddressed in this study.
Keywords: B cell, antibody, transcription factor, ubiquitin

Introduction
B cells differentiate to plasma cells in response to B cell receptor stimulation by antigen or
polyclonal stimulation such as lipopolysaccharide. En route of the differentiation take place
recombination of antibody class switch regions and somatic hypermutation of antigen-binding
coding regions. Little is known about how class switch recombination and somatic hypermutation
are deployed specifically upon B celll activation. Since Bach2 is required for both class switch
recombination and somatic hypermutation, we hypothesized that Bach2 activity should be tightly
controlled in activated B cells. To explore this possibility, we tried to identify Bach2-interacting
proteins and examine their role in regulating Bach2 activity.

Results
We ﬁrst expressed epitope (FLAG and HA) tagged Bach2 in Bal17 mature B cell line. We puriﬁed
Bach2 complex from these cells using anti-epitope antibodies (affinity purification). Using mass
spectrometry analysis, we identiﬁed two proteins that show similar domain structure to Keap1 with
BTB and Kelch-like repeat domains. Keap1 is an adaptor protein for E3 ubiquitin ligase Cullin3
and mediates recognition of its substrate Nrf2. Thus, we named these two new proteins KeapX
and KeapY and hypothesized that they might mediate poly-ubiquitination of Bach2. To test thisidea, we overexpressed Bach2 and His6-Ub together with or without KeapX and KeapY, separately
or in combination. In normal cultured cells, we observed little polyubiquitination of Bach2.
However, co-expression of KeapX and KeapY strongly promoted poly-ubiquitination of Bach2.
Importantly, Neither of them promoted polyubiquitination of Bach2 when expressed individually.
For the cooperative action, we found that BTB domain of the Keap-like molecules was essential.
Considering the known function of BTB domain in homo-dimer formation, our data may suggest that
KeapX and KeapY form a heterodimer depending on their respective BTB domain, which may thenrecognizes Bach2 for recruiting to Cullin3 E3 ligase.

- 108 -

We are now trying to generate KeapX and KeapY deﬁcient mice using gene knockout strategy. We
are also carrying out knockdown of these genes in cultured B cells and primary spleen B cells using
RNAi. We have successfully identified RNAi sequence that give us efficient knockdown of thesegenes. We are now examining changes in Bach2 protein level and class switch recombination after
knockdown.
To understand whether Bach2 protein levels affect plasma cell differentiation and class switch
recombination, we utilized heterozygous Bach2+/- mice lacking one of the two copies of Bach2
gene. Although this kind of experiment would not allow predict phenotypic changes in KeapX/Y
deﬁcient mice, it should give us insight how Bach2 protein level is important during the process of B
cell activation and plasma cell differentiation.
First we confirmed that B cell differentiation process itself was not grossly affected by theheterozygous knockout of Bach2. We isolated spleen B cells from these mice and stimulated themwith LPS to induce class switch recombination and plasma cell differentiation. We found that
Bach2+/- B cells underwent less class switch recombination. We also found that differentiation toplasma cells was promoted compared with wild-type B cells. Taken together, these observations
suggest that amount of Bach2 protein in B cells regulate the frequency of antibody class switch andplasma cell differentiation.

Discussion & Conclusion
In this study, we showed that Keap1-related molecules KeapX and KeapY form heterodimer,
generating an adaptor for Cullin3 to bind to and polyubiquitinate Bach2. Involvement of Cullin 3 wassuggested because it was also present in the puriﬁed Bach2 complex. Also, we conﬁrmed that KeapX
and KeapY interact with Cullin3 in immunoprecipitation assays when they were overexpressed incultured cells. Most importantly, a heterodimeric nature of substrate recognition adaptor is not known
thus far for Cullin 3 E3 ligase. Keap1 is known to function as a homodimer. Because there are morethan 20 proteins with BTB and Kelch-like repeats, our observation suggests that these protein may
generate a vast protein network for protein ubiquitination. This possibility should be explored in thenear future.

References
There has been no publication describing above project. Function of Bach2 was reported:
Ochiai, K., Muto, A., Tanaka, H., Takahashi, S. and Igarashi, K. Regulation of the plasma celltranscription factor Blimp-1 gene by Bach2 and Bcl6. Int. Immunol. 20, 453-460 (2008)- 109 -

Regulation of soma/germ-line distinction by small RNAs.
Kunio Inoue
Department of Biology, Graduate School of Science, Kobe University
kunio@kobe-u.ac.jp

Abstract
Our present study shows that an evolutionarily conserved RNA-binding protein DAZ-like (Dazl)
can relieve the miR-430-mediated repression of tdrd7 mRNA, playing an important role in germline/
somatic cell distinction in zebraﬁsh.
Keywords: zebraﬁsh, germ cell, microRNA

Introduction
Recently, many studies have suggested that a plenty of small RNA, such as microRNA, play crucial
roles in control of gene expression. Our previous study showed that microRNA (miRNA) miR-430
represses germline mRNAs in somatic cells during zebraﬁsh embryogenesis.

Results
During zebraﬁsh embryogenesis, miR-430 contributes to restrict Nanos1 and TDRD7 to primordial
germ cells (PGCs) by inducing mRNA deadenylation, mRNA degradation, and translational
repression of nanos1 and tdrd7 mRNAs in somatic cells. Although miR-430 is also expressed in
PGCs, the nanos1 and tdrd7 3’UTRs include cis-acting elements that allow activity in PGCs evenin the presence of miRNA-mediated repression. In the present study, we indicate that a germlinespeciﬁc RNA-binding protein DAZ-like (Dazl) can relieve the miR-430-mediated repression of tdrd7
mRNA. Dazl counteracts miRNA-mediated deadenylation and mRNA degradation by binding to its- 110 -

target sequences in tdrd7 3’UTR. Dazl also enhances its own expression. Thus, these results indicatethat Dazl acts as an “ant-miRNA factor” during vertebrate germ cell development.

Discussion & Conclusion
Our present study shows that control of miRNA function plays a crucial role in germline/somatic
cell distinction at least in zebraﬁsh. Our data also suggest that miRNA-mediated regulation can be
modulated on speciﬁc target mRNAs through the poly(A) tail control.

References
1. Mishima, Y., Giraldez, A.J., Takeda, Y., Fujiwara, T., Sakamoto, H., Schier, A.F., & Inoue, K. (2006)
Differential regulation of germline speciﬁc mRNAs in soma and primordial germ cells by zebraﬁshmiR-430. Current Biology 16, 2135-2142.

- 111 -

Comprehensive understanding of molecular mechanismsthat can control all sequentially happens in the ocular dominancecolumns from developmental stage to adult.
Koichi Tomita
Center for Genetic Analysis of Behavior, National Institute for Physiological Sciences
ktomita@nips.ac.jp

Abstract
This study aims at comprehensive clariﬁcation of the molecular mechanisms which regulate several
sequential events in ocular dominance (OD) columns from developmental stage to adulthood. A
molecular chaperone that accumulates and activates many kinds of factors is preferentially expressedin ipsilateral OD columns at all stages examined. The salient molecular chaperone offers a chance toidentify molecules that control several sequential events in OD columns, which might be carried out
via yeast two-hybrid isolation of binding molecules to the molecular chaperone at different ages.
Here, ipsilateral OD column specificity of the chaperon was first confirmed (Fig.1). Second,through a full length cDNA library screening of the visual cortex two isoforms of the chaperone
were identiﬁed; long and short forms (Fig.2). Because the short form is likely to show ipsilateral OD
column speciﬁcity, a plan is to use the short form as bait in yeast two-hybrid screening.
Keywords: Ocular dominance (OD) column, molecular chaperone, molecular mechanisms

Introduction
Each of biological events never happens exclusively, but is considered as one of components thatline in chain. What sequentially happen in the brain from birth to adulthood are to form coarse brain
structure during development, to sculpt the functional brain by experience in early postnatal life,
and to exhibit a full brain function in adult. Therefore, these should be ideally analyzed in one hand,
although that has been practically impossible. A challenge of this study is analyzing many sequential
events that take place in the model system, OD columns, at once.

Results
The alternating bands of ipsi- and contralateral eye preference that lie in the visual cortex ofhigher mammals are called ocular dominance (OD) columns, important functional units to achieve
depth perception. Those which take place successively in OD columns such as start of ipsi- andcontralateral eye OD column segregation during development, refinement to form adult like map
in early postnatal life, and a full function of depth perception in adult cannot be put apart. A goal ofthis study is to identify comprehensive molecular mechanisms which control the several sequential
events in OD columns. A molecular marker speciﬁc for ipsilateral OD columns that I have originallyisolated can help to achieve such analysis. Ipsilateral OD column specific molecule encodes a- 112 -

molecular chaperone which can recruit and activate many client proteins including regulatory factors
and signaling factors. This intriguing ﬁnding proposes an idea that a developmental factor to initiate
segregation of ipsi- and contralateral OD columns, a reﬁnement factor to set up adult like map and asignaling factor to achieve depth perception appear and disappear on this chaperone sequentially one
after another.

I have employed yeast two-hybrid system to isolate these molecules which might bind to theipsilateral OD speciﬁc chaperone at different stages. Before start of yeast two-hybrid screening, I
figured out two important molecular characters of the chaperone. First, I repeated experiments to
conﬁrm the ipsilateral speciﬁc expression of the chaperone (Fig.1). OD maps are ﬁrst visualized by
the optical imaging method and thereafter the same area is subjected to in situ hybridization studiesof the chaperone. The overlaid image of the chaperone in situ hybridization pattern onto OD maps
shows clear preferential expression of the chaperone for left OD columns in the left hemisphereand for right OD columns in the right hemisphere, meaning a favorite of ipsilateral OD columns
with the chaperone (Fig.1). Second, the original clone of the chaperone corresponds to a part of thefull length clone. Therefore, complete molecular structure of the chaperone was clariﬁed (Fig.2). A
cDNA library of the visual cortex that theoretically contains full length of genes was prepared. The
screening of the cDNA library by the original partial clone found two isoforms of the full length- 113 -

chaperon; long and short forms (Fig.2). The sequence analysis of both isoforms clearly reidentiﬁes
the sequence results of the original partial clone, namely they encode the molecular chaperone.
Recently, I have investigated ipsilateral OD column specificity of these isoforms by using in situ
hybridization and have recognized that the short form seems to show high ipsilateral speciﬁcity. The
results of strong ipsilateral preference of the short form were ﬁrst shown in one animal, which is
now reinvestigated in different animals. Recently, I have started the preparation of yeast two-hybrid
screening, where I am planning to use the short form chaperone as bait.

Discussion & Conclusion
OD columns are well studied models to look for the mechanisms underlying formation of functional
brains in early postnatal life. However, none of the mechanisms to initiate OD column segregation
during development or to perform depth perception in adult OD columns can be addressed before.
Although this study has not been completed, the goal of this study originally pursues comprehensive
understanding of molecular mechanisms which control these sequential events in OD columns. Here,I reconﬁrmed the ipsilateral speciﬁc expression of the molecular chaperone which can recruit andactivate many client proteins including regulatory factors and signaling factors (Fig.1). The screeningof the visual cortex cDNA library found two isoforms of the full length chaperone; long and shortforms (Fig.2). This new finding prompts an analysis of ipsilateral OD column specificity of two
isoforms, which gave me a preliminary result; preferential expression of the short form for ipsilateral
OD columns. Based on this intriguing result I am planning to carry out yeast two-hybrid screening
by using the short form of the chaperone as bait, which might enable me to isolate molecules that
control several sequential events in OD columns,
In early postnatal life, neurons of contralateral OD columns have stronger neuronal activity
compared to those of ipsilateral columns. There has been a Hebbian rule that active area has a highchance to gain more than silent area. Then, contralateral OD columns can occupy most of the visualcortex and ipsilateral OD columns lose area, but this is not true. An asymmetric activity between
contra- and ipsilateral OD columns could be canceled, where the ipsilateral OD column specificmolecular chaperone might protect widespread of contralateral OD columns in the visual cortex. In
the research ﬁeld of OD columns or maybe beyond it, this interesting expression of the chaperon
not only offers a challenge of comprehensive analysis of several sequential events but also includes
biological signiﬁcance.

- 114 -

Efﬁcient Synthesis of Biologically Active Nitrogen-Containing
Heterocyclic Compounds by the Carboamination Reactions
Takuya Kurahashi
Department of Material Chemistry, Graduate School of Engineering, Kyoto University
tkuraha@orgrxn.mbox.media.kyoto-u.ac.jp

Abstract
A new nickel-catalyzed reaction of alkynes with N-arylphthalimides to provide isoquinolones has
been developed.
Keywords: Heterocyclic compounds, Transition-metal catalyst, Cycloaddition

Introduction
A direct oxidative addition of a low valent transition-metal catalyst into C–N bond generates theactive species (C–M–N), which may perform a carboamination with alkyne. The reaction will form
C–C and C–N bond simultaneously. It should be useful transformation to prepare nitrogen-atom
containing compound, however, has not been studied well, because of the difﬁculty of such oxidative
addition.

Results
We postulated that a nickel(0) with electron-donating phosphine ligands will attack an amide
nucleophilically; the addition may afford the C–M–N species via decarbonylation, and might allow
insertion reaction to alkynes. Indeed, we have developed a new nickel-catalyzed reaction of alkynes
with N-arylphthalimides to provide isoquinolones. The isoquinolone skeleton is widely found invarious natural product and medicinal drug that exhibits a broad range of biological properties. It
was demonstrated that amide C–N bonds are susceptible to nucleophilic attack of Ni(0) complex,
which allow intermolecular addition to alkynes via decarbonylation. A key step in the reaction is
migratory extrusion of CO. The extruded CO should be removed from the reaction system for an
efﬁcient catalytic cycle, since it interacts very strongly with Ni and saturates the coordination sites,
thus interfering with regeneration of an active catalyst or causing the reverse. We postulated that an
active species (C–Ni–N) might be generated alternatively via an oxidative addition of a carbamate
to Ni(0) and decarboxylation. This would allow for robust carboamination of alkynes, since CO2
coordinates to a nickel complex much less strongly than CO. Thus, we have also developed a newnickel-catalyzed reaction of alkynes with isatoic anhydrides to provide quinolones. Quinolone
represents an important class of naturally occurring alkaloids that display a wide range of biologicalactivities, and are useful intermediates in the synthesis of a variety of hetero- and carbocyclic
compounds. We demonstrated that carbamates are susceptible to oxidative addition of a Ni(0)complex, which allows intermolecular addition to alkynes via decarboxylation.

- 115 -

Discussion & Conclusion
It was revealed that amide C–N bonds are susceptible to nucleophilic attack of Ni(0) complex,
which allow intermolecular addition to alkynes via decarbonylation. The reaction was applied forfacile synthesis of isoquinolones. The isoquinolone skeleton is widely found in various natural
product and medicinal drug that exhibits a broad range of biological properties. It was also
discovered that carbamates are susceptible to oxidative addition of a Ni(0) complex, which allows
intermolecular addition to alkynes via decarboxylation. Thus, it opens the way for facile synthesisof quinolones, which represent an important class of naturally occurring alkaloids that display a wide
range of biological activities, and are useful intermediates in the synthesis of a variety of hetero- andcarbocyclic compounds.

References(1) “Nickel-Catalyzed Decarboxylative Carboamination of Alkynes with Isatoic Anhydrides”
Yoshino, Y.; Kurahashi, T.; Matsubara, S. J. Am. Chem. Soc. 2009, 131, ASAP.
(2) “Nickel-Catalyzed [4 + 2] Cycloaddition of Enones with Alkynes”
Koyama, I.; Kurahashi, T.; Matsubara, S. J. Am. Chem. Soc. 2009, 131, 1350.
(3) “Nickel-Catalyzed Decarbonylative Addition of Anhydrides to Alkynes”
Kajita, Y.; Kurahashi, T.; Matsubara, S. J. Am. Chem. Soc. 2008, 130, 17226.
(4) “Nickel-Catalyzed Decarbonylative Addition of Phthalimides to Alkynes”
Kajita, Y.; Kurahashi, T.; Matsubara, S. J. Am. Chem. Soc. 2008, 130, 6058.

- 116 -

Studies on cytokinetic block in cellular senescence
Eiji Hara
The Cancer Institute of Japanese Foundation for Cancer Researcheiji.hara@jfcr.or.jp

Abstract
In order to understand how cellular senescence is induced and maintained in cultured humanprimary cells, we sought signaling pathway(s) that promote production of reactive oxygen species(ROS). Here, we found that both Ras/Raf/MEK pathway and PI3-kinase pathway are required for
production ROS in senescent cells. Furthermore, we also found that E2F/DP1 transcription factors
are involved in ROS production. These results indicate that there are several different pathways that
activate ROS production in cellular senescence.
Keywords: Cellular senescence / ROS / Cytokinesis

Introduction
Oncogenic proliferative signals are coupled to a variety of anti-proliferative signals, such as
apoptotic cell death or senescence cell cycle arrest1, 2, 3. Thus, both apoptosis and senescence arethought to act as important tumor suppression mechanisms. Unlike apoptotic cells, senescent cells are
viable for long periods of time4. It is therefore important to understand the molecular mechanism(s)that maintain senescence cell-cycle arrest. Recently, we found that sustained production of ROS
irreversibly block cytokinesis in senescent cells4. However, molecular mechanism(s) underlying
this process remained unclear. In this study, we aimed to clarify the mechanisms that irrevocably
promote ROS production in senescent cells.

Results
To seek mechanistic insight into the role of the p16INK4a-RB axis in production of ROS, we ﬁrstly
tested whether E2F/DP, downstream transcription factor complex, may have a role in this process.
Towards this end, we utilized RNA interference (RNAi) approach to stably knock-down the levels of
E2Fs or DP proteins. Although normal human diploid ﬁbroblasts (HDFs) express multiple membersof E2F family proteins, they mainly express DP1 but not other members of DP family proteins. We,
thus, knocked-down DP1, rather than E2Fs in HDFs. As expected, E2F/DP activity was dramaticallyreduced in DP1 knock-down HDFs. Interestingly, we observed that the intracellular levels of ROS
were signiﬁcantly increased in this setting, indicating that the p16INK4a-RB pathway provokes ROS
production through blocking the E2F/DP transcription factors. Moreover, we found that the levelsof Manganase superoxide dismutase (MnSOD) and Glutathione peroxidase (GPX), the enzyme
known to be involved in ROS production, are altered in E2F/DP1 knocked-down cells, as well as
in senescent HDFs. Together, these results strongly suggest that the activation of the p16INK4a-RB- 117 -

pathway causes ROS production through altering the expression of E2F/DP target genes involved
in ROS production. These results signiﬁcantly advance our understanding of how p16INK4a tumor
suppressor promotes ROS production in senescent cells.
We secondly asked if other signaling pathway may involve ROS production in senescent cells. To
this end, we took an advantage of using SVts8 cells, a conditionally immortalized human ﬁbroblast
cell line that express temperature-sensitive mutant of SV40 large antigen (tsLT) and elevated levelof the endogenous telomerase. Svts8 cells proliferate indefinitely at the permissive temperature,
because tsLT binds to and inactivates both pRb and p53. However, when shifted to the nonpermissive temperature, tsLT is inactivated and a senescence-like cell cycle arrest is induced. Using
SVts8 cells, in conjunction with a panel of chemical inhibitors, we tested which signaling pathway(s)are required for the onset of cellular senescence. SVts8 cells were cultured at non-permissive
temperature for 5 days in the presence of these chemicals. These cells were subsequently cultured
at permissive- temperature for another 5 days after removing chemicals inhibitors

Interestingly,

although control cells remained arrested even after shifting back to the permissive temperature,cells treated with several inhibitors were able to reinitiate cell proliferation upon shifting back to
the permissive temperature. The same experiments were repeated three times and found that 25
out of 260 chemicals were able to revoke cell cycle arrest when shifting back to the permissive
temperature, indicating that these chemicals are potent inhibitors of the onset and/or maintenance of
cellular senescence. Indeed, we noted that production of intracellular levels of ROS was substantially
blocked when SVts8 cells were treated with these chemicals at non-permissive temperature. Thesechemicals were classiﬁed into two groups; one blocks Ras/Raf/MEK kinase pathway and the otherblocks PI-3 kinase pathway, suggesting that both pathways are required for the onset of irrevocable
ROS production in senescent cells (see Figure).
We previously reported that a ptoteasome-dependent degradation of the WARTS/ LATS1 protein,a mitotic exit network (MEN)-kinase required for cytokinesis, was strikingly enhanced by elevationof ROS level, leading to an irreversible cytokinetic block in senescent cells4.

- 118 -

However, we wereunable to override this cytokinetic block by over-expression of WARTS/LATS1, implying that otherprotein(s) required for cytokinesis may also be de-stabilized by ROS in senescent cells. We therefore
sought such factor(s). So far, we found that Lats2, another member of Last1 family, were also destabilized by ROS in senescent cells. Further investigation is on going.

Discussion & Conclusion
Our results reveal that the induction of p16INK4a expression activates RB, thereby blocking E2F/DP1
transcription activity, leading to an irrevocable ROS production in senescent cells. We also foundthat both Ras / Raf / MEK kinase pathway and PI-3 kinase pathway are involved in this process.
These results signiﬁcantly advance our understanding of how p16INK4a expression causes elevationof the intracellular levels of ROS which required for the onset of irreversible cell-cycle arrest. We,
however, do not understand how these three different pathways (p16INK4a –RB/ E2F/DP1-pathway,
Ras/Raf/MEK kinase pathway and PI-3 kinase pathway) are integrated into the ROS production insenescent cells. Further investigations are required to resolve this issue.

References
1. Lowe, S.W., Cepero, E. and Evan, G. Nature, 432: 307-315 (2004).
2. Sharpless, N.E. and DePinho, R.A. Nature, 436, 636-637 (2005).
3. Serrano, M. and Blasco, M.A. Curr. Opin. Cell Biol., 13: 748-753 (2001).
4. Takahashi, A. et al., Nature Cell Biol., 8: 1291-1297 (2006).

- 119 -

Physiological functions of D-serine in mammals and development of
its enzymatic assay system
Tohru Yoshimura
Graduate School of Bioagricultural Sciences, Nagoya University
yosimura@agr.nagoya-u.ac.jp

Abstract
We have found a novel enzyme, eukaryotic D-serine dehydratase, catalyzing a dehydration of
D-serine to pyruvate and ammonia in Saccharomyces cerevisiae. The enzyme is highly speciﬁc toD-serine. We have applied the enzyme to an enzymatic D-serine assay system, which is probably

useful for the clinical examination of neurological disorders .
Keywords: D-serine, D-serine dehydratase, D-serine assay, neurological disorder

Introduction
Several D-amino acids occur in eukaryotes and have various physiological functions. For example,

D-aspartate is distributed in the mammalian central nerve system and suggested to be involved in
the hormone regulation and teststerone synthesis. D-Serine occurs primarily in the mammalianbrain and modulates brain function as a coagonist of the NMDA receptor. To understand themolecular mechanism of the physiological functions of D-amino acids, our group has been studying
the eukaryotic enzymes related to the D-amino acid metabolism. We have found a novel D-serine
dehydratase in Saccharomyces cerevisiae and established an enzymatic D-serine assay system withusing the enzyme.

Results
We have found that the uncharacterized gene of Saccharomyces cerevisiae, ygl196, encoding
D-serine dehydratase. Ygl196wp, which we name DsdSC (D-serine dehydratase of S. cerevisiae),

depends on pyridoxal 5’-phosphate (PLP) and catalyzes the dehydration of D-serine to produce
pyruvate and ammonia (1). DsdSC is the first D-serine dehydratase found in eukaryotes, andpredicted to have a motif similar to that of the N-terminal domain of the bacterial alanine racemase.
D-Serine dehydratase had been already found in several bacteria. Bacterial D-serine dehydrataseshowing a little activity for L-serine is also a pyridoxal enzyme, but the enzyme structure iscompletely different from that of DsdSC. Bacterial enzyme and DsdSC are probably evolved
convergently from different ancestral proteins. DsdSC acts efficiently on D-serine. D-Threonine,
D-allo-threonine, and β-Cl-D-alanine also serve as substrates with catalytic efﬁciencies, which areabout 3, 1, and 2% of D-serine, respectively. L-Serine, L-threonine, and D-, L-alanine are inert as
substrates.
DsdSC lost the D-serine dehydrase activity by the EDTA treatment, and fully re-activated by the- 120 -

$.-#)!((-%('!$+!) -()('!$ -')(










$%& $%"





%*&"!$$%& $%"














)) -'%$(%*&"!$



 

 







 

 











 

- , ' 

 



 

 

- , ' 

 









 





'!$









    '!$

presence of 2.5 - 5.0 mM Zn2+. Over 10 mM, zinc showed inhibitory effects. Little recovery wasobserved with Mg2+, Mn2+, Ca2+, Ni2+, Cu2+, K+, or Na+. Atomic absorption analysis revealed thatthe enzyme contains 1.0 Zn atom per enzyme monomer. DsdSC is probably the first example ofa eukaryotic D-serine dehydratase and a specifically zinc-dependent pyridoxal enzyme as well.
Activities toward D-threonine and D-allo-threonine were also decreased below the detection limit
by the EDTA-treatment and recovered by the addition of Zn2+. On the other hand, α, β-eliminationof β-Cl-D-alanine was slightly increased rather than decreased by the EDTA treatment. These
results suggest that zinc is implicated in the step of OH elimination. We separately analyzed therates of Cα-H abstraction and OH elimination of D-threonine in D2O with 1H NMR, and found thatzinc accelerated both steps. If OH elimination is a rate-limiting step, zinc accelerates the rates ofD-serine and D-threonine dehydration. Because DsdSC catalyzes the elimination of OH group of

threonine irrespective of the stereochemistry of the C3 carbon, OH group probably interacts withzinc indirectly. The tryptophan fluorescence of the enzyme was changed by the EDTA-treatment
and returned to the original one by the addition of zinc. On the bases of these results, we speculatethat zinc maintains the enzyme structure to be favorable for the elimination of OH group from thesubstrate.
Behavior of D-serine is related to the clinical states of the patients of various neurologicaldisorders such as schizophrenia, Alzheimer’s disease, and amyotrophic lateral sclerosis (ALS). We
developed an enzymatic method for the D-serine assay with DsdSC exhibiting the strict substratestereospecificity (2). In this method, pyruvate produced from D-serine by the DsdSC reaction
is assayed with lactic dehydrogenase and NADH. Under the conditions, we could measure 10– 200 µ M D-serine. The method was not affected by the contamination of other amino acids
including L-serine and bovine serum. The D-serine contents obtained with the enzymatic methodin human urine and various tissues of the scrapie-infected hamster agreed with those obtained by aconventional HPLC method. Pyruvate formed from D-serine with DsdSC can be also assayed by the- 121 -

coupling method with pyruvate oxidase. We could assay 1 – 10 µM D-serine with xylenol orange bydetermining the amount of H2O2 produced during the pyruvate oxidase reaction.

Discussion & Conclusion
Putative proteins, which are homologous to DsdSC have been found in various eukaryotic microbes,
zebra fish, and birds. The enzyme from cellular slime molds and chicken have been confirmed
to have a D-serine dehydrase activity. D-Serine dehydratase is a main pathway of the D-serine
degradation in these organisms. In mammals, D-amino acid oxidase bears the similar role. There isa signiﬁcant relationship between D-serine and central nervous system diseases. For example, theamount of D-serine and the relative ratio of D-serine to (D+L)-serine are signiﬁcantly decreased in
the serum and frontal lobe of schizophrenia or Alzheimer’s disease patients, and an excess amountof D-serine is produced in the spinal ﬂuid of ASL patients. D-Serine is expected to be a diagnostic
biomarker for such diseases. Currently D-serine and other D-amino acids are assayed by HPLC after
the amino acids are derivatized to ﬂuorescent diastereomers. The HPLC-method is highly sensitiveand enables the comprehensive analysis of D- and L-amino acids. However, it is time-consuming, andrequires expensive equipments and a proﬁcient analyzer. Our enzymatic assay method with DsdSC isprobably useful for the clinical examination of such neurological disorders.

References(1) Ito, T., Hemmi, H., Kataoka, K., Mukai, Y., and Yoshimura T (2008) Biochemical J., 409, 399-406.
(2) Ito, T., Takahashi, K., Naka, T., Hemmi, H., and Yoshimura T. (2007) Anal. Biochem., 371, 167-172.

- 122 -

Functional Role of Heme-Protein Interaction
in Monooxygenase Cytochrome P450
Takashi Hayashi
Graduate School of Engineering, Osaka University
thayashi@chem.eng.osaka-u.ac.jp

Abstract
Monooxygenase cytochrome P450cam is one of the attractive heme-containing enzymes, where
the heme is non-covalently bound in the protein interior. In our study, we prepared a P450cam
protein reconstituted with an artiﬁcial “one-legged” heme (7-despropionated heme) to understandthe functional role of the 7-propionate side chain. From the present results, we conclude that the
7-propionate side chain plays an essential role in the process of elimination of the water cluster fromthe substrate-binding site upon d-camphor binding, and forms a barrier against entry of bulk water tomaintain the high d-camphor afﬁnity.
Keywords: P450cam, Heme, Monooxygenase, Enzyme Mechanism

Introduction
Cytochrome P450cam is a heme-containing enzyme which catalyzes hydroxylation of d-camphor.
The heme is bound in the heme pocket via noncovalent interactions, where two heme-propionate side
chains interact with Arg, His and/or Asp residues. The peripheral heme-propionate side chains havebeen long regarded simply as anchors for connecting the heme prosthetic group to the protein matrix.
However, we have recently proposed that the propionate side chains play an important role in theregulation of the enzymatic function. Our current work supported by Novaltis Foundcation clearly
demonstrated that the heme-7-propionate side chain regulates the substrate binding afﬁnity.

Results
To understand the functional role of the heme-7-propionate side chain in the P450cam protein
interior, we prepared the protein reconstituted with an artificial one-legged heme which has
a 7-methyl group at the position of the
7-propionate side chain (Figure 1). The insertionof the one-legged heme into apoP450cam
was carried out by the method described in
our previous paper. The reconstitution was
confirmed by ESI-TOF mass spectrometry.
Purified reconstituted P450cam exhibits theferrous CO-bound P450cam spectrum withthe characteristic Soret band at 446 nm,- 123 -

indicating that the thiolate of Cys357 is ligated to the heme iron of the one-legged heme. In contrast,
unexpectedly, the UV-vis spectrum of the reconstituted ferric P450cam in the presence of 1 mM
d-camphor is similar to that of the typical d-camphor-free low-spin ferric P450cam species withmaxima at wavelengths of 417, 540 and 570 nm, whereas the wild type ferric protein usually has aspectrum characteristic of the d-camphor-bound high-spin species under the same conditions. The
content of the high-spin state of the reconstituted protein in the presence of 1 mM d-camphor isestimated to be 26%. From the UV-vis spectroscopic study, it is found that the d-camphor afﬁnity forthe substrate-binding site of the reconstituted protein is approximately 3 mM at 20 °C, pH 7.4. Fromthese results, the removal of heme-7-propionate was found to dramatically decrease the d-camphor
affinity by approximately three orders of magnitude, suggesting that the 7-propionate side chain
plays a role in the high afﬁnity of cytochrome P450cam for its substrate, d-camphor.
Although the affinity of the one-legged heme-reconstituted P450cam for reduced putidaredoxin(Pdx) is essentially the same as that of wild type P450cam, the process of electron transfer fromreduced Pdx to ferric reconstituted P450cam is much slower. As a result of this slowed firstelectron transfer in the reconstituted protein, the rate constant of the NADH-driven hydroxylation of
d-camphor is only 8% of that determined for the wild type protein. These ﬁndings are supported by
the spectroscopic data that indicate the predominant presence of the less reactive low-spin species
in the reconstituted protein. These results indicate that the removal of the 7-propionate side chain
dramatically decreases the d-camphor afﬁnity.

Results
The X-ray structure of the reconstituted P450cam at a resolution of 1.8 Å reveals that theasymmetric one-legged heme is incorporated into the heme pocket in the same plane and in
essentially the same conformation as the heme of the wild type protein. In the vicinity of the
7-propionate side chain, in the wild type protein there is a unique hydrogen bonding tetrad network
comprising Arg299, 7-propionate, Asp297 and Gln322. Conformational differences are clearlyobserved for Asp297 and Gln322, which evidently deviate from their respective positions in thenative structure, whereas, notably, the conformation of Arg299 is almost same as that of the wild
type protein. The d-camphor substrate binds to the substrate-binding site with nearly the same
conformation as that of the wild type protein. However, thesubstrate-binding site is not fully occupied with d-camphor.
The F o – F c map of the substrate-binding site clearlyshows electron densities that fit to a camphor moleculeand two water molecules with occupancies of 0.25, 0.75,
and 0.75, respectively. In the d-camphor-free structure, atleast two distinct water molecules are located within thesubstrate-binding site; one water molecule is coordinatedto the heme iron at a distance of 2.54 Å. Furthermore, in
the reconstituted protein, two additional water molecules- 124 -

are identified near Tyr96 and Asp297, and a single water molecule occupies the position of the
7-propionate carboxylate. As a result, a unique array of water molecules extending from the Tyr96
residue to the outside of the protein is observed in the crystal structure (Figure 2). In addition, it isnoted that the Asp297 residue is remarkably ﬂipped and its side chain Oγ1 and Oγ2 atoms are locatedwithin hydrogen bonding distance of the special water molecules. The hydrogen bonding interaction
between the characteristic water molecules and the Asp297 residue disconnects the Gln322 residue
from the Asp297 residue due to the cleavage of the O(Asp297)–HN(Gln322) interaction.

Discussion & Conclusion
The resting state in P450cam has a unique water cluster at the substrate-binding site and thereplacement of water cluster with d-camphor is the initial step of the catalytic cycle. Removal of
the heme-7-propionate side chain in the heme prosthetic group dramatically lowers the d-camphor
afﬁnity for the P450cam substrate-binding site. As a result, we found a water molecule array frombulk to the substrate-binding site without causing any major changes in the protein structure with
the notable exception of conformational changes occurring at Asp297 and Gln322 residues. It is
proposed that the heme-7-propionate side chain plays an essential role in an aqua gate of the water
cluster in the substrate-binding site and a barrier against entry of bulk water molecules. Therefore,
the combination of the propionate side chain with Asp297, Arg299 and Gln322 acts as a regulation
system of the water molecule expelling/repelling event at the edge of the substrate-binding site in theprotein during the enzymatic reaction toward the d-camphor oxidation. The present study is the ﬁrst
example that experimentally indicates the functional roles of the heme-7-propionate side chain in
P450cam.

References
1. A Role of the Heme-7-Propionate Side Chain in Cytochrome P450cam as a Gate for Regulating theAccess of Water Molecules to the Substrate-Binding Site
T. Hayashi, K. Harada, K. Sakurai, H. Shimada, S. Hirota,
J. Am. Chem. Soc. 131, 1398–1400 (2009).

2. Evaluation of the Functional Role of the Heme-6-Propionate Side Chain in Cytochrome P450cam
K. Harada, K. Sakurai, K. Ikemura, T. Ogura, S. Hirota, H. Shimada, T. Hayashi,
J. Am. Chem. Soc. 130, 432–433 (2008).

- 125 -

Basic research for the olfactory disease occurrence
and its degenerative therapy
Akio Tsuboi
Laboratory for Molecular Biology of Neural Systems
Research Institute for Frontier Medicine
Nara Medical University
atsuboi@naramed-u.ac.jp

Abstract
Neurosphere assays are standard studying these stem-like cells in normal tissues.
To isolate neural stem cells (NSCs) from the mouse olfactory epithelium, we made neurospheresfrom the olfactory mucosa by a serum-free culture system. Then we succeeded to differentiate thoseto olfactory neurons and astrocytes in media, not supplemented with mitogens such as EGF and FGF.
Keywords: olfactory system, neural stem cell, neurosphere, neuronal differentiation

Introduction
Neurosphere assays have been a standard for identifying stem cell populations, allowing for
isolation and characterization of NSCs1. These assays consist of a selective serum-free culture
system that enables NSCs to proliferate and generate neurospheres, while the more differentiatedcells die. In this study, we adopted this assay to the olfactory system to obtain epithelial NSCs.

Results
Five-weeks-old mice were anesthetized with pentobarbital and sacrificed by bleeding. Olfactory
mucosa were dissected, treated with Dispase II, and separated into the olfactory epithelium and thelamina propria. Both samples were treated with collagenase 1A and centrifuged. The pelleted cells
were suspended and incubated in DMEM-hormone mix, supplemented with epidermal growth factor(EGF) and basic ﬁbroblast growth factor (bFGF) in a concentration of 10 ng/ml, respectively 2,3,4.
After cultivating for 10 days, a sphere-like aggregate appears
mainly from the lamina propria. The sphere was transferred
into a new petridish to progress to grow largely. The sphere was
immunostained with Nestin (stem cell marker), indicating NSCs
(Fig. 1).
To differentiate the neurosphere to neuronal and glial cells,
dissociated sphere cells (NSCs) were spread into the poly-Dlysine-coated dish containing DMEM-hormone mix without
growth factors. Some cells were immunostained with TuJ1
(neuronal cell marker), and the others with GFAP (astrocyte- 126 -

F i g u re 1 .
A sphere-like aggregate, termed
neurosphere, appears mainly from thelamina propria. The neurosphere was
immunostained with Nestin (stem cellmarker), indicating NSCs.

marker) (Fig. 2). These results indicate that the neurospherefrom the olfactory mucosa has a capacity to differentiate into
neuronal and glial cells.

Discussion & Conclusion
F i g u re 2 .
Differentiate of the neurosphere into
neuronal and glial cells. Some cells were
immunostained with TuJ1 (neuronal cellmarker; red), and the others with GFAP
(astrocyte marker; blue).

Neurosphere assays were ﬁrst developed by Reynolds & Weiss(1992) to isolate NSCs from the mouse striatum 5, and remain
to be a standard for determining the presence of these stemlike cells in normal tissues, because they can determine whether
a particular cell possesses self-renewing and multi-potential

capabilities

1,6. Under these conditions, there exist usually cells that are grown in serum-free

medium and on non-adherent plates, because both unknown serum factors and adherence promote
differentiation 5,6. In addition, mitogens including EGF 4,5 and/or FGF 4,7, are also used frequently
because they facilitate NSC proliferation. The neurospheres formed under these conditions can beserially dissociated and plated to generate additional neurospheres. Furthermore, NSCs within theneurospheres can differentiate into neurons, astrocytes, and/or oligodendrocytes. This has been
showed by markers speciﬁc for neurons (TuJ1), astrocytes (GFAP) and oligodendrocytes (Oli4) 7.
The discovery of NSCs breaks ground a new avenue to potential treatments for degenerative
diseases such as Parkinson and devastating cancers such as GBM, which has a mean lifespanof 14.6 months after diagnosis 8. The best ways of assessing these types of stem cells are due toneurosphere assays. Progress in this area will help us to move forward to studying novel therapies
for neurological diseases.

References
1. Reynolds BA, Rietze RL. Neural stem cells and neurospheres
Nat Methods;2:333–336 (2005).

－ Re-evaluating the relationship.

2. Sanai N, Tramontin AD, Quinones-Hinojosa A et al. Unique astrocyte ribbon in adult human brain
contains neural stem cells but lacks chain migration. Nature 427:740–744 (2004).
3. Ignatova TN, Kukekov VG, Laywell ED et al. Human cortical glial tumors contain neural stem-likecells expressing astroglial and neuronal markers in vitro. Glia 39:193–206 (2002).
4. Gritti A, Frolichsthal-Schoeller P, Galli R et al. Epidermal and ﬁbroblast growth factors behave as
mitogenic regulators for a single multipotent stem cell-like population from the subventricular regionof the adult mouse forebrain. J Neurosci 19:3287–3297(1999).
5. Reynolds BA, Weiss S. Generation of neurons and astrocytes from isolated cells of the adultmammalian central nervous system. Science 255:1707–1710 (1992).
6. Vescovi AL, Galli R, Reynolds BA. Brain tumour stem cells. Nat Rev Cancer 6:425–436 (2006).
7. Gritti A, Parati EA, Cova L et al. Multipotential stem cells from the adult mouse brain proliferate andself-renew in response to basic ﬁbroblast growth factor. J Neurosci 16:1091–1100 (1996).
8. Stupp R, Mason WP, van den Bent MJ et al. Radiotherapy plus concomitant and adjuvanttemozolomide for glioblastoma. N Engl J Med 352:987–996 (2005).

- 127 -

Pre-mRNA splicing mechanism of human Dystrophin gene
Akila Mayeda
Institute for Comprehensive Medical Science (ICMS), Fujita Health University
mayeda@fujita-hu.ac.jp

Abstract
To elucidate splicing mechanisms of extremely large introns in higher eukaryotes, we analyzed
the human dystrophin (DMD) gene that is the largest known human gene. We have obtained
experimental evidence supporting a novel mechanism termed ‘nested-intron splicing’, i.e., multiple
splicing of internal nested-introns preceding the eventual splicing at the authentic 5’ and 3’ splicesites.
Keywords: Splicing, Large intron, Dystrophin gene

Introduction
The essentia l pre-mRNA splicing is highly discriminatory and faithful. Thus a defect andmisregulation in this process cause disorders in cell functions, often with severe clinicalconsequences. Huge introns (>100 kb) are prevalent among important human genes. We do notknow yet how such huge introns are precisely spliced−−demanding to ignore many good splice sites
in the intron. We have obtained experimental data that provide a clue to solve this enigma.

Results
In this grant, we proposed innovative hypothesis to elucidate the splicing mechanism of huge intronthat is prevalent in the human genome. Using the human DMD gene that is associated with theserious muscular dystrophy, we obtained
experimental evidence supporting a novel
mechanism, termed ‘nested intron splicing’,i.e., multiple splicing of internal nested
introns prior to the eventual splicing at theauthentic 5’ and 3’ splice sites (Fig. 1).
We obtained the following experimental
results that strongly support this hypothesis[Ref. 2]; (i) Computer analysis of theextremely large intron 7 (109,574 nt) of
the human DMD gene revealed thepresence of 6,161 and 6,936 potential 5’
and 3’ splice site sequences, respectively.

Fig. 1. The model of multi-step nested-splicing in large intron
7 of DMD pre-mRNA.
The extremely large intron 7 of the DMD pre-mRNA isgradually shorten by multi-step nested splicing and thesufficiently shortened intron is eventually spliced out
between the authentic 5’ and 3’ splice sites.

- 128 -

(ii) Using a method to selectively
detect lariat introns by RT-PCR acrossthe branch site [Ref. 1], we obtained
evidence for at least two nested-splicing
processes within intron 7 (Fig. 2A).
(iii) We detected branching of a lariat
intron between the authentic splice sites,
however series of RT-PCR experiments
revealed that this intron lariat is
discontinuous (either Y-shaped orcontaining discontinuous sequences;Fig. 2A). (iv) We successfully isolated
lariat RNAs with highly-puriﬁed E.
coli recombinant 3’-5’exoribonuclease(RNase R) that degraded all the linear
and Y-shaped (open-lariat) RNAs.
Surprisingly we detected the samebranching after the RNase R digestion,

Fig. 2. Detection of splicing-dependent lariat RNAs from intron 7of DMD gene transcripts.

suggesting that the lariat is intact and(A) Detection of two lariat nested-introns in the intron 7
and whole lariat intron 7 (partial). The primer sets (arrows)
on the intron 7 used for RT-PCR assays are depicted. Fromthe position of primer sets SII and AII, we estimated thatthe branched product contains ~158 nt and ~156 nt from thebranch site (to 5’ side) and from the 5’ splice site (to 3’ side),
respectively. (B) Time-course detection of lariat RNAs fromintron 7 during the differentiation of myoblast. The RT-PCR
products of lariat RNAs were quantitated. (C) Two possiblemodels to detect lariat RNAs from the nested introns.

contains

discontinuous

sequences(not Y-shaped). These data indicatethat the detected lariat RNA product isthe shortened, nested-spliced, intron
7. (v) We observed the accumulation
of

ﬁnal

mRNA

duringthedifferentiation of myoblast, and the accumulation rate of lariat RNAs from the nested introns and ﬁnal
intron was faster than that of ﬁnal mRNA (Fig. 2B). We thus could exclude a trivial possibility thatthe detected lariat RNAs from the nested introns are re-splicing byproducts of excised whole lariat
intron 7 that is generated by a conventional one-step splicing (Fig. 2C).

Discussion & Conclusion
The mechanism of the nested-intron splicing may have important implications for understanding
splicing of extremely long introns in general. Our results will also provide new insights into thegeneral mechanisms of splice site selection, exon definition, and initiation and termination of
splicing.
Furthermore, it will provide considerable insight into many unsolved splicing defects in the DMD
gene that were found in cases of Duchenne muscular dystrophy.

- 129 -

References
1. H. Suzuki, Y. Zuo, J. Wang, M.Q. Zhang, A. Malhotra & A. Mayeda (2006). Characterization of RNase
R-digested cellular RNA source that consists of lariat and circular RNAs from pre-mRNA splicing.
Nucleic Acids Res. 34, e63.
2. H. Suzuki, J. Wang, Screaton, G.R., M.Q. Zhang & A. Mayeda. Nested introns in an intron: evidenceof multi-step splicing in a large intron. Manuscript in preparation.

- 130 -

Small molecules that induce the cell adhesion
Motonari Uesugi
Institute for Integrated Cell-Material Sciences, Kyoto University
uesugi@scl.kyoto-u.ac.jp

Abstract
During an image-based phenotype screening of our chemical library, we noted a small moleculethat boosts the adhesion of human cells to plastic cell culture plates. Chemical and cell biologicalexperiments suggest that the diaryldispirotripiperazine derivative (adhesamine) targets selective cellsurface glycosaminoglycans, especially heparan sulfate, for increasing cell adhesion. Unlike polyL-lysine, adhesamine induces apparently normal cell adhesion accompanied with organized actin
structures and activation of focal adhesion kinase and ERK1/2 mitogen-activated protein kinases.
Adhesamine may ﬁnd its use as a cell-attaching reagent for cell engineering and basic cell biology.
Keywords: Cell adhesion, Diaryldispirotripiperazine, Adhesamine, Glycosaminoglycan, HeparansulfateIntroduction
Interactions between cell and extracellular matrix are pivotal for survival of adherent cells both invivo and in vitro.1 In vitro maintenance of adherent cells can be promoted by coating culture plateswith artiﬁcial substrates. Artiﬁcial substrates, however, may cause abnormal cell-spreading, leading
to physiologically irrelevant outcomes. 2
We report the discovery of the ﬁrst non-peptidic organic molecule whose simple addition induces
apparently physiological cell adhesion to culture plates. Chemical and cell biological experimentssuggest that adhesamine is most likely to exert its cell-adhesion activity by interacting with selective
sulfated glysosaminoglycans (GAGs) to modulate cell surface properties and intercellular signaling
pathways.

Results
Discovery and Characterization of Adhesamine-Induced Cell Adhesion
During an image-based phenotype screening of our chemical library, we found a small moleculethat induces cell adhesion to culture plates. This molecule, we named adhesamine (1) seemed topromote cell adhesion of both adherent HepG2 and ﬂoating Jurkat cells with no obvious cytotoxicity(Figure 1). The growth of the cells was actually slightly enhanced in a dose-dependent manner, andthe cell morphology appeared normal just as well-maintained HepG2 and Jurkat cells. Visualizationof actin cytoskeltons and focal adhesions indicate that the adhesamine-induced contacts of the cells
to the plates initiate a normal schedule of cell adhesion.3,4 Cytoskeletal disruption inhibited theadhesamine-mediated cell adhesion in a dose-response manner, just as they impaired the ﬁbronectin-- 131 -

mediated cell adhesion (Figure 1). These data collectively
suggest that 1 induces cell adhesion similar to thoseinduced by native extracellular matrix.
Cell-surface Glycosaminoglycans as Possible Targets
To analyze the target of adhesamine (1), we synthesized
fluorescent probe (2) of 1. Fluorescence microscopic
observation of molecule 2 revealed its localization on thecell surface. The cell-surface localization of positively
charged molecule 2 suggested glycosaminoglycans(GAGs) as potential targets of adhesamine. GAGs include
five major components ― heparin, heparan sulfate,chondroitin sulfate, keratan sulfate, and hyaluronic acid.
Excess amounts of each GAG were added to the medium,and their ability to squelch the activity of adhesamine was
examined.5 Heparin, heparan sulfate, and keratan sulfate
decreased adhesamine-induced cell adhesion.
We carried out isothermal titration calorimetric (ITC) measurements of the binding of adhesamine
to each GAG. Clear exothermic interactions were observed for heparin, heparan sulfate, and keratan
sulfate with Kd values of 0.39, 4.67, and 5.85 µM, respectively. Titrations with chondroitin sulfate
and hyaluronic acid failed to display signals strong enough for Kd estimation. These data suggested
that adhesamine interacts selectively with heparin, heparan sulfate, and keratan sulfate on the cellsurface. Since the interaction with heparin is the most tight, we focused on heparin as a model for
detailed analyses of the interaction.
Cell-surface Heparan Sulfate as a Target
Jurkat cells were treated with GAG-degrading enzymes
to conﬁrm that cell adhesion with adhesamine is mediatedby cell-surface heparan sulfate. Degradation of cellsurface heparan sulfate chains reduced the adhesamineinduced cell attachment (Figure 2).
To further validate the interaction of adhesamine withheparan sulfate, we used well-characterized CHO-K1
cell mutants deﬁcient in glycosaminoglycan synthesis.6-9
Mutant cells lines were less responsive to adhesamine
than parental CHO-K1 cells (Figure 2). These data
indicate that the adhesamine-induced cell adhesion is aheparan sulfate proteoglycan-dependent process.
Adhesamine Induces Phosphorylation of FAK and ERK
Cell adhesion mediated by integrins and cell surface heparan sulfate proteoglycans generates
intercellular signals that stimulate a number of non-receptor kinases. Phosphorylation of focal- 132 -

adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK) were monitored in thepresence of adhesamine. We found that the phosphorylation of FAK and ERK is occurred 5 h after
adhesamine stimulation (Figure 2). Activation of FAK and ERK plays important roles in cell motility,
cell growth, cytoskeletal organization, and adhesion-dependent cell survival.10,11 The phosphorylationof these two kinases may account for the cytoskeltal-organizing and growth-promoting activities ofadhesamine.

Discussion & Conclusion
To our knowledge, adhesamine represents the first non-peptidic organic molecule whose simple
addition induces adhesion of cells to culture plates. Remarkably, adhesamine not only enhancesadhesion of adherent HepG2 cells but also renders ﬂoating Jurkat cells adherent to its substrates.
Chemical and cell biological analyses indicate that adhesamine binds to selected cell-surface
glycosaminoglycans, especially heparan sulfate, to exert its adhesion-enhancing activity. However,it remains unclear how the interaction of adhesamine with heparan sulfate promotes cell adhesion.
Adhesamine may bridge between heparan sulfate proteoglycans and plastic/glass surfaces byinteracting both of them. However, it is unlikely that adhesamine simply alters physical properties ofcell surface through electrostatic interactions. Unlike poly-L-lysine, adhesamine induces apparently
normal cell adhesion accompanied with organized actin structures, focal adhesion, activation of focal
adhesion kinase/ERK kinases. Such normal adhesion similar to that to extracellular matrix is usuallyinduced by clustering of integrins and heparan sulfate. One possibility is that the dumbbell-shaped
molecule may acts as a bridge among heparan sulfate proteoglycans, leading to the clustering of
heparan sulfate proteoglycans.

References
1. Howe, A. K., Aplin, A. E., and Juliano, R. L. (2002). Anchorage-dependent ERK signaling-mechanisms and consequences. Curr Opin Genet Dev 12, 30-35.
2. Bershadsky, A., Chausovsky, A., Becker, E., Lyubimova, A., and Geiger, B. (1996). Involvement of
microtubules in the control of adhesion-dependent signal transduction. Curr Biol 6, 1279-1289.
3. Huo, X., Xu, X. J., Chen, Y. W., Yang, H. W., and Piao, Z. X. (2004). Filamentous-actins in humanhepatocarcinoma cells with CLSM. World Journal of Gastroenterolgy 10, 1666-1668.
4. Parsey, M. V., and Lewis, G. K. (1993). Actin polymerization and pseudopod reorganization accompany
anti-CD3-induced growth arrest in Jurkat T cells. J Immunol 151, 1881-1893.
5. Mahalingam, Y., Gallagher, J. T., and Couchman, J. R. (2007). Cellular adhesion responses to theheparin-binding (HepII) domain of ﬁbronectin require heparan sulfate with speciﬁc properties. J Biol
Chem 282, 3221-3230.
6. Esko, J. D., Stewart, T. E., and Taylor, W. H. (1985). Animal cell mutants defective inglycosaminoglycan biosynthesis. Proc Natl Acad Sci U S A 82, 3197-3201.
7. Zhang, L., and Esko, J. D. (1995). Accumulation of a pentasaccharide terminating in alpha-Nacetylglucosamine in an animal cell mutant defective in heparan sulfate biosynthesis. J Biol Chem 270,
12557-12562.

- 133 -

8. Bame, K. J., and Esko, J. D. (1989). Undersulfated heparan sulfate in a Chinese hamster ovary cellmutant defective in heparan sulfate N-sulfotransferase. J Biol Chem 264, 8059-8065.
9. Bai, X., and Esko, J. D. (1996). An animal cell mutant defective in heparan sulfate hexuronic acid
2-O-sulfation. J Biol Chem 271, 17711-17717.
10. Gilmore, A. P., Burridge, K. (1996). Molecular mechanisms for focal adhesion assembly throughregulation of protein-protein interactions. Structure 4, 647-651.
11. Zhao, J. H., Reiske, H., and Guan, J. L. (1998). Regulation of the cell cycle by focal adhesion kinase.
J Cell Biol 143, 1997-2008.

- 134 -

Analysis of the signaling system for a novel lung cancer oncogene,
EML4-ALK, and its clinical application
Hiroyuki Mano
Jichi Medical University
hmano@jichi.ac.jp

Abstract
EML4-ALK is a fusion-type protein tyrosine kinase that is generated in human non–small celllung cancer (NSCLC) as a result of a recurrent chromosome inversion, inv(2)(p21p23). In thisproject, we have established transgenic mouse lines that express EML4-ALK specifically in lungalveolar epithelial cells. All transgenic mice examined developed hundreds of adenocarcinoma
nodules in both lungs within a few weeks after birth, conﬁrming the potent oncogenic activity of thefusion kinase. Although such tumors underwent progressive enlargement in control animals, oral
administration of a small-molecule inhibitor of the kinase activity of ALK resulted in their rapid
disappearance. These data provide experimental support for the treatment of this intractable cancer
with ALK inhibitors.
Keywords: Lung cancer, Oncogene, Protein-tyrosine kinase, EML4-ALK

Introduction
We recently developed a cDNA expression library system in retrovirus, and with this technique we
could discover a novel fusion-type protein-tyrosine kinase EML4-ALK in non-small cell lung cancer(NSCLC) (1). A small inversion within the short arm of chromosome 2 was found to result in theligation of EML4 and ALK, leading to the production of a fusion protein consisting of the aminoterminal portion of EML4 and the intracellular region of the protein tyrosine kinase ALK (Fig. 1).
Although the inv(2)(p21p23) rearrangement responsible for the fusion event occurs recurrently in
NSCLC patients (2-4), it has remained to be demonstrated that EML4-ALK plays an essential role inthe carcinogenesis of NSCLC harboring the fusion gene.

Results
To explore the in vivo function of EML4-ALK fusion kinase in the pathogenesis of NSCLC, we
have constructed an expression cassette for EML4-ALK cDNA driven by a promoter fragment of
surfactant protein C gene (SPC), which is known to be speciﬁcally expressed within type II alveolar
cells in lungs (5). With this expression construct, we then generated several independent lines ofsuch transgenic mice. Surprisingly, all these mice developed hundreds of adenocarcinoma nodules inboth lungs, proving that EML4-ALK is essential for the pathogenesis of NSCLC with this oncogene(6). To observe the development of NSCLC in the transgenic mice, we performed a series of CT
scans of the chest. Multiple large nodules, some with inﬁltrative proﬁles of NSCLC, were detected- 135 -

in the lungs of progeny mice. Other progeny with similar CT ﬁndings were subjected to pathologicalexamination, conﬁrming that such CT proﬁles reﬂected tumor expansion and inﬁltration in the lungs.
Examination of other organs of these mice failed to detect metastatic tumor nodules.
Such nodules were then resected, and transplanted into the shoulder of nu/nu mice to examine thetransformed characteristics of the nodules. As expected, cells in these nodules successfully formed
subcutaneous tumors in the nu/nu mice.
Since EML4-ALK is a kinase activity–dependent transforming molecule, any compounds thatspeciﬁcally suppress EML4-ALK activity would become a molecular targeted therapy for NSCLC
positive for EML4-ALK. To address this possibility, an ALK inhibitor was administered orally to thetransgenic mice that were periodically examined with CT scanning. After ~one month of treatment
with the inhibitor, almost all of the NSCLC nodules disappeared in every mouse examined, proving
for the ﬁrst time that ALK inhibitors could become an effective drug against EML4-ALK–positive
lung cancer (Fig. 2).

Such drastic effect of the compound was further examined in another in vivo system, that of loading
mice with a large number of EML4-ALK–positive cells. Mouse 3T3 ﬁbroblasts expressing EML4-- 136 -

ALK undergo transformation and generate subcutaneous tumors when injected into nu/nu mice.
Such 3T3 cells were therefore injected intravenously into nu/nu mice, and the ALK inhibitor wasadministered to half of these animals.
All untreated mice died within 1 month of injection with the 3T3 cells. Postmortem examination ofthese mice revealed extensive dissemination of EML4-ALK–positive cells into the lungs (>60% oflung tissue was occupied with the transformed 3T3 cells in all mice). Pathological examination of thelungs revealed many nodules of various sizes that were ﬁlled with the 3T3 ﬁbroblasts. In a separate
experiment, we conﬁrmed that injection of parental 3T3 cells did not induce the formation of such
nodules in the lungs or affect the survival of mice.
Peroral administration of the ALK inhibitor markedly improved the outcome of mice injected withthe transformed 3T3 cells, with all animals in the treatment group surviving the 1-month observation
period. The treated mice were also subjected to pathological analysis after this period, revealing
the absence of 3T3 nodules from the lungs and again demonstrating the high efﬁcacy of the ALK
inhibitor.

Discussion & Conclusion
We have shown here that the EML4-ALK fusion kinase plays an essential role in lungtumorigenesis. Hundreds of adenocarcinoma nodules developed simultaneously within a few weeks
after birth in all independent lines of EML4-ALK transgenic mice examined. Given that the promoter
fragment of SPC becomes active only at a late stage of gestation (7), only a short period of EML4ALK expression appears to be sufﬁcient for full transformation. Although we did not examine TP53
and RB1 for possible abnormalities in the adenocarcinoma nodules of the transgenic mice, with bothof these genes being frequently inactivated in human lung cancers (8), it is likely that only one (orat most a few) additional genetic event is required to generate cancer in EML4-ALK–expressing
alveolar epithelial cells.
Given the rapid development of NSCLC induced by EML4-ALK, the tumor cells are likely
dependent for growth on the tyrosine kinase activity of the fusion protein. Such “oncogene-addiction”(9) provides a potential target for the development of treatment strategies. We therefore tested
whether inhibition of the enzymatic activity of EML4-ALK might reduce the tumor burden in thetransgenic mice. The ALK inhibitor examined proved to be a promising candidate for the treatment
of EML4-ALK–positive tumors. Further, given the high sensitivity of the tumors in the mice to the
ALK inhibitor, our transgenic mice would be one of ideal means to examine the in vivo activity of
any compounds or other reagents to suppress ALK.

References
1) Soda M, Choi YL, Enomoto M, Takada S, Yamashita Y, Ishikawa S, Fujiwara S, Watanabe H,
Kurashina K, Hatanaka H, Bando M, Ohno S, Ishikawa Y, Aburatani H, Niki T, Sohara Y, Sugiyama Y& Mano H: Identiﬁcation of the transforming EML4-ALK fusion gene in non-small-cell lung cancer.
Nature 448: 561-566, 2007.

- 137 -

2) Choi YL, Takeuchi K, Soda M, Inamura K, Togashi Y, Hatano S, Enomoto M, Hamada T, Haruta H,
Watanabe H, Kurashina K, Hatanaka H, Ueno T, Takada S, Yamashita Y, Sugiyama Y, Ishikawa Y &Mano H: Identiﬁcation of novel isoforms of the EML4-ALK transforming gene in non-small cell lungcancer. Cancer Res 68: 4971-4976, 2008.
3) Inamura K, Takeuchi K, Togashi Y, Nomura K, Ninomiya H, Okui M, Satoh Y, Okumura S, Nakagawa
K, Soda M, Choi YL, Niki T, Mano H & Ishikawa Y: EML4-ALK fusion is linked to histologicalcharacteristics in a subset of lung cancers. J Thorac Oncol 3: 13-17, 2008.
4) Takeuchi K, Choi YL, Soda M, Inamura K, Togashi Y, Hatano S, Enomoto M, Takada S, Yamashita
Y, Satoh Y, Okumura S, Nakagawa K, Ishikawa Y & Mano H: Multiplex reverse transcription-PCR
screening for EML4-ALK fusion transcripts. Clin Cancer Res 14: 6618-6624, 2008.
5) Zhao B, Magdaleno S, Chua S, Wang YL, Burcin M, Elberg D, Finegold M, Tsai S & DeMayo FJ:
Transgenic mouse models for lung cancer. Exp Lung Res 26: 567-579, 2000.
6) Soda M, Takada S, Takeuchi K, Choi YL, Enomoto M, Ueno T, Haruta H, Hamada T, Yamashita Y,
Ishikawa Y, Sugiyama Y & Mano H: A mouse model for EML4-ALK-positive lung cancer. Proc Natl
Acad Sci U S A 105: 19893-19897, 2008.
7) Korfhagen TR, Glasser SW, Wert SE, Bruno MD, Daugherty CC, McNeish JD, Stock JL, Potter SS &Whitsett JA: Cis-acting sequences from a human surfactant protein gene confer pulmonary-speciﬁc
gene expression in transgenic mice. Proc Natl Acad Sci U S A 87: 6122-6126, 1990.
8) Testa JR, Liu Z, Feder M, Bell DW, Balsara B, Cheng JQ & Taguchi T: Advances in the analysis ofchromosome alterations in human lung carcinomas. Cancer Genet Cytogenet 95: 20-32, 1997.
9) Sharma SV & Settleman J: Oncogene addiction: setting the stage for molecularly targeted cancer
therapy. Genes Dev 21: 3214-3231, 2007.

- 138 -

Dysfunction in cardiovascular system using micethat lost a novel membrane-associated estrogen receptor
Yoichi Mizukami
Center for Gene Res. Yamaguchi Univ.
mizukami@yamaguchi-u.ac.jp

Abstract
To investigate the physiological functions of G protein-coupled receptor 30 (GPR30), we produced
KO mice for GPR30. Mice homozygous for GPR30 KO are not burned in C57/BL6 and ICR mice.
We tried to produce the transgenic mice whose GPR30 expression was suppressed using RNAinterference (RNAi) vector inserted green ﬂuorescent protein (GFP) gene as a marker gene. No mice
showed a suppression of GPR30 expression greater than 60% compared with wild-type controls.
Next, we observed the embryos of transgenic mice at 12.5 days after fertilization. Twenty-one
percent of embryos injected with the dsRNA for GPR30 were dead in the uterus, and the expressionof GFP was increased 40-fold compared with surviving embryos. The fertilized eggs injected withthe dsRNA for GPR30 were cultured in vitro, and cell division was observed microscopically. Allof the fertilized eggs injected with the dsRNA for GPR30 arrested by the 4-cell stage, althoughfertilized eggs injected with control DNA developed beyond the8-cell stage. These observations
indicate that GPR30 may be essential for the development of mouse embryos.
Keywords: GPR30, transgenic mouse, estrogen, GPCR, plasma membrane

Introduction
During screening for orphan GPCRs in response to oxidative stress, we found that the mRNA for
G protein-coupled receptor 30 (GPR30) is rapidly induced in rat embryonic cardiomyocyte-derived
H9c2 cells by hypoxia/reoxygenation stimulation (1). Fluid shear stress as well as oxidative stressalso leads to GPR30 mRNA expression in human endothelial cells (2).

Recently, Revankar et al.

and Filardo et al have reported that the sex steroid hormone E2 is a ligand for GPR30, and that theactivation of GPR30 in COS cells results in an increase in intracellular calcium concentrations (3,4).
Although increasing attention has been paid to the biological activities of GPR30, many experiments
involving GPR30 were carried out using cancer cell, and the physiological roles of GPR30 in vivo
remained unknown. We tried to produce mice in which GPR30 expression is suppressed in order
to elucidate the function of GPR30 in vivo. During the study, we have found that GPR30 might beinvolved in the maintenance of cell division during the development of preimplantation embryos,
and suggested that GPR30 mediated the E2-induced the Ca2+ transients in early embryos.

- 139 -

Results
To examine the function of GPR30 in vivo, we produced mice homozygous for GPR30 KO,
however, GPR30 KO mice were not burned in 200 newborn mice. Next we produced the transgenic
mice expressing the dsRNA for GPR30 using GFP expression system as a marker. No transgenic
mice showing a significant suppression of GPR30 expression were born, we examined GPR30
expression in embryos injected with dsRNA. Approximately 21 % of embryos injected with thedsRNA had died by E12.5, and all of the dead embryos expressed GFP at a greater than 40-foldhigher level than other embryos. In embryos injected with the dsRNA for another orphan receptor,
TPRA40, or control embryos, no dead embryos were observed at E12.5. The expressions of the
GPR30 protein and mRNA were hardly detected since the dead embryos were already damaged.
These findings suggest that the introduction of the dsRNA for GPR30 may result in embryoniclethality before E12.5.
To examine the effects of GPR30 in early embryos, fertilized eggs injected with dsRNA were
cultured in vitro for 4 days after fertilization. First, we examined GPR30 expression in fertilized
eggs 4 days after fertilization by immunocytochemistry using anti-GPR30 antibody. GPR30
expression was observed at 4 days in fertilized eggs injected with control DNA, and the expression
was signiﬁcantly decreased in eggs injected with the dsRNA for GPR30 as compared with control
eggs. Sixty-nine percent of embryos injected with the dsRNA for GPR30 were arrested at the 2-cellstage and 31% were arrested at the 4-cell stage. In eggs injected with control vector, 75% of theembryos had developednbeyond the 8-cell stage at 4 days. These findings suggest that GPR30
is already expressed in early embryos, and is required for the development of embryos during
preimplantation. .
Recently, two groups show that GPR30 is a transmembrane receptor for estrogen (17 β -E2, E2)
which induces nongenomic response including Ca2+ transients and activation of signal transduction.
We determined the intracellular Ca2+ concentrations ([Ca2+]i) in the presence of estrogen to examine
whether GPR30 pathway is involved in the mitosis of early embryos, because an increase of in[Ca2+]i is sufficient for driving mitosis in early embryos. E2 stimulation induced Ca 2+ transients
immediately after the stimulation in eggs injected with empty vector or mock control eggs. The Ca2+transients was indected in eight of nine eggs at least two days after the injection of RNAi for GPR30,
although Ca2+ ionophore, A23187 cause the generous increase of [Ca2+]i . These observations suggestthat Ca2+ mobilization induced by E2 may be mediated by GPR30 in early embryos

Discussion & Conclusion
During the production of dsRNA-mediated knock down mice for GPR30, we have found that
GPR30 play a role in the driving mitosis in early embryos, and may be implicated in E2-induced theCa2+ transients in the fertilized eggs. Recently, GPR30 was shown to be a novel receptor of E2 (3,4),
and the relationship between E2 and GPR30 hasdreceived attentios. The treatment of early embryoswith an inhibitor of E2 leads to an inhibition of development from the 4-cell preimplantation embrys- 140 -

to the blastocyst (5), a findins consistent with our present data that fertilized eggs injected withthe dsRNA for GPR30 are arrested by the 4-cell-stage. Classical receptors for E2, the mRNAs for
estrogen receptors a and b, are also detected in 4-celf embryos (6,7), but growth arrest during early
embryogenesis are not observed it mice deficienf in both estrogen receptors α and β (8). Theseobservations indicate that the effect of E2 on embryogenesis may be mediated, at least in part, by
GPR30. The Ca2+ transients for early embryos are sufficient events to drive mitosis, and often
observed during nuclear emvelop breakdown. In mouse oocyte, E2 resulted in the Ca2+ oscillationin immediately after the fertiliozation. We determined [Ca2+]i in a number of fertilized eggs in thepresence of E2, but Ca2+ oscillation were unobserved under our conditions. It is necessary for at least
two days to express exogenous genes after the microinjection, and the occurrence of Ca2+ oscillation
may be affected by the progression of cell division. How does GPR30 regulate embryogenesis?
It is well known that E2 enhances cell proliferation through the release of growth factors (3). In
GPR30-deﬁcient cells, the release of HB-EGF is strongly inhibited in the presence of 17b-E2 (3,9).
There is the possibility that growth factor secretion via GPR30 may be present in embryogenesis,
since growth factors released by the autocrine/paracrine mechanism are prerequisite for cell
division of early embryos (10). The present study provides new insights into the role of GPR30 inembryogenesis.

References
1. Kimura, M., Mizukami, Y., Miura, T., Fujimoto, K., Kobayashi, S., and Matsuzaki, M. (2001) J Biol
Chem 276(28), 26453-26460
2. Takada, Y., Kato, C., Kondo, S., Korenaga, R., and Ando, J. (1997) Biochem Biophys Res Commun
240(3), 737-741
3. Filardo, E. J., and Thomas, P. (2005) Trends Endocrinol Metab
4. Revankar, C. M., Cimino, D. F., Sklar, L. A., Arterburn, J. B., and Prossnitz, E. R. (2005) Science
307(5715), 1625-1630
5. Gupta, J. S., Dey, S. K., and Dickmann, Z. (1977) Steroids 29(3), 363-369
6. Hiroi, H., Momoeda, M., Inoue, S., Tsuchiya, F., Matsumi, H., Tsutsumi, O., Muramatsu, M., andTaketani, Y. (1999) Endocr J 46(1), 153-158
7. Hou, Q., and Gorski, J. (1993) Proc Natl Acad Sci U S A 90(20), 9460-9464
8. Couse, J. F., and Korach, K. S. (1999) Endocr Rev 20(3), 358-417
9. Filardo, E. J., Quinn, J. A., Bland, K. I., and Frackelton, A. R., Jr. (2000) Mol Endocrinol 14(10), 16491660
10. Adamson, E. D. (1993) J Cell Biochem 53(4), 280-287- 141 -

Analyses of DMRT1 gene knockout medaka,
which reverse sex in genetic male
Masaru Matsuda
Utsunomiya University
matsuda@cc.utsunomiya-u.ac.jp

Abstract
To analyze the function of DMRT1, we searched and found four missense mutations on DMRT1
exon 2 region in a medaka mutant library. One mutation named allele DMRT1b, where arginine issubstituted with cysteine exhibited a clear phenotype. All XX individuals looked like normal females.
Conversely, XY medaka homozygous for allele b (b/b) developed ovary, whereas heterozygous(b/+) XY medaka developed testis. These results suggest that DMRT1 is required for testiculardevelopment in the medaka, Oryzias latipes.
Keywords: Sexual differentiation, medaka, sex-reverse

Introduction
All species, whose sex is genetically determined, have sex-determining genes—which work as theﬁrst switch in deciding the direction of sexual development of the gonad. Only two sex-determining
genes have been found in vertebrates, SRY/Sry in mammals and DMY in the medaka, Oryzias latipes(1). Further investigations for the sex-determining genes in the Oryzias species have suggested thateach species may have its own unique sex-determining gene (1). Conversely, many genes that areimportant for gonadal sex differentiation in mammals are thought to be common among vertebrates.

Results
There are two strategies to analyze functions of DMRT1 gene. Gain-of-function and Loss-offunction of genes. But in gain of function, explanation of the results obtained is usually complicated,because it is difficult to control gene expression temporally and spatially in these experiments.
Therefore, we try to a Loss-of-function experiment. For loss-of-function of the DMRT1, we screened
mutations of DMRT1 ex2 region in medaka mutants library for TILLING (Targeting Induced Local
Lesions In Genomes) (2). Approximately 6,000 samples of genomic DNA library were amplified
by PCR and the PCR products of DMRT1 exon 2 region were assayed using high resolution
melting (HRM) to detect heteroduplex of PCR products. Then, mutations were conﬁrmed by directsequencing method. Finally, we found four missense mutations on DMRT1 exon 2 region, which
include ATG start codon and a part of DM domain. We named allele a, b, c and d, respectively.
We produced F1 progeny using artiﬁcial insemination with frozen sperm and wild type eggs. Since
frozen sperm contain both mutant allele and wild type allele, F1 progeny that have mutant allele haveto be selected by genotyping. Then we mated heterozygous female to heterozygous male to obtain- 142 -

homozygous mutant. They were analyzed sex chromosome types and phenotypes. For genotyping of
DMRT1 allele a, direct sequencing of PCR products were used. For genotyping of DMRT1 allele b,
SNPs detection by TaqMan probes, which were speciﬁc to wild type and mutant type, were used. For
genotyping of DMRT1 allele c and d, the PCR-RFLPs (Restriction Fragment Length Polymorphisms)
method was used. The sex chromosome types were conﬁrmed by presence or absence of the sexdetermining gene, DMY by using PCR. To elucidate phenotypes of the mutants, we checked gonadby histological analyses at 30 days after hatching.

Results
Homozygous mutants of allele a and d looked like normal in gonadal section of 30 days after
hatching. XX individuals showed female secondly sex characteristics and were fertile, whereas XY
individuals showed male secondly sexual characteristics, and were fertile. In allele b mutant, wherearginine is substituted with cysteine, most of individuals showed no secondly sex characteristics.
Only few individuals laid eggs both in XX and XY, and one XY medaka laid eggs. Therefore, wecrossed the XY female to an inbred male and the female progeny were back-cross to an inbred
male again. Then, we selected heterozygous offspring. By brother-sister mating of the offspring,
we obtained homozygous mutants and analyzed what happen in the homozygous mutants. All
XX individuals looked like normal females. Conversely, XY medaka homozygous for allele b (b/b) developed ovary, whereas heterozygous (b/+) XY medaka developed testis (Figure 1). In allele
c mutant, where arginine is substituted with cysteine, most of individuals showed secondly sex
characteristics and were fertile. But, a few XY individuals showed no secondly sex characteristics
and were sterile. Histological analysis at 30 days after hatching demonstrated XY gonads havevariations. We could ﬁnd four types of gonad; many oocytes, a few oocytes in testis, small testis,developed testis.

Figure 1. Gonad histology of the DMRT1b mutants.
30 days after hatching gonads of normal XX (A) and XY (C), andmutants XX (B) and XY (D). (C) The gonadal region is outlined bydotted lines. (D) XY DMRT1b mutants have ovary.

- 143 -

Discussion & Conclusion
These results suggest that a mutation in DMRT1 causes male to female sex reversal in the medaka
and that DMRT1 has a critical function in testis development in the medaka. However, the gonadal
phenotype of homozygous mutant bb is different from that of homozygous mutant cc. This may
be cause various degree of DMRT1 function between allele b and allele c. Further analysis of themutants will reveal mechanisms of gonadal sex reverse.
In homozygous mutant of allele b, XY medaka develop to female, suggesting that the sex ofindividuals could be determined not by the presence or absence of DMY but by the genotype of
DMRT1 in this mutant line. This means that DMRT1 determines the sex here, suggesting that we canmake the gene for sex determination.

References(1) M. Matsuda, Sex determination in the teleost medaka, Oryzias latipes. Annual Review of Genetics 39:
293-307, 2005.
(2) Mitani H, Kamei Y, Fukamachi S, Oda S, Sasaki T, Asakawa S, Todo T, Shimizu N. The medaka
genome: why we need multiple fish models in vertebrate functional genomics. Genome Dynamics
2:165-82, 2006

- 144 -

Development of novel approach for liver failure using genetically
modiﬁed hepatocytes
Kazuo Ohashi
Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University
ohashi@abmes.twmu.ac.jp

Abstract
In the present study, we attempted to transduce hepatocyte growth factor (HGF) gene, a potent antiapoptotic gene, to the primary hepatocytes and used the transduced cells for liver tissue engineering.
We investigated whether HGF-transduced hepatocytes could survive at higher rates resulting in thecreation of larger liver tissues.
Keywords: Liver tissue engineering, hepatocyte transplantation, regenerative medicine

Introduction
Liver tissue engineering is an emerging ﬁeld in which a functional liver system is created in vivousing isolated hepatocytes to treat liver diseases. Our lab has recently developed procedures tocreate liver tissues under the kidney capsule space that functionally persist for over 200 days inmice. However, in the previous series of experiments, we have realized that signiﬁcant portion ofhepatocytes underwent the process of cell death during the first 48 hours period after hepatocyte
infusion. These ﬁndings led us to develop a procedure that could provide protective effects to thehepatocytes during the cell death process. In the present study, we attempted to transduce hepatocyte
growth factor (HGF) gene, a potent anti-apoptotic gene, to the primary hepatocytes and used thetransduced cells for liver tissue engineering. We investigated whether HGF-transduced hepatocytes
could survive at higher rates resulting in the creation of larger liver tissues.

Results
Methods: Donor hepatocytes were isolated and puriﬁed from human alpha-1 antitrypsin (A1AT)
transgenic mice (provided by Dr. Bumgardner, OSU) with the FVB/N genetic background. The
HGF gene transduction to the hepatocytes were carried out using adenoviral vectors (Ad), either
Ad-CAG-HGF or Ad-CAG-null (for control), based on our previously developed non-culture
Ad-infection system (Cell Transpl 15: 1-12, 2006). After this vector infection step, liver tissue
engineering procedures were performed to the wild FVB/N mice by transplanting the hepatocyte
with EHS-matrix under the kidney capsule spaces. Functional volume of the engineered liver tissues
was assessed by periodic measurement of serum A1AT levels of the recipients and histologicalexamination. In order to assess therapeutic potential, lethal liver failure was induced to the tissue
engineered mice by performing 2/3 hepatectomy and ligation of right portal pedicles.
Results: Tunnel staining of the transplanted hepatocytes revealed that the number of apoptotic- 145 -

hepatocytes was signiﬁcantly fewer in the HGF-hepatocyte group as compared with control group.
The serum A1AT levels in the HGF-hepatocyte group were significantly higher than those in thecontrol-hepatocyte group at any time points throughout the experimental period. Histologicalexamination revealed that HGF-transduced hepatocytes engineered thicker liver tissues comparedwith control hepatocytes. The Kaplan-Meier analysis showed signiﬁcantly improved survival in the
HGF-hepatocyte group, compared to that in the control group.

Discussion & Conclusion
The present study demonstrates that HGF-transduced hepatocytes are less susceptible to apoptotic
cell death and are able engraft at higher rates under the kidney capsule site. The present study also
conﬁrmed that the liver tissue engineering approach could provide therapeutic effects on severe formof liver failures. These data provides evidence that hepatocyte-based therapy in combination with
HGF-gene therapy represents an important step forward in advancing hepatocyte transplantation andliver tissue engineering.

- 146 -

References
Kuge H, Ohashi K, Yokoyama T, Hiromichi K, Hisanaga M, Koyama F, Bumgardner GL, Kosai K-I,
Nakajima Y. Genetic modification of hepatocytes towards hepatocyte transplantation and liver tissue
engineering. Cell Transplantation 15: 1-12, 2006
Ohashi K. Liver tissue engineering: the future of liver therapeutics. Hepatology Res 38: S76-87, 2008.
Pipe SW, High KA, Ohashi K, Ural AU, Lillicrap D. Progress in the molecular biology of inherited
bleeding disorders. Haemophilia 14 (S3): 130-137, 2008.
Ohashi K, Tatsumi K, Utoh R, Takagi S, Shima M, Okano T. Engineering liver tissues under the kidney
capsule site provides therapeutic effects to hemophilia B mice. Cell Transplantation, in press 2009.

- 147 -

III.
Reports from the Recipients of Grants
for International Meetings- 148 -

International Symposium on Biologically Active Peptides: Peptide Diversity(First Meeting of the Japan Branch of the International Neuropeptide Society)
1. Representative
Kazuhiro Takahashi, the chairman of organizing committee

2. Opening period and Place
August 31-September 1, 2008, Gonryo Alumni Hall, Sendai

3. Number of participants
110 persons (7 persons from foreign countries)

4. The total cost
One million and eight-hundred-fifty thousand yen

5. Main use of the subsidy
Support for travel expenses within Japan and accommodation of invited speakers fromforeign countries.

6. Result and Impression
The International Neuropeptide Society is open to all scientists interested in and conducting
research with neuropeptides, including all biologically active peptides. The International
Neuropeptide Society consists of seven Societies and nine Branches throughout the world. The
participating Societies are the Winter Neuropeptide Society, the Summer Neuropeptide Society,the European Neuropeptide Club, the Invertebrate Neuropeptide Society, the Bacterial/Antibiotic
Peptide Group, the Venom Peptide Group, and the Plant Peptide Group. The establishedbranches include China, India, Africa, South America, Asia minor, Australia, Canada, andMiddle East.
The Japan Branch of the International Neuropeptide Society has been recently established.
This Symposium was the 1st Meeting of Japan Branch the International Neuropeptide Society. The
Symposium consisted of 7 sessions and one plenary lecture. The plenary lecture was given by Dr.
Abba J. Kastin, the President of the International Neuropeptide Society. The sessions were as follows:
1. Pain; 2. Hypothalamo-pituitary peptides/comparative endocrinology; 3. Appetite and energy- 149 -

metabolism; 4. Neuroactive peptides/ Blood brain barrier; 5. Calcitonin gene-related peptide andadrenomedullin; 6. Gastrointestinal tract and pancreas; 7. Novel aspects of neuropeptides. 23
lectures were given in these 7 sessions by top-grade researchers in the peptide research fromforeign counties and Japan.
This Symposium has given a unique chance to all the Japanese peptide researchers to get
together and exchange their novel scientific findings and opinions. Furthermore, this symposium
will enable the Japanese researchers to communicate internationally via invited speakers fromforeign countries. The next meeting (2nd Meeting of Japan Branch the International Neuropeptide
Society) will be held in Osaka or Kyoto, 2010 with the chairperson of Dr. Naoto Minamino (TheNational Cardiovascular Research Center).

7. Additional description
7.1 Number of participating countries: 5 countries
7.2 Approvals
The Japan Branch of the International Neuropeptide Society was officially established on
28th August 2008, and this Symposium was approved to be the First Meeting of the Japan
Branch of the International Neuropeptide Society by the Japan Branch Committee and theInternational Neuropeptide Society.
7.3 Satellite symposiumInternational Symposium on Neuropeptides and Neuroendocrinology was held on 29th
August 2008 in Tokyo in co-operation with the 35th Meeting of the Japan Neuroendocrine
Society and the 23rd Japan Pituitary Meeting.

- 150 -

The CMDS 2008 Conference Report
1.

Title of the Conference
The 4th International Conference on Coherent Multidimensional Spectroscopy(CMDS 2008)

2.

Representative
Yoshitaka TANIMURA
Chairman, Local Organizing Committee

3.

Conference Period and Location
Period: August 27-30, 2008
Location: Fukui Institute for Fundamental Chemistry, Kyoto University
Kyoto, Japan

4.

Number of Participants
A total of 101 scientists and students participated.

5.

Total Cost of the Conference¥5,875,000

6.

The Use of the Fund
The majority of the fund was used to support a part of travel expenses and local
expenses for young scientists and student participants who presented their work at theoral or poster sessions. The fund was also used for the cultural activities.

7.

The Overview of the Conference and Comments
The International Conference on Coherent Multidimensional Spectroscopy (CMDS) is
held every two years.

The CMDS2008 was the fourth in a row of workshops,

initially launched by M. Cho in Seoul (Korea) in 2002, and then continued in 2004 by
J. Wright in Wisconsin (USA), and in 2006 by P. Hamm in Rigi Kulm (Switzerland).
The primary mission of the Conference is to foster the interdisciplinary work inexperimental and theoretical coherent multidimensional vibrational and electronic
spectroscopy. The participated were not only those who have been actively engaged in
the field, but also young scientists and students who were eager to learn about thelatest developments in the field. Throughout the sessions, the active and informative- 151 -

discussions among the participants were noticed.
<Program>
Forty 30-min lectures were delivered by the invited and selected speakers in the 14
oral sessions during the three and a half day conference period. The presented lectures
include “Ultrafast vibrational dynamics of bulk liquid H2O and H2O in DNA” byThomas Elsaesser (Max-Born Institute, Germany), “Multidimensional spectroscopy
with quantum laser fields: New insights and effects” by Shal Mukamel (U. of
California-Irvine, USA), “Two dimensional electronic spectroscopy of light harvesting
complexes”

byG.rahamFleming(U.

ofCalifornia-Berkeley,

USA),“Multidimensional optical fourier transform spectroscopy of semiconductor quantum
well excitons and biexcitons” by Keith Nelson (MIT, USA), and “2D-IR spectroscopy:
From peptides to proteins” by Peter Hamm (U. of Zurich, Switzerland). Thirty-fourposters were also presented in the two 100-min poster sessions. The discussed topics
were multidimensional spectroscopy of biological systems, molecular liquids,
solvation, proton transfer, semiconductors, excitons, photosynthesis, surfaces,interfacial systems, and the classical and quantal dynamics of complex dissipative
systems.

New experimental and simulation methods for multidimensional

spectroscopy were also discussed.

This conference program provided theopportunities to exchange the ideas and knowledge among the experimental andtheoretical researches to propel the advancement of coherent multidimensional
vibrational and electronic spectroscopy.
<Cultural Activities>
Majority of young participants were the first time visitors to Japan while the senior
participants have visited Japan several times. We organized the following threedifferent cultural activities to accommodate their interests as well as to provide
opportunities to interact with Japanese young scientists and students.
1) Visit to Kinkaku-ji Temple and Ryoan-ji Temple led by Kyoto University graduate
students. This excursion is for the first timers. The students escorted them tothe temples using Kyoto City bus and explained the history and significance of thehistorical temples.
2) Visit to Sanzein Temple in Ohara and Zazen experience at a Zen Temple led by
Kyoto University graduate students and Prof. Tahara.
3) Hike to Daimonji mountain and visit to Heian Shrine led by Profs Tanimura andAndo.

- 152 -

8.

Others(a) Break down of the participants by the countries of their affiliations.
Number of
Nationality of Participants Other than theCountry
Participants
Country of the Affiliation
Japan
47
Philippine 1, China 1
USA

19

China 2, Japan 1

Switzerland

6

Italy 2, Iran 1

Germany

5

Russia 1, USA 1

The Netherlands

4

Russia 1

UK

3

China 1

Sweden3

Canada3

China 1, Germany 1

France2

Canada 1

Korea

2

Czech Republic

2

China

1

Taiwan

1

India

1

Israel1

Austria

1(b) Post Symposium for Young Scientists
The Post-Symposium was organized by Prof. Kaoru Ohta (Kobe University) and Prof.
Kazuya Watanabe (Kyoto University).

It was held at Kyoto University on August

31st to bring together the CMDS 2008 participating young scientists for exchanging
their ideas and research results.

Since the atmosphere of the symposium was friendly

and informal, many questions were raised and good comments were made by theyoung participants. The financial support for this symposium was also provided by the
Global Center of Excellence Program at Kyoto University.
(c) Advertisement
We acknowledge the financial supports from Novartis foundation by conference WEB
page, circular and poster. From time to time, we informed to the supported young
scientists and student participants that most of their local expenses were warmly
supported from Novartis foundation.

All of supported people were deeply

appreciated Novartis’s support with full respect.

- 153 -

- 154 -- 155 -28th Sapporo Cancer Seminar: International Symposium“TGF-β signaling and cancer”
1. Representative
Kohei Miyazono, President
2. Opening period and Place
June 26 - 27, Hokkaido University Conference Hall (Sapporo, Japan)
3. Number of participants
155 (including 22 from outside Japan)
4. The total cost
13,340,000 yen
5. Main use of the subsidy
Support for travel expenses of invited speakers
6. Result and Impression
The Sapporo Cancer Seminar (SCS) is a conference series with a history back to 1981,
and held every year. Dr. Hiroshi Kobayashi, the founder of the seminar and now Professor
Emeritus of Hokkaido University, inspired originally by The Gordon Research
Conferences and established SCS in Japan to provide greater opportunities for cancer
researchers to share their knowledge in a relaxed but academic atmosphere.
Following this tradition, the present international symposium, the 28th SCS entitled“TGF-β signaling and cancer”, has been carried out successfully with plenty of excellent
talks and fruitful discussions. The signaling by TGF-β and its family molecules has avariety of functions and play important roles in many diseases, especially in cancer. The
functions of the signaling system in the genesis and the progression of cancer have beenreported to be complex and intense. In the present conference, we discussed such wide
spectra of functions of TGF-β family signaling in cancer, including potential applicationsof the signaling to therapeutics, with attendance of a lot of world-class investigators.
The followings are the titles of the talks given in this conference:<Session 1. Molecular mechanisms of TGF-β signaling>
1. Bidirectional roles of TGF-β signaling in cancer
2. Regulation of TGF-β receptor activity: Location, location and location
3. TGF-β receptor function in cancer cell behavior
4. TMEPAI makes a novel negative feedback loop of TGF-β signaling
5. Identification of Smad2/3 binding regions by ChIP-chip reveals involvement of TFAP2
and ETS in TGF-β-induced transcription- 156 -

<Session 2. TGF-β signaling and carcinogenesis>
1. Molecular mechanism for TGF-β induced epithelial-mesenchymal transition
2. TGF-β receptor signaling in angiogenesis
3. Smad2 and Smad3 signaling activated by TGF-β is important for vascular stability
4. SnoN in tumorigenesis: Identity crisis
5. Inhibitor of Growth 2 as a novel regulator of TGFβ-dependent signaling and responses
6. RNAi-based dissection of Dpp-induced epithelial plasticity in a Drosophila cell culture
model
7. Smad7 cooperates with Smad6 to induce TGF-β-mediated anti-infl ammatory effects
through interaction with the adaptor protein, Pellino-1<Session 3. TGF-β signaling and cancer metastasis>
1. Novel mechanism of colon cancer invasion: Role of CCR1+ bone-marrow derived cell
2. Extracellular matrix proteins and tumor microenvironment
3. Transforming growth factor beta (TGFβ): Role in bone metastases due to breast cancer,
prostate cancer and melanoma
4. TGFβ1 suppresses colitis-associated colon cancer by preventing pre-clinicalinflammatory state of colon mucosal epithelium
5. TGF-β signaling during human carcinogenesis: the shift from Cterminallyphosphorylated Smad3 to linker phosphorylated Smad3 pathway
6. Transcriptional induction of MMP10 by TGF-β mediated by MEF2 and class II HDACs<Session 4. Development and application of TGF-β-based therapeutic agents>
1. In vivo optical imaging of tumor microenvironment and TGF-β signaling
2. Antagonizing TGF-β to restore anti-tumor immune responses
3. Manipulation of TGF-β signaling for cancer treatment
4. Tumor-stromal interaction in aggressive pancreatic ductal adenocarcinoma in mice-As apossible therapeutic target for human pancreatic cancer
Beside these talks, 30 more posters were presented and discussed.
7. Additional description
Number of participating countries: 7 countries, including Japan, USA, Korea, China, The
Netherlands, Sweden, and Hungary- 157 -

- 158 -- 159 -- 160 -- 161 -- 162 -- 163 -- 164 -第 22 期（2008 年度）助成事業報告
当財団は、1987 年 9 月 3 日に文部大臣より認可を受けて設立して以来、寄附行為に定めた研究助成を中心とした事業を行ってきました。2008 年度は、下記に示したノバ
ルティス研究奨励金、研究集会助成、海外留学生受入れ助成の総額 5,740 万円の助成を行っております。
ノバルティス研究奨励金45 件 （1 件
研究集会助成6 件 （1 件
海外留学生受入れ助成 5 件 （1 件

100 万円）
40 万円）
200 万円）

4,500 万円
240 万円
1,000 万円
総額 5,740 万円

2008 年度ノバルティス研究奨励金贈呈者
この事業は、生物・生命科学およびそれに関連する化学の領域において、我が国で行われる創造的な研究に対し、助成することを目的としています。
( 受付順、 敬称略、 所属 ・ 職位は申請時。 助成金額 ： 1 件 100 万円）
番号

氏

名

タケシマ

ヒロシ

1

竹島

浩

コ ダ ニ

シ ン ヤ

2

小谷

真也

3
4
5

6

7

8

9

10

研究機関名
京都大学
静岡大学

部 門
大学院薬学研究科 ・生体分子認識学分野
創造科学技術大学院
農学部 （兼務） 応用
生物化学講座

役職

研究テーマ

教授

TRIC チャネルの生理機能

助教

土壌微生物間の気菌糸誘導シグナル物質の探索と機能解析

ゼブラフィッシュを用いた繊毛研究員 の発生維持機構にかかわる分
子メカニズムの解析
キタガワ
ヒ ロ シ
ガン抑制遺伝子 EXT ファミリー
教授
北川 裕之 神戸薬科大学 生化学教室に属する EXTL2 の機能解析
大学院医学研究科
細胞間コミュニケーションを介し
イ ガ キ
タ ツ シ
神戸大学
細胞生物学分野
特命助教 た上皮の異常細胞排除機構の井垣 達吏
G-COE 井垣研究室
解明
大学院薬学研究科創生理活性天然物環状デプシペ
ド イ
タカユキ
東北大学
薬化学専攻反応制御 教授 プチドを基にしたペプチドミメテ
土井 隆行
化学分野
ィクスの創製大学院理学研究科
遷移金属錯体／有機分子二成
テ ラ ダ
マサヒロ
化学専攻境界領域化
東北大学教授 分リレー触媒系の開発による含寺田 眞浩
学講座反応有機化学
窒素化合物の高度分子変換研究室
脱ミエリン病の治療標的分子の
ヤマウチ
ジュンジ
国立成育医療 薬剤治療研究部 分
室長 探索からミエリン形成シグナル
山内 淳司
センター研究所 子薬理研究室を探る
医学部歯学部附属病
ミヤハラ
ノブアキ
アレルギー性気道炎症におけ岡山大学
院 呼吸器 ・ アレル
助教
宮原 信明
るロイコトリエン B4 の役割
ギー内科
高病原性インフルエンザウイル
医学部機能制御医学
イ マ イ
ユ ミ コ
スによる致死性呼吸不全 ・ 多
秋田大学
講座情報制御学 ・ 実 教授
今井 由美子
臓器不全における細胞膜イノシ
験治療学分野トールリン脂質の役割
オオモリ

ヨシヒロ

大森

義裕

大阪バイオサイエ
発生生物学部門ンス研究所- 165 -

番号

氏
ヤマグチ

ユ ウ ジ

11

山口

裕史

ヤマシタ

タク

12

山下

沢

イチカワ

ヨシヤス

13

市川

善康

ラ

タク ミ

14

江良

択実

シノハラ

ミ

15

篠原

美都

ヨ シ ダ

カズヒロ

16

吉田

和弘

17

ベアテ
ハイジッヒ
タ ナ カ

モ ト マサ

18

田中

元雅

19

井上 貴美子
トヨフク

トシヒコ

20

豊福

利彦

ヨシモト

トモヒロ

21

善本

知広

サ ト ウ

ア キ コ

22

佐藤

明子

ダイトク

ヒ ロ アキ

23

大徳

浩照

アンドウ

カ オ リ

24

安藤

香織

ス ズ キ

モトシ

25

鈴木

元

マルヤマ

ア キ コ

26

丸山

明子

タカムラ

ヒ ロヨシ

27

高村

浩由

サカモト

ワタル

28

坂本

亘

ワ

ヒ デ キ

29

三輪

秀樹

エ

イノウエ

ミ

名

キ

研究機関名

ト

ミ

コ

部 門
役職
研究テーマ
大学院 医学研究科
Wnt 阻害剤 ・ Dickkopf １がアン
感覚器 ・ 形成医学講
チエイジング （抗しわ、 美白、
名古屋市立大学
准教授
座 加齢 ・ 環境皮膚色素 ･ 毛髪 ･ 部位特異的皮膚科学分野
再生） に及ぼす影響
大学院薬学研究科
最小単位分子ガスによって制大阪大学
分子薬科学専攻 分
助教
御される転写機構の解明
子反応解析学分野
理学部・応用理学科・
シグマトロピー反応を用いた含
高知大学
教授
天然物化学研究室
窒素生物活性物質の合成研究
発生医学研究センタ
胚葉幹細胞の誘導と増幅そし熊本大学
ー 器官形成部門
教授て医療応用への技術開発
神経発生分野大学院・医学研究科・
G 蛋白質による精子幹細胞の京都大学
遺伝医学講座 ・ 分子 助教 ニッシェへのホーミング制御機
遺伝学教室
構の解明
大学院理学研究科
医薬品開発への貢献を志向す
千葉大学
基盤理学専攻 化学
助教 る新規不斉カルベン配位子のコース 有機金属研究室
開発
組織プラスミノーゲン活性化因
医科学研究所東京大学
特任助教 子による骨髄由来細胞を介したフロンティア研究拠点
血管新生機構の解明
脳科学総合研究セン ユニット 新規プリオン様細胞質遺伝因理化学研究所
ター 田中研究ユニット リーダー 子の機能解析
バイオリソースセン
誘導性生殖幹細胞の樹立を目
理化学研究所 ター 遺伝工学基盤 研究員 的とした生殖細胞 - 体細胞間技術室
エピゲノムプロファイルの作製
微生物病研究所
個体発生におけるセマフォリン
大阪大学准教授
感染病態分野の臓器形成メカニズムの解析
HGF を用いた術後腸管癒着の兵庫医科大学 免疫学・医動物学講座 准教授
予防
理学研究科生命理学
視細胞の生と死のシグナル伝
名古屋大学 専攻 細胞形態形成 准教授
達経路の解明学講座
大学院生命環境科学
アポトーシス促進因子 BAD の研究科/先端学際領域
アルギニンメチル化と Akt シグ
筑波大学
講師
研究センター 生命情ナルのクロストークに関する研
報機能研究アスペクト
究
工学部応用化学科物
プロセス化学を志向したα - ア
岐阜大学
教授
質変換２
ミノホスホン酸の不斉合成
大学院医学系研究科
GILC1 遺伝子産物の肺癌の発
名古屋大学
講師
分子腫瘍学分野
生と悪性化に及ぼす影響
発ガン抑制含硫二次代謝物質
生物資源学部
福井県立大学講師 グルコシノレート生合成制御の
植物環境学分野
分子基盤
大学院自然科学研究
海洋プランクトンが生産する超岡山大学
科分子科学専攻有機 助教 炭素鎖有機分子の合成と構造
化学研究室
に関する研究資源生物科学研究所
ライブイメージングによる葉緑体
岡山大学
環境シグナル伝達機
教授
チラコイド形成初期過程の検証
構グループ
大学院医学系研究科
情動記憶における扁桃体抑制
群馬大学
助教
遺伝発達行動学分野  
性局所回路の役割の解明  

- 166 -

番号
30

31
32
33

34

35

36

37

38

39
40

41

42

43

44

45

氏

名

研究機関名

部 門
難治疾患研究所ケ イ ジ
敬司 東京医科歯科大学 MTT プログラム 分
子病態分野

研究テーマ
アンジオテンシン変換酵素２
ク バ
久場
特任講師 (ACE2) による自然免疫シグナ
ル抑制のメカニズムの解明
核膜孔複合体蛋白質 Rae1 のリチャード・
フロンティアサイエン 特任准教
金沢大学
機能と細胞内動態及びがん発
ウォング
ス機構
授
症メカニズムの解析
ヤマガタ
カ ズ ヤ
大学院医学薬学研究膵β細胞におけるＨＮＦ転写因
熊本大学
教授
山縣 和也
部病態生化学分野
子ネットワークの役割の解明
植物の機械刺激センサーとして
イ イ ダ
ヒデトシ
教育学部 生命科学
教授 はたらく機械受容性カルシウム
飯田 秀利 東京学芸大学
分野
チャネルの研究
小胞体ストレスなどで活性化さオヤ ドマリセイイチ
疾患ゲノム研究セン
徳島大学教授 れる eIF2 αリン酸化シグナル
親泊 政一
ター 生体機能分野
による代謝制御機構の解明
胸腺上皮幹細胞の維持ならび
ゴ イ ツ カリョウ
生命科学研究所 発
教授 に再生に関与する転写因子ネ
後飯塚 僚 東京理科大学
生及び老化研究部門
ットワークの解明
医学部 ・ 分子生命科
ハラ
ヒロミツ
Carma1 によるリンパ球活性化
佐賀大学学講座 ・ 生体機能制 准教授
原 博満の制御機構の解明
御学分野
大学院共生科学技術
低分子 G-q リガンドを基盤とす
ナガサワ
カ ズ オ
研究院 ・ 生命機能科
准教授 る G-quadruplex 形成配列の探
長澤 和夫 東京農工大学
学部門 ・ 生命有機化
索法開発
学分野
大学院人間総合科学
脂肪酸組成を基盤とした生活
マツザカ タカシ
研究科疾患制御医学
筑波大学
助教 習慣病の病態解明および新規
松坂 賢
専攻 臨床医学系内分治療法の開発泌代謝 ・ 糖尿病内科
イマイズミ
カズノリ
医学部 解剖学講座
骨軟骨組織における小胞体スト
宮崎大学
教授
今泉 和則
分子細胞生物学分野
レス応答と制御法開発
超高速電子環状反応による生
タ ナ カ
カツノリ
大学院理学研究科
大阪大学
助教 体高分子の標識化と蛍光 CD
田中 克典
深瀬研究室
観測を用いた構造変化解析
先端医学研究機構
血管発生 ・ 成熟機能の調節メ
ナ カ ガ ワ オサム
生命システム医科学
教授 カニズムと疾患機序における転中川 修 奈良県立医科大学
分野 循環器システ
写調節の意義ム医科学
心筋梗塞に対する次世代治療
ツ ツ イ
マ サ ト
准教授 戦略の開発を目指した基盤研
筒井 正人 産業医科大学 医学部 薬理学
究
加齢医学研究所
・特紡錘体チェックポイント関連分
タ ナ カ
コウゾウ
東北大学
定領域研究推進支援 准教授 子による染色体分配制御ネット
田中 耕三
センター
ワークの解明大学院医歯薬学総合
腫瘍壊死因子によるミトコンドリ
カ マ タ
ヒデアキ
広島大学
研究科探索医科学講 准教授 アのエネルギー代謝制御を介し
鎌田 英明
座医化学研究室た肝細胞死と肝発癌の連関
アンジオポエチン－ 1 受容体
フクハラ
シゲ ト モ
国立循環器病福原 茂朋
循環器形態部室長 Tie2 のトランス結合形成機構とセンター研究所
その生物学的意義の解明- 167 -

役職2009 年度研究集会助成金贈呈集会
この事業は、生物・生命化学およびそれに関連する化学の領域において、我が国で開催される国際性豊かな研究集会の運営費の一部を援助することを目的としています。
（受付順、敬称略、所属・職位は申請時。助成金額：1 件 40 万円）
番号

研 究 集 会 名

開催期日
（開 催 地）助 成 先 代 表 者
所 属・職 位氏

名1

第 29 回札幌がんセミナー国北海道大学
2009/7/13 ～ 7/14
際シンポジウム 「ピロリ菌と胃遺伝子病制御研究所
( 札幌）
がん」
教授

畠山 昌則

2

第 5 回オートファジーに関す
京都大学 大学院
る国際会議 ： オートファジー ： 2009/9/24 ～ 9/28
農学研究科
分子機構、 細胞 ・ 生理機能（大津）教授と病理 ・ 病態

阪井 康能

3

第 9 回日本蛋白質科学会年会

熊本大学 大学院
2009/5/20 ～ 5/22
医学薬学研究部（熊本）教授

山縣 ゆり子4

第 11 回外分泌腺機能国際
シンポジウム徳島 09
外分泌 – メカニズムと病気

徳島大学 大学院
2009/7/23 ～ 7/25 ヘルスバイオサイエンス
（徳島）
研究部
教授

細井 和雄

5

東京大学 大学院
第 16 回シトクロム P450 国際 2009/6/21 ～ 6/25 農学生命科学研究科
会議
（沖縄）
教授

祥雲 弘文

6

国際生理学会第 36 回世界大会 サテライトシンポジウム

2009/8/1（京都）- 168 -

京都大学 大学院
薬学研究科
教授

辻本 豪三

2008 年度海外留学生助成金贈呈者
この事業は、生物・生命科学およびそれに関連する化学の領域において、創造的な研究に携わるアジアの国々および地域から日本への留学生（留学中を含む）に対して、旅費・滞在費を助成することを目的としています。
（受付順、敬称略、所属・職位は申請時。助成金額：1 件 200 万円）
番号
1

2

3

4

氏
名
Baizhumanova
  Ainur

国 名

職

位

所属機関
研究テーマ
名古屋大学 大学院
Results of anemia
医学系研究科prevention programs in
Kazakhstan PhD Student
Kazakhstan and lessons
ヴァイジュマノア
社会生命科学講座
learned  アイノル（教授 坂本純一）
山梨大学 大学院
中華人民 山梨大学 大学院
医学工学研究科 医学工学総合教育部博 自己免疫疾患発症誘Huhehasi Wu
共和国
博士課程在籍中 士課程、 先進医療科学 因としての細胞内２本フホハス ウ 内モンゴル 国立感染症研究所
専攻人体病理研究室 鎖ＤＮＡの役割の解析
自治区 生体防御部研究生（教授 鈴木幸一）
Department of Cell
KITIPONG
Pharmacology, Faculty UV 照射による免疫抑SOONTRAPA Kingdom of
PhD Student
of Medicine, Kyoto
制機構における PGE2
キティポン
Thailand
Universityの役割の解析
スーントラパ（教授 成宮 周）Farzana DIBA
Doctoral  ファルザナ
Bangladesh course    ディバ
student

Soil Science laboratory,
Division of Environmental
土壌からの N2O 放出
Resources, Graduateに及ぼす無機化、 硝
School of Agriculture,化、 脱窒の影響
Hokkaido University.

（教授 波多野隆介）

5

  SAXENA
     Nishant  サクセーナ     ニシャント

INDIA

PhD Student

National Institute ofAdvanced Industrial
Science & Technology
細胞老化 ・ 癌化 ・ 環(AIST), Research Institute
境ストレス制御におけfor Cell Engineering, Cell
るストレスタンパク質モ
Proliferation Research
ータリン
Group

（グループ長ワダワ レヌー）- 169 -

第 22 期（2008 年度）財務報告

貸借対照表

収支計算書

2009 年 3 月 31 日現在

2008 年 4 月 1 日から 2009 年 3 月 31 日まで（単位：円）（単位：円）

科目
Ⅰ資産の部
１． 流動資産
現金預金
未収収益
有価証券
前払費用
流動資産合計
２． 固定資産(1) 基本財産
基本財産合計(2) 特定資産
特定資産合計(3) その他固定資産
什器備品
電話加入権
その他有価証券
その他固定資産合計
固定資産合計資産合計
Ⅱ負債の部
１． 流動負債
未払金
預り金
流動負債合計
負債合計
Ⅲ正味財産の部
１． 指定正味財産
指定正味財産合計
２．一般正味財産
正味財産合計正味財産合計
負債及び正味財産合計負債
及び正味財産合計

金額

14,827,601
6,116,347
5,986,646
433,455
27,364,049

728,540,000
10,020,000
275,01276,44031,820,484
32,171,936
760,711,936
798,095,985

55,079,658
39,66655,119,324
55,119,324

694,780,000
48,196,661
742,976,661
798,095,985- 170 -

科
目
Ⅰ事業活動収支の部
１． 事業活動収入
基本財産運用収入
特定財産運用収入
寄付金収入
雑収入
事業活動収入計
２． 事業活動支出
事業費支出
ノバルティス研究奨励金
研究集会助成金財団年報発行
選考費用
その他事業費
海外留学生受入れ助成
管理費支出
事業活動支出計
事業活動収支差額
Ⅱ投資活動収支の部
１． 投資活動収入
有価証券売却収入
基本財産取崩収入
投資有価証券取崩収入
投資活動収入計
２． 投資活動支出
基本財産取得支出
固定資産取得支出
他投資活動支出
投資活動支出計
投資活動収支差額
当期収支差額
前期繰越収支差額
次期繰越収支差額

決算額

27,923,981
157,517
40,343,000
4,777,108
73,201,606
64,292,325
45,000,000
2,800,000
1,572,953
4,619,202
300,170
10,000,000
16,229,272
80,521,597△ 7,319,991

21,73589,600,000
10,000,000
99,621,735
100,000,000
360,150
8,317,410
108,677,560△ 9,055,825△ 16,375,816
20,441,025
4,065,209

役員名簿（理事・評議員）

理事

会
2009 年 9 月 3 日現在 ( 順不同、 敬称略 )

職 名

氏 名

現

職

就任年月日

常勤・非常勤理事長 金 子

章 道 畿央大学大学院健康科学研究科教授
慶應義塾大学名誉教授

2003年 6月 10日

非常勤理

茂 隆 早稲田大学理工学術院教授
東京大学名誉教授

1999年 6月 4日

非常勤

石 川

裕 子 ノバルティス ファーマ株式会社
常務取締役 人事･コミュニケーション本部長

2004年 6月 7日

非常勤

大 島

泰 郎 共和化工株式会社 環境微生物学研究所長
東京工業大学名誉教授

1997年 6月 8日

非常勤

1999年 6月 4日

非常勤

事 浅 野

黒

川

清 政策研究大学院大学教授
東京大学名誉教授

園 田

孝 夫 財団法人大阪腎臓バンク 理事長
大阪大学名誉教授

1999年 6月 4日

非常勤

眞 崎

知 生 東京女子医科大学国際統合科学インスティ 1999年 6月 4日
テュート 特任教授
筑波大学名誉教授、京都大学名誉教授

非常勤マックス ･
ブルガー

1987年 9月 16日

非常勤

眞 弓

忠 範 神戸学院大学薬学研究科教授
大阪大学名誉教授

2004年 6月 7日

非常勤

三 谷

宏 幸 ノバルティス ファーマ株式会社
代表取締役社長

2007年 9月 5日

非常勤

村 崎

光 邦 CNS薬理研究所長
北里大学名誉教授

2001年 6月 1日

非常勤

2005年 6月 13日

非常勤

2006年 6月 5日

非常勤

1998年 2月 10日

非常勤

森
監

ノバルティス サイエンスボード議長
バーゼル大学教授

美 和 子 北海道医療大学客員教授
北海道大学名誉教授

事 中 嶋

徳 三 中嶋徳三公認会計士事務所
公認会計士

松 本 秀三郎 元ノバルティス ファーマ株式会社
常勤監査役- 171 -

評 議 員 会
2009 年 9 月 3 日現在 ( 順不同、敬称略 )
職 名

氏

名

現

職

就任年月日

常勤･非常勤2002年 2月 7日

非常勤

評 議 員 赤池 紀扶 熊本保健科学大学リハビリテーション学科教授 1999年 6月 4日
銀杏学園理事 ・ 副学長
九州大学名誉教授

非常勤

評議員会 黒岩 常祥 立教大学極限生命情報センター長
議 長
東京大学名誉教授

赤沼 安夫 財団法人朝日生命成人病研究所名誉所長

2001年 6月 1日

非常勤

浅 島

1999年 6月 4日

非常勤

遠藤 政夫 山形大学名誉教授

1997年 6月 8日

非常勤

小 川

聡 国際医療福祉大学三田病院長
慶應義塾大学名誉教授

2001年 6月 1日

非常勤川嵜 敏祐 立命館大学糖鎖工学研究センター長
京都大学名誉教授

1999年 6月 4日

非常勤

川島 博行 元新潟大学大学院医歯学総合研究科教授

2001年 6月 1日

非常勤

北1999年 6月 4日

非常勤

後藤 勝年 JST サテライト茨城センター長

2001年 6月 1日

非常勤榊2001年 6月 1日

非常勤

柴崎 正勝 東京大学大学院薬学系研究科教授

2005年 6月 13
日

非常勤

冨 永

1998年 6月 5日

非常勤

中西 重忠 財団法人大阪バイオサイエンス研究所所長
京都大学名誉教授

1999年 6月 4日

非常勤

長田 敏行 法政大学生命科学部学部長
東京大学名誉教授

2005年 6月 13日

非常勤西川 武二 慶應義塾大学名誉教授
日本ワックスマン財団常務理事

2001年 6月 1日

非常勤

西宗 義武 大阪大学微生物病研究所特任教授

1999年 6月 4日

非常勤

水野 美邦 順天堂大学医学部教授

1999年 6月 4日

非常勤

誠 東京大学大学院総合文化研究科客員教授
東京大学名誉教授

徹 神戸市立医療センター中央市民病院
病院長

佳 之 豊橋技術科学大学学長
東京大学名誉教授

健 昭和大学附属豊洲病院
外科客員教授- 172 -

事務局便りご寄附のお願い
当財団は、 生物 ・ 生命科学およびそれに関連する化学の領域における創造的な研究に対して助成し、 学術の進展と福祉の向上に寄与することを目的としております。
これらの事業は、 基本財産の運用益および寄附金にて行われており、 当財団は事業の趣旨にご賛同いただける方々からのご寄附を募っております。
なお、 当財団はご寄附をいただく方々へご配慮するために、 特定公益増進法人の認定を受けております。
特定公益増進法人とは、 公益法人等のうち教育または科学の振興、 文化の向上、 社会福祉への貢献その他公益の増進に著しく寄与するものとして認定された法人をいいます。
これらの法人に対して、 個人または法人が寄附を行った場合は、 下記の税法上の優遇措置を受けることができます。
優遇措置の概略
個人 ： 支 出 し た 寄 附 金 （ そ の 年 の 総 所 得 額 の 40 ％ を 限 度 と す る ） の う ち、
5 千円を超える部分について寄附金控除が認められます。
また、 上記の寄附金控除の対象になっている寄附金は、 個人住民税の寄附金控除が受けられます。
法人 ： 支出した寄附金は、 通常一般の寄附金の損金算入限度額と同額まで、別枠で損金に算入できます。
ご寄附は、 随時受付けております。
詳しくは、 財団事務局までお問合せ下さい。
（電話 ： 03-5464-1460、 E-メール ： novartisfound.japan@novartis.com）

事務局より
平成 20 年度は、財団にとりまして百年に一度といわれる出来事が重なった年でした。
ひとつ目は金融危機でありまして、 財団の活動資金の元となります基本財産の運用益が目減りし、 助成事業を含めた財団の財政状況に少なからず影響を与えました。
もうひとつは公益法人の制度改革でありまして、 こちらは明治 29 年 (1898 年 ) の制定以来 110 年ぶりの改定とのことですが、 当財団も新公益財団法人に移行するべく認可を受ける必要が出てまいりました。
財団を取り巻く環境に変化は出てきておりますが、 事務局といたしましてはこれまで
通り、 学術の発展に少しでも寄与できるよう微力ながら取り組んで行きたいと考えております。
今後ともご指導、 ご支援の程、 よろしくお願い申し上げます。
事務局長 松田光陽〒 106-0031 東京都港区西麻布 4-16-13
西麻布 28 森ビル 10F
Tel:03-5464-1460 Fax:03-5467-3055
E- メール ： novartisfound.japan@novartis.com
ホームページ :http://www.novartisfound.or.jp- 173 -