Annual Report (2013)

No.25
The NOVARTIS Foundation (Japan)
for the Promotion of Science

平成 25 年度

財団年報 第 25 号

Contents
Ⅰ . Introduction はじめに ............................................................................................ 6
Akimichi Kaneko, MD, PhD
Chairman of the Board of Trustees

Ⅱ . Reports from the Recipients of Novartis Research Grants
研究報告 .................................................................................................................. 8
1. Ageing effects on the neurophysiological parameters of human brain ......................... 9
Yoshikazu Ugawa
Fukushima Medical University, Department of Neurology

2. Synthetic study of non-alkaloidal diterpene isolated from the genus Aconituma ...... 12
Takahiro Suzuki
Tokyo University of Science, Faculty of Pharmaceutical Sciences

3. Bio-Inspired Total Synthesis of Tryptophan-Based
Dimeric Diketopiperazine Alkaloids ...................................................................... 15
Hayato Ishikawa
Kumamoto University

4. Regulation of vascular integrity by AM-RAMP system and its application ............... 19
Takayuki Shindo
Department of Cardiovascular Research,
Shinshu University Graduate School of Medicine

5. Development of novel molecular imaging method for visualization ofclinical condition of intravital lipids ........................................................................ 22
Hideaki KANO
University of Tsukuba

6. Molecular mechanisms of corniﬁcation of the epidermis. .......................................... 25
Masatake Osawa
Graduate School of Medicine, Gifu University

7. Analysis for the molecular mechanisms
causing paroxysmal cough in pertussis ................................................................... 29
Yasuhiko Horiguchi
Research Institute for Microbial Diseases, Osaka University

8. Analysis of mice deﬁcient in molecules involved
in intracellular polarized transport ............................................................................ 32
Akihiro HARADA
Osaka University

9. Investigation of molecular signal output from a dividing nucleus to cell cortex ........ 35
NAKANO KENTARO

Graduate School of Life and Environmental Sciences,
University of Tsukuba-1-10. The role of transcription factor IкB-ζ in immune homeostasis .................................... 38
Takashi Maruyama
Graduate School of Life Sciences, Tohoku University

11. Study of dense-core vesicle synthesis on the membrane of Golgi complex ............... 40
Tetsushi Sadakata
Advanced Scientific Research Leaders Development Unit, Gunma University

12. Stress response, homeostasis and cancer ...................................................................... 43
Toshiharu Sakurai
Kinki University, Faculty of Medicine,
Department of Gastroenterology and Hepatology

13. Regulation of cerebral vasospasm by growth factors and matricellular proteins......... 47
Hidenori Suzuki
Mie University Graduate School of Medicine

14. Centriole biogenesis and Chromosomal instability ..................................................... 50
Daiju Kitagawa
National Institute of Genetics

15. The role of specific cell adhesion molecules in
dopaminergic innervation of striatal neurons ........................................................... 53
Akaike Akinori
Department of Pharmacology, Graduate School of Pharmaceutical Sciences,
Kyoto University

16. In vivo imaging reveals molecular mechanisms ofadult common diseases based on chronic inflammation ........................................... 56
Satoshi Nishimura
Department of Cardiovascular Medicine, TSBMI, The University of Tokyo

17. The structural study of the human centromeric chromatin .......................................... 59
Hitoshi Kurumizaka
Waseda University, Graduate School of Advanced Science & Engineering

18. An intrinsic molecular mechanism governing
the development of root nodule primordia ............................................................... 62
Takuya Suzaki
National Institute for Basic Biology

19. Identification and characterization of novel skeletal muscle-derived
angiogenesis growth factor ....................................................................................... 65
Yasuhiro Izumiya
Department of Cardiovascular Medicine, Faculty of Life Sciences,
Kumamoto University

20. Molecular mechanism of self-renewal and proliferation of a hematopoietic cell
due to HMGA2 expression ....................................................................................... 69
Kazuhiko Ikeda
Fukushima Medical University-2-

21. The biological and pathological signiﬁcance of Stox1 transcriptional factor .............. 71
Keizo KANASAKI
Kanazawa Medical University

22. Novel role of neuronal Ca2+ sensor-1 as a survival factor
activated in cardiomyocytes under stress. ................................................................ 74
Tomoe Nishitani
National Cerebral and Cardiovascular Center

23. Paternal uniparental disomy 14 and related disorders: placental gene expression
analyses and histological examinations .................................................................... 77
Masayo Kagami
Department of Molecular Endocrinology,
National Research Institute for Child Health and Development

24. Mechanism of dNTPs supply at beginning of DNA replication................................... 80
Hiroyuki Niida
Hamamatsu University School of Medicine

25. Characterization of mammalian homologue of Unc119 as a tumor suppressor ........... 82
Kentaro Nakagawa
Tokyo Medical and Dental University

26. Epigenetic involvement in a mouse model ofE-cadherin-p53-deﬁcient diffuse-type gastric cancer ............................................... 85
Shu Shimada
Division of Molecular Oncology, Graduate School of Medical and Dental Sciences,
Tokyo Medical and Dental University

27. Characterization and generation of organ-speciﬁc vascular cells
from human iPS cells towards development of novel in vitro models
for intractable vascular diseases ............................................................................... 89
Kenji Osafune
Center for iPS Cell Research and Application (CiRA), Kyoto University

28. Fine mapping of autophagy-related proteins during autophagosome formation in
Saccharomyces cerevisiae ........................................................................................ 92
Kuninori Suzuki
University of Tokyo

29. Synthesis of fatty acids having various carbon frameworks ........................................ 95
Takanori Iwasaki
Department of Applied Chemistry, Graduate School of Engineering,Osaka University

30. Analyses of structural dynamics of a G protein-coupled receptor
using site-directed ﬂuorescent labeling. ................................................................... 98
Hisao Tsukamoto
Institute for Molecular Science

31. Regulation of the generation and maintenance of memory T cells in the intestine.... 101
Naoto Ishii
Tohoku University Graduate School of Medicine-3-32. Studies on the regulatory mechanisms underlying cell
cycle-dependent centrosome replication................................................................. 104
Takaya Satoh
Department of Biological Science, Graduate School of Science,
Osaka Prefecture University

33. microRNA-mediated regulation of vasculogenesis ................................................... 107
Yuichiro Mishima
Institute of Molecular and Cellular Biosciences, The University of Tokyo

34. The role of molecular motor for regulating T cell activation ..................................... 110
Akiko Hashimoto-Tane
RIKEN Research Center for Allergy and Immunology

35. Generation of Batf reporter/conditional knock-out mice forelucidation of Batf function during in vivo immune responses ............................. 113
Wataru Ise
Immunology Frontier Research Center (IFReC), Osaka University

36. A study on the molecular mechanisms of mitochondrial regulationby the genes responsible for early-onset familial forms of
Parkinson’ s disease .............................................................................................. 115
Yuzuru Imai
Department of Neuroscience for Neurodegenerative Disorders,Juntendo University Graduate School of Medicine

37. Fundamental study for long intergenic RNA (linc RNA) andepigenetic regulation by tumor suppressor p53 using ES/iPS cells tocontrol cancer stem cells......................................................................................... 118
Tomoaki Tanaka
Chiba University Graduate School of Medicine

38. Drugs discovery for CKD patients using transporter manipulation ........................... 121
Takaaki ABE
Tohoku University Graduate School of Meidicine

39. Molecular mechanism of semaphorin receptor activation
implicated in axon-pathﬁnding ............................................................................... 123
Terukazu Nogi
Graduate School of Medical Life Science, Yokohama City University

40. Promoter-based super improvement of gene expression
in an antibody production ....................................................................................... 126
Masakiyo Sakaguchi
Okayama University Graduate School of Medicine, Dentistry, andPharmaceutical Sciences

41. Analysis of a novel mechanism of tumorigenesis
involving cMyc proto-oncogene product ............................................................... 130
Yoshihiro Kawasaki
Institute of Molecular and Cellular Biosciences, The University of Tokyo-4-

Ⅲ . Reports from the Recipients of Grants for International Meetings
研究集会報告 ....................................................................................................... 133
1. The 8th Annual Conference of OOTR ......................................................................... 134
Masakazu Toi
OOTR (Organisation for Oncology and Translational Research)

2. The 22nd CDB Meeting “RNA Sciences in Cell and Developmental Biology II” ..... 135
Toshifumi Inada
Professor, Tohoku University, Graduate School of Pharmaceutical Sciences

3. 20th International Symposium on Molecular Cell Biology of Macrophages .............. 136
Shigeo Koyasu
Department of Microbiology and Immunology,
Keio University School of Medicine

4. Plant and Microbes Adaptations to the Cold 2012: Toward risk assessment andmanagement of sustainable agriculture in the cool and cold regions ..................... 137
Naoyuki Matsumoto (Hokkaido University)
Ryozo Imai (NARO Hokkaido Agricultural Reseach Center)

5. The 18th Young Asian Biochemical Engineers' Community (YABEC) ...................... 138
Takeshi Omasa
Chairman, Organizing Committee of YABEC 2012
Professor, The University of Tokushima

6. The 8th 3R Symposium(International Symposium on DNA Replication, Recombination and Repair) ...... 139
Kaoru Sugasawa
Professor, Biosignal Research Center,
Organization of Advancd Science and Technology, Kobe University

Ⅳ . The 26th (ﬁscal year 2012) Promotion Report
第 26 期 （2012 年度） 助成事業報告 ................................................................ 143
Ⅴ . The 26th (ﬁscal year 2012) Financial Report
第 26 期 （2012 年度） 財務報告 ........................................................................ 147
Ⅵ . List of the Board Members
役員名簿 ............................................................................................................... 150
Ⅶ . Information from the Secretariat
事務局便り ............................................................................................................ 152-5-

Introduction

Akimichi Kaneko, MD, PhD
Chairman of the Board of Trustees
This booklet includes research and meeting reports written by the 2012 grantees. The
Foundation was originally established on September 4, 1987 with basic assets of JPY 1
billion donated by Ciba-Geigy AG, Switzerland for the purpose of contributing to academic
development and thus improving public health and welfare by means of promoting creative
research and international exchange in the field of life science and related chemistry. Since
then, the Foundation has granted nearly JPY 1.8 billion to approximately 1,500 researches andinternational exchange activities. The Foundation supports basic researches in the field of life
science and related chemistry. They may not develop directly into applications, but accumulationof new findings and theories will open a road to application research and business someday.
The tireless effort of researchers including the present grantees is the driving force topromote and to keep the high standard of the Japanese life science. I strongly believe that theaim of the Foundation is to help keeping their activity and we will do our best toward this goal.
We are encouraged by warm acknowledgements to the Foundation written in many publicationsof grantees.
I sincerely appreciate the assistance and warm encouragement extended by the membersof the Board of Trustees, the auditors, the Board of Councilors and the Selection Committee.
The powerful support by the Novartis Pharma KK enabled us to sustain our activity without
interruption.

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はじめに
代表理事 金 子 章 道
ここに 2012 年度にノバルティス科学振興財団研究助成金を受けられた方々の研究報告を収録いたしました。 当財団は 1987 年 9 月 4 日、 スイス、 チバガイギー社からの 10 億円のご寄附をもとに、 「生物・生命科学および関連する化学の領域において、創造的な研究ならびに国際交流への助成を行うことにより、 学術の振興を図り国民の健康と福祉の向上に寄与する」ことを目的に設立されました。 爾来 26 年間に約 1,500
件、 金額にしておよそ 18 億円の助成を行ってまいりました。 当財団がご提供する研究費は、 研究に要する総費用のうち微々たるものかもしれませんが、 研究をスムーズに遂行するための役に立てていただくのがその目的です。 事務局に寄せられた発表
論文の謝辞の中にも受賞者のそうしたお気持ちが垣間見られ、 大変嬉しく思っております。
当財団が助成の対象とする研究は生物 ・ 生命科学および関連する化学の領域における基礎的な研究です。 その成果が即応用につながらなくても、 新発見や新しい理論が応用研究のアイディアとして役立つことになるもしれません。 基礎的な発見や理論を積み上げることが、 いつの日か新分野を開き、 新産業を生み出すもとになるものと信じております。
この年報は受賞者の皆様の素晴らしい研究がまとめられたエッセイ集です。 研究者
お一人おひとりの努力の結晶は我が国の学術水準を発展させていく原動力です。 こ
れらの優れた研究を選考していただいた選考委員の皆様をはじめ、 財団の活動を支えて下さっている関係者の皆様に深く感謝いたします。

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II.
Reports from the Recipients ofNovartis Research Grants-8-Ageing effects on the neurophysiological parameters of human brain
Yoshikazu Ugawa
Fukushima Medical University, Department of Neurology
ugawa-tky@umin.net

Abstract
To study ageing influence on the corticospinal tract in humans, we measured the corticoconus
motor conduction time (CCCT) in 100 healthy volunteers. Age was significantly correlated with L1level latency reflecting total peripheral nerve conduction, but it was not significantly correlated with
CCCT. Central motor conduction was found to be relatively less affected by aging compared with
peripheral motor conduction. This is probably because the peripheral nerves are more often affectedby several accidental influences, such as injury, compression and metabolic disorders effects(diabetes mellitus and so on) as compared with the central nervous system protected by bones (skull
or spine).
Key words : ageing, corticospinal tract, peripheral nerves, transcranial magnetic stimulation

Introduction
Anti-ageing medication is one big problem, especially in Japan, because of high average lifetime. The central nervous system is usually affected by ageing and its involvement often produces
clinical symptoms, such as memory loss, dementia, difficulty in locomotion and so on. Motorsystem involvement may be a serious issue to be assessed. The corticospinal tract, main central
motor pathway, must be affected by ageing. The objective assessment of its function is requisite forassessing the ageing effect. In the present investigation, therefore, we investigated aging effects on
the corticospinal function by measuring the corticoconus motor conduction time (CCCT) in various
aged normal subjects.

Results
Subjects.
Subjects consisted of 100 healthy volunteers (41 men and 59 women) with age and body height(mean 6 SD) of 52.9 6 21.6 (range 20–90) years and 160.4 6 10.4 (132–189) cm, respectively. All
subjects were able to engage in normal tasks of daily life without assistance, even the most elderly.
Informed consent to participate in our study was obtained from all subjects. The protocol used
was approved by the ethics committee of the Faculty of Medicine, University of Tokyo, and theexperiments were conducted in accordance with the ethical standards of the Declaration of Helsinki.
Methods.
Motor evoked potentials (MEPs) were recorded from the right tibialis anterior muscle (TA) withsubjects sitting comfortably on a bed.

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Magnetic stimulation was conducted using a monophasic stimulator (Magstim 200; MagstimCo., Ltd., Whitland, UK). For cortical stimulation, the edge of a round coil (20-cm diameter,
0.98T; Magstim) was placed over Cz (international 10–20 system), with induced currents flowing
mediolaterally over the contralateral leg motor area.16,17 MEP onset latency was measured in theactive condition (cortical latency).
For the conus medullaris stimulation, the magnetic augmented translumbosacral stimulation(MATS) coil was always placed from the midline to the contralateral side of the body (the opposite
side from the recorded muscle) to prevent non-target parts of the coil from activating distal
peripheral nerves for the target TA.
The CCCT, conventional CMCT, and cauda equina conduction time (CECT) values were obtained
as follows. The CCCT was obtained by subtracting the L1-level latency from the cortical latency, theconventional CMCT by subtracting the L5-level latency from the cortical latency, and the CECT bysubtracting the L5-level latency from the L1-level latency.
Results
The correlations between each conduction parameter and age are depicted in Figures 1and 2. All
measured conduction parameters except CCCT had a significant positive, linear correlation withage. On the other hand, no significant correlation was found between CCCT and age (P: 0.946).

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Discussion & Conclusion
In this study we obtained the following findings: L5- and L1-level latencies, cortical latency,
conventional CMCT, and CECT depended on age, whereas CCCT was relatively independent ofthese variables. Thus, CCCT, a measure most directly reflecting corticospinal tract conduction, did
not depend on age. In contrast, L1-level latency, reflecting the entirety of peripheral motor nerve
conduction, depended significantly on age. Based on these findings, we conclude that, althoughcorticospinal tract conduction is affected slightly by aging, peripheral motor conduction is strongly
affected by aging.
One plausible explanation for the lack of ageing effects on the corticospinal tracts is that the brain
and spinal cord are protected by cranial bones and the vertebral column, respectively. Moreover,the corticospinal tract is surrounded by central nervous system tissue, except for the pyramidal
decussation portion, and bathed in cerebrospinal fluid. The blood–brain barrier may also be involvedin the protection of the corticospinal tract. In contrast, peripheral nerves can be directly affected byminor trauma or injuries, due to their relative lack of protection. Based on these anatomical features,
we believe that the corticospinal tract is less likely to be damaged by the various events occurring
on a daily basis over the course of a long lifespan.

References
1. Furubayashi T, Mochizuki H, Terao Y, Arai N, Hanajima R, Hamada M, Matsumoto H, Cortical
hemoglobin concentration changes underneath the coil after single-pulse transcranial magnetic
stimulation: A near-infrared spectroscopy (NIRS) study. J Neurophysiol. 2012 Dec 28
2. Matsumoto H, Konoma Y, Shimizu T, Okabe S, Shirota Y, Hanajima R, Terao Y, Ugawa Y.
Aging influences central motor conduction less than peripheral motor conduction: A transcranial
magnetic stimulation study. Muscle Nerve. 2012 Dec;46(6):932-936
3. Shirota Y, Hamada M, Terao Y, Ohminami S, Tsutsumi R, Ugawa Y, Hanajima R. Increased
primary motor cortical excitability by a single-pulse transcranial magnetic stimulation over the
supplementary motor area. Exp Brain Res. 2012 Jun;219; 339-49

一般の皆様へ
加齢が人の神経系にどのような影響を与えるかは、一般国民の関心事の一つである。認知症など高次機能に関する影響が重要であるが、歩行・姿勢などに影響を及ぼす運動神経系に対する加齢の影響も非常に重要である . しかし、ヒトの中枢運動系を客観的に評価できる検査法は従来存在しなかった。ところが、近年磁気刺激を用いた検査により、ヒトの中枢運動系を客観的に評価できるようになった。
本研究では、その方法を用いて様々な年齢の被験者で中枢運動系の評価を行い、ヒトの運動系に対する加齢の影響を検討した。その結果、末梢運動神経系は加齢とともに機能が低下する事が証明されたが、中枢運動系は加齢による大きな影響はなかった。
この理由としては、物理的に骨・髄液・脳血管関門に守られている脊髄・脳に比べて、末梢神経は特に守られてないため、外傷・代謝性影響など影響を受けやすい事が大きなファクターになっているのではないかと考えた。

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Synthetic study of non-alkaloidal diterpene
isolated from the genus Aconitum
Takahiro Suzuki
Tokyo University of Science, Faculty of Pharmaceutical Sciences
suzuki-t@rs.noda.tus.ac.jp

Abstract
We have achieved the construction of an unprecedented pentacyclic framework of non-alkaloidal
diterpene, atropurpuran, by an intramolecular reverse electron demand Diels-Alder reaction ofmasked o-benzoquinone. We also have achieved the first synthesis of a tetracyclo[5.3.3.0 4,9.04,12]
tridecane skeleton and development of a concise approach to the atropurpuran skeleton.
Key Words : Total synthesis, Diels-Alder reaction, Diterpene

Introduction
Aconitum is a genus of flowering plants that produce a variety of poisons and compounds of
medicinal importance. The fascinating bioactivities of these compounds arise from C19 andC20 diterpene alkaloids, such as aconitine, hetisine, and atisine. Isolation of non-alkaloidal
diterpenes from the genus Aconitum, however, has rarely been reported. In 2009, Wang and coworkers reported the isolation of the structurally unique non-alkaloidal diterpene atropurpuran
from Aconitum hemsleyanum var. atropurpureum.1 The structure of atropurpuran features
an unprecedented cage-like skeleton, tetracyclo[5.3.3.0 4,9.0 4,12]tridecane, which includes twobicyclo[2.2.2]octane units.

Results
Despite the intriguing biosynthetic and structural properties of the tetracyclo[5.3.3.0 4,9.0 4,12]
tridecane skeleton, there are no previous reports that describe efforts to synthesize this compound.
Consequently, at the outset of our synthetic investigation on atropurpuran, we attempted to establish
a methodology for the construction of the tetracyclic skeleton. Our strategy towards the synthesisof the tetracyclic skeleton was to utilize a reverse electron demand Diels–Alder (REDDA) reactionof masked o-benzoquinone (MOB). The REDDA reaction with MOB has recently emerged as a
powerful tool for the construction of highly functionalized complex molecules. We envisaged thatthe intramolecular REDDA reaction of MOB 3 would directly provide an anti-Bredt compound 4.
(Fig. 1)

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The preparation of REDDA precursor was started from o-eugenol (Fig. 1). Easily scalable tetralone
1 was successfully converted to phenol 2 by removal of Benzyl group, reduction with DIBAL,
and silyl etherification. Oxidative dearomatization of phenol 2 with hypervalent iodine reagent,
PhI(OAc)2 afforded MOB 3. The REDDA reaction of MOB 3 (mesitylene, 180 °C, 1h) afforded
Diels-Alder adduct 4 in high yield almost as a single diastereomer. This result established that we
had achieved the first artificial synthesis of the tetracyclo [5.3.3.04,9.04,12]tridecane skeleton. Next,
we proceeded to the construction of pentacyclic carbon framework of atropurpuran. Deprotectionof common intermediate 1 with BCl3 in CH2Cl2 and addition of allylmagnesium bromide gave triallylated compound, of which ring-closing metathesis with Grubbs 2nd generation catalyst andoxidative dearomatization with PhI(OAc)2 gave MOB 5. REDDA reaction of the tricyclic MOB
5 was achieved to construct the pentacyclic Diels-Alder adduct 6 in high yield (75%). Finally,hydrogenation of 6 with Pearlman’s catalyst and subsequent dehydration (Tf 2O, pyridine) afforded 7
to complete the construction of the pentacyclic framework of atropurpuran, despite the lack of somefunctionality.2

Next, we were embarked on the synthetic study toward the total synthesis of atropurpuran (Fig.
2). Carboxylation of common intermediate 1 afforded the corresponding β-keto ester, which was
subjected to Rh-catalyzed reverse prenylation of active methyne position to give ketone 8. Additionof allylmagnesium bromide to ketone 8 gave anti-adduct 9 in excellent yield (95%). However,
all attempts for further transformation from anti-adduct 9 was failed due to the steric hindranceof dimethyl group. Despite of our efforts to obtain syn-adduct 10, any condition of allyl group
addition was failed resulting in low yield of 10 (up to 27%). Fortunately, we found that anti-adduct 9
underwent base-promoted isomerization to give syn-adduct 10 by treatment of NaH. We postulated
that the isomerization of syn-adduct 10 was caused by a retro-aldol/aldol reaction. Ring-closing
metathesis of syn-adduct 10, reduction of ester with LiAlH4, and TPAP oxidation of the resulting
alcohol afforded aldehyde 11. Introduction of vinyl group as a dienophile to aldehyde 11 wassuccessively proceeded by Grignard addition in diastereoselective manner. TES protection of thesecondary alcohol, removal of Bn group with lithium naphthalenide, and oxidative dearomatizationof the resulting phenol provided MOB 12. The REDDA reaction of MOB 12 afforded the DielsAlder adduct, which was treated with TBAF to give diol 13 in 49% overall yield. The pentacyclic
structure was determined by NOESY experiment of diol 13. At the same time, the stereochemistryof secondary alcohol generated by addition of vinylmagnesium bromide was also determined.

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Discussion & Conclusion
In summary, we have achieved the construction of the pentacyclic framework of atropurpuran by
an intramolecular REDDA reaction of MOB. Characteristic features of the present study are: 1)
the first synthesis of a tetracyclo[5.3.3.04,9.04,12]tridecane skeleton and 2) a concise approach to theatropurpuran skeleton. These results demonstrate the power of the REDDA reaction by using MOB
for the construction of anti-Bredt and cage-like complex molecules. Use of this methodology insynthetic efforts toward atropurpuran is currently underway. Further synthetic plan is that oxidationof methyl group would be achieved by Barton reaction and Wittig reaction of acetal carbonyl groupwould give atropurpuran.

References
1) P. Tang; Q.-H. Chen; F.-P. Wang; “Atropurpuran, a novel diterpene with an unprecedented
pentacyclic cage skeleton, from Aconitum hemsleyanum var. atropurpureum”, Tetrahedron Lett.
2009, 50, 460-462.
2) T. Suzuki; A. Sasaki; N. Egashira; S. Kobayashi; “A Synthetic Study of Atropurpuran:Construction of a Pentacyclic Framework by an Intramolecular Reverse-Electron-Demand DielsAlder Reaction”, Angewandte Chemie, Int. Ed. 2011, 50, 9177-9179.

一般の皆様へ
トリカブト (Aconitum) 属の植物は、強力な神経毒であるアコニチンをはじめとして、根に多くのアルカロイドを含み、古来より狩猟の際の毒や、神経痛などの鎮痛薬としても広く用いられていた。
2009 年にトリカブト属の植物から単離が報告されたアトロプルプランは、珍しいことにトリカブト由来
であるにもかかわらず、窒素を含まないテルペノイドである。また構造的にも、前例のない複雑な 5環性骨格を有している。その構造と生合成経路についての興味から、アトロプルプランの合成研究を開始した。現在までに炭素骨格の合成を達成しており、今後は官能基変換により全合成を達成したい。

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Bio-Inspired Total Synthesis of Tryptophan-Based
Dimeric Diketopiperazine Alkaloids
Hayato Ishikawa
Kumamoto University
ishikawa@sci.kumamoto-u.ac.jp

Abstract
Direct bio-inspired dimerization reactions along with an originally proposed biosynthetic pathway
from commercially available amine-free tryptophan derivatives in aqueous acidic media weredeveloped to provide symmetric and asymmetric dimeric compounds. In addition, concise two-pot
or three-step syntheses of naturally occurring dimeric diketopiperazine alkaloids (+)-WIN 64821(1), (–)-ditryptophenaline (2) and (+)-naseseazine B (3) were accomplished in total yield of 20%,
13% and 20%, respectively.
Key Words : alkaloids, biomimetic synthesis, ditryptophenaline, naseseazine B, WIN 64821

Introduction
A large number of tryptophan-based dimeric diketopiperazine alkaloids, which show fascinating
biological activities, have been isolated from fungi. For instance, (+)-WIN 64821 (1) and(–)-ditryptophenaline (2) were isolated from Aspergillus sp. and reported to be a competitive
substance P antagonist with respect to human neurokinin-1 and the cholecystokinin B receptor [1].
In 2009, (+)-naseseazine B (3) possessing a C3sp3–C7sp2 bridge was isolated from Fijian actinomycete
Streptomyces sp [2]. Herein, we describe direct bio-inspired dimerization reactions along with our
proposed biosynthetic pathway from commercially available amine-free tryptophan derivatives inaqueous acidic media and concise three-step total syntheses of 1, 2 and 3.

Results

To achieve bio-inspired dimerization reaction in aqueous acidic media for synthesis of 1, 2 and 3,
suitable oxidants and acids were screened with commercially available L-tryptophan ethyl ester (4)- 15 -

as substrate (Figure 1, Table 1). When the reaction was carried out in aqueous CH3SO3H solution in
the presence of 1.2 equivalent of Mn(OAc)3 as oxidant, high conversions were observed (entry 1).
Thus, dimeric compounds 5, 6, and 7 were obtained in 19%, 11%, 28% yield, respectively. CH3SO3H
was also a suitable acid for VOF3 as oxidant, providing almost the same result as Mn(OAc)3 (entry2). Interestingly, V2O5 in aqueous CH3SO3H solution was the most effective combination for forming
C3 symmetric compounds (entry 3). Thus, when 4 was treated with 0.65 equiv of V2O5 at –15 °C
for 129 h, C3 asymmetric compounds 5 and 6 were obtained, each in 28% yield. The dimerization
reactions described above supplied the desired dimeric compounds in multigram amounts by a onestep procedure.

With large amounts of these key dimeric intermediates of proposed biosyntheses in hand, ourfocus moved to concise syntheses of (+)-1, (–)-2 and (+)-3. For the dehydration condensation reaction
toward the synthesis of (+)-1, 4-(4, 6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride(DMT-MM), developed by Kunishima and co-workers [3], was employed because the reagent canbe used in environmentally friendly alcohol as solvent without any additives such as bases. Thus,
treatment of resolved 5 in ethanol with 2.0 equiv of N-Boc-phenylalanine in the presence of 2.0
equiv of DMT-MM (0 °C, 4 h) in an open flask cleanly provided 8 in superb conversion (Scheme 1).
Initial attempts to convert 8 directly to 1, including removing the Boc group and diketopiperazine
formation in a single step provided (+)-1 in 59% yield under an argon atmosphere at 230 °C inneat conditions. Further improvement of conversion was accomplished under vacuum, because the- 16 -

reaction side products such as isobutene, carbon dioxide and ethanol could be removed immediatelyfrom the reaction vessel (0.1 mbar, 30 min, 73%). Furthermore, the dehydration condensation
reaction and Boc deprotection followed by intramolecular amide formation could be conducted in a
one-pot operation (70%).
After completing the synthesis of (+)-1, the same procedure was applied to the synthesis of (+)-3(Scheme 2). Thus, the dehydration condensation reaction with 7 (1 equiv) and N-Boc-L-proline (2equiv) in the presence of DMT-MM (2 equiv, EtOH, 0 °C, 4 h) was followed by diketopiperazine
formation (neat, 230 °C, 30 min, 0.1 mbar) to provide (+)-3 in superb yield (70%).

On the other hand, DMT-MM was not an effective coupling reagent for 6 to synthesize (–)2. Surprisingly, when the same reaction conditions were applied to 6 with N-Boc-methyl-Lphenylalanine, the starting material 6 was completely recovered. In addition, N-Boc-methylL-phenylalanine ethyl ester was obtained as the only isolatable product. After several attempts
to convert 6 to 9, we found that O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium
hexafluorophosphate (HATU) and Et 3N in combination gave the desired coupling reaction inmoderate yield (Scheme 3). Thus, treatment of 6 with N-Boc-methyl-L-phenylalanine (2 equiv)in the presence of HATU (2 equiv, 5 equiv of Et 3N, DMF, 0 °C, 72 h) provided 9 in 45% yield.
Deprotection and diketopiperazine formation of 9 (neat, 230 °C, 30 min, 0.1 mbar) then furnished(–)-2 (quantitative yield).

Discussion & Conclusion
We have developed direct bio-inspired dimerization reactions along with our proposed biosynthetic
pathway from commercially available amine-free tryptophan derivatives in aqueous acidic media
to provide symmetric and asymmetric dimeric compounds. Then, concise two-pot or three-- 17 -

step syntheses of naturally occurring dimeric diketopiperazine alkaloids (+)-WIN 64821 (1),(–)-ditryptophenaline (2) and (+)-naseseazine B (3) were accomplished in total yields of 20%, 13%
and 20%, respectively. The present synthesis has several noteworthy features. 1) Tryptophan-based
symmetric and asymmetric dimeric key intermediates can be prepared on a multigram scale in
one step. 2) The developed oxidation reaction was carried out in acidic aqueous solution without
deactivation of metal oxidants. 3) Protection of the primary amine can be avoided by salt formationin acidic water in the synthesis scheme. 4) In two-pot operations, the reaction media are only
environmentally friendly water and ethanol for the preparation of (+)-1 and (+)-3. 5) Satisfactory
total yields are obtained compared with previous reported synthesis. 6) No special care is needed
to exclude water or air. 7) Effective one-pot diketopiperazine formation was discovered using the
Kunishima coupling protocol followed by Boc deprotection and intramolecular amide formation
under vacuum.

References
1) C. J. Barrow, P. Cai, J. K. Snyder, D. M. Sedlock, H. H. Sun, R. Cooper, J. Org. Chem. 1993, 58,
6016–6021.
2) R. Raju, A. M. Piggott, M. Conte, W. G. L. Aalbersberg, K. Feussner, R. J. Capon, Org. Lett.
2009, 11, 3862–3865.
3) M. Kunishima, A. Kitao, C. Kawachi, Y. Watanabe, S. Iguchi, K. Hioki, S. Tani, Chem. Pharm.
Bull. 2002, 50, 549–550.

一般の皆様へ
天然から得られる含窒素天然有機化合物（アルカロイド）には、多種多様な化学構造および顕著な生物活性を有するものも数多く、新規医薬品開発の観点からも注目されている。本研究ではこれらアルカロイド類の効率的かつ統一的な全合成法を確立し、量的供給並びに誘導体合成研究から新
たな医薬品シーズとして発展させる事を目的とした。WIN 64821、ditryptophenaline、naseseazine B
は細菌類から見出されたアルカロイドであり、その特異な構造と生物活性が注目されている。今回、これらアルカロイドの生合成が酸性水溶液中で進行しているという着想を得、フラスコ内で再現する事に成功した。本工程を鍵段階として、市販のトリプトファン誘導体から全行程 3 段階合成を達成した。

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Regulation of vascular integrity by AM-RAMP system
and its application.
Takayuki Shindo
Department of Cardiovascular Research, Shinshu University Graduate School of Medicine
tshindo@shinshu-u.ac.jp

Abstract
Adrenomedullin (AM) is a peptide involved in both the pathogenesis of cardiovascular diseases
and circulatory homeostasis. We have shown knockout mice of RAMP2 (RAMP2-/-), one of
the accessory proteins of AM-receptor, are lethal at mid-gestation with the similar vascularabnormalities of AM-/-.
We generated vascular endothelial cell-specific RAMP2 knockout mice (E-RAMP2-/-). A small
number of E-RAMP2-/- can survive until adulthood. In adult E-RAMP2-/-, spontaneous occurrenceof vasculitis lesions was detected throughout the body. Furthermore, E-RAMP2-/- showed severeorgan fibrosis with enhanced oxidative stress.
We next generated drug-inducible E-RAMP2-/- (DI-E-RAMP2-/-). After the induction of thegene-deletion in adult, DI-E-RAMP2-/- showed remarkable vascular leakage and malformation ofendothelial cells.
In conclusion, RAMP2 is the key determinant of the vascular functions of AM; RAMP2 isessential for the vaso- and organ-protective effects of AM.
Key Words : Endothelial cell, Adrenomedullin, RAMP2Introduction
Revealing the mechanisms underlying the functional integrity of the vascular system could makeavailable novel therapeutic approaches. Adrenomedullin (AM) is a peptide involved in both thepathogenesis of cardiovascular diseases and circulatory homeostasis. We have shown RAMP2, oneof the accessory proteins of AM-receptor, plays critical roles in AM’s vascular functions.

Results
We previously showed that knocking out the AM or RAMP2 causes vascular abnormalities and isembryonically lethal. In this study, to analyze the pathophysiological roles of vascular AM-RAMP2
system directly, we generated vascular endothelial cell (EC)-specific RAMP2 knockout mice
(E-RAMP2-/-).
Most of E-RAMP2-/- were lethal at later-gestation with systemic edema. The surviving E-RAMP2-/adults exhibited thinning of the aortic wall and enlarged aortic diameters. Electron microscopic
observation revealed that the aortic smooth muscle layer was in disarray in E-RAMP2-/- mice, andthat the endothelial cells were detached from the basement membrane and severely deformed.

- 19 -

Vasculitic lesions developed spontaneously in the surviving E-RAMP2-/- adults, and severe
infiltration and accumulation of inflammatory cells was observed in blood vessels within the majororgans, including the live, kidneys, and lungs.
In 6-month-old E-RAMP2-/- mice, we detected elevated levels of oxidative stress in the majororgan. Levels of 4-HNE immunostaining, which reveals the presence of peroxides of unsaturated
fatty acids, were increased in E-RAMP2-/-. More interestingly, organ damage developed
spontaneously in E-RAMP2-/- adults. In particular, the livers of E-RAMP2-/- mice appeared
cirrhotic. Pathological analysis revealed that the cirrhosis-like changes were not caused by damage
to parenchymal hepatocytes, but by severe fibrosis along the vasculature within the liver, most
likely due to dysfunction of the sinusoidal ECs (Fig).
We next used EAhy926 ECs to generate EC lines stably
overexpressing RAMP2. H 2O2-treatment has been shown to
induce EC senescence. We found that after incubation for 24
h in the presence of H2O2 plus AM, control cells were strongly
stained by SA-β-gal, but cells overexpressing RAMP2 wereresistant to staining.
As only a small number of adult E-RAMP2-/- was obtained,
next we generated d r ug-inducible E-R A MP2-/- (DI-ERAMP2-/-). With this model, we are able to selectively delete RAMP2 gene from the ECs ofadult mice through administration of tamoxifen. Interestingly, the tamoxifen-treated mice showedremarkable body weight gains with systemic edema. The cause of the edema was enhanced vascularpermeability and plasma leakage, as evidenced by the extravascular leakage of Evans Blue dye.
We also confirmed that at the chronic stage after the gene-deletion, DI-E-RAMP2-/- mice
exhibited vascular damage with enhanced perivascular inflammation that was the same as that of
adult E-RAMP2-/-. With DI-E-RAMP2-/-, therefore, we were able to clarify both the acute andchronic effects of RAMP2 deletion in the adult, and demonstrate that RAMP2 is essential for EC
viability, vascular integrity, and homeostasis.

Discussion & Conclusion
In this study, we were able to clarify both the acute and chronic effects of RAMP2 deletion in theadult, and to demonstrate that the AM-RAMP2 system is essential for vascular integrity and organ
homeostasis. Early after RAMP2 gene deletion, ECs show morphological changes. The resultant EC
deformation causes detachment, barrier dysfunction, enhanced vascular permeability and edema,
which in turn promote the attachment and infiltration of inflammatory cells. Chronic vascularinflammation induces vascular damage and accelerated senescence, and enhances oxidative stressand organ fibrosis. Finally, the accumulated disorders cause chronic organ dysfunction with aging.
The results obtained using these models demonstrate that vascular EC integrity is essential for organ
homeostasis.

- 20 -

References
1. Shindo T, et al.
Adrenomedullin-RAMP2 system in cardiovascular development and homeostasis
Curr Hypertens Rev. 2011; 7 (4) 217-27.
2. Koyama T, Shindo T, et al.
Vascular endothelial adrenomedullin-RAMP2 system is essential for vascular integrity and organ
homeostasis
Circulation. 2013 Feb 19;127(7):842-53.
3. Shindo T, et al.
Regulation of adrenomedullin and its family peptide by RAMP system
Curr Prot Pept Sci. 2013 in press

一般の皆様へアドレノメデュリン (AM) は、血管をはじめ、全身の組織で広範に産生され、多彩な生理活性を有する因子あり、臨床応用も期待されている。我々は、AM による血管保護作用、臓器保護作用が、調節タンパク RAMP2 によって制御されていることを見出した。RAMP2 は、新たな治療標的分子として期待される。

- 21 -

Development of novel molecular imaging method for visualization ofclinical condition of intravital lipids
Hideaki KANO
University of Tsukuba
hkano@bk.tsukuba.ac.jp

Abstract
The regulation of lipolysis in adipocytes involves coordinated actions of lipid droplet (LD) andLD-associated proteins such as perilipin. Here, we studied the cellular origin and physiologicalsignificance of micro LDs (mLDs) that emerge in the cytoplasm during active lipolysis indifferentiated 3T3-L1 adipocytes. Multiplex coherent anti-Stokes Raman scattering (CARS)
microscopy demonstrated that mLDs receive the fatty acid (FA) moiety of triglyceride from preexisting LDs during lipolysis through vibrational imaging of CD bonds.
Key Words : Lipolysis, lipid droplet, CARS, Raman

Introduction
Coherent anti-Stokes Raman scattering (CARS) microscopy has been applied widely to cell andtissue imaging ex and in vivo [1]. CARS microscopy, however, provides only a monochromatic
image using a single vibrational frequency such as CH 2 stretch vibrational mode. In order toobtain full spectral information on vibrational modes, we used a novel light source, nanosecond
supercontinuum or white-light laser, which facilitated ultra-broadband (>3000cm-1) multiplex CARS
microspectroscopy. Owing to the narrow spectral bandwidth of the pump/probe laser in the CARS
process, the vibrationally resonant CARS imaging in the fingerprint region is performed with highmolecular specificity [2].

Results
The light source is a Q-switched microchip Nd:YAG laser. Fundamental output of the 1064- nm
radiation is firstly divided into two. One is used as a pump laser for the CARS process, and theother is introduced into a photonic crystal fiber to generate white-light supercontinuum, which isalso used as a Stokes laser for the CARS process. Although the spectral profile of the white-lightsupercontinuum ranges from visible to near infrared (NIR), only NIR spectral components areused for the sample excitation. After removing redundant components from excitation pulses, The
two radiations are introduced into a modified inverted microscope. The sample is placed upon a
PZT stage for raster scanning. Forward propagating CARS signal is collected and introduced into aspectrometer.
Figure shows the results of multi-modal and multiplex CARS and third-order sum-frequency(TSF) imaging of a living adipose cell, which is cultured with deutrated (D-) palmitate. In the basal- 22 -

state, D-palmitate was detected in thepreexisting large LDs. During lipolytic
stimulation, mLDs containing both
CH and CD bonds appeared in thecytoplasm, and increased with time.
This result suggests that mLDs receive
FAs from preexisting LDs, directly
by fragmentation of large LDs and/or Figure. Multiplex CARS and TSF imaging of a living adipose cell before (basal) andindirectly by re-esterification of FAs. after lipolytical stimulation. The images at the top and middle rows correspond to the

CARS images at CH and CD stretch vibrational modes, respectively. The image at the

Similar behavior is also found in the bottom row shows the TSF image. Image acquisition time is about 8 min.
TSF images (bottom). The dynamical

behavior of the lipolytic process is clearly visualized with multi-modal and multiplex CARS and
TSF imaging.
We next examined whether mLDs are formed as a consequence of the fragmentation of large LDs
or formed by FA re-esterification. To address this issue, we inhibited the FA re-esterification and
TAG synthesis by an acyl-CoA synthetase (ACS) inhibitor, triacsin C, and a DAG acyltransferase(DGAT) inhibitor, 2-bromooctanoate, and visualized the appearance of mLDs. mLDs did not appear
even after 32 min or 64 min of lipolytic stimulation, when re-esterification of FAs was inhibited.
This result indicates that mLDs are formed by re-esterification of FAs derived from central LDs or
somewhatand possibly in part from the media[3].

Discussion & Conclusion
The present study provides important findings in understanding molecular mechanisms oflipolysis in adipocytes: during lipolysis, significant amount of FAs generated by TAG hydrolysis
are re-esterified, leading to formation of mLDs. Multiplex CARS microspectroscopy elucidated
the origin of the TAG contents in mLDs due to its high sensitivity to lipids abundant in long-chainhydrocarbons by tuning the laser wavelengths to CH stretch vibrational mode, and has proven to
be an excellent tool for LD tracking. The deuterium in a CD bond is heavier than hydrogen, placing
CD vibrational stretching frequencies into near 2100 cm-1, which is an otherwise silent region of thebiological Raman spectra. By incubating differentiated adipocytes with deuterium-labeled palmitate
and replacing some CH bonds with CD bonds, it is possible to selectively image a specific chemicalbond (i.e. CD bond) using CARS contrast. The multiplex CARS microspectroscopy enabled us toobtain simultaneous imaging of CD and CH stretch vibrational modes, and revealed that newly
appearing mLDs are formed as a consequence of the fragmentation of large LDs and/or the reesterification of intracellular FAs.

- 23 -

References
1. C. L. Evans, X. S. Xie, Annu. Rev. Anal. Chem., 1 (2008) 883-909.
2. M. Okuno, H. Kano, P. Leproux, V. Couderc, J. Day, M. Bonn, and H. Hamaguchi, Angew.
Chem. Int. Ed., 49 (2010) 6773-6777.
3. T. Hashimoto, H. Segawa, M. Okuno, H. Kano, H. Hamaguchi, T. Haraguchi, Y. Hiraoka, S.
Hasui, T. Yamaguchi, F. Hirose, T. Osumi, J. Cell Sci. 125, 6127 (2012).

一般の皆様へ近年、肥満による糖尿病、高血圧、脂質異常症など生活習慣病が増加しています。肥満は脂肪細胞に脂肪が過剰に蓄積した状態であり、それによる細胞機能異常が、脂肪組織そのものに炎症性変化を引き起こすこと、そして炎症の慢性化がメタボリックシンドロームと関連があることが、最近の研究でわかってきました。しかしながら、細胞の機能異常と組織の病態との関連については、未だ不明な点が多いのが現状です。そこで本研究では、生体内脂肪そのものの機能を捉え、脂肪の病態を可視化できる新しい方法を開発することで、脂肪組織の病態を見る、新しい研究アプローチを試みました。代謝産物である脂肪は、これまで生きたまま可視化することは困難でしたが、今回開発したコヒーレント・ラマン (CARS) 分光イメージングを用いることで、非標識にて脂肪の分子構造及びその機能を可視化することができました。本手法により、細胞内の脂肪代謝過程において、細胞質に可溶化した脂肪酸が、再度リサイクルされる経路がある、という知見を得ることが出来ました。

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Molecular mechanisms of cornification of the epidermis.
Masatake Osawa
Graduate School of Medicine, Gifu University
masaosaw@gifu-u.ac.jp

Abstract
Epidermal barrier formation requires a coordinated process where keratinocytes undergo drastic
morphological and biochemical changes to form highly keratinized denucleated cell layers. Despite
critical importance of epidermal barrier in maintaining healthy skin, the underlying molecularmechanisms are poorly understood. Here we found that autophagy is activated in differentiating
epidermal keratinocytes in vivo and this activation is partly mediated by the induced expression of
Nix (Bnip3l), a member of the BH3-only family, which selectively induces mitochondrial autophagy(mitophagy). Induction of terminal differentiation in cultured keratinocytes results in significant
upregulation of Nix expression and subsequent activation of mitophagy. Conversely, knockdownof Nix attenuates keratinocyte differentiation and autophagy induction. Taken together, our datastrongly suggest an important role of Nix in promoting keratinocyte terminal differentiation andsubsequent mitochondria clearance by mitophagy. Our data would provide a new insight into
understating molecular basis of skin barrier formation.
Key Words : Epidermis, Autophagy, Skin barrier formation, Atopic dermatitis.

Introduction
The skin epidermis serves as a critical barrier that protects the body from various environmental
insults and dehydration. Defective skin barrier formation represents a common cause of variety ofskin inflammatory diseases including psoriasis and atopic dermatitis, which are affecting about
10-30 percent of the human population. Therefore, understanding molecular details of skin barrier
formation has huge implications for developing effective therapeutic treatments for people sufferingsuch skin disorders.
Terminal differentiation of keratinocytes within the epidermis involves extensive cellular
remodeling, whereby keratin filaments and lipids are cross-linked to form rigid bundles andcell organelles including nucleus and mitochondria are eliminated to induce apoptosis-like cell
death, which all together result in formation of an impenetrable barrier in the outermost layerof the epidermis. Despite extensive studies of this process, the mechanism by which epidermal
keratinocytes are allowed to achieve the dramatic cellular remodeling is largely unclear. One
candidate mechanism that enables such quick remodeling might be autophagy, a process that
promotes cellular self-cannibalization. Autophagy is an intracellular bulk degradation systemthat delivers cytoplasmic organelles and proteins to the lysosome, where they are degraded to bequickly eliminated damaged and unnecessary cell components1. Accumulating evidence indicates- 25 -

that autophagy has been implicated in wide range of the cellular remodeling that occurs duringorganogenesis2, thereby enabling developing cells to rapidly differentiate or change their fate inorder to acquire new functions. Based on these facts, we hypothesize that the induction of autophagy
may play an important role in promoting epidermal terminal differentiation. Therefore, the focusof this study is to obtain an experimental proof for the involvement of autophagy in the regulationof epidermal terminal differentiation and barrier formation. Our study will uncover a novel role ofautophagy in regulating epidermal barrier formation and provide new insights into development ofnovel therapeutic strategies for skin disorders.

Results
To test whether autophagy is induced during epider mal development, we perfor med
immunohitochemical staining of the mouse embryonic skin using a specific antibody against
an autophagosomal protein, LC3. Whereas LC3 positive cells were hardly detectable in E15.5
epidermis, strong LC3-positive cells were readily detected in the granular layer of E17.5 epidermis
and these positive cells showed the accumulation of LC3 in cytoplasmic puncta, which is a typicalsign of autophagy. To further confirm the induction of the autophagy, westernblot analysis was
carried out using the LC3 antibody that distinguishes the cytosolic (LC3-I) and autophagosome
membrane bound forms of LC3 (LC3-II). Consistent with the immunohistochemical analysis, theaccumulation of LC3-II was seen in freshly isolated E17.5 epidermis. Hence these data clearly
indicate that autophagy is induced in the granular layer cells of developing epidermis.
To further gain insights into understanding molecular mechanisms of the autophagy induction,
we explored the upstream molecule responsible for in the induction of the autophagy in theepidermis. Previously, we performed trasncriptome analysis of developing epidermis to identify
genes whose expression is changed in associated with epidermal differentiation3. Among dozens
of such molecules, Nix (also called as Bnip3l) became focus of our study due to its importantroles in the induction of autophagy and autophagy-mediated mitochondria clearance. In fact,
immunohistochemical staining of embryonic skin revealed that Nix expression was detected in
the granular layers, the region where coincided with autophagy induction. Thus these data suggest
potential correlation between Nix expression and autophagy induction in the epidermis.
Previously, Nix has been implicated in mitochondria clearance in reticulocytes. As eliminationof mitochondria also occurs during epidermal cornification, we hypothesized that Nix-mediatedautophagy might contribute to mitochondria clearance in the epidermis. In support of thispossibility, immunofluorescence confocal microscopic examination revealed colocalization of Nix
protein and mitochondria within LC3-positive autophagosome. While the exact function of Nix is
not clarified, our observation strongly suggests that epidermal autophagy may participate in the
mitochondria clearance in the differentiating epidermal cells.
To address the function of Nix in epidermal keratinocytes, loss-of-function analysis was performed
using cultured keratinocytes. To recapitulate terminal differentiation process of keratinocytes in- 26 -

vitro, we carried out keratinocytes suspension culture. Consistent with the in vivo observation,
the induction of keratinocyte differentiation caused dramatic upregulation of Nix expression andsubsequent activation of autophagosome formation. In contrast, knockdown Nix expression by
specific shRNAs resulted in attenuation of keratinocyte differentiation and impaired autophagosome
formation. Hence, it has been clearly suggested from these data that Nix plays an important role inepidermal differentiation and autophagy.

Discussion & Conclusion
Here, we show that autophagy is activated in differentiating epidermal keratinocytes in vivo.
Although physiological function of autophagy is still unclear, our in vitro analyses suggest apotential link between epidermal development and autophagy formation. In support of this notion,
the importance of autophagy in organogenesis has already been demonstrated in various tissues
development including cardiogenesis, hematopoiesis and osteogenesis, where rapid cell remodelingis required for executing proper development. As epidermal development involves extensive cell
remodeling, it is conceivable that autophagy may also play a role in this process. However, without
in vivo functional analysis, it could be hard to exclude the possibility that, despite the extensive
autophagy activation, autophagy exhibits limited function in epidermal development. In fact, froman evolutionary perspective, autophagy is thought to be evolved as a means for surviving against
various stresses, and it has been also demonstrated that autophagy play a crucial role in maintaining
quality control of cells by eliminating damaged organelles and misfolded proteins. These functionsof autophagy are particularly important for post-mitotic or long-lived cells. As in the epidermis,
the cells undergoing autophagy are mostly confined to the granular layer, a layer consisting of
post-mitotic cells, an alternative scenario that may explain the role of autophagy would be that
autophagy plays a role in conferring quality control and stress resistance to granular layer cells. Thispossibility is also supposed to be likely because cells within the outermost layer of the epidermis arecontinuously exposed to environmental stimuli and stresses such as UV damage, oxidative stress,
toxic chemical exposure and microbe infection. If this is the case, autophagy may be needed for the
epidermal cells to cope effectively with such stresses, and defective autophagy might result in cell
damages and impaired homeostasis, which ultimately causing skin abnormalities such as cancerous
transformation or chromic inflammation.
In either case, further studies are needed to clarify the physiological role of autophagy andmolecular function of Nix in the epidermis. To address these issues, generation of combinational
knockout mice of Nix and its related gene, Bnip3, has been currently underway in our laboratory.

- 27 -

References
1. Mizushima N, Komatsu M. Autophagy: renovation of cells and tissues. Cell. 11;147:728-41, 2011.
2. Di Bartolomeo S, Nazio F, Cecconi F. The role of autophagy during development in higher
eukaryotes. Traffic. 11(10):1280-9, 2010.
3. Moriyama M, Durham AD, Moriyama H, Hasegawa K, Nishikawa SI, Radtke F, Osawa M.
Multiple Roles of Notch Signaling in the Regulation of Epidermal Development. Dev Cell 14:594-406, 2008.

一般の皆様へ
皮膚の健康は皮膚の角質層のバリア機能によって保たれており、その異常はさまざまな皮膚病の原因になります。角質層は上皮細胞がダイナミック変化を起こすことによって形成されますが、その詳細なメカニズムは明らかにされていません。私たちは、オートファイジーという現象に注目し、角質層形成機構を明らかにすることを目標にして研究を進めています。この研究から得られる成果は、皮膚病の新たな治療法開発に役立つと思われます。

- 28 -

Analysis for the molecular mechanisms
causing paroxysmal cough in pertussis
Yasuhiko Horiguchi
Research Institute for Microbial Diseases, Osaka University
horiguti@biken.osaka-u.ac.jp

Abstract
Key Words : Whooping cough, Bordetella, Animal model

Introduction
Pertussis (whooping cough) is a highly contagious, acute respiratory disease caused by thepathogenic bacterium, Bordetella pertussis (1). Clinical manifestations of the disease include
paroxysmal coughing, pneumonia, apnea, and hypoxia. The mechanism by which the bacteria
establish the disease is poorly understood. It is not known either how the organisms cause theparoxysmal coughing. A major obstacle to analyzing the course of whooping cough is a lackof suitable animal models. Notably, hardly any models that reproduce the paroxysmal cough inwhooping cough have been described (2). In this study, we tried to establish an animal model thatcan be readily utilized and reproduce the coughing in human whooping cough, and by using themodel, we analyzed the course of the disease including the coughing.

Results
In this study, we carried out a series of experiments for animal infections with the combinations
between various experimental animals and species and strains of Bordetella, and eventually, could
establish an appropriate model for whooping cough by using B. bronchiseptica and a commonlyused small animal (hereinafter referred to as “Ax”). B. bronchiseptica, which is closely related to B.
pertussis, producing nearly identical virulence factors except pertussis toxin, has Ax as its natural
host. Axs infected with 103 cfu of B. bronchiseptica had a persistent cough which was observed
as early as day 5 postinfection and its frequency reached a peak at day 9. The total number ofcoughs for 5 days from day 6 to 11 of infection ranged from 56 to 242/30 min (5 min x 6 days). The
frequency of coughing of the animal infected with wild-type B. bronchiseptica was 140 coughs/30
min on average. We considered this coughing to be paroxysmal. Histological screenings revealed noapparent pathological changes in the trachea of infected animals. In contrast, Axs given SS mediumor the nonpathogenic phase III variants of B. bronchiseptica hardly exhibited any coughing. Mutant
strains deficient in genes for known major virulence factors caused the paroxysmal coughing
indistinguishable from that caused by wild type strain, implying that unknown factors might beinvolved in the coughing. Similar coughing was also observed in Axs infected with B. pertussis
inoculated at 108 cfu/Ax. During the course of these experiments, we unexpectedly isolated a- 29 -

mutant strain of B. bronchiseptica, tentatively named ΔC, which colonized the trachea but did notcause coughing paroxysms. To identify the mutated gene in ΔC, which might be responsible forthe cough in infected Axs, we sequenced the whole genome of the mutant and compared it withthat of the parental strain. The first screening of mutation sites revealed single-base substitutions
at 41 sites and deletion/insertion mutations at 6 sites. These regions of the ΔC and parental strain
genomes were directly sequenced and eventually, we found a single-base insertion and a singlebase substitution in geneA and geneB (tentative names) of ΔC, respectively. It was predicted that theformer mutation should result in a translational frameshift and the latter should cause an aminoacid substitution. When geneA but not geneB in the wild type strain was disrupted by insertionalmutagenesis, the resultant variant failed to cause coughing paroxysms. On day 15 postinoculation,
the geneA-deficient strain was recovered from the trachea to a similar extent to the wild-type strain,indicating that the strain colonized the trachea but did not cause coughing. Moreover, when geneA
was complemented, ΔC regained the ability to cause the paroxysmal coughing. From these results,
we concluded that geneA, directly or indirectly, to be involved in the coughing in infected Axs.

Discussion & Conclusion
We utilized Ax model focusing on coughing paroxysms in this study, by using B. bronchiseptica
instead of B. pertussis as the pathogen. B. bronchiseptica shares with B. pertussis various virulence
factors that are highly homologous in amino-acid sequence. Several animal species including pigs
and dogs exhibit a persistent cough when infected with B. bronchiseptica. This prompted us to use
B. bronchiseptica for the rat model to explore the pathogenesis of B. pertussis.
In addition, Axs were found to respond to B. bronchiseptica infections with a persistent cough.
A large amount of B. pertussis, although the infection did not persist, caused a similar cough,which assured us that Axs infected with B. bronchiseptica could be a model of human whooping
cough. We also isolated ΔC, a spontaneous mutant strain, which did not cause coughing in Axs. A
comparison of the genomes of ΔC and the parental strain revealed DNA mutations in two distinct
genes, tentatively designated geneA and geneB. Notably, the present results imply that geneA is
the more probable candidate involved in the coughing of infected Axs. The coughing is a common
manifestation of the diseases caused by Bordetellae including B. pertussis, B. parapertussis, and B.
bronchiseptica. In a future study, we hope to clarify the mechanism responsible for the coughingparoxysms associated with B. bronchiseptica infections and eventually those seen in humanwhooping cough by analyzing the functions of geneA-like genes in B. pertussis.

References
1. Mattoo, S. & Cherry, J. D. Molecular pathogenesis, epidemiology, and clinical manifestations ofrespiratory infections due to Bordetella pertussis and other Bordetella subspecies. 18, 326–382(2005).
2. Elahi, S., Holmstrom, J. & Gerdts, V. The benefits of using diverse animal models for studying
pertussis. Trends Microbiol 15, 462–468 (2007).

- 30 -

一般の皆様へ
百日咳は百日咳菌（Bordetella pertussis）の上部気道感染によって起こる伝染性の疾病です。百日咳の特徴的な症状は激しい咳発作ですが、この咳発作の原因も発症機序もこれまで全く不明でした。
本研究課題では、咳発作を再現する動物モデルを作製し、百日咳菌類縁の気管支敗血症菌の感染による咳発作発症機構の解析を行いました。その結果、気管支敗血症菌の特定のある遺伝子が、咳発作発症に関与していることが明らかになりました。この遺伝子は百日咳菌にも存在するため、現在、
この遺伝子の機能を解析して、咳発作の発症機構を理解すべくさらに研究を進めています。

- 31 -

Analysis of mice deficient in molecules involved in
intracellular polarized transport
Akihiro HARADA
Osaka University
aharada@acb.med.osaka-u.ac.jp

Abstract
Polarized intracellular transport is required for the establishment and maintenance of cell polarity.
We generated knockout mice of various genes involved in polarized transport to know how they
are involved in this process. Several knockout mice showed unexpected results which are beingsummarized for publication.
Key Words : Cell polarity, knockout mice, polarized transport

Introduction
Cell polarity is crucial for the development and function of epithelial cells and neurons. To
maintain cell polarity, polarized intracellular transport is essential, but the research of polarizedtransport has been carried out using cell lines, thus, how it relates to the development, function, andpathogenesis of tissues is largely unknown. To elucidate this, we generated and analyzed knockout
mice of various genes involved in polarized transport.

Results
We have already generated knockout mice of Rab8a, Rab8b, syntaxin3, SNAP23, annexin13,
MAL2, FAPP1, FAPP2, PKD1, and PKD2. We are now mainly analyzing Rab8a, Rab8b, syntaxin3,
SNAP23, FAPP2, PKD1, and PKD2 knockout mice regarding their function in polarized transport.
Rab8a,b:
We have already analyzed and published about Rab8a single knockout mice in Nature. They
showed abnormalities in apical transport. However, Rab8a has a closely related molecule, Rab8b.
Thus, we generated Rab8b knockout. As Rab8b knockout mice were almost normal, we generated
Rab8a and Rab8b double knockout mice to know their redundancies. As expected, we obtained data
which implied their redundancies and we are currently submitting the manuscript for publication.
Syntaxin3:
Syntaxin3 has been known to be involved in apical and axonal transport in the epithelial cells andneurons, respectively. When we generated knockout mice which lacked syntaxin3 in the whole body,
we were not able to find adult mice, suggesting syntaxin3 null mice were embryonic lethal. We are
currently analyzing when and how they die during development. On the other hand, we generated
neuron- and small intestine- specific knockout mice and currently analyzing their phenotype.
Some results of knockout mice supported the previous observations, but other results apparently- 32 -

contradicted the previous ones. We are currently analyzing why these contradictions occurred.
SNAP23:
SNAP23 has been known to be necessary for membrane fusion and secretion of enzymes andhormones. When we generated knockout mice of SNAP23, they were embryonic lethal. Thus, we
generated neuron-, endocrine pancreas-, and exocrine pancreas- specific knockout mice. Like insyntaxin3 knockout mice, some results of knockout mice supported, but others did not support theprevious findings. We are currently submitting the manuscript for publication.
FAPP2:
FAPP2 has been known to be necessary for apical transport. It has also known to be necessary fortransport of glycolipids to the trans-Golgi network. Though FAPP2 knockout mice showed no grossphenotype, it showed some defects in lipid transport. Analysis of this knockout mice was done with
Dr. DeMatteis (TIGEM, Italy) and the result will be published shortly.
We also generated FAPP1, a closely related molecule to FAPP2. To know the possible functional
redundancies, we are now crossing FAPP1 and FAPP2 knockout mice.
PKD1, 2:
PKD1, 2 have been known to be necessary for basolateral transport and other biological activities.
When we generated knockout for each molecule, we were not able to see gross phenotype. We are
currently generating double knockout mice, but the double knockout mice seemed to be embryoniclethal. To know their function in vivo, we are now making double floxed mice (PKD1 floxed/floxed; PKD2 floxed/floxed). After establishment of double floxed mice, we will delete PKD1 andPKD2 genes altogether by infection of adenovirus which encodes cre recombinase or by crossing
double floxed mice with transgenic mice which express cre in a tissue-specific manner. Since they
are expressed abundantly in the lung, we will introduce lung explant culture for infection of Cre byadenovirus to see the developmental defects in the lung.
In addition to analyzing knockout mice, we established a gene trap system which could identify
novel genes involved in axonal elongation or distribution of synaptic vesicles. In this system, weused PC12 cell, which is a cell line derived from rat pheochromocytoma. We infected retrovirus
to PC12 and screened infected cells by puromycin which was expressed only when the inserted
retrovirus was within the transcriptionally active regions.
After infection, drug-resistant colonies were picked up and they were further processed formorphological abnormalities. By this strategy, we identified several genes whose loss affected themorphology of PC12 cells or the distribution of synaptic vesicle-like small vesicles. These results
were published in FASEB Journal.

Discussion & Conclusion
We identified essential genes for polarized transport or cell polarity by gene targeting in the mouseand the gene trap in the cell lines. Using gene targeting, we were able to confirm the previous
results in some cases, but in most cases of gene targeting, our results contradicted the previous- 33 -

results. Above all, we were not able to see in many knockout mice. This clearly showed that there isa functional redundancy between genes, but in some cases such as Rab8a, the previous results were
not correct probably by technical limitations. We will pursue which genes are truly involved in cell
polarity by continuing our efforts. We will, at the same time, search for the novel essential genes incell polarity by screening C. elegans. We already identified a number of genes which caused defects
in intestinal cell polarity in their absence. We are currently generating knockout mice for thesegenes to confirm their general importance in cell polarity and we hope the results will be publishedin a few years.

References
1. Hashimoto Y, Muramatsu K, Kunii M, Yoshimura S, Yamada M, Sato T, Ishida Y, Harada R,
Harada A.
Uncovering genes required for neuronal morphology by morphology-based gene trap screening
with a revertible retrovirus vector. FASEB Journal, 26:4662-4674 (2012), doi: 10.1096/fj.12207530.

一般の皆様へ細胞の向き（極性）はその細胞や組織、個体の機能や発生に重要であるが、細胞の極性がどのようにして生じて維持されるかは良く分かっていない。本研究の目的は細胞の極性に関わる遺伝子を欠損するマウスを作製解析し、細胞の極性の出来るメカニズムを解明するものである。解析により、ある遺伝子については以前通りの役割が確認されたが、大多数については以前報告された機能が見られなかったため、その原因を解明中である。更に他にも重要な遺伝子が予想されるため、線虫を用いて新たな遺伝子をスクリーニングし、それらの遺伝子の欠損マウスも作製解析中である。

- 34 -

Investigation of molecular signal output
from a dividing nucleus to cell cortex
NAKANO KENTARO
Graduate School of Life and Environmental Sciences, University of Tsukuba
knakano@biol.tsukuba.ac.jp

Abstract
The existence of all life is based on cell proliferation, which usually accompanies the processof cell division. After replicating its genome and enlarging its body, the mother cell divides intodaughter cells without fail. Here we have studied the molecular systems supporting this fundamental
process for life using fission yeasts and ciliate Tetrahymena. We found that two molecular systems
were involved to ensure cytokinesis in fission yeast Schizosaccharomyces pombe. On the other
hand, it was demonstrated that Tetahymena divides into two cells by unexpected and unidentified
molecular mechanism. In future comparative study on cytokinesis using evolutionary divergent
organisms will be indispensable to understand the fundamental cellular phenomenon for life.
Key Words : actin, myosin, cytokinesis, fission yeast, Tetrahymena

Introduction
In the final stage of the cell cycle, cleaving a mother cell into two daughters ensures completionof both genome segregation and organelle distribution for life. This step called as cytokinesis inthat requires the cell to solve a spatial problem (to divide in the correct place, orthogonally to theplane of chromosome segregation) and a temporal problem (to coordinate cytokinesis with mitosis).
Defects in the spatiotemporal control of cytokinesis may cause cell death, or increase the risk oftumor formation. The principal focus of this study will be the molecular mechanisms of cytokinesis
in the mitotic cycle using unicellular model organisms.

Results
To investigate the molecular mechanism of cytokinesis, we mainly used the fission yeast
Schizosaccharomyces pombe. Because, this simple model has
contributed significantly to our understanding of how the cell cycle
is regulated, and serves as an excellent model for studying aspects ofcytokinesis. The position of the division site is set by the location of
the interphase nucleus and depends on a PH-domain protein Mid1p andproteins that confer cell polarity. A contractile ring (CR) is assembled
at the center of the cell during mitosis at the end of anaphase, theconstriction of this ring is thought to guide synthesis of the septum thatbisects the cell (see the right cartoon). In this great opportunity given- 35 -

by the Novartis found (Japan), I have firstly tried to reveal the molecular mechanism how actin andmyosin II assemble into a ring under the control of Mid1p. Previous reports from our lab and others
have mentioned that Rng2p is possible to link myosin II accumulation to the cortical Mid1p in theprospective division site. A member of my lab and I have investigated a physiological interaction
among those proteins using a pull-down assay of cell extracts and Yeast Two-hybrid system.
However, at this moment, their interaction has not been fully uncovered yet for reporting here. On
the other hand, even in cells lacking Mid1p-activity, myosin II can associate with a cable-like actin
filaments (F-actin) elongating from the cell cortex during anaphase, resulting in CR formation
at the significant rate. In the course of study, we have succeeded to characterize the N-terminal
actin-binding domain of Rng2p that can induce myosin II binding to F-actin in vivo and in vitro(manuscript in preparation). Thus, in S. pombe cell division is accomplished with Mid1p-dependentand –independent systems. This duplicate system for ensuring cell division may be conserved inanother organism. To investigate this possibility, we have isolated genes encoding a counterpart ofthose proteins form another species of fission yeast S. japonicas and are now trying to characterize
their cellular function.
In addition, we studied the molecular mechanism of cytokinesis in the ciliate Tetrahymena.
Eukaryote can be broadly classified into opistkont and bikont. Animal cells and fungi including
yeasts are grouped in the former, while plant cells, algae and ciliate belong to the latter.
Interestingly, no gene corresponding to myosin II is conserved in bikont. However, it had well been
known that many ciliate divide like animal cells. To challenge to solve this mystery-shrouded issue,
we have studied function of actin and its binding proteins, and myosins unrelated to myosin II in
Tetrahymena. Actin-depolymerizing factor (ADF) is one of the most important proteins controlling
actin dymamics in eukaryotic cells. Suppressing cellular function of ADF causes severe defect incytokinesis in opistkont. We have evaluated ADF-activity in cytokinesis of Tetrahymena cell by
deleting the corresponding gene. As a result, the cells continue to divide by binary fission even inlacking ADF-activity (Shiozaki et al., in press). Moreover, administration of actin-inhibitory drug
did not affect on cytokinesis of this organism (manuscript under revision). After determination ofcellular localization using green fluorescence protein, all of 13 gene products of myosin were notfound in a cleavage furrow in a dividing cell of Tetrahymena (manuscript in preparation). Taken
together, we concluded that this organism probably accomplishes binary fission independently of theactomyosin-based system.

Discussion & Conclusion
In this study we arrive at the conclusion that S. pombe adopts two independent mechanisms, butclosely related each other, in CR formation for cytokinesis. Rng2p is involved in both molecularpathways via its different structural domains. On the other hand, Tetrahymena cell seems to divide
without the actomyosin-based system. At this moment we do not have a clue as to this unexpectedphenomenon. Very recently, a foreign group has reported that the kinase complex called Sid2p-- 36 -

Mob1p, a member of the NDR kinase family which is implicated in growth control and cytokinesis
in multicellular eukaryotes as part of the Salvador-Warts-Hippo tumor suppressor network, may be
involved in positional control of division site in Tetrahymena. This kinase family plays importantrole for CR maturation following the Mid1p-dependent actin and myosin II recruitment in S. pombe.
Therefore, identification of downstream substrate of Sid2p-Mob1p may shed light on some of themysteries about the conserved molecular mechanism of spatio-temporal control for cytokinesis in
eukaryote.

References
1. Shiozaki, N., K. Nakano, Y. Kushida, T. QP Noguchi, T. QP Uyeda, D. Wloga, D. Dave, K.
Vasudevan, J. Gaertig, & O. Numata. ADF/cofilin is not essential but critically important for
actin activities during phagocytosis in Tetrahymena thermophile. Eukaryotic cell, in press.

一般の皆様へ
核ゲノムを複製して細胞が増殖する際に、細胞は適切な位置で分裂する。分裂中の細胞のくびれにみられる「収縮環」は、骨格筋と同様にアクチン繊維とミオシンを主成分とした分子装置であり、細胞体を分裂させる力を発生させると考えられている。分裂細胞では、分配された核ゲノムを確実に娘細胞に伝承するために、適切な位置に一過的に収縮環が形成される。細胞表層から離れた空間に存在する分裂核が、どのようにして細胞表層での収縮環形成の位置を規定しうるのか、それらを結びつける分子シグナルの実体は不明な点が多い。そこで本研究では分裂酵母などの単細胞真核生物の実験系の利点を活用し、核と細胞表層をつなぐ分子シグナルの実体の解明を目指した。この研究
成果は、生命活動の根本を支える細胞増殖のしくみや、生物史を通して母細胞から娘細胞へとゲノムが伝承されてきた過程を深く理解するのに貢献できる。

- 37 -

The role of transcription factor IкB-ζ in immune homeostasis
Takashi Maruyama
Graduate School of Life Sciences, Tohoku University
tmaru@life.biology.tohoku.ac.jp

AbstractIкB-ζ plays an important role in immune homeostasis. This study showed that IкB-ζ is importantin the immune suppressive function of Tregs. Furthermore, IкB-ζ is not required for the stability offorkhead box p3 (Foxp3) gene expression.
Key Words : IкB-ζ, Foxp3, Treg

Introduction
Nuclear transcriptional regulator IкB-ζ–deficient mice develop Sjögren’s syndrome (SS) with age.
Several studies have shown that SS is a T-cell dependent autoimmune disease. An Id3-deficient SS
mouse model exhibits defects in the generations and suppression of the Foxp3+ Tregs. This study
focused on the role of IкB-ζ in immune homeostasis through the generation, function, and stabilityof Tregs.

Results
A previous study (Okuma et al., Immunity. 3: 450-460, 2013) showed that IкB-ζ–deficient micedeveloped SS and exhibited splenomegaly and lymphadenopathy. In addition, these mice showedabsolute numbers of T cells, whereas the number of CD44high memory/effector-type T cells in the
peripheral lymph nodes significantly increased. These phenotypes can be found in each generationof the IкB-ζ–deficient mice. These observations indicate that IкB-ζ-deficient mice were unable tomaintain immune homeostasis.
The CD4+ Tregs express Foxp3 and play an important role in maintaining immune homeostasis.
Scurfy mice deficient in functional Foxp3 undergo massive inflammation of the whole body and die
within 4 weeks.
Unexpectedly, this study showed that the numbers of Foxp3+ Tregs and CD4+ T cells in the
peripheral lymph nodes were similar to those in the wild type (WT) and IкB-ζ–deficient mice. To
check the transcriptional activity of IкB-ζ in relation to Foxp3 gene expression, a Foxp3 reporter
assay was performed. The results showed that the overexpression of IкB-ζ did not alter the Foxp3
reporter activity in human embryonic kidney (HEK)293 cells.
To analyze the suppression ability of Tregs, naïve CD4+ T cells were co-cultured with Tregs
for 3 days, which showed that IкB-ζ–deficient Tregs suppressed the expansion of naïve CD4+ T
cells, although slightly lower than that in WT Tregs. Then, the immune-suppression associatedwith co-stimulatory molecules (cytotoxic T-lymphocyte antigen 4 [CTLA-4] and glucocorticoid-- 38 -

Induced tumor necrosis factor receptor [GITR]) on Tregs were examined to address whether the
low suppressive ability of IкB-ζ–deficient Tregs could be attributed to the expression levels of costimulatory molecules. This experiment showed that the expression levels were similar in the WT
and IкB-ζ–deficient Tregs. In addition, production of the immune regulatory cytokine, Interleukin(IL)-10, was similar between WT and IкB-ζ-deficient Tregs.
Next, the stability of the Foxp3 gene expression in Tregs was examined. This is considered as
one of the mechanisms behind the alteration in immune homeostasis in IкB-ζ–deficient mice.
Carboxyfluorescein succinimidyl ester (CFSE)-labeled CD4+CD25+ Tregs cultured for 3 days under
TCR and a low dose of IL-2 stimulation showed that the Foxp3 expression level was similar in both
WT and IкB-ζ–deficient Tregs at each cell division. To examine the Foxp3 gene expression stability
in vivo, adoptive WT or IкB-ζ-deficient Tregs were transplanted into Rag2-/- mice. Four weeks later,
the Foxp3 expression levels in CD4+ T cells was similar in both the WT and IкB-ζ-deficient Tregs.
This study also found that natural Tregs express IкB-ζ, but the expression level was significantly
lower than that in the Th17 cells. The SOCS1 expression level, which is required for the Foxp3 gene
expression stability, was similar in both the WT and IкB-ζ-deficient Tregs. These results indicatedthat IкB-ζ is not required for the Foxp3 gene expression stability.

Discussion & Conclusions
Tregs strongly expressed CD25 (IL-2 receptor α); therefore, these cells can predominantly
proliferate in inf lammatory environments (IL-2–rich regions) such as the lymph nodes. The
percentage of Tregs was similar in the WT and IкB-ζ–deficient mice; however, the role of IкB-ζ inthe generation of Tregs from naïve CD4+ T cells remains unresolved.
IкB-ζ-deficient Tregs have less suppressive ability; thus, it is necessary to check the suppressive
ability in vivo by using an animal model, such as that showing inflammatory bowel disease.
However, the molecular mechanisms responsible for this low suppressive ability in IкB-ζ–deficient
Tregs remain unclear.
The stability of Foxp3 gene expression in IкB-ζ–deficient Tregs is similar between WT and IкB-ζ–
deficient mice.
Another interesting results is regarding IкB-ζ and Th17. It was previously shown that IкB-ζ
interacts with RORγt to promote Th17 differentiation. Accordingly, in IкB-ζ-deficient mice, naïve
CD4+ T cells failed to generate Th17 cells. Thus, IкB-ζ accelerates the differentiation of both Tregs
and Th17 cells.

一般の皆様へ
本研究では、シェーグレン症候群様の自己免疫疾患を引き起こす遺伝子欠損マウス (IкB-ζ 欠損マウス ) において、その原因の 1 つとして免疫抑制能力の低下が示唆された。今後、その詳しい分子メ
カニズムについて明らかとする必要がある。

- 39 -

Study of dense-core vesicle synthesison the membrane of Golgi complex
Tetsushi Sadakata
Advanced Scientific Research Leaders Development Unit, Gunma University
sadakata-1024@umin.ac.jp

Abstract
We found that somal Ca2+-dependent activator protein for secretion 2 (CAPS2) is associated with
the Golgi membrane, and mediates binding and recruitment of the GDP-bound form of ARF4 andARF5 to the Golgi membrane. CAPS2 knockdown and over-expression of CAPS2-binding-deficient
ARF4/5 both induced accumulation of the dense-core vesicle (DCV) resident protein chromogranin
A around the Golgi apparatus. CAPS2 knockout mice have dilated trans-Golgi structures whenviewed by electron microscopy. These results for CAPS2 strongly support our idea that the CAPS
family proteins exert dual roles in DCV trafficking, mediating trafficking at both the secretion site
for exocytosis and at the Golgi complex for biogenesis.
Key Words : CAPS, chromogranin, Golgi complex

Introduction
The CAPS family consists of two members (CAPS1 and CAPS2) and regulates exocytosis ofcatecholamine- or neuropeptide-containing DCVs at secretion sites such as nerve terminals. A
large fraction of CAPS1 protein, however, is localized in the cell soma, and we have recently
shown the possible involvement of somal CAPS1 in DCV trafficking in the trans-Golgi network(TGN). CAPS1 and CAPS2 are differentially expressed in various regions of the mouse brain but
exhibit similar expression patterns in other tissues such as the spleen. Thus, in the present study weanalyzed whether CAPS2 displays similar subcellular localization and functional roles in the cellsoma as CAPS1.

Results
An exogenously expressed CAPS2 PH domain fused with the f luorescent protein YPet andsyntaxin 6 (Stx6), a TGN membrane trafficking protein were colocalized in αT3-1 pituitary
gonadotrope cells. We found that the CAPS2 constructs containing both C2 and PH domain (fulllength and ΔC-Terminal) exhibited a diffuse distribution pattern throughout the cytoplasm, but ΔNTerminal and ΔC2, which contain a PH domain but lacked the C2 domain, accumulated around theGolgi in a pattern similar to that of the PH domain alone.
To verify whether, like CAPS1, endogenous CAPS2 associates with the Golgi membrane, we
analyzed CAPS2-immunoaffinity-purified microsomal fractions from P21 mouse cerebellum bywestern blotting with antibodies for various Golgi membrane-associated proteins. The western
blot results indicated that CAPS2-immunoaffinity purification enriched the Golgi marker proteins- 40 -

p115, GM130, GS28, vti1a, vti1b, Stx6, and VAMP4 as well as vesicle-trafficking-related proteins
SNAP25 and ARF.
We next investigated the ability of CAPS2 to bind to the small GTPase ARF family (ARF1–6)
using a co-immunoprecipitation assay. We found that only the class II ARFs (ARF4 and ARF5)
were co-immunoprecipitated with anti-CAPS2 antibody. Interestingly, the binding of CAPS2 to
ARF4 and ARF5 was preferentially observed with the GDP-locked form (T31N) but not with the
GTP-locked form (Q71L).
Finally, we analyzed the in vivo effect of CAPS2 knockout on the Golgi appearance by utilizing
CAPS2 knockout mice. Electron microscopic analyses showed that in the CA1 pyramidal cell layerof the hippocampus, approximately half of the identifiable cisternae of the Golgi stack, which werepredominantly on the trans side, were dilated in CAPS2 knockout, compared with wild-type mice.

Discussion & Conclusion
Our findings indicate that CAPS2 binds to the Golgi membrane and interacts with the class II ARF
small GTPases ARF4/5 in a GDP/GTP-state dependent manner, demonstrating the involvementof CAPS2 in the recruitment of ARF4/5 onto the Golgi membrane. Our data demonstrate that therecruitment of ARF4/5 via CAPS2 is indispensable to DCV trafficking. Firstly, CAPS2 knockdown
resulted in the accumulation of ChgA around the Golgi apparatus. Secondly, overexpression of
CAPS2-binding-deficient ARF5 mutants induced accumulation of ChgA in the Golgi. Thirdly,
CAPS2 knockdown caused abnormal ARF5 distribution around cell soma, and finally CAPS2
knockout mice had dilated structures in the Golgi cisternae. Collectively, these findings suggest that
CAPS2 is required for DCV trafficking in the Golgi complex by recruiting the class II ARF small
GTPases, in a GDP-bound dependent manner, to the Golgi membrane.

References
1. Reduced axonal localization of a Caps2 splice variant impairs axonal release of BDNF and causes
autistic-like behavior in mice.
Tetsushi Sadakata, Yo Shinoda, Megumi Oka, Yukiko Sekine, Yumi Sato, Chihiro Saruta, Hideki
Miwa, Mika Tanaka, Shigeyoshi Itohara, Teiichi Furuichi
Proceedings of the National Academy of Sciences of the United States of America. 109 (51),p21104-21109 (2013)
2. Autistic-like behavioral phenotypes in a mouse model with copy number variation of the CAPS2/CADPS2 geneTetsushi Sadakata, Yo Shinoda, Megumi Oka, Yukiko Sekine, Teiichi Furuichi.
FEBS Letter. 587(1), p54-59 (2013)
3. Calcium-dependent activator protein for secretion 2 (CAPS2) interacts with the class II ARF
small GTPases and regulates dense-core vesicle trafficking.
Tetsushi Sadakata, Yukiko Sekine, Megumi Oka, Makoto Itakura, Masami Takahashi, Teiichi
Furuichi.
FEBS Journal. 279(3), p384-394 (2012)
4. Interaction of CAPS1 with the class II ARF small GTPases is required for dense-core vesicle
trafficking in the trans-Golgi network.
Tetsushi Sadakata, Yo Shinoda, Yukiko Sekine, Chihiro Saruta, Makoto Itakura, Masami
Takahashi, Teiichi Furuichi.
Journal of Biological Chemistry. 285(49), p38710-38719 (2010).

- 41 -

一般の皆様へ
有芯小胞はインスリンやドーパミンなどを内包し、生命活動や疾患に関わる重要な構造物である。
しかし、有芯小胞による細胞からの分泌メカニズムについては詳しく解明されていない。今回我々は CAPS2 タンパク質が有芯小胞の分泌にどのように関与しているかを細胞生物学的に明らかにした。
今後様々な応用発展の可能性を秘めた結果であると考えている。

- 42 -Stress response, homeostasis and cancer
Toshiharu Sakurai
Kinki University, Faculty of Medicine, Department of Gastroenterology and Hepatology
sakurai@med.kindai.ac.jp

Abstract
Most hepatocellular carcinomas (HCCs) develop in the context of severe liver fibrosis caused bychronic liver inflammation, which also results in accumulation of reactive oxygen species (ROS).
We examined whether the stress activated protein kinase p38α and cold-inducible RNA-binding
protein (CIRP) control ROS metabolism and development of fibrosis and cancer in mice given
thioacetamide (TAA). Liver-specific p38α ablation enhanced ROS accumulation, which was exerted
through the reduced expression of anti-oxidant protein heat shock protein (HSP) 27. CIRP-deficiency
decreased ROS accumulation and attenuated TAA-induced hepatocarcinogenesis. Intriguingly, therisk of human HCC recurrence is positively correlated with ROS accumulation in liver.
Key Words : CIRP, p38, liver fibrosis, ROS, SOX2, Gankyrin

Introduction
HCC is the most common form of liver cancer and the third leading cause of cancer deaths
worldwide and is usually associated with a very poor prognosis (1). The risk of hepatocarcinogenesis
depends on background liver factors, of which chronic inflammation is a major one. Stress activatedprotein kinases (MAPK/SAPK) play a pivotal role in modulating numerous cellular responses,
including cell proliferation, and metabolism (2,3). One of the SAPKs, p38α, the major p38 isoform,
is activated in response to inflammation and oxidative stress (4-7). CIRP is induced by cellularstresses such as UV irradiation and hypoxia (8-10). In response to the stress, CIRP migrates fromthe nucleus to cytoplasm, and affects expression of its target mRNAs posttrascriptionally (11-13).

Results
TAA model may be suitable for studying the relationship between inflammation and cancer.
TAA can induce liver cirrhosis and cancer of the bile ducts when given to rats over a period ofseveral months (14). However, as described here we found that mice given TAA for 10 months
develop HCC rather than cholangiocellular carcinoma subsequent to appearance of severe liver
fibrosis, thus providing a model that closely mimics the natural history of human HCV-related liverdisease. In addition, the histology of the TAA-exposed rat liver was reported to resemble humanliver cirrhosis (15). Thus, the mouse TAA model may be suitable for studying the relationshipbetween ROS accumulation, liver fibrogenesis and hepatocarcinogenesis.
Enhanced ROS accumulation and fibrogenesis in p38αΔhep mice.
We used a conditional p38α ”floxed” strain to generate p38α Δhep mice, lacking p38α in liver- 43 -

parenchymal cells. After 8 weeks of TAA treatment, we observed inflammation, HSC activation andformation of fibrotic septa as assessed histologically or by immunohistochemistry with a specificantibody against α-SMA. p38α Δhep mice exhibited more TAA-induced liver damage assessed by ALT
release, and hepatocyte apoptosis measured by a TUNEL assay, relative to controls. In addition,
there were higher numbers of α-SMA-positive cells, higher levels of hydroxyproline and larger
fibrotic areas in p38α Δhep mice compared with control mice. Loss of p38α significantly enhanced
expression of the mRNAs for col1α1, TIMP1, TGF-β1 and PDGFb. p38α Δhep mice were found tohave higher levels of oxidized protein in comparison to controls. To evaluate the contribution ofoxidative stress to TAA-induced liver damage and fibrosis, we placed a group of mice on chowdiet supplemented with the antioxidant butylated hydroxyanisole (BHA). p38α Δhep mice kept on
this diet showed a significant reduction in TAA-induced liver injury and fibrosis. Thus, loss of
p38α enhances TAA-induced cell death and fibrogenesis through mechanisms that may depend on
ROS accumulation. Adenoviral transduction of HSP27 in p38α Δhep liver prevented TAA-induced
ROS accumulation, liver damage and fibrogenesis. Loss of p38α significantly enhanced expressionof stemness factor SOX2 mRNA and protein. p38α Δhep mice exhibited a significant increase inGankyrin expression.
Enhanced hepatocarcinogenesis in p38α Δhep mice and attenuated hepatocarcinogenesis in
CIRP-deficient mice.
Tumor size and area were considerably larger in p38α Δhep mice relative to similarly treated controls.
In contrast, TAA-treated CIRP-deficient mice harbored less tumor load than control mice.
Association between risk of HCC recurrence and ROS accumulation in human liver.
Intrahepatic HCC development after hepatectomy is caused by de novo HCC development and/or
metastasis from the resected HCC. The risk of the former depends on background liver factors suchas liver fibrosis, while the risk of the latter mainly depends on characteristics of resected HCC (16).
In mouse models, HSP25-mediated inhibition of ROS accumulation is involved in liver fibrogenesis
and can subsequently affect de novo HCC development. We examined whether this hypothesis isapplicable to humans, focusing on non-cancerous liver tissues rather than cancers to assess thepotential for de novo HCC development or rapid progression of lesions that were undetectable orpre-neoplastic at the time of resection. In patients exhibiting HCC recurrence after hepatectomy,
ROS levels in the non-tumor tissues, but not in tumors, were significantly higher than in those
without HCC recurrence. In addition, patients with low ROS accumulation in non-cancerous liver
had a prolonged recurrence-free survival. (17-19)

Discussion & Conclusionp38α plays a critical role in liver fibrogenesis and hepatocarcinogenesis through the controlof HSP27 expression and ROS accumulation in the mouse TAA model which may be suitable
for studying the pathogenesis of inflammation-related HCC development. CIRP also controls
ROS accumulation and hepatocarcinogenesis in mice given TAA. Correspondingly, elevated
ROS accumulation in the liver are associated with increased risk of human HCC development or- 44 -

recurrence. Pluripotency-associated transcription factors such as SOX2 and Nanog are known as
regulators of cellular identity in embryonic stem cells (20). Gankyrin, a liver oncoprotein, wasreported to mediate dedifferentiation and facilitate the tumorigenecity of rat hepatocytes (21).
Deletion of p38α upregulates expression of SOX2 and Gankyrin, which may be involved in cancer
stem cell maintenance.

References
1. Thorgeirsson SS, Grisham JW. Molecular pathogenesis of human hepatocellular carcinoma. Nat
Genet 2002;31:339-46.
2. Chang L, Karin M. Mammalian MAP kinase signalling cascades. Nature 2001;410:37-40.
3. Dhillon AS, Hagan S, Rath O, Kolch W. MAP kinase signalling pathways in cancer. Oncogene
2007;26:3279-90.
4. Cuenda A, Rousseau S. p38 MAP-kinases pathway regulation, function and role in humandiseases. Biochim Biophys Acta 2007;1773:1358-75.
5. Park JM, Greten FR, Wong A, et al. Signaling pathways and genes that inhibit pathogen-induced
macrophage apoptosis--CREB and NF-kappaB as key regulators. Immunity 2005;23:319-29.
6. Sakurai T, He G, Matsuzawa A, et al. Hepatocyte necrosis induced by oxidative stress and IL-1αrelease mediate carcinogen-induced compensatory proliferation and liver tumorigenesis. Cancer
Cell 2008;14:156-65.
7. Wagner EF, Nebreda AR. Signal integration by JNK and p38 MAPK pathways in cancer
development. Nat Rev Cancer 2009;9: 537-49.
8. Fujita J. Cold shock response in mammalian cells. J Mol Microbiol Biotechnol. 1999
Nov;1(2):243-55.
9. Wellmann S, Bührer C, Moderegger E, et al. Oxygen-regulated expression of the RNA-binding
proteins RBM3 and CIRP by a HIF-1-independent mechanism. J Cell Sci. 2004 Apr 1;117(Pt9):1785-94. Epub 2004 Mar 16.
10. Yang C, Carrier F. The UV-inducible RNA-binding protein A18 (A18 hnRNP) plays a protectiverole in the genotoxic stress response. J Biol Chem. 2001 Dec 14;276(50):47277-84.
11. De Leeuw F, Zhang T, Wauquier C, et al. The cold-inducible RNA-binding protein migrates fromthe nucleus to cytoplasmic stress granules by a methylation-dependent mechanism and acts as atranslational repressor. Exp Cell Res. 2007 Dec 10;313(20):4130-44.
12. Morf J, Rey G, Schneider K, et al. Cold-inducible RNA-binding protein modulates circadiangene expression posttranscriptionally. Science. 2012 Oct 19;338(6105):379-83.
13. Yang R, Weber DJ, Carrier F. Post-transcriptional regulation of thioredoxin by the stressinducible heterogenous ribonucleoprotein A18. Nucleic Acids Res. 2006 Mar 2;34(4):1224-36.
14. Gupta DN. Production of cancer of the bile ducts with thioacetamide. Nature 1955;175:257.
15. Zimmermann T, Müller A, Machnik G, et al. Biochemical and morphological studies on
production and regression of experimental liver cirrhosis induced by thioacetamide in Uje: WIST
rats. Z Versuchstierkd 1987;30:165-80.
16. Imamura H, Matsuyama Y, Tanaka E, et al. Risk factors contributing to early and late phaseintrahepatic recurrence of hepatocellular carcinoma after hepatectomy. J Hepatol 2003;38:200-7.
17. Mine H, Sakurai T, Kashida H, Matsui S, Nishida N, Nagai T, Hagiwara S, Watanabe T, Kudo
M. (2013) Association of Gankyrin and stemness factor expression in human colorectal cancer.
Dig Dis Sci.
18. Sakurai T, Kudo M, Umemura A, He G, Elsharkawy AM, Seki E, Karin M. (2013) p38α Inhibits
Liver Fibrogenesis and Consequent Hepatocarcinogenesis by Curtailing Accumulation of
Reactive Oxygen Species. Cancer Res. 73(1):215-24.

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19. Nagata Y, Kudo M, Nagai T, Watanabe T, Kawasaki M, Asakuma Y, Hagiwara S, Nishida N,
Matsui S, Kashida H, Sakurai T. (2013) Heat shock protein 27 expression is inversely correlatedwith atrophic gastritis and intraepithelial neoplasia. Dig Dis Sci. 58:381-8
20. Lengerke C, Fehm T, Kurth R, et al. Expression of the embryonic stem cell marker SOX2 inearly-stage breast carcinoma. BMC Cancer 2011;11:42.
21. Sun W, Ding J, Wu K, Ning BF, Wen W, Sun HY, et al. Gankyrin-mediated dedifferentiation
facilitates the tumorigenicity of rat hepatocytes and hepatoma cells. Hepatology 2011;54:125972.

一般の皆様へ
ストレス応答蛋白は外敵から細胞を守るのに重要な役割を果たしています。p38 という蛋白はストレス応答蛋白 HSP27 の量を増加させ活性酸素種を抑制することで、肝臓における慢性炎症と癌の発生を抑制します。また別のストレス応答蛋白 CIRP は活性酸素種の蓄積を増幅させることで癌の発生を促進します。ヒトにおいて肝臓がんの再発率と活性酸素種が相関します。将来、HSP27 や CIRPの蛋白量を増やしたり、減らしたりする薬剤を開発することで肝癌を予防することが可能になるかもしれません。

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Regulation of cerebral vasospasm by growth factors
and matricellular proteins
Hidenori Suzuki
Mie University Graduate School of Medicine
sakurai@med.kindai.ac.jp

Abstract
The purpose of this study was to assess whether imatinib mesylate (imatinib), an inhibitor of thetyrosine kinases of platelet-derived growth factor receptors (PDGFRs), prevents cerebral vasospasm
after subarachnoid hemorrhage (SAH) in rats, and to elucidate if tenascin-C (TNC), a matricellular
protein, is involved in the mechanism. Imatinib significantly prevented post-SAH neurologicalimpairments and vasospasm. SAH caused PDGFR-β upregulation, PDGFR activation, mitogenactivated protein kinases activation, and TNC upregulation in the spastic cerebral arteries, all ofwhich were significantly suppressed by imatinib treatment. Recombinant TNC reversed the antivasospastic effects and protein expression changes by imatinib.
Key Words : vasospasm, subarachnoid hemorrhage, platelet-derived growth factor, tenascin-C

Introduction
Cerebral vasospasm remains a major cause of poor outcome after subarachnoid hemorrhage(SAH). We reported that tenascin-C (TNC) was induced after SAH in a clinical setting, associatedwith cerebral vasospasm. TNC is a matricellular protein, and exerts diverse functions through directbinding to cell surface receptors, other matrix proteins, and soluble extracellular factors such as
growth factors and cytokines. As platelet-derived growth factor (PDGF), which is a potent inducerof TNC, has been involved in the pathogenesis of vasospasm, we hypothesized that the inhibitionof tyrosine kinases of PDGF receptors (PDGFRs) prevents experimental vasospasm after SAH via
inhibiting TNC induction.

Results
First, imatinib mesylate (imatinib; 10 or 50mg/kg body weight), an inhibitor of the tyrosine
kinases of platelet-derived growth factor receptors (PDGFRs), was administered intraperitoneallyto rats undergoing subarachnoid hemorrhage (SAH) by endovascular perforation, and the effects
were evaluated by neurobehavioral tests and India-ink angiography at 24-72 hours post-SAH.
Western blotting and immunohistochemistry were performed to explore the underlying mechanisms
on cerebral arteries at 24 hours post-SAH. Recombinant tenascin-C (TNC) was administeredintracisternally to imatinib-treated SAH rats, and the effects were evaluated by neurobehavioral
tests, India-ink angiography and immunohistochemistry at 24 hours post-SAH. Both dosages ofimatinib significantly prevented post-SAH neurological impairments and vasospasm at 24-72- 47 -

hours. SAH caused PDGFR-β upregulation, PDGFR activation, mitogen-activated protein kinases
activation, and TNC upregulation in the spastic cerebral arteries, all of which were significantly
suppressed by imatinib treatment. Osteopontin (OPN), another matricellular protein, was also
upregulated mildly after SAH, but its levels were not significantly affected by imatinib. In SAH rats,
TNC immunoreactivity was markedly induced in the smooth muscle cell layers of spastic cerebral
arteries at 24 hours post-SAH, but not in control animals. The TNC immunoreactivity decreased at
72 hours, as vasospasm improved: OPN immunoreactivity, on the other hand, was more induced in
the arterial wall at 72 hours after SAH. Recombinant TNC reversed the anti-vasospastic effects andprotein expression changes by imatinib.
Second, two dosages (1 and 10 μg) of TNC were administered intracisternally to healthy rats,
and the effects were evaluated by neurobehavioral tests and India-ink angiography at 24, 48, and72 hours after the administration. Western blotting and immunohistochemistry were performed to
explore the underlying mechanisms on constricted cerebral arteries after 24 hours. The effects of
toll-like receptor 4 (TLR4) antagonists (LPS-RS), c-Jun N-terminal kinase (JNK), p38 inhibitors(SP600125 and SB203580) and recombinant OPN on TNC-induced vasoconstriction were evaluatedat 24 hours. Higher dosages of TNC induced more severe cerebral arterial constriction, which
continued for more than 72 hours. TNC administration also upregulated TLR4, and activated JNK
and p38 in the smooth muscle cell layer of the constricted cerebral artery. LPS-RS blocked TNCinduced TLR4 upregulation, JNK and p38 activation, and vasoconstrictive effects. SP600125 andSB203580 abolished TNC-induced TLR4 upregulation and vasoconstrictive effects. In addition,
recombinant TNC-induced prolonged contractions of rat basilar arteries were reversed byrecombinant OPN, although recombinant OPN itself had no effect on the vessel diameter.
Third, imatinib (50mg/kg body weight) was administered intraperitoneally to rats undergoing
SAH by endovascular perforation, and the effects were evaluated by neurobehavioral tests, Indiaink angiography, Western blotting, terminal deoxynucleotidyl transferase-mediated uridine
5-triphosphate-biotin nick end-labeling staining and immunohistochemistry. Recombinant TNC
was administered intracisternally to imatinib-treated SAH rats, and the effects were also evaluated.
Imatinib significantly prevented cerebral vasospasm at both 24 and 72 hours post-SAH, but
improved neurobehavioral tests only at 24 hours. SAH caused PDGFR activation, mitogen-activatedprotein kinases activation, TNC upregulation and caspase-dependent neuronal apoptosis in the
cerebral cortex, all of which were significantly suppressed by imatinib treatment at 24 hours, but not
at 72 hours post-SAH. Recombinant TNC reversed the anti-apoptotic effects and protein expression
changes in brain by imatinib.

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Discussion & Conclusion
We demonstrated for the first time that treatment by imatinib mesylate, an inhibitor of the tyrosine
kinases of platelet-derived growth factor receptors, prevented cerebral vasospasm after subarachnoid
hemorrhage at least partly via inhibiting the upregulation of TNC. We also demonstrated for the firsttime that TNC induced prolonged cerebral arterial constriction via TLR4 and activation of JNK and
p38 in healthy rats. TNC may be involved in the pathogenesis of chronic vasoconstrictive diseases,
such as cerebral vasospasm, and may provide a novel therapeutic approach to treat such diseases.
Further investigations may prove that TNC provides a novel therapeutic approach against cerebral
vasospasm.

References
1. Fujimoto M, Suzuki H, Shiba M, Shimojo N, Imanaka-Yoshida K, Yoshida T, Kanamaru K,
Matsushima S, Taki W. Tenascin-C induces prolonged constriction of cerebral arteries in rats.
Neurobiol Dis 2013; 55:104-109
2. Suzuki H, Shiba M, Fujimoto M, Kawamura K, Nanpei M, Takeuchi E, Matsushima S,
Kanamaru K, Imanaka-Yoshida K, Yoshida T, Taki W. Matricellular protein: a new player incerebral vasospasm following subarachnoid hemorrhage. Acta Neurochir Suppl 2013; 115:213-218
3. Shiba M, Suzuki H, Fujimoto M, Shimojo N, Imanaka-Yoshida K, Yoshida T, Kanamaru K,
Matsushima S, Taki W. Imatinib mesylate prevents cerebral vasospasm after subarachnoid
hemorrhage via inhibiting tenascin-C expression in rats. Neurobiol Dis 2012; 46:172-179

一般の皆様へ
脳動脈瘤破裂によるくも膜下出血は、現在でも死亡率は約 50％と高く、しかも生存者の 30% 以上に身体的障害を残す重篤な脳血管障害です。くも膜下出血患者の予後を改善するためには、2 次的に生じる脳血管攣縮や脳損傷を克服する必要があります。本研究では、炎症性サイトカインの 1 つである血小板由来増殖因子と近年注目されているマトリックス細胞蛋白と呼ばれる特殊な蛋白の 1 つであるテネイシン C が、くも膜下出血後に生じる脳血管攣縮や脳損傷の発生に関与していることを明らかにしました。今後、更に研究を進め、これらの成果をくも膜下出血に対する新たな治療法の開発につなげたいと考えています。

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Centriole biogenesis and Chromosomal instability
Daiju Kitagawa
National Institute of Genetics
dkitagaw@nig.ac.jp

Abstract
Formation of a new centriole next to each existing one is fundamental for centrosome duplicationand thus for genome integrity. Recent studies indeed highlighted that centriole amplification can be
an origin of genome instability in tumor formation. We demonstrated that MCPH7/STIL is essential
for centriole formation and also that increased expression levels of STIL promote formation ofmultiple new centrioles per pre-existing one. Therefore, regulated STIL levels and/or function
ensure the “only-one rule” of centriole formation. In order to identify proteins which modulate STIL
function, we employed the combined approach using MS-based proteomic analysis and a functional
RNAi screening in human cells. As a result, we identified a tumor suppressor with a RNA-binding
motif as a STIL-interacting protein that has a role in limiting the number of centrioles.
Key Words : Chromosomal instability, Centrosome, Centriole

Introduction
The mechanisms of centrosome duplication have been a long-standing mystery in biology. Like thegenetic material, centrosome duplication must occur once per cell cycle, such that the two resulting
centrosomes assemble a bipolar spindle during mitosis. Aberration in the number of centrosomes
is implicated in genome instability and tumor formation. The centrosome is composed of a pair ofcentrioles surrounded by pericentriolar material (PCM). Centrioles are also essential for generating
cilia and flagella across eukaryotic evolution. Despite being crucial for genome integrity and many
biological processes, the mechanisms controlling centriole copy number remain incompletely
understood.

Results
Centrioles are barrel-shaped microtubule-containing structures characterized by a universal
9-fold radial symmetry that they also impart to cilia and flagella. In most species, the centriole isorganized around a cartwheel that comprises a central hub from which 9 spokes emanate outwards
towards the microtubules. The molecular mechanisms and structural principles governing centriole
assembly remain poorly understood.
To elucidate how the onset of centriole formation takes place in higher eukaryotes with a particularemphasis on SAS-6, a protein required for centriole formation from worm to human, we used
a combination of multi-disciplinary approaches including cell biology, biochemistry, genetics,
biophysics and structural biology (1-3). Our findings revealed how SAS-6 is targeted to the assembly- 50 -

site of centrioles and timely modified (2), and further demonstrated that oligomerization of SAS6 homodimers is at the root of the 9-fold symmetry of the cartwheel and thus of centrioles (3).
These works represented the first molecular and structural insights into the mechanisms governing
this fundamental process. Even though we demonstrated that SAS-6 self-assembly is a key step
to initiate centriole formation, we hypothesized that something as upstream should facilitate
this process based on the fact that SAS-6 self-assembly alone does not seem to be sufficient tospontaneously form central part of cartwheel.
To identify such upstream regulator, we first analyzed the MCPH7 protein STIL, which has beensuggested to be related to the centriole proteins SAS-5 in C. elegans and Ana2 in Drosophila, bothof which are essential for centriole formation. Although whether STIL is required for centriole
formation in human cells has not been addressed, we found that siRNA-mediated depletion of
STIL results in a failure of centriole formation, with mitotic cells harboring usually ≤2 Centrin-3(a centriole marker) positive foci instead of the usual four, as is the case for cells depleted of SAS6 (1). A similar failure of centriole formation was observed using two distinct siRNAs directedagainst STIL. We conclude that STIL is required for centriole formation in human cells. This
also lends support to the notion that SAS-5/Ana2/STIL is an evolutionarily conserved module
critical for centriole formation. Importantly in addition, we found that centriolar localizationof STIL, at least in part, is critical for SAS-6 loading to centrioles, suggesting that STIL could
facilitate self-assembly of SAS-6 at the assembly site of a new centriole. Indeed, overexpressionof STIL led to the simultaneous formation of multiple new centrioles through SAS-6 recruitment
at the assembly site, indicating that STIL is a critical regulator to control the copy number of newcentrioles. Furthermore, we demonstrated that some STIL-depleted cells assemble an asymmetricbipolar spindle in which less pronounced astral microtubules emanate from one spindle pole (1).
Importantly, we found in addition that whereas spindle position is normal in those STIL-depleted
cells with a symmetric bipolar spindle, spindle position is randomized in the subset of cells withasymmetric spindles. Therefore, STIL is also critical for proper spindle positioning as well as
centriole formation in human cells.
Next, we set out to decipher the mechanisms limiting the number of newly formed centrioles
and maintaining genome integrity. We reasoned that such suppressors for centriole amplification
would physically interact with and directly control critical centriolar regulators/components suchas SAS-6 and STIL. Accordingly, we employed the combined approach using MS-based proteomic
analysis and a functional RNAi screening in human cells, which led to the identification of
RBM14, a tumor suppressor, as a STIL-interacting protein that has a role in limiting the numberof centrioles. Depletion of this protein by siRNA in human cells induces dramatic amplification ofcentriolar intermediates through STIL function, and also results in multipolar spindle formation as aconsequence. We are currently focusing on the underlying molecular mechanisms in more detail.

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Discussion & Conclusion
Overall, we identified STIL as a novel regulator for centriole formation. STIL seems to have a
critical role in the initiation of centriole formation most likely through the recruitment of SAS6 at the assembly site of an emerging centriole. Furthermore, our data suggest that the expression
levels of STIL and its interactor, RBM14, regulate the copy number of centrioles. It will be therefore
tempting to investigate how they interact and mutually modulate their function/expression toproperly coordinate this process.
We expect that our findings will make unique contributions to our understanding of centrosome
duplication. Cancer cells often exhibit aberrations in centrosome number, which could be acause of tumorigenesis. Furthermore, dysfunction of centrosomes is associated with humanneurodevelopmental disorder and infertility. Therefore, centrosome duplication offers importanttherapeutic and diagnostic opportunities in oncology and stem cell biology.

References
1. Daiju Kitagawa, Gregor Kohlmaier, Debora Keller, Petr Strnad, Fernando R Balestra, Isabelle
Flückiger and Pierre Gönczy (2011) Spindle positioning in human cells relies on proper centriole
formation and on the microcephaly proteins CPAP and STIL. J Cell Sci, 124, 3884-93.
2. Daiju Kitagawa, Isabelle Flückiger, Jolanta Polanowska, Debora Keller, Jérôme Reboul and Pierre
Gönczy (2011) PP2A phosphatase acts upon SAS-5 to ensure centriole formation in C. elegans
embryos. Dev Cell, 20, 550-562.
3. Daiju Kitagawa, Ioannis Vakonakis, Natacha Olieric, Manuel Hilbert, Debora Keller, Vincent
Olieric, Miriam Bortfeld, Michèle C. Erat, Isabelle Flückiger, Pierre Gönczy and Michel O.
Steinmetz (2011) Structural basis of the 9-fold symmetry of centrioles. Cell, 144, 364-375.

一般の皆様へ近年、中心小体の過剰複製と細胞がん化の関与が指摘されており、実際に複数のがん抑制遺伝子
が中心小体複製に関与することが報告されています。よって、中心小体複製の素過程における知見
は細胞がん化メカニズムの解明にも寄与することが期待されています。また、今後の抗癌剤の標的探索という観点からも有益であると考えられます。なぜなら、現在までに微小管重合形成を阻害する
抗癌剤は多数開発されており、今後も細胞分裂に必須である中心小体複製、紡錘体形成を標的にした有効な抗癌剤の開発が期待されているからです。さらには、本研究により、中心小体形成不全が原因と推定される遺伝病、繊毛病、男性不妊症など種々の疾病の原因解明に向けて基礎的な知見が提供されることが期待されます。

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The role of specific cell adhesion molecules
in dopaminergic innervation of striatal neurons
Akaike Akinori
Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Kyoto University
aakaike@pharm.kyoto-u.ac.jp

Abstract
The main pathological feature of Parkinson disease is a selective degeneration of the nigrostriatal
dopaminergic pathway. However, the precise mechanisms by which dopaminergic axons innervate
striatal neurons are still unclear. In this study, we demonstrated that integrin α5β1 expressing in
dopaminergic neurons participate in dopaminergic innervation of striatal neurons. This finding
is beneficial for cell transplantation of dopaminergic neurons into the striatum of patients with
Parkinson disease.
Key Words : dopaminergic innervation; integrin; Parkinson disease; embryonic stem cell

Introduction
The main pathological feature of Parkinson disease is a selective degeneration of the nigrostriatal
dopaminergic pathway, and regeneration of the neuroprojection is one of the promising therapeutic
approaches. However, the precise mechanisms by which dopaminergic axons innervate striatal
neurons are still unclear. Integrins, heterodimers of α and β subunits that function as cell adhesion
molecules, play an important role in embryonic development of central nervous system. In thisstudy, we investigated the involvement of integrins in dopaminergic innervation of striatal neurons.

Results
We established a novel method to evaluate dopaminergic innervation of striatal neurons by using
primary dissociated cells (Wakita et al., 2010). Mecencephalic and striatal cells were dissected fromrat embryos, and then a mecencephalic cell region was formed adjacent to a striatal cell region ona plastic coverslip. After paired-culturing for 11 days, dopaminergic neurons in the mesencephaliccell region extended their axons to the striatal cell region. Immunofluorescent microscopy showedthat dopaminergic axons elongated along striatal neurons and formed synapses with striatal neurons.
Integrin β1 subunit plays a central role in the function of integrin. Neutralizing integrin β1
antibody suppressed dopaminergic innervation of the striatal neurons. Next, we examined the role of
RGD-binding integrins in dopaminergic innervation of striatal neurons. Inhibition of RGD-binding
integrins by the small peptide RGDS and an integrin α5β1/αvβ3-blocking peptide ATN-161 (AcPHSCN-NH2) suppressed dopaminergic neurite outgrowth to the striatal cell region. To identify the
integrin α subunit, we examined the effect of selective integrin-blocking peptides and neutralizing
integrin α antibody. As a result, an integrin α5β1-blocking peptide A5-1 (VILVIF) and neutralizing- 53 -

integrin α5 antibody attenuated the dopaminergic innervation of striatal neurons, while an integrin
αvβ3-blocking peptide cyclo-RGDfV had no effect.
Integrin α5 and β1 subunits were expressed at the axonal growth cones and somas of dopaminergic
neurons. In addition, dopaminergic axonal outgrowth was enhanced by coating with fibronectin, aligand of integrin α5β1. To confirm that the integrin α5 subunit expressing in dopaminergic neurons
participate in dopaminergic innervation of striatal neurons, we employed RNAi to knockdown ofintegrin α5 in mesencephalic cells. First, we prepared lentiviral vector expressing short hairpin
RNA (shRNA) designed against rat integrin α5. As determined by immunoblot analysis, the shRNA
decreased expression of endogenous α5 integrin to almost 40%. By infection of mesecepalic
cells with lentiviral integrin α5 shRNA, the dopaminergic innervation of striatal neurons was
siginificantly suppressed.
To explore a ligand of integrin α5β1, we focused on chondroitin sulfate and L1 cell adhesion
molecule. It has been reported that chondroitin sulfate is expressed in and around the dopaminergic
axons (Charvet et al., 1998). However, treatment with chondroitinase ABC that degrades chondroitin
sulfate did not affect the dopaminergic innervation. The L1 cell adhesion molecule can bind withintegrin α5β1, and promotes axon growth and cell migration (Thelen et al., 2002). Coating with
L1 protein tended to enhance dopaminergic axonal outgrowth. With respect to a ligand of integrin
α5β1, further studies are required.
Last, we produced mouse integrin α5-overexpressing embryonic stem cells for cell transplantationin Parkinson disease model mice. Integrin α5 cDNA was prepared from mouse whole brain. Integrin
α5 cDNA was then subcloned into viral vector. Mouse embryonic stem cells infected by retroviral
vector exhibited no fluorecense of marker protein because of gene silencing. Embryonic stem
cells infected by lentiviral vector exhibited faint fluorescence, but the fluorescence intensity wasincreased during neural differentiation. At presence, we are examining the effect of overexpressionof integrin α5 on dopaminergic differentiation. In the next experiment, we will transplant embryonicstem cell-derived integrin α5-overexpressing dopaminergic neuons into Parkinson disease model
mice.

Discussion & Conclusion
In this study, we established an in vitro model for the quantitative analysis of the dopaminergic
innervations of striatal neurons using primary dissociated neruons. Pharmacological analysis
revealed that dopaminergic axons innervate striatal neurons through integrin α5β1. Selective
knockdown of integrin α5 in mesencephalic cells with use of lentiviral vector demonstrated that
integrin α5β1 expressing in dopaminergic neurons participate in dopaminergic innervation of
striatal neurons. This result suggests that integrin α5-overexpressing dopaminergic neurons promote
the striatal innervation. Furthermore, we produced mouse integrin α5-overexpressing embryonicstem cells. Transplantion of embryonic stem cell-derived integrin α5-overexpressing dopaminergic
neuons into Parkinson disease model mice is expected to ameliorate motor dysfunction. In
conclusion, our findings are beneficial for cell transplantation of dopaminergic neurons into thestriatum of patients with Parkinson disease.

- 54 -

References
1. Wakita S, Izumi Y, Matsuo T, Kume T, Takada-Takatori Y, Sawada H, Akaike A. (2010)
Reconstruction and quantitative evaluation of dopaminergic innervation of striatal neurons in
dissociated primary cultures. J Neurosci Methods. 192:83-89.
2. Charvet I, Hemming FJ, Feuerstein C, Saxod R. (1998) Mosaic distribution of chondroitin andkeratan sulphate in the developing rat striatum: possible involvement of proteoglycans in theorganization of the nigrostriatal system. Brain Res Dev Brain Res. 109:229-244.
3. Thelen K, Kedar V, Panicker AK, Schmid RS, Midkiff BR, Maness PF. (2002) The neural celladhesion molecule L1 potentiates integrin-dependent cell migration to extracellular matrix
proteins. J Neurosci. 22:4918-4931.

一般の皆様へ
黒質 - 線条体ドパミン神経が変性するパーキンソン病において、ドパミン神経細胞を線条体に細胞移植しようと試みられている。しかし、ドパミン神経細胞が線条体神経細胞と相互作用し神経突起を伸ばす機序については明らかになっていない。本研究では、ドパミン神経細胞に発現する細胞接着因子であるインテグリン α5β1 がその相互作用に重要であることを見出した。本研究の成果は、将来的に、パーキンソン病患者に対するドパミン神経細胞移植において、線条体の広範囲にドパミ
ン神経突起を伸長させ効率的に神経支配する目的で活用されることが期待できる。

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In vivo imaging reveals molecular mechanisms ofadult common diseases based on chronic inflammation
Satoshi Nishimura
Department of Cardiovascular Medicine, TSBMI, The University of Tokyosnishi-tky@umin.ac.jp

Abstract
To elucidate the underlying mechanisms of adult common diseases based on chronic inflammation
including metabolic and thrombotic disease, it is vital to examine the multi-cellular kinetics inliving animals.
Therefore, we developed in vivo imaging technique based on single- and multi-photon microscopy,
and we assessed dynamic immune and inflammatory cellular interplay in diseased conditions.
Key Words : In vivo imaging, inflammation, adult common diseases, metabolic syndrome

Introduction
Obesity associates with higher risks for various diseases, including atherosclerotic cardiovasculardisease, type 2 diabetes and cancer. Moreover, recent studies have shown that obesity induces
chronic inf lammation within visceral adipose tissue, which appears to lead to metabolic
abnormalities and inflammation in distant tissues. Inflammation is considered to play a pivotal rolein the development of metabolic diseases.

Results
CD8+ effector T cells contribute to macrophage recruitment and adipose tissue inflammationin obesity
Recently, we found that large numbers of CD8+ effector T cells infiltrated epididymal adipose
tissue in obese mice, whereas the numbers of CD4+ helper and regulatory T cells were diminished.
The infiltration of CD8+ T cells preceded the accumulation of macrophages during diet-inducedobesity. Immunological and genetic depletion of CD8+ T cells reduced inf lammatory (M1)macrophage infiltration and adipose tissue inf lammation, and ameliorated systemic insulin
resistance. The transfer of CD8+ T cells to CD8-deficient mice enhanced inflammation in obese
adipose tissue, demonstrating the importance for CD8+ T cells in obese conditions. Coculture andother in vitro experiments revealed major interactions between CD8+ T cells, macrophages, andhypertrophied adipocytes. Several in vitro studies have shown that obese adipose tissue directly
activates CD8+ T cells, which subsequently promotes recruitment and activation of macrophages,
thereby initiating and maintaining the inflammatory cascades in obese adipose tissue. Infiltration of
CD8+ T cells is, therefore, essential for the initiation and development of adipose inflammation.

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Platelet Kinetics in Thrombus Formation, and Inflammatory Status
It is well known that platelets play an important role in inf lammatory diseases, such as
atherosclerosis. However, the molecular and cellular mechanisms of platelet kinetics in vivo are
unknown, because experimental methodology to analyze kinetics of individual platelets in living
animals is not well established. We have already established an in vivo visualization system, whichenabled us to analyze thrombus formation at the individual platelet level. We can analyze platelet
kinetics in vessels of various sizes, including large-sized arteries, veins, and small capillaries. In unstimulated conditions, single platelet moved in a “stop-and-go” manner along the vessel wall andinteracted with endothelial cells. By inducing the production of ROS (reactive oxygen species) bylaser injury, we can trigger thrombus formation in vessels of all sized including small capillaries
and the carotid arteries and observe the kinetics of individual platelets. We could evaluate not
only thrombus formation but also platelet functions at the individual platelet level. After laser
injury, platelets started to adhere to the vascular wall, and aggregated. The thrombus caused
decreased blood flow and the vessel was subsequently completely occluded. Using this technique,
we elucidated that Lnk (adapter protein) regulates integrin signaling leading to stabilization ofdeveloping thrombus in vivo. We established the efficient culture system of human iPS-derived
platelets, and we confirmed the functional role. These artificial platelets can circulate, andcontribute to their thrombus formation in vivo, indicating the clinical usefulness considering the celltherapy for future.
In addition, we elucidated the contribution of inflammatory cytokines, ROS, and integrin signaling
to our thrombosis models. Therefore, our imaging technique had very broad applications, such as
evaluation of anti-platelet drugs in vivo, stem cell-derived platelet function in vivo, and the effect ofgenetic mutations on platelet functions.

Discussion & Conclusion
Cellular interrelationships associated with chronic inflammatory disease
Cellular reactions in inflammation are particularly dynamic and involve multiple cell types.
Consequently, analysis of the multi-cellular dynamic processes involved in inf lammation
by in vivo imaging using intact tissues in living animals offers a powerful tool. Our results
demonstrated the advantages and potential of our imaging technique to analyze multi-cellularinteractions in inflammation in vivo. Using our new imaging technique to analyze the complex
cellular interactions within obese adipose tissue, we were able to show that visceral adipose tissue
obesity is an inflammatory disease. Our technique could be used to evaluate potential therapeuticinterventions against inflammation in obesity. Clearly, further studies are needed to identify what
initiates the cascade of inflammatory responses in obesity. To address these important questions, itwill be necessary to combine in vivo visualization technologies with genetic and pharmacologicalinterventions.
In summary, using our imaging system can be a powerful tool to analyze the inflammatory- 57 -

conditions including metabolic syndrome and thrombosis. We clarified the mechanism by whichthrombi are rapidly formed by discoid platelets on undisputed endothelium. The initial platelet
aggregation subsequently leads to irreversible integrin- and actin-dependent thrombus development.
Inflammatory cytokine signaling in endothelial cells (ECs) played pivotal role in discoid platelet
aggregations in vivo. In addition, Our imaging revealed close spatial and temporal interrelationships
between angiogenesis and adipogenesis in obese adipose. In addition, increased inflammatory cellinteractions in obese adipose were visualized and we found that large numbers of CD8+ effector T
cells infiltrated into obese adipose, and these cells were essential for the initiation and developmentof adipose inflammation.

References
1. Nishimura S, Manabe I, et al. Diabetes. 2007,56:1517-1526.
2. Nishimura S, Manabe I, et al. J Clin Invest, 2008, 118(2): 710-721.
3. Nishimura S, Manabe I, et al. Nature Medicine 2009, 15(8):914-920
4. Nishimura S, and Takizawa H, Takayama N, et al., J Clin Invest, 2010,120(1): 179-190.
5. Takayama N, Nishimura S, et al.,J Exp Med, 2010; 207(13):2817-2830
6. Nishimura S, Manabe I, et al. Blood, 2012, 119(8):e45-56.

一般の皆様へ
生活習慣病がどうしておきるのか、どうやって治療をしたらいいのか、そのためには、からだのな
かで何が起きているかをみる技術はとても有用です。私は、光を用いてカラダの中を直接みて、肥満や血管の傷害がどのように影響を及ぼすかを、検討してきました。免疫・炎症といった過程が「見る」ことにより、「わかり」ます。

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The structural study of the human centromeric chromatin
Hitoshi Kurumizaka
Waseda University, Graduate School of Advanced Science & Engineeringsnishi-tky@umin.ac.jp

Abstract
The centromere functions as a chromosome site for kinetochore assembly. However, architecture of
the centromeric chromatin has not been understood. CENP-B is known to bind to the CENP-B box
DNA sequence, which is located in the centromeric chromatin region containing histone H3 variant
CENP-A. However, how CENP-B efficiently binds to the CENP-A nucleosome is not known. In
the present study, we tested the CENP-B binding to the CENP-A nucleosome, and found that Nap1
(nucleosome assembly protein 1) significantly stimulates the CENP-B binding to the CENP-A
nucleosome in vitro.
Key Words : CENP-A, CENP-B, Nap1, centromere, nucleosome

Introduction
The centromere is the chromosome region for kinetochore assembly. CENP-A is the centromerespecific histone H3 variant that is widely conserved among eukaryotes, and is an essential
component for the formation of functional kinetochores (1-6). On the other hand, CENP-B is thecentromeric protein that specifically binds to a 17 base-pair DNA sequence, known as the CENP-B
box (7). The CENP-B binding to the CENP-A nucleosome may be important for formation of thefundamental architecture of the centromeric chromatin. However, the mechanisms of efficient
CENP-B binding to the CENP-A nucleosome remain unclear.

Results
CENP-B is the only sequence-specific DNA-binding protein found in the centromere-specificproteins. The CENP-B function in the centromere formation has remain elusive, but the CENP-B
binding mechanism to chromatinized DNA may be important for understanding the centromericchromatin architecture. To clarify the CENP-B binding mechanism, we performed the in vitro
CENP-B binding assay with the CENP-A nucleosome. To do so, we purified the human histones(H2A, H2B, CENP-A, and H4) and the DNA-binding domain of CENP-B (CENP-B DBD), as
recombinant proteins. CENP-B DBD contained the N-terminal amino acid residues 1-129 ofhuman CENP-B. We first reconstituted CENP-A nucleosome with a 192 base-pair DNA, in whicha CENP-B box sequence was located at the entry/exit regions of the nucleosome. We then tested
CENP-B binding to the CENP-A nucleosome by an electrophoretic mobility shift assay (EMSA),
using a polyacrylamide gel. The CENP-B DBD was incubated with the reconstituted CENP-A
nucleosome under physiological salt conditions. In this assay, we detected only a small amount of- 59 -

the specific complex with the CENP-B DBD bound to the CENP-B box sequence of the CENP-A
nucleosome. This result indicated that CENP-B non-specifically interacts with the nucleosomal
DNA, and forms aggregates, which may be inactive for functional centromeric chromatin formation.
However, we found that the CENP-B DBD properly bound to the CENP-A nucleosome, and thespecific complexes were formed in the presence of Nap1, an acidic histone chaperone, which
reportedly inhibits non-specific histone binding to DNA. Since another histone chaperone sNASP
did not promote the CENP-B binding, this CENP-B DBD assembly activity may be specific for
Nap1. Furthermore, we found that incubation with excess Nap1 dissociates the CENP-B DBD from
specific complexes with the CENP-A nucleosomes. Eviction of the CENP-B DBD from the specificcomplex was not observed in the presence of excess amounts of sNASP. We next tested whether
the Nap1-mediated CENP-B assembly may occur on the canonical nucleosome containing histone
H3, instead of CENP-A. We then found specific CENP-B assembly was efficiently observed in the
canonical H3 nucleosome containing the CENP-B box within it. We therefore conclude that Nap1,
but not sNASP, promotes the specific binding of CENP-B to the CENP-B box in a nucleosomal
context by suppressing non-specific aggregate formation. We further investigated whether Nap1
can regulate the association of CENP-B with chromosomes in vivo. Consistent with in vitro results,
we found that the interaction between Nap1 and CENP-B was detected by immunoprecipitation. To
test whether Nap1 evicts CENP-B non-specifically bound to chromatin in vivo, we established the
CENP-B eviction assay using the Nap1 tethering to a chromosome and CENP-B overexpression. For
this purpose, we construct the mutant CENP-B box that abolishes the specific CENP-B binding. A
chromatin-immunoprecipitation (ChIP) analysis showed that the non-specific binding of CENP-B
to the mutant CENP-B box was increased under conditions where CENP-B was overexpressed. Wethen found that co-expressed Nap1 with CENP-B significantly reduced the non-specific CENP-B
binding to the mutant CENP-B box selectively, whereas no significant reduction of the specific
CENP-B binding to the host centromere with canonical CENP-B boxes was observed under thesame conditions. We therefore conclude that the selective inhibition of non-specific CENP-B-DNA
binding by Nap1 may regulate proper CENP-B binding to chromosomes in vivo.

Discussion & Conclusion
It has been reported that Nap1 eliminates non-nucleosomal histone-DNA interactions, andpromotes correct nucleosome assembly in vitro (8). In the present study, we found that human Nap1
significantly inhibits non-specific CENP-B binding to the CENP-A or canonical H3 nucleosomesin vitro. In addition, Nap1 also promotes eviction of the CENP-B improperly bound to chromatin invivo. Nap1 is an acidic histone chaperon that may simply bind electrostatically to the basic CENP-B
DBD, and non-specifically inhibit the CENP-B-DNA interaction. However, we do not think thisis the case, because another acidic histone chaperone, sNASP, affected neither specific nor nonspecific CENP-B binding to nucleosomes in vitro and chromosomes in vivo. These studies haveled us to propose a novel Nap1 function: the regulation of specific CENP-B loading at centromeric- 60 -

chromatin by the inhibition of non-specific CENP-B binding to other chromosome loci. Thus, Nap1
may indirectly promote the specific assembly of CENP-A nucleosomes, a key structural element ofactive centromeres (9).

References
1. Stoler,S., Keith,K.C., Curnick,K.E. and Fitzgerald-Hayes,M (1995) A mutation in CSE4, anessential gene encoding a novel chromatin-associated protein in yeast, causes chromosome
nondisjunction and cell cycle arrest at mitosis. Genes Dev., 9, 573-586.
2. Buchwitz,B.J., Ahmad,K., Moore,L.L., Roth,M.B. and Henikoff,S. (1999) A histone-H3-likeprotein in C. elegans. Nature, 401, 547-548.
3. Takahashi,K., Chen,E.S. and Yanagida,M. (2000) Requirement of Mis6 centromere connector for
localizing a CENP-A-like protein in fission yeast. Science, 288, 2215-2219.
4. Blower,M.D. and Karpen,G.H. (2001) The role of Drosophila CID in kinetochore formation, cellcycle progression and heterochromatin interactions. Nat. Cell Biol., 3, 730-739.
5. Goshima,G., Kiyomitsu,T., Yoda,K. and Yanagida,M. (2003) Human centromere chromatinprotein hMis12, essential for equal segregation, is independent of CENP-A loading pathway. J.
Cell Biol., 160, 25-39.
6. Régnier,V., Vagnarelli,P., Fukagawa,T., Zerjal,T., Burns,E., Trouche,D., Earnshaw,W. and
Brown,W. (2005) CENP-A is required for accurate chromosome segregation and sustained
kinetochore association of BubR1. Mol. Cell Biol., 25, 3967–3981.
7. Masumoto,H., Masukata,H., Muro,Y., Nozaki,N. and Okazaki,T. (1989) A human centromere
antigen (CENP-B) interacts with a short specific sequence in alphoid DNA, a human centromericsatellite. J. Cell Biol., 109, 1963-1973.
8. Andrew,A.J., Chen,X., Zevin,A., Stargell,L.A. and Luger,K. (2010) The histone chaperone Nap1
promotes nucleosome assembly by eliminating nonnucleosomal histone DNA interactions. Mol.
Cell, 37, 834-842.
9. Tachiwana H, Miya Y, Shono N, Ohzeki J, Osakabe A, Otake K, Larionov V, Earnshaw WC,
Kimura H, Masumoto H, Kurumizaka H. (2013) Nap1 regulates proper CENP-B binding to
nucleosomes. Nucleic Acids Res. 41, 2869-2880.

一般の皆様へ
本研究の目的は、セントロメア領域の構築原理を解明することである。生物は、細胞の増殖と分裂を繰り返して生命活動を維持しており、細胞分裂時に遺伝情報を担うゲノム DNA を正確に継承する必要がある。この機構が破綻するとダウン症やガン化に関与することが示されており、正確な遺伝子の維持・継承機構の解明は非常に重要である。これまでに、セントロメアとよばれる特異的な領域が、ゲノム DNA の含まれる染色体の均等分配に重要であることが示されており、セントロメアの構築原理を明らかにすることは非常に重要である。本事業の助成によって得られた研究成果から、
セントロメア構築原理の一端に重要な知見を与えることができた。

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An intrinsic molecular mechanism governing
the development of root nodule primordia
Takuya Suzaki
National Institute for Basic Biology
tsuzaki@nibb.ac.jp

Abstract
Nodulation is a form of de novo organogenesis that occurs mainly in legumes. In the present study,
we investigated the detailed patterns of auxin response during nodule development in L. japonicus.
In addition, we characterized a novel nodulation-deficient mutant, tricot. Taken together, ouranalyses will contribute further to our understanding of the genetic mechanism regulating nodule
development.
Key Words : Auxin, Nodulation, Root nodule symbiosis

Introduction
During the course of plant evolution, mainly leguminous plants have acquired the ability to form
de novo structures called root nodules. Two classic phytohormones, cytokinin and auxin, playessential roles in diverse aspects of cell proliferation and differentiation. Although recent genetic
studies have established how activation of cytokinin signaling is crucial to the control of cortical celldifferentiation, the physiological pathways through which auxin might act in nodule development arepoorly characterized. Moreover, our understanding of molecular genetic mechanism of nodulation isstill limited by the low number of nodulation-related genes that have been identified.

Results
During early nodule development, the host plant root is infected by soil bacteria collectively calledrhizobia that induce dedifferentiation of some cortical cells; these cortical cells then proliferate toform the symbiotic root nodule primordium.
In the present study, we investigated the detailed patterns of auxin response during nodule
development in Lotus japonicus. In order to determine the precise distribution of auxin response
during nodule development in L. japonicus, we created stable transgenic plants that expressed a GFP
and nuclear localization signal (NLS) fusion protein (GFP-NLS) under the control of DR5, which
is a highly active synthetic auxin responsive element. Our analyses showed that auxin response
predominantly occurred in dividing cortical cells and that cytokinin signaling and NODULE
INCEPTION, a key transcription factor in nodule development, positively regulated this response.
Additionally, we found that auxin response was inhibited by a systemic negative regulatory
mechanism called autoregulation of nodulation (AON). The basis of AON is systemic long-distancesignaling between root and shoot. In L. japonicus, HYPERNODULATION ABERRANT ROOT

- 62 -

FORMATION 1 (HAR1), which encodes a leucine-rich repeat receptor-like kinase, is hypothesized
to function in shoots where it recognizes and responds to root-derived signals involved in thenegative regulation of nodulation. Here, we showed that the har1 mutant has increased and morewidespread response of auxin in cortical cells than the WT. This indicates that auxin response prior
to cortical cell division is controlled by the AON mechanism (including HAR1) and it determines
not only the auxin response level but also the site of auxin response. Analysis of the constitutive
activation of LjCLE-RS genes, which encode putative root-derived signals that function in AON, in
combination with determination of auxin response patterns in proliferating cortical cells, indicatedthat activation of LjCLE-RS genes blocked the progress of further cortical cell division, probably
through controlling auxin response (Suzaki et al., 2012).
Recent studies using spontaneous nodule formation 2 (snf2) mutant line of L. japonicus haveshown that activation of cytokinin signaling is crucial to the control of nodule organogenesis(Tirichine et al., 2007). The snf2 phenotype was demonstrated to result from a gain-of-function
mutation of the LOTUS HISTIDINE KINASE 1 (LHK1) gene that encodes a putative cytokinin
receptor. The mutation triggers formation of nodule-like structures (defined as spontaneous nodules)in the absence of rhizobia, due to the constitutive activation of LHK1. Here we focused on thedownstream part of the cytokinin signaling pathway. We identified a mutant tricot (tco) in a screen
for mutants that could suppress the snf2 phenotype. The tco mutation prevented snf2-dependent
spontaneous nodule formation, suggesting that TCO positively regulates nodule organogenesis in thedownstream part of the cytokinin signaling pathway. Nodule development and rhizobial infection
process were also impaired in the tco mutant. Additionally, auxin reporter analysis showed there aresome defects relevant to auxin regulation in the tco mutant. The shoot phenotype of the tco mutant
also indicated that TCO is involved in the maintenance of the shoot apical meristem (SAM). We
used map-based cloning and found that TCO encodes a putative glutamate carboxypeptidase withpossible orthology to Arabidopsis ALTERED MERISTEM PROGRAM 1. An expression analysis
showed that TCO is expressed in meristematic regions of nodules and in the SAM (Suzaki et al.,2013).

Discussion & Conclusion
In comparison with cytokinin, relatively little is known of the role of auxin in nodule
development. In the present study, however, we have uncovered the site of auxin action during
nodule development. In addition, our data provide evidence for the existence of a novel finetuning mechanism that controls nodule development in a cortical cell stage-dependent manner.
Our analyses of tricot mutant have not only identified a novel gene for regulation of nodule
organogenesis but also provided significant additional evidence for a common genetic regulatory
mechanism in nodulation and shoot apical meristem formation. This new data will contribute
further to our understanding of the evolution and genetic basis of nodulation.

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References
1. Suzaki T, Yano K, Ito M, Umehara Y, Suganuma N, Kawaguchi M. (2012) Positive and negative
regulation of cortical cell division during root nodule development in Lotus japonicus isaccompanied by auxin response. Development 139:3997-4006.
2. Suzaki T, Kim CS, Takeda N, Szczyglowski K, Kawaguchi M. (2013) TRICOT encodes an
AMP1-related carboxypeptidase that regulates root nodule development and shoot apicalmeristem maintenance in Lotus japonicus. Development 140:353-361.
3. Tirichine L, Sandal N, Madsen LH, Radutoiu S, Albrektsen AS, Sato S, et al. (2007) A gainof-function mutation in a cytokinin receptor triggers spontaneous root nodule organogenesis.
Science 315:104-107.

一般の皆様へダイズやエンドウなどのマメ科植物に代表される一部の植物は、根に「根粒」と呼ばれるこぶ状の器官をつくる能力を獲得することによって、土の中にいるバクテリアの一種である根粒菌と共生していることが知られています。根粒内部では、植物は根粒菌が固定した大気中の窒素を栄養源として受け取ることができます。本研究では植物ホルモンの 1 つであるオーキシンと根粒形成の制御関係を明らかにし、さらに根粒形成を制御する新たな遺伝子を単離しました。得られた知見は基礎的なものですが、将来的にはマメ科以外の有用な作物に応用することで、窒素肥料いらずの低化学肥料農業の実現や栄養の少ない土地での農業展開への貢献が期待されます。

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Identification and characterization of novel skeletal muscle-derived
angiogenesis growth factor
Yasuhiro Izumiya
Department of Cardiovascular Medicine, Faculty of Life Sciences, Kumamoto University
izumiya@kumamoto-u.ac.jp

Abstract
It has been proposed that skeletal muscle secretes factors, referred to as “myokines,” that
influence the behavior of remote cells. Based upon microarray analysis, we have identified 5 novel
muscle-secreted factors (MSFs), encoding secreted proteins, that are upregulated during Aktmediated muscle growth. The present research performed in vivo and in vitro assays to assess theangiogenesis regulatory properties of these MSFs. We have shown that MSF1 and 5 play a role inpromoting revascularization under conditions of ischemic stress. MSF1 and 5 expressions in muscle
were up-regulated by Akt1 transgene activation during muscle hypertrophy and by ischemic injury.
Administration of MSF1 and 5 improved revascularization in ischemic limbs of wild type mice.
Key Words : angiogenesis, growth factor, skeletal muscle, hypertrophy

Introduction
It has been proposed that skeletal muscle secretes factors, referred to as “myokines,” that influence
the behavior of neighboring or remote cells 1). We have reported several myokines regulated by
Akt signaling. Myogenic Akt signaling controls both fiber hypertrophy and VEGF synthesis 2).
In addition to VEGF, it is likely that other as-yet-unidentified angiogenesis regulators contributeto the increase in capillary density in response to Akt1 induction in skeletal muscle. Basedupon microarray analysis, we have identified 5 novel MSFs, encoding secreted proteins, that are
upregulated during Akt-mediated muscle growth. The present research performed in vivo and invitro assays to assess the angiogenesis regulatory properties of these MSFs.

Results
In vivo evaluation of MSF angiogenic properties
Angiogenic properties of MSFs were evaluated. As shown in Figure 1A, ischemic/normal LDBF
ratio was significantly increased 7 days after MSF 5 overexpression and improvement of bloodflow recovery was persisted at each of the subsequent time points (14 and 28 days). As shown in
Figure 1B, adeno-MSF1-treated mice showed a modest but statistically significant increase in flow
recovery at 28 days after hind limb surgery. On the other hand, overexpression of MSF2, 3 or 4 inskeletal muscle had no effect on blood flow recovery in this model. These results suggest that MSF
1 and 5, but not MSF 2, 3, and 4 functions as an angiogenesis-regulatory protein.

- 65 -

To examine the extent of angiogenesis

Figure 1

at the microcirculatory level, CD31
staining was performed. As shownin Fig 1C, capillary density wassignificantly increased in mice adductor
muscle transferred with adeno-MSF
5 on 14 days after operation. These
results suggest that MSF 5 functions as
a pro-angiogenic factor.

Gene transfer of MSF1 and 5 promotes angiogenesis in ischemic hind limbs.
Quantitative analysis of the ischemic/nonischemic LDBF ratio in the MSF1, 2, 4, 5 andβ-Gal-treated(control) mice.

Characterization of angiogenic MSF expression
Based upon the results of in vivo angiogenesis
assay, MSFs that show in vivo angiogenic properties
were examined to understand their expression
properties. mRNA expression prof ile of MSF
5 in adult mouse tissue was analyzed by semiquantitative RT-PCR. As shown in Figure 2, MSF
5 mRNA was abundantly expressed in skeletal

MSF 5 increases capillary density in WT mice.

muscle, kidney and placenta, and moderately
expressed in adipose tissue, suggesting thatskeletal muscle is one of the major sources ofExpression profile of MSF 5 in adult mouse tissue.
cDNAs from different mouse tissue was synthesized and analyzed by
RT-PCR using MSF 5-specific primer pairs.

MSF5. Expression profile of MSF 1 in adultmouse tissue is under investigation.

Expression profile of MSF 1 and 5 during physiological muscle growth and wasting was also
evaluated. RNA was isolated from gastrocnemius muscle in various rodent models, including forced
treadmill exercise test (Acute Ex), skeletal muscle atrophy model (denervation, starvation, acute
uncontrolled diabetes (DM), chronic renal failure (CRF), tumor-bearing (Tumor)). The levels ofmRNA expression were evaluated by quantitative real-time PCR (Table 1). mRNA expression of
MSF 1and 5 were 6.8- and 1.9-fold, respectively, up-regulated in gastrocnemius muscle from skeletal
muscle-specific Akt1 transgenic
mice compared with that from

Table 1
Summary of mRNA expression of MSFs in gastrocnemius muscle during physiological muscle
growth and wasting.

control mice. MSF 1 mRNA
expression was modestly upreg ulated in t umor-bear ing
model, and down regulated in starvation model. Transcript expression of MSF 5 was up-regulated in
denervation model, and decreased in acute exercise and another atrophy model.
Although MSF 1and 5 are commonly up-regulated by Akt1, their expression profile in various
physiological conditions seems to be different.Gene expression of these MSFs in functional
overloaded skeletal muscle and skeletal muscle from myostatin deficient mouse, that shows- 66 -

excessive muscle growth, is under investigation.
For protein detection, I have produced anti-peptide antibody against MSF 5. Peptide sequencecorresponding to immunogenic regions of MSF 5 was determined by the prediction software(Accelrys Inc.). Adenoviral vector expressing Flag-tagged MSF 5 was transfected into 293T cells.
After 2 days, the cell pellets and media fractions were collected and analyzed by western blot using
anti-SNFL and anti-FLAG antibodies. MSF 5 protein was detected both in the cell lysates and in
the media by anti-FLAG antibody; however, anti-SNFL antibody recognized only MSF 5 protein
form cell lysates, but not culture media. It is possible that peptides sequence recognized by antiSNFL antibody is modified during protein secretion. Therefore, I am producing another antibody
that recognizes different peptide sequence from anti-SNFL antibody. Production of anti-peptide
antibodies to MSF 1 is under processing.

Discussion & Conclusion
It has been proposed that skeletal muscle secretes factors, referred to as “myokines,” that influence
the behavior of neighboring or remote cells 1). The present research shows that MSF1 and 5 play arole in promoting revascularization under conditions of ischemic stress. MSF1 and 5 expressions inmuscle were up-regulated by Akt1 transgene activation during muscle hypertrophy and by ischemic
injury. Administration of MSF1 and 5 improved revascularization in ischemic limbs of wild type
mice.
Using this inducible transgenic system, we recently demonstrated that Akt1-mediated skeletal
muscle growth attenuates cardiac remodeling after MI, and is associated with an increased capillarydensity in the heart 3). We found that the improvement in left ventricular remodeling resulting frommuscle growth accompanied by an increase in capillary density in the heart. We found that levelsof a number of angiogenic growth factors are increased in serum following transgene activation
including VEGF-A, SDF-1, FGF-1 and FGF2. This increase in angiogenic growth factor production
was associated with an increase in the activating phosphorylation of eNOS at Ser1177 in heart.
Because eNOS is pro-angiogenic, these data are consistent with the increase in myocardial capillarydensity in response to Akt1 induction in skeletal muscle.
In conclusion, the proposed study identified novel angiogenic factor that may have utility fortherapeutic angiogenesis.

References
1. Pedersen BK, Febbraio MA. Muscles, exercise and obesity: skeletal muscle as a secretory organ.
Nat Rev Endocrinol. 2012;8(8):457-465.
2. Takahashi A, Kureishi Y, Yang J, Luo Z, Guo K, Mukhopadhyay D, Ivashchenko Y, Branellec D,
Walsh K. Myogenic Akt signaling regulates blood vessel recruitment during myofiber growth.
Mol Cell Biol. 2002;22(13):4803-4814.
3. Araki S, Izumiya Y, Hanatani S, Rokutanda T, Usuku H, Akasaki Y, Takeo T, Nakagata N,
Walsh K, Ogawa H. Akt1-mediated skeletal muscle growth attenuates cardiac dysfunction andremodeling after experimental myocardial infarction. Circ Heart Fail. 2012;5(1):116-125.

- 67 -

一般の皆様へ
慢性心不全患者は高頻度に骨格筋委縮を呈し、それが独立した予後不良因子となることが知られています。一方、骨格筋量の維持・増加を目的とした運動療法は心不全患者における QOL や予後の改善に寄与することが報告されていますが、その有用性の機序は良くわかっていません。近年、運動により骨格筋から様々な因子が分泌され、遠隔臓器の機能制御に寄与することが明らかになってきました。私たちは、任意の時期に骨格筋肥大を誘導できるマウスを作成し、骨格筋の肥大が血管新生因子の発現亢進を介して心筋梗塞後のリモデリングを抑制することを昨年報告しました。本研究では新規の血管新生因子候補をいくつか同定することができました。本研究で得られた成果は心血管疾患患者に対して、筋力トレーニングを主体とした運動療法を行うことの理論的根拠を与えるものとなりうると考えられます。

- 68 -

Molecular mechanism of self-renewal and proliferation ofa hematopoietic cell due to HMGA2 expression
Kazuhiko Ikeda
Fukushima Medical University
kazu-ike@fmu.ac.jp

Abstract
Myeloproliferative neoplasms (MPNs) are clonal hematological disorders characterized byproliferative hematopoiesis. Clonal expansion mechanism has not been fully understood in the
MPNs. Here we study the role of HMGA2 on pathogenesis of MPNs. We found that HMGA2
expression seems like to contribute to proliferative hematopoiesis with conferring clonal growthadvantage in association with some signaling pathways and let-7 micro RNA that negatively
regulates HMGA2 expression.
Key Words : Hematopoietic stem cell, HMGA2, clonal expansion

Introduction
Myeloproliferative neoplasms (MPNs) are clonal hematological disorders characterized byproliferative hematopoiesis and sometimes develop secondary myelofibrosis (MF) or acute myeloid
leukemia (AML). It remains largely unknown how an abnormal clone acquire a clonal growthadvantage with accumulation of some genetic abnormalities that contributes to disease progression.
In this study we focused on the role of HMGA2 in the pathogenesis of MPNs with the respect of
clonal growth advantage.

Results
HMGA2 expression is negatively regulated by let-7-family micro RNAs. Either downregulation of
let-7 or removal of let-7-binding sites from HMGA2 is thought to cause overexpression of HMGA2.
Since overexpression of HMGA2 has been reported in hematopoietic cells from patients with MPNs,
we generated transgenic mice that overexpress HMGA2 without let-7-binding sites (∆Hmga2 mice).
∆Hmga2 mice showed not only proliferative hematopoiesis but also clonal growth advantage at thelevel of a hematopoietic stem cell (Ikeda K et al, 2011). Then we have been investigating molecularmechanism that causes abnormal hematopoiesis in the presence of HMGA2 overexpression using
the ∆Hmga2 mice. Young adult ∆Hmga2 mice showed deregulated expression let-7 in BM andspleen cells compared with wild-type (WT) mice. The difference between ∆Hmga2 mice and WT
mice were greater in spleen (Ikeda K et al, 2012). We have also studied the influence of aging in the
background of HMGA2 overexpression, because HMGA2 overexpression may be associated withmyelofibrosis, which often occurs after long course of other types of MPN such as polycythemia
vera or essential thrombocytosis. Interestingly, althogh hematopoiesis of old ∆Hmga2 mouse shows- 69 -

a distinct feature from that of young ∆Hmga2 mice, myelofibrosis has not been seen for up to 18
months until now (Ikeda K et al, manuscript in preparation). Moreover, hematopoiesis under Hmga2
overexpression might contribute to some signaling pathways in the young and old hematopoiesis.
We are currently performing experiments using hematopoietic cells from ∆Hmga2 mice and celllines expressing HMGA2 to confirm such novel findings.

Discussion & Conclusion
In this study, we have found that HMGA2 overexpression alone can cause a proliferative
hematopoiesis mimicking MPN, at least in part caused by activation of some signaling pathways
involving JAK-STAT or PI3K-AKT. Although some mutations including the JAK2V617F, has beenfound in patients with MPNs, 30-40% patients do not show any known mutations that activate
signaling pathway. Thus, HMGA2 overexpression is a candidate abnormality, which possibly causes
proliferative hematopoiesis in MPNs. In the aged mice, Hmga2 overexpression caused abnormal
hematopoiesis without myelofibrosis, suggesting that long-term Hmga2 overexpression can cause
secondary hematopoietic abnormality in addition to proliferative hematopoiesis. Finally, Hmga2
overexpression may further influence let-7 expression in a tissue specific manner, which possibly
contributes to such abnormal hematopoiesis. We believe we will clarify a detailed HMGA2-involved
mechanism in the pathogenesis of MPNs and hematopoiesis in the near future.

References
1. Ikeda K, Mason PJ, Bessler M. 3’UTR-truncated Hmga2 cDNA causes MPN-like hematopoiesis
by conferring a clonal growth advantage at the level of HSC in mice. Blood. 2011;117:5860-5869.
2. Ikeda K, Harada K, Ogawa K, Mason PJ, Bessler M, Takeishi Y. Expression of Let-7 Micro RNAs
in Transgenic Mice Overexpressing Truncated Hmga2. Blood (ASH Annual Meeting Abstracts).
2012;120:3470.
3. Ikeda K, Ogawa K, Takeishi Y. The role of HMGA2 in the proliferation and expansion of ahematopoietic cell in myeloproliferative neoplasms. Fukushima J Med Sci. 2012;58:91-100.

一般の皆様へ
骨髄増殖性腫瘍は成熟した血液が増加する疾患で、一部は予後の悪い骨髄線維症や急性白血病に移行します。このように病気が進行してしまう理由はよくわかっていない面が多く残されています。私たちは、HMGA2 とよばれる遺伝子が、この疾患の形成や進行に関係していると考えており、
HMGA2 を増減させる因子や、逆に HMGA2 によって影響をうける物質の経路を調べています。このことが将来診断や治療の役に立つことを期待しています。

- 70 -

The biological and pathological significance ofStox1 transcriptional factor
Keizo KANASAKI
Kanazawa Medical University
kkanasak@kanazawa-med.ac.jp

Abstract
Key Words : Diabetes, metabolic syndrome, kidney fibrosis

Introduction
Storkhead-box protein 1 (Stox1) was identified using a genome-wide screening methods and single
nucleotide polymorphism (SNPs) analyses, as gene associated with pregnancy disorder (1-4). The
function of Stox1 is unknown. The Stox1 protein contains a winged helix domain, related to FOX
family, and is speculated to be a transcription factor. There are several transcriptional splice variantsof human Stox1 and most of these involve the exon 3. We made stox1 deficient mice (stox1-/-) andanalyzed; preliminary analysis reveals stox1 would be key transcriptional factor to modulate renin
expression.

Results
Stox1 has been originally reported as a candidate gene associated with preeclampsia. Preeclampsia
experienced women has been reported to develop metabolic, cardiovascular, and chronic kidneydisease in her later life. Therefore we hypothesized that stox1 deficiency may be shared molecularmechanisms between preeclampsia and these health problem. To prove this hypothesis we performedexperiment using stox1-/- mice, inducing type 1 or type2/metabolic syndrome models mice.
1) Type I diabetic mice (129sv background): we injected streptozotocin (50mg/kgBW) for 5
consequence days and confirmed type 1 diabetes in stox1-/- and control mice (both 129sv
background). We found after 4 weeks, some of the diabetic stox1-/- mice (129sv) developedmassive proteinuria and ascites whereas nothing happened in control diabetic mice. Bloodpressure, blood sugar levels were not different between all the groups. Histological analysis
reveals that diabetic stox1-/- mice displayed significant interstitial fibrosis when compared tocontrol diabetic mice. Glomerular hypertrophy was not change in the groups but some mice
exhibited massive mesangiolysis and increased deposition of mesangial matrix in diabetic stox1-/mice. Urine albuminuria was significantly increased in diabetic stox1-/- mice when compared tocontrol diabetic mice. Therefore basically we stox1-/- would be the gene responsible for diabetic
nephropathy deterioration. Kidney renin is higher in diabetic stox1-/- mice.
2) Type I diabetic mice (c57Bl6 background): we tried to analyze often used diabetic mice, C57Bl6
background. We backcrossed stox1-/- mice (129sv background) with wild type C57bl6 mice for- 71 -

8 generation to make stox1-/- mice (C57Bl6 background). We successfully made the mice andinduced same experiment using STZ injection. Again there were no differences in blood pressure
and blood sugar between diabetic stox1-/- mice or control diabetic mice. However we did findno difference in albuminuria, glomerulosclerosis, and tubulo-interstitial fibrosis in all groups
analyzed. different from 129sv mice, kidney renin was no changed in C57Bl6 stox1-/- mice.
3) Metabolic syndrome mice: we analyzed metabolic syndrome mice by feeding high fat diet
(30% fat in energy) using C57Bl6 mice (129sv background mice are high fat diet resistant it isreasonable to use C57Bl6 background). We found massive deposition of lipid droplet in control
high fat fed mice but in stox1-/- high fat fed mice, there is obviously less lipid droplet in the tubule.
Lipid droplet have shown to play protective roles in kidney damage and less drop let in stox1-/- mice
may be pathological significance, even though we could not find significant histological evidenceof kidney damage in this metabolic syndrome models.

Discussion & Conclusion
Our analysis revealed that stox1 could play protective roles in kidneys under diabetic conditions.
Some interesting lines of evidence are upcoming such as strain specific sensitivity of stox1
deficiency in type 1 diabetic mice. Also in high fat fed mice lipid droplet formations are less instox1 deficient mice compared with control mice with high fat diet. When thinking about the sharedmolecular mechanisms between preeclampsia and metabolic syndrome and diabetes, some possible
connected mechanisms can be hypothesized from stox1 deficiency mice such as vascular defects,
endothelial dysfunction and biochemical abnormality such as autophagy deficiency. Therefore stox1
deficiency could be involved in the organ dysfunction in diabetes or metabolic syndrome. We are
now analyzing much detailed molecular mechanisms involvement including microRNA profilings.

References
1. van Dijk M, Mulders J, Poutsma A, Konst AA, Lachmeijer AM, Dekker GA, Blankenstein MA,
Oudejans CB: Maternal segregation of the Dutch preeclampsia locus at 10q22 with a new memberof the winged helix gene family. Nat Genet 2005;37:514-519
2. Berends AL, Bertoli-Avella AM, de Groot CJ, van Duijn CM, Oostra BA, Steegers EA: STOX1gene in pre-eclampsia and intrauterine growth restriction. Bjog 2007;114:1163-1167
3. Iglesias-Platas I, Monk D, Jebbink J, Buimer M, Boer K, van der Post J, Hills F, Apostolidou S,
Ris-Stalpers C, Stanier P, Moore GE: STOX1 is not imprinted and is not likely to be involved in
preeclampsia. Nat Genet 2007;39:279-280; author reply 280-271
4. Kivinen K, Peterson H, Hiltunen L, Laivuori H, Heino S, Tiala I, Knuutila S, Rasi V, Kere J:
Evaluation of STOX1 as a preeclampsia candidate gene in a population-wide sample. Eur J Hum
Genet 2007;15:494-497- 72 -

一般の皆様へ
糖尿病患者数は年々増加しており、予備軍を含めると日本では 2000 万人に達すると考えられます。
糖尿病合併症症例も増加しており、その中でも糖尿病性腎症による透析導入は全体の 44% にものぼります。したがって、その対策は急務ですが、画期的治療薬はなく、新しい治療標的を見つける事が必要です。今回の検討では糖尿病と妊娠高血圧腎症の間にある共通点に注目し、妊娠高血圧腎症
とかかわりが強いたんぱく質 stox1 に注目して検討しています。現在までに stox1 の働きが十分でないと糖尿病状態で腎臓を保護できない可能性が分かってきております。今後治療標的になりうるかを更に検討いたします。

- 73 -

Novel role of neuronal Ca2+ sensor-1 as a survival factor
activated in cardiomyocytes under stress.
Tomoe Nishitani
National Cerebral and Cardiovascular Center
tomoen@ri.ncvc.go.jp

Abstract
In this study, we assessed the role of neuronal Ca2+ sensor-1 (NCS-1) in cardiac survival understress. We found that NCS-1-/- myocytes were more susceptible to various kinds of stressors, withhaving reduced Akt activity. Using RNA interference, we identified phosphatidylinositol 4-kinase(PI4K) as an NCS-1 target that promotes cell survival. NCS-1 accelerates phosphoinositide turnoverand increases stimulation-elicited Ca2+ levels, which in turn activate PI3K/Akt pathway as well as
mitochondrial ATP synthesis. These would promote cardiomyocyte survival under stress.
Key Words : Calcium sensor, heart, survival, stress

Introduction
Dysregulation of Ca2+ homeostasis in cardiomyocytes often results in heart failure. Identifying
molecular targets that regulate cardiomyocyte survival is therefore of therapeutic importance.
Neuronal Ca 2+ sensor-1 (NCS-1) is a small EF-hand Ca 2+-binding protein that is important forneuronal functions. Using NCS-1-/- mice, we recently identified a cardiac function for NCS-1 asa positive regulator of Ca2+ signals in immature and diseased hearts1. Since the mortality rate inneonatal NCS-1-/- mice was high and we previously reported that NCS-1 is involved in neuronalsurvival2, we hypothesized that NCS-1 may be cardioprotective, especially under stress.

Results
Cultured neonatal mouse ventricular myocytes (NMVMs) or whole hearts from the wild-type(WT) and NCS-1-/- groups were subjected to several kinds of stressors (serum/glucose depletion,oxidative stress, and ischemia-reperfusion injury), and the resistance to stress-induced toxicity wasevaluated by measuring the time to cessation of spontaneous beating, LDH release, live/dead cellstaining and infarct size.
Long-term culture in serum-free media resulted in decrease in the spontaneous beating rate of
NMVMs much faster in Ncs1-/- group compared to WT group, and Ncs1-/- myocytes ultimately
ceased their spontaneous beating and morphological change occurred after 1 week of treatment. Cell
viability analysis by counting the condensed chromosome in nuclei using Hoechst 33258-staining
revealed that more apoptosis was induced in Ncs1-/- myocytes than WT myocytes. Similar
vulnerabilities were also detected in Ncs1-/- group when we used H2O2 and 2-deoxyglucose to deplete
cellular glucose as stressors.

- 74 -

Such cytotoxicity was largely prevented by WT NCS-1 overexpression but not by the low Ca 2+binding NCS-1 mutant E120Q, suggesting that NCS-1 protects cardiomyocytes from stress-induced
cytotoxicity in a Ca 2+ -dependent manner. In NMVMs, a major survival pathway—PI3K/Akt
signaling—was significantly reduced in the NCS-1-/- group, demonstrating that NCS-1 is importantfor cardiomyocyte survival.
To clarify the molecular mechanism of NCS-1-mediated cardiomyocyte survival, we focusedon phosphatidylinositol 4-kinase (PI4K), a molecule located upstream of the PI3K/Akt pathway,
and known to interact with NCS-1 in neurons3. Immunoprecipitation and immunofluorescenceanalysis revealed that NCS-1 interacts and co-localized with PI4K in the heart. Furthermore, NCS1 increased the amount of the activation product PI4P, suggesting that PI4K is indeed activatedby NCS-1 in the heart. By measuring Kir3.1 K+ current, which was used as a PI(4,5)P2 sensor, wefound that NCS-1 promotes the production of a downstream signal molecule PI(4,5)P2 as well as
PI4P. Furthermore, PI4K RNA silencing decreased cardiomyocyte resistance to stress-induced
cytotoxicity as observed for NCS-1-/-, indicating that PI4K is a target of NCS-1, which promotes
cardiomyocyte survival under stress.
Phosphoinositide turnover affects receptor stimulation-elicited Ca 2+ signaling, a process involvedin various cellular responses, we next compared the intracellular Ca 2+ levels between WT and
NCS-1-/- myocytes before and after receptor stimulation. Receptor-stimulation-elicited intracellular
Ca 2+ increase was significantly diminished in NCS-1-/- myocytes under extracellular Ca 2+-free
condition. Since increased Ca 2+ signals are reported to be required for activation of PI3K/Akt
pathway, we analyzed the stimulation-elicited translocation of EGFP-Akt-PH, which was used as anindicator of PI3K/AKt pathway activation. Compared to WT group, plasma membrane localizationof EGFP-Akt-PH was much less in NCS-1-/- group after receptor-stimulation. Furthermore, because
increase in Ca2+ signaling also affects mitochondrial ATP syntheses, we measured intracellular ATP
levels. We found that ATP levels were also considerably lower in NCS-1-/- myocytes. These results
are consistent with the notion that stress-induced survival signaling is less activated in NCS-1-/myocytes.
We finally compared the susceptibility of whole hearts to ischemia-reperfusion injury using
Langendorff apparatus. The infarct area were significantly larger in NCS-1-/- hearts than that of WT
hearts (39% ± 3.5% and 22% ± 4.1% for NCS-1-/- and WT mice, respectively; n = 6 for each). As
observed for NMVM, phosphorylation levels of Akt and its downstream molecules GSK3beta andmTOR were both largely decreased in KO hearts, demonstrating that NCS-1 plays an important rolein stress-tolerance in whole heart.

Discussion & Conclusion
We found that NCS-1 promotes cardiomyocyte survival under stress: in NCS-1-/- hearts, Akt -amajor survival factor- was less activated upon stimulation with stressors. The NCS-1 effects are
partially mediated by the activation of PI4K, which accelerates phosphoinositide turnover. Although,- 75 -

the level of PI(4,5)P2 is reported to decrease in the animal model of heart failure possibly becauseof jeopardizing cardiac cell function3, decrease in phosphoinositides does not necessarily lead toinhibited Akt activation4. We found that NCS-1 modestly increases receptor stimulation-elicited
Ca2+ levels, which itself can activate PI3K/Akt pathway5. Increase in cytosolic Ca 2+ level is also
involved in mitochondrial ATP synthesis, and we indeed detected that both activation of PI3K/Akt
pathway and cytosolic ATP levels were decreased in NCS-1-/- myocytes.
In conclusion, we identified a novel role of NCS-1 as a survival factor activated in cardiomyocytes
under stress.

References
1. Nakamura TY, Jeromin A, Mikoshiba K, Wakabayashi S. Neuronal calcium sensor-1 promotes
immature heart function and hypertrophy by enhancing Ca2+ signals. Circ. Res. 2011;109:512-523
2. Nakamura TY, Jeromin A, Smith G, Kurushima H, Koga H, Nakabeppu Y, Wakabayashi S,
Nabekura J. Novel role of neuronal Ca2+ sensor-1 as a survival factor up-regulated in injured
neurons. J. Cell Biol. 2006;172:1081-1091
3. Ziegelhoffer A, Tappia PS, Mesaeli N, Sahi N, Dhalla NS, Panagia V. Low level of sarcolemmal
phosphatidylinositol 4,5-bisphosphate in cardiomyopathic hamster (UM-X7.1) heart. Cardiovasc.
Res. 2001;49:118-126
4. Chu KM, Minogue S, Hsuan JJ, Waugh MG. Differential effects of the phosphatidylinositol
4-kinases, PI4KIIalpha and PI4KIIIbeta, on Akt activation and apoptosis. Cell Death Dis.
2010;1:e106
5. Yang F, Feng L, Zheng F, Johnson SW, Du J, Shen L, Wu CP, Lu B. GDNF acutely modulates
excitability and A-type K(+) channels in midbrain dopaminergic neurons. Nat. Neurosci.
2001;4:1071-1078

一般の皆様へ
心筋梗塞、心不全などの心疾患は、現在、世界でも死因の上位を占める重篤な社会問題である。
心筋細胞の生死がどのような経路で調節されるか理解することが心不全の予防に重要であるが、その全容は未だ明らかでない。本研究では、心筋細胞の生存に関わる因子を明らかにすることを目的とし、NCS-1 というタンパク質が重要な役割を担うことを見出した。NCS-1 は、筋収縮や細胞死などに重要な働きをもつ細胞内カルシウムを鋭敏に察知する、“ カルシウムセンサー ” と呼ばれる一群のタンパク質の１つである。ストレスにより細胞内カルシウム濃度が上昇すると、NCS-1 およびその関連分子が活性化され、最終的に心筋細胞の生存を促す経路が活性化させることがわかった。本研究をさらに深めることにより、各種心疾患の予防につながる可能性がある。

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Paternal uniparental disomy 14 and related disorders: placental gene
expression analyses and histological examinations
Masayo Kagami
Department of Molecular Endocrinology,
National Research Institute for Child Health and Development
kagami-ms@ncchd.go.jp

Abstract
Placental study of two cases with UPD(14)pat, and a case with maternal microdeletion involving
DLK1, DMRs and MEG3 showed as follows: 1) In the placentas of UPD(14)pat revealing
placentomegaly, the expression of DLK1 and RTL1 increased, even though in the placenta of a casewith maternal microdeletion revealing placentomegaly, only the expression of RTL1 increased.
2) Electron microscopic study showed hyperplasia of the endothelial cells and the pericytes in
the placentas of UPD(14)pat. 3) Expression analysis using fresh placental samples of UPD(14)patshowed excessive RTL1 expression because of a synergic effect between the biallelic activation of
RTL1 and loss of functional microRNA-containing RTL1as as a repressor for RTL1. These results
indicate that excessive RTL1 expression causes placentomegaly and RTL1 expression is regulated
through an RNAi mechanism.
Key Words : UPD(14)pat, placenta, RTL1, histology

Introduction
Although recent studies in patients with UPD(14)pat and other conditions affecting thechromosome 14q32.2 imprinted region have successfully identified underlying (epi)genetic factors
for the development of UPD(14)pat phenotype, several matters including regulatory mechanism(s)for RTL1 expression, imprinting status of DIO3, and placental histological characteristics remain tobe elucidated.

Results
We first performed molecular studies using fresh placental samples from two patients with
UPD(14)pat. After correction for the amount of expression positive cells, RTL1 expression level
was assessed to be ~5 times higher in the placental samples of the two patients than in the control
placental samples, whereas DIO3 expression level was evaluated to be similar between the placental
samples of the two patients and the control placental samples. We next carried out histologicalstudies using the above fresh placental samples and formalin-fixed and paraffin-embedded placental
samples obtained from a patient with a maternally derived microdeletion involving DLK1, the
IG-DMR, the MEG3-DMR, and MEG3. Terminal villi were associated with swollen vascularendothelial cells and hypertrophic pericytes, together with narrowed capillary lumens. DLK1, RTL1,- 77 -

and DIO3 proteins were specifically identified in vascular endothelial cells and pericytes, and thedegree of protein staining was well correlated with the expression dosage of corresponding genes.

Fig 1. Quantitative real-time PCR analysis using placental
samples. For a control, a pooled RNA sample consistingof an equal amount of total RNA extracted from threefresh control placentas was utilized.
A. Relative mRNA expression levels for DLK1, RTL1, andDIO3 against GAPDH (mean ± SE) and lack of MEGs
expression (indicated by arrows) (mir433 and miR127
are encoded by RTL1as) in the placental samples ofcases 1 and 2.
B. Relative mRNA expression levels for DLK1, RTL1,
and DIO3 against GAPDH (mean ± SE), in the equalamount of expression positive placental cells (vascularendothelial cells and pericytes) of cases 1 and 2(corrected for the difference in the relative proportionof expression positive cells between the placental
samples of cases 1 and 2 and the control placental
samples, on the assumption that the DLK1 expression
level is "simply doubled" in the expression positive
placental cells of case 1 and 2).

Fig 2. Histological examinations.
LM: light microscopic examinations;
EM: electron microscopic
examinations; DLK1, RTL1, and DIO3:
immunohistochemical examinations forthe corresponding proteins. The arrows
and arrowheads in the EM findings
indicate endothelial cells and pericytes,
respectively.

Discussion & Conclusion
These results argue for RTL1as-encoded microRNAs functioning as a repressor for RTL1
expression, and argue against DIO3 being a paternally expressed gene. Furthermore, it isinferred that DLK1, RTL1, and DIO3 proteins, especially RTL1 protein, play a pivotal role in thedevelopment of vascular endothelial cells and pericytes.

References
1. Kagami, M., Matsuoka, K., Nagai, T., Yamanaka, M., Kurosawa, K., Suzumori, N., Sekita, Y.,
Miyado, M., Matsubara, K., Fuke, T. Kato F, Fukami M, Ogata T. (2012) Paternal uniparental
disomy 14 and related disorders: Placental gene expression analyses and histologicalexaminations. Epigenetics, 7 (10) : 1142–1150, 2012.

- 78 -

一般の皆様へ
14q32.2 領 域にはインプリンティング 遺伝 子が 多 数 存 在し、14 番 染 色体父 親 性ダイソミー(UPD(14)pat) は、羊水過多、ベル型の小胸郭、腹壁異常、胎盤過形成を示す。インプリンティン
グ遺伝子は胎盤・胎児の発育に重要な役割を果たし、マウスにおいて、本領域の父性発現遺伝子
Rtl1 は胎盤の成長・機能に関与することが明らかとなっているが、ヒトでは検討されていない。我々は UPD(14)pat 表現型を示す患者の胎盤解析で、UPD(14)pat 胎盤での発現量は、正常に比較し、父性発現遺伝子の RTL1 は 4-5 倍、DLK1 の発現量は約 2 倍を示し、母性発現遺伝子である MEG3,
MEG8, snoRNAs, microRNAs の発現は消失していた。DLK1 抗体、RTL1 抗体を用いた免疫染色では RTL1 については発現量が著しく増加していたが、胎盤過形成を認める微小欠失症例では DLK1の発現は正常と同程度であった。以上の結果から胎盤過形成は RTL1 の過剰発現によることがあきらかとなった。これまで RTL1 の発現制御はマウスにおいて検討されており、母性発現遺伝子である
Rtl1as 上の microRNAs が父性発現遺伝子の Rtl1 の発現を trans に抑制していることが明らかとなっていたが、ヒトでは検討されていなかった。本研究で、upd(14)pat 胎盤において、RTL1 発現量が正常コントロールに比較し 4-5 倍程度発現していることが明らかとなった。これは、14 番染色体がともに父親に由来することから、RTL1as 上の microRNA の発現がなく、RTL1 の発現抑制がなくなり、1 コ
ピーあたりの RTL1 の発現量が 2-2.5 倍となり、両アレルから 2 コピーの RTL1 が発現し、4-5 倍の発現量になったと推測される。以上の結果はヒトにおける RTL1 の発現制御が RTL1as 上の microRNAによって RNAi のメカニズムで行われていることを明らかにした初めての報告となった- 79 -

Mechanism of dNTPs supply at beginning of DNA replication
Hiroyuki Niida
Hamamatsu University School of Medicine
niidah@hama-med.ac.jp

Abstract
DNA を複製する際に原料となる dNTPs は細胞周期の S 期に合成され S 期後期に最大濃度となる。このため S 期初期には DNA 合成局所特異的な dNTPs 供給機構が存在すると考えられ、その仕組みに付いて検討を行った。
Key Words：DNA 複製、dNTPs

Introduction
生物にとり DNA の安定性を維持することは、癌をはじめとする疾患を防ぐ為に重要である。
DNA 損傷は DNA 複製過程において多数発生するため、DNA 複製を円滑に遂行することが安定生維持の基礎となる。最近の研究から、DNA 複製が行われる際にその原料となるデオキシリ
ボ核酸 (dNTPs) が十分に供給されないと DNA に多くの損傷が発生し、発癌の原因となることが報告された (Bester AC et al. Cell. 2011 )。

Results
dNTPs 濃度は細胞周期に依存して厳密にコントロールされており、S 期後期に濃度は最大となる。これは dNTPs 産生経路の律速酵素、リボヌクレオチドレダクターゼ (RNR) の調節サブユ
ニットが S 期に発現してくるに伴い酵素活性を獲得、増大する為であることが明らかにされている。DNA 複製が開始する前段階、G1 期において dNTPs 濃度は低い状態で保たれている。一方、dNTPs を基質とし DNA ポリメラーゼにより触媒される、DNA 複製速度は S 期初期から後期にかけて基本的に一定であると考えられている。このことから申請者は DNA 複製開始時において、
DNA ポリメラーゼに dNTPs を効率よく供給する機構が存在するものと予想した。この機構は発癌予防において重要な働きを担っていることが予想され、癌の発症リスクを減じる為の知見を与えると考えられる。
研究の方法としてはまず RNR が DNA 複製マシナリーに局在することを示すために PCNA とRNR の細胞内局在を免疫染色法により検討した。HeLa 細胞をコラーゲンコートしたカバースラ
イド上において培養し、Toriton X 処理を行い可溶性画分を除去した。このクロマチン結合サン
プルを RNR の触媒サブユニットである RRM1 と PCNA に対する抗体で免疫染色を行った。この結果 RRM1 と PCNA の共局在を確認することができた。
次に RNR が複製マシナリーに局在する分子機構を明らかにするために、DNA 複製過程に関わる分子との相互作用を検討した。293 細胞に HA タグを付けた RRM1 および調節サブユニット
RRM2 を強制発現させ抗 HA 抗体で RNR 複合体を精製し LC/MS により RNR と相互作用する分子の同定を試みた。しかしながらこの解析によって RNR 複合体に含まれる DNA 複製関連因- 80 -

子を同定することが出来なかった。
このため RNR と結合しうる分子のスクリーニングを既知の DNA 複製関連因子に絞り、それらに対する抗体をもちいて IP-Western blot を行った。この解析から DNA 複製前にライセンシングを行う pre-replicative complex の Cdc6 と MCM ヘリカーゼのサブユニット MCM6 を RNR と相互作用しうる分子として同定することが出来た。
RNR が Cdc6 および MCM6 と相互作用することから、RNR は Cdc6 依存的に DNA 複製起点にリクルートされ、ここで MCM6 と結合することで DNA 複製開始時に局所的に dNTPs を産生する機構が存在することが考えられた。この仮説を証明するために RNR の複製起点への局在を MCM4 origin における ChIP assay を行うことにより、RNR の DNA 複製起点への局在を検討した。この結果 RNR が MCM4 origin に局在していることが示された。

Discussion & Conclusion
RNR は dNTPs de novo 合成経路において NDPs から dNDPs への還元を触媒する、経路の律速酵素である。dNTPs は細胞増殖の際に染色体 DNA が複製されるための原料であり、哺乳動物細胞内においては細胞周期依存的に厳密にその濃度が制御されている。申請者は DNA 二重鎖切断修復時において dNTPs 合成が Tip60 依存的に損傷部位にリクルートされた RNR が行っていることを報告している（Niida et al. Genes & Development 2010）。本研究において RNR がDNA 複製起点に局在し、pre-RC complex の構成因子 CDC6 と G1/S 期特異的に結合することが見出された。このことは初期 DNA 合成開始時において DNA 複製部位に局所的な dNTPs 供給を行っていることを予想させる。DNA 合成が開始し複製マシナリーが鋳型 DNA上を新規 DNAを合成しながら移動する時には MCM6 との結合を介し RNR も移動して行くのかもしれない。本
研究をさらに進めるためには RNR と CDC6 および MCM6 との結合領域を決定し、その結合を妨げる様な点変異体を細胞内に導入した時の異常を測定することが重要であろうと考えられる。
現在この点について検討を進めている。

References
1. Bester AC, Roniger M, Oren YS, Im MM, Sarni D, Chaoat M, Bensimon A, Zamir G, Shewach
DS, Kerem B.: Nucleotide deficiency promotes genomic instability in early stages of cancer
development. Cell. 145:435-446. 2011.
2. Niida H, Katsuno Y, Sengoku M, Shimada M, Yukawa M, Ikura M, Ikura T, Kohno K, Shima
H, Suzuki H, Tashiro S, Nakanishi M.: Essential role of Tip60-dependent recruitment ofribonucleotide reductase at DNA damage sites in DNA repair during G1 phase. Genes Dev.
24:333-338. 2010.

一般の皆様へ
遺伝情報がコードされている DNA の安定性を維持することは癌などの疾患を防ぐために非常に重要です。今回の研究では DNA 合成酵素を至適な条件下で機能させるために必要なデオキシリボヌク
レオチド (dNTPs) を効率よく DNA 合成酵素に供給する仕組みについて研究を行いました。まだ全容は明らかとなっていませんが、とても重要な機構であると考えており研究を進めております。

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Characterization of mammalian homologue of Unc119
as a tumor suppressor
Kentaro Nakagawa
Tokyo Medical and Dental University
kentaro_nakagawa.mbc@tmd.ac.jp

Abstract
Mammalian homolog of Caenorhabditis elegans Unc119 (Munc119) functions as a specificmyristoyl receptor. Previous reports suggest its implication in the primary cilia formation. We
identified Munc119 as a putative binding partner of RASSF6, which is a tumor suppressor andlocalized at the primary cilia in epithelial cells. In this study we revealed that Munc119 interacts
with MDM2 and enhances its auto-ubiquitination and degradation to stabilize p53 and that its
depletion impairs p53-mediated G1/S arrest, delays DNA repair after DNA damage and eventually
results in polyploidy. These findings have revealed a novel function of Munc119 as a tumor
suppressor.
Key Words : Cell cycle; p53; RASSF; tumor suppressor

Introduction
Unc119 was discovered in Caenorhabditis elegans (C.elegans) based on the recessive mutation
affecting locomotion. Human homologue Munc119 was identified as a retina-specific gene 4(HRG4). Munc119 expression is reduced in human colon, stomach, and breast cancers. Munc119
interacts with various proteins and plays versatile roles in the primary cilia formation, trafficking oftransducinα, regulation of endocytosis, and activation of src family kinases. We identified Munc119
as a putative interactor with a tumor suppressor RASSF6 and analyzed the tumor suppressive role ofMunc119 in this study.

Results
Mammalian homolog of Munc119 is a RASSF6 interactor.
RASSF6 expression is suppressed in human cancers and is known as a tumor suppressor.
RASSF6 is closely related with the tumor suppressive Hippo pathway, but our previous findings
suggest that it exerts a tumor suppressive role independently of the Hippo pathway. To clarify themolecular mechanism behind RASSF6-mediated tumor suppression we performed a yeast twohybrid screening using RASSF6 as a bait on human kidney cDNA library and obtained Munc119 asa positive clone. We confirmed the co-immunoprecipitation of endogenous RASSF6 and Munc119
from rat liver and the co-localization of these proteins in rat kidney. As the in silico data support
that Munc119 expression is reduced in human cancers, we tested the possibility that Munc119 alsofunctions as a tumor suppressor.

- 82 -

Munc119 induces G1/S arrest in HeLa cells.
RASSF6 expression causes apoptosis and G1/S arrest in HeLa cells. In contrast Munc119 did not
induce apoptosis. Munc119 coexpression and depletion did not influence RASSF6-induced apoptosis
in HeLa cells. However Munc119 depletion enhanced cell proliferation and BrdU incorporation in
HeLa cels. In the cell cycle analysis, Munc119 expression induced G1/S arrest, while its depletion
promoted the cell cycle progression in HeLa cells.
Munc119 regulates cell cycle via p53.
We recently revealed that RASSF6-mediated apoptosis and cell cycle regulation depend on p53(the paper under revision). Therefore we examined whether p53 is involved in Munc119-mediatedcell cycle regulation. Munc119 expression did not induce G1/S arrest in p53-negative H1299 cells.
Munc119 depletion increased BrdU incorporation in p53-positive U2OS cells but not in p53-negative
SaOS2 cells. In Munc119-depleted HeLa cells, p53 degradation was facilitated. MG132 treatment
recovered p53 protein expression. We confirmed in experiments using heterologous HEK293FT
cells that Munc119 interacted with MDM2, the most important E3 ligase for p53, and enhanced
auto-ubiquitination.
Munc119 depletion impairs DNA repair and causes polyploidy.
We tested the idea that Munc119 depletion impairs G1/S checkpoint and leads to the genomic
instability. We treated HeLa cells with VP-16 to induce DNA damage and immunostained γ-H2AX
as a marker of DNA damage. γ-H2AX was detected immediately after the VP-16 treatment but
remarkably diminished at 24 h after VP-16 removal in the control cells. In contrast, γ-H2AX signals
remained in the Munc119-depleted HeLa cells. Munc119 depletion induced polyploid cells in VP-16treated HeLa cells.

Discussion & Conclusion
In this study we confirmed that Munc119 is a bona fide interactor of RASSF6. Unlike RASSF6,
Munc119 does not cause apoptosis, but plays a role in the cell cycle regulation. Our data suggestthat Munc119 interacts with MDM2 and regulates p53 protein expression. We propose a model thatupon DNA damage Munc119 facilitates MDM2 auto-ubiquitination and stabilizes p53 to induce G1/
S arrest. As Munc119 depletion impairs p53-mediated G1/S arrest and causes polyploidy, Munc119
could function as a tumor suppressor. It is highly important to test Munc119 expression in humancancer samples in the near future. We have recently found that RASSF6 exerts its tumor suppressiveroles depending on p53. We used p53-compromized HeLa cells for most experiments of this study
and cannot so far clarify whether and how Munc119 influences RASSF6-induced apoptosis and cell
cycle regulation. However now that p53 is important for the function of Munc119 as well as thatof RASSF6, we need to test how Munc119 works in p53-intact cells for further studies. It will be
intriguing to examine whether the cell cycle regulations by Munc119 and RASSF6 correlate with theprimary cilia formation.

- 83 -

一般の皆様へ
DNA の損傷や染色体分裂の異常は発がんやがんの悪性化に密接に関係しています。網膜変性症の原因遺伝子産物である Munc119 が、このような遺伝子に異常を生じた細胞の分裂を抑制し、腫瘍抑制因子として働きうることを私たちは明らかにしています。生命の設計図のおさめられている染色体を守る仕組みの解明につながることが期待されます。

- 84 -

Epigenetic involvement in a mouse model of E-cadherin-p53-deficientdiffuse-type gastric cancer
Shu Shimada
Division of Molecular Oncology, Graduate School of Medical and Dental Sciences,
Tokyo Medical and Dental University
shimada.monc@tmd.ac.jp

Abstract
Diffuse-type gastric cancer (DGC) exhibits rapid disease progression and a poor patient prognosis.
However, there is no optimal therapeutic strategy for DGC. We have reported an E-cadherin/p53
double conditional knockout (DCKO) mouse line that recapitulates human DGC histologically andmolecularly (Shimada et al., 2012). Furthermore, we have established DGC cell lines from primarytumors and lymph node metastases of the DCKO mice. In this study we identified that epigenetic
mechanism can play a significant role in diffuse-type gastric carcinogenesis, and demonstrated thatsome epigenetic drugs had tumor suppressor activity in vitro and in vivo using the mouse gastriccancer cell lines.
Key Words : Diffuse-type gastric cancer

Introduction
Although several types of anti-cancer drugs have been developed, none of them dramatically
improve overall survival of cancer patients. This is because human tumor cell lines commonlyused in laboratories bear little resemblance to primary cancer due to over passage, but xenograftsof human cancer specimens are not appropriate for drug testing because of cost and effort. We
have established a mouse model of DGC and cell lines from mouse DGC. Then, we clarified themechanism of DGC and screened anti-cancer molecules using the low-passage mouse cancer celllines, and then assessed the effectiveness of these candidates in vivo.

Results
Establishment of cell lines from DGC of the DCKO mice
We have established mouse gastric cancer cell lines (GC) from primary DGC and lymphatic
dissemination of the DCKO mice. We also utilized mouse gastric epithelial cell lines (GE) derived
from gastric mucosae of fetal p53-null mice (Fukamachi et al., 2004) as control. We examinedbiological traits of the GC cell lines comparing with those of the GE cell lines: 1) The former had
30-fold more sphere-forming capacity than the latter in serum-free condition using Ultra-Low
Attachment Culture Dishes (Figure 1A). 2) When subcutaneously injected to immunocompromised
mice with Matrigel, only 100 cells of the GC cell lines immediately induced large tumors, while
even 105 cells of the GE cell lines did not (Figure 1B). 3) The GC cell lines were resistant to- 85 -

Figure 1

5-Fluorouracil and Paclitaxel, cytotoxic agents generally used in patients with advanced gastriccancer (Figure 1C).
Epigenetic involvement in DGC formation
To identify what molecular mechanism plays an important role in DGC formation of the DCKO
mice, we performed microarray analysis of normal gastric mucosae and primary gastric cancer,
and compared their gene expression patterns. The results indicated that the expression levels of
frequently-methylated genes in several types of cancer including stomach cancer (Ohm et al.,
2 0 0 7 ) we r e d ow n - r e g u l a t e d
whe r e a s t ho s e of e pige ne t ic
modulators were up-regulated
in mouse DGC. We conducted
reverse-transcription polymerase
chain reaction (RT-PCR) andmethylation-specific PCR (MSP)of Mgmt and Sfrp5, reported asf requently-methylated genes,
and validated down-regulated
e x p r e s s io n ( Fig u r e 2 A) a n d
DNA methylation (Figure 2B)of t hese t wo genes i n mousecancer lesions. Quantitative RTPCR (qRT-PCR) also revealed
overexpression of Dnmt3b (DNAme t hylt r a n sfe r a s e), S u v39h1(H3K9 methyltransferase) andEzh2 (H3K27 methyltransferase)- 86 -

in primary gastric cancers and transplanted tumors (Figure 2C). These data suggest that epigenetic
alteration can be involved in diffuse-type gastric carcinogenesis.
Evaluation of epigenetic drugs in vitro
Next, we assessed tumor suppressor activity of epigenetic drugs, that is, 5-Aza-2’-deoxycytidine(AZA) as DNA methyltransferase inhibitor, Trichostatin A (TSA) and sodium butyrate (NaB) as
histone deacetylase inhibitors, Tranylcypromine (TCP) as Lsd1 (H3K4 demethylase) inhibitor,
Chaetocin as Suv39h1 inhibitor,
a nd 3 -D e a z a ne pla no ci n A(DZN) as Ezh2 inhibitor, usingt he G C a nd GE cell li nes.
T he dose -re s p on se cu r ve s
showed that AZA attenuatedproliferation only in the GC
cell lines (Fig u re 3A), andmor pholog ic a l ch a nge s ofthe mouse cancer cell lines
to epithelial phenotype wereo b s e r ve d b y t r e a t m e n t of
TSA, NaB and DZN, implying
dif ferent iat ion i nduct ion(Figure 3B). We also exposed
the GC cell lines to the six
epigenetic inhibitors describedabove in low dose (IC80) under
sphere-for ming conditions,because sphere -for m i ng
property of cancer cells correlates tumorigenicity in vivo in this experimental system, as often
reported in other studies. Remarkably, TSA, NaB and Chaetocin blocked self-renewal of the GC celllines almost completely (Figure 3C).
Evaluation of epigenetic drugs in vivo
Finally, two weeks after we subcutaneouslytransplanted 105 cells of GC1 in nude mice, we
intraperitoneally administered vehicle (10%dimethyl sulfoxide), TSA (10 mg kg-1), NaB(1200 mg kg-1) and Chaetocin (0.5 mg kg-1)
to these DGC-bearing mice every day. The
tumors of treated mice grew less rapidly than

- 87 -

those of control mice, and their volumes were strikingly reduced five and six weeks after medicalcare started (Figure 4). We also found the same results in GC3 (data not shown).

Discussion & Conclusion
We succeeded in culturing mouse DGC in vitro, and the cell lines exhibited high sphere-forming
and tumorigenic activity. The DCKO mice and their cancer cell lines provide us a relevant approachto research and development of DGC both in vivo and in vitro, respectively. Using this mousebased system, we identified that epigenetics contributed to DGC formation, and demonstrated that
epigenetic agents could be efficient in this preclinical trial.
Certainly, a critical problem still remains that only epigenetic inhibitors can not lead to complete
remission of DGC in this report. We are now investigating gene expression profiles of GC andGE cell lines, and have detected some oncogenic pathways activated in DGC of the DCKO mice.
Combining epigenetic agents with drugs targeted to such signaling pathways, we will achieve
radical treatment of DGC not only in the DCKO mice but also in human beings.

References
1. Fukamachi H, Ito K, Ito Y. Runx3-/- gastric epithelial cells differentiate into intestinal type cells.
Biochem Biophys Res Commun. 2004 321(1):58-64.
2. Ohm JE, McGarvey KM, Yu X, Cheng L, Schuebel KE, Cope L, Mohammad HP, Chen W,
Daniel VC, Yu W, Berman DM, Jenuwein T, Pruitt K, Sharkis SJ, Watkins DN, Herman JG,
Baylin SB. A stem cell-like chromatin pattern may predispose tumor suppressor genes to DNA
hypermethylation and heritable silencing. Nat Genet. 2007 39(2):237-42.
3. Shimada S, Mimata A, Sekine M, Mogushi K, Akiyama Y, Fukamachi H, Jonkers J, Tanaka H,
Eishi Y, Yuasa Y. Synergistic tumour suppressor activity of E-cadherin and p53 in a conditional
mouse model for metastatic diffuse-type gastric cancer. Gut. 2012 61(3):344-53.

一般の皆様へ
胃がんは、依然として発症数と死亡数が多いがんの一つです。胃がんの中でも、スキルス胃がんに代表される未分化型胃がん（DGC）は、浸潤と転移が著しく、予後が悪いことが知られています。我々は世界で最初に DGC のマウスモデルを作製し、その胃がんから細胞株を樹立することにも成功しました。それらを利用して、DGC ではエピジェネティクスが重要な働きをしており、エピジェネティクス阻害薬が実験室レベルのみではなく、生体レベルでも DGC の治療に有効であることがわかりました。今後は、この実験系を利用して、更なるメカニズムの解明と臨床への早期応用を目指していきます。

- 88 -

Characterization and generation of organ-specific vascular cells fromhuman iPS cells towards development of novel in vitro models for
intractable vascular diseases
Kenji Osafune
Center for iPS Cell Research and Application (CiRA), Kyoto University
osafu@cira.kyoto-u.ac.jp

Abstract
In this study, we aimed to characterize organ-specific vascular cells from cerebral artery, aorta,
pulmonary artery and renal glomeruli in order to develop the directed differentiation methods
from human iPS cells into the organ-specific vascular cell types. These methods could be used toestablish novel in vitro disease models for intractable vascular disorders with patient-derived iPS
cells.
Key Words : iPS cell, organ-specific vascular cell, in vitro model

Introduction
Among the most remarkable advances in iPS cell research is the embodiment of disease modelingusing patient-derived iPS cells. It has been shown that the in vitro iPS model successfully
recapitulates some aspects of intractable disorders, suggesting the feasibility that the iPS model
can provide a platform for studies aiming at both understanding pathological mechanisms anddiscovering new drug compounds. To create novel iPS disease models for intractable vasculardisorders, the specificity of vascular cells in each organ or tissue need to be characterized, and the
directed differentiation protocols from human iPS cells into organ-specific vascular cell types are
required.

Results(1) Characterization of organ-specific vascular cells.
We first tried to isolate vascular endothelial cells from mouse brain, lung, aorta and kidney tissues.
However, the tissue samples were so small and we found it very difficult to obtain enough amountof mRNA or protein from them. Then, we used the vascular samples removed from adult micro
mini pigs. The vascular tissue samples including both endothelia and smooth muscle cells frompig aorta, pulmonary artery and renal artery were collected and examined by Mass Spectrometry.
The results showed that these three samples shared the expression of many cytoskeleton proteins,
such as smooth muscle actin, cardiac muscle actin, skeletal muscle actin and actinin, while several
molecules specifically expressed in each of three vascular types were also identified, which could be
used to characterize and distinguish the three vascular types. We are currently examining the detailof both temporal and spatial expression patters of the candidate molecules using embryonic and- 89 -

adult mouse vascular samples. In addition, we are now trying to isolate only vascular endothelial
cell samples from adult micro mini pigs to focus on the endothelial analyses.
(2) Development of directed differentiation methods from human iPS cells into organ-specificvascular cells.
We performed high-throughput chemical screening to identify potent chemical compounds that
efficiently induce vascular endothelial cells from human iPS cells. To prepare the starting materials
for the screen, we used the previously-reported differentiation protocol into vascular endothelia
using two step treatment, 1st step with BIO (6-bromoindirubin-3’-oxime) and 2nd step with VEGF
(vascular endothelial growth factor; Homma K. et al., 2010). We examined the effects of adding
each of around 6,000 different chemicals into the 2nd step, in order to identify compounds thatsynergistically promote the differentiation into vascular endothelia with VEGF. Out of 6,000
chemicals examined, we found one hit compound that could increase the induction rate of vascularendothelia from human iPS cells from 10% up to 20%. We are optimizing the differentiation
protocols for vascular endothelia using the hit compound, examining the mechanisms of action of
the compound and characterizing the endothelial cells induced by the compound treatment.
(3) Derivation of patient-specific iPS cells from intractable vascular disorders.
We generated iPS cell lines from the patients with three intractable vascular disorders, intracranial
aneurysms associated with autosomal dominant polycystic kidney disease (ADPKD), microscopic
polyangiitis (MPA), and malignant hypertension, by reprogramming dermal fibroblasts or T cellswith retrovirus or episomal vector method. We examined the following tests in each cell line derived
from patients to select suitable iPS cell clones for further investigation, (1) silencing of transgene forreprogramming (2) pluripotent marker gene expression, such as OCT4, NANOG, SSEA3, SSEA4,
TRA1-60 and TRA1-81 (3) multipotent differentiation potential into three embryonic germ layers
using embryoid body (EB) and teratoma formation (4) karyotype analyses (5) transcriptome (6)methylation analyses (7) mutational analyses of causative genes. Then, we confirmed that thesepatient-derived iPS cell lines can be differentiated into both vascular endothelia and smooth muscle
cells using the previously-reported vascular differentiation protocol (Homma K. et al., 2010).

Discussion & Conclusion
In Mass Spectrometry analyses of adult micro mini pig vascular samples, most of the identified
molecules were cytoskeleton proteins which are normally expressed in muscular cells. We guessedthat the majority of cells included in the vascular samples were smooth muscle cells rather
than endothelia, and then smooth muscle-related genes were mainly identified. These results
indicated that the purification of endothelial cells from the samples was required to characterize
endothelial signatures of organ-specific vasculature. We are now isolating the endothelial cellsby flow cytometry sorting using an endothelial cell surface marker, VE-cadherin. In conclusion,
vascular cells may be different in protein expression patterns among the organs or tissues they
are belonging to. In this study, we also identified the candidate chemical compound that promotes- 90 -

the differentiation of human iPS cells into vascular endothelia, and succeeded in deriving patientspecific iPS cell lines from the three intractable vascular disorders.

References
1. Sone M. et al., Different differentiation kinetics of vascular progenitor cells in primate and mouseembryonic stem cells. Circulation. 2003; 107(16): 2085-8.
2. Yurugi-Kobayashi T. et al., Adrenomedullin/cyclic AMP pathway induces Notch activation anddifferentiation of arterial endothelial cells from vascular progenitors.
Arterioscler Thromb Vasc Biol. 2006; 26(9): 1977-84.
3. Homma K. et al., Sirt1 plays an important role in mediating greater functionality of human ES/
iPS-derived vascular endothelial cells. Atherosclerosis. 2010; 212(1): 42-7.

一般の皆様へ
本研究は、特定の臓器の血管にしか発生しない難治性の血管疾患に対する iPS 疾患モデルを作製するために、脳動脈、大動脈、肺動脈、腎動脈の血管細胞の遺伝子発現の特徴などを解明し、その情報を基にヒト iPS 細胞から各臓器特異的な血管細胞の作製法開発を目指すものである。

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Fine mapping of autophagy-related proteins during autophagosome
formation in Saccharomyces cerevisiae
Kuninori Suzuki
University of Tokyo
kuninori@k.u-tokyo.ac.jp

Abstract
Autophagy is a bulk degradation system mediated by biogenesis of autophagosomes understarvation conditions. Here, we visualized forming autophagosomes as cup-shaped structures using
fluorescence microscopy by enlarging a selective cargo of autophagosomes, and finely mapped the
localizations of autophagy-related (Atg) proteins. The results obtained by this observation suggestthat Atg proteins play individual roles at spatially distinct localizations during autophagosome
formation. These findings will facilitate detailed investigations of the function of each Atg protein
during autophagosome formation.
Key Words : autophagy; autophagosome; autophagy-related (ATG) genes; endoplasmic reticulum
exit sites (ERES); yeast

Introduction
Autophagy is a bulk degradation system widely conserved among eukaryotes from yeast to
mammals. When eukaryotic cells are faced with nutrient starvation, they degrade cytoplasmic
materials to obtain building blocks that help them adapt to severe environmental conditions.
Currently, over 30 ATG genes have been identified as necessary for various types of autophagy
in yeast. Among them, 18 ATG genes are essential for autophagosome formation upon starvation(Mizushima et al., 2011; Nakatogawa et al., 2009). These Atg proteins are involved in autophagosome
formation as components of several discrete functional units, but their interrelationships are not yet
well understood.

Results
Among Vps (vacuolar protein sorting) proteins, which are involved in sorting of Prc1
(carboxypeptidase Y; CPY) to the vacuole (Raymond et al., 1992), Vps15, Vps30, and Vps34 areessential for autophagosome formation as components of phosphatidylinositol (PtdIns) 3-kinase
complex I (Kametaka et al., 1998; Kihara et al., 2001; Obara et al., 2008). We investigated the
localizations of representative GFP-fused Atg and Vps proteins in GAC cells expressing 2×mCherryAtg8 (Ch-Atg8), and finely mapped the localization of Atg/Vps proteins during autophagosome
formation. Atg13 and Atg17, and PtdIns 3-kinase complex I were localized to a dot at the junction
between the forming autophagosome and the vacuolar membrane, which we termed VICS. By
contrast, Atg1, Atg8, and the Atg16–Atg12–Atg5 complex labeled the VICS and the forming- 92 -

autophagosome. We measured the fluorescence intensity along the forming autophagosome andfound that the profiles of these proteins and Ch-Atg8 overlapped extensively. The Atg2–Atg18
complex and Atg9 were distributed as two or three dots associated with the forming autophagosome;
some of these dots were apparently distinct from the VICS. Time-lapse microscopy showed that
Atg2-GFP was initially visualized as a dot and subsequently appeared as multiple dots at the edge ofthe forming autophagosome.
In mammalian cells, physical connections between forming autophagosomes and the endoplasmic
reticulum (ER) have been observed by electron microscopy (Hayashi-Nishino et al., 2009; YlaAnttila et al., 2009). We found that the forming autophagosome was localized in the close proximity
to the ER. When we labeled the ER lumen with GFP-HDEL (Okamoto et al., 2006) and observed
cells with three-dimensional fluorescence microscopy, we found that the forming autophagosome
was associated with the ER at two or three sites. Despite close association of the forming
autophagosome with the ER, neither ER transmembrane nor lumenal proteins were ever detected at
the forming autophagosome.
ER exit sites (ERES), which are subdomains of the ER specifically responsible for the productionof COPII vesicles, can be labeled by fluorescent protein-tagged Sec13 or Sec16 (Connerly et al.,2005). We visualized the localization of ERES in GAC cells by three-dimensional fluorescencemicroscopy, using Sec13-GFP as a marker, and found that the forming autophagosome was localized
in the close proximity to ERES at two or three sites per cell. The same result was obtained when
Sec16-GFP was used. Association of the forming autophagosome with ERES labeled with Sec16GFP was clearly detected in 79% of the cells (38 cells were examined three-dimensionally). Sec16,
which is essential for ER-to-Golgi transport, is thought to be involved in organization of ERES asa scaffold (Connerly et al., 2005). Previously, our group showed that a temperature-sensitive sec162 mutant is defective in autophagy at the non-permissive temperature (Ishihara et al., 2001). These
data suggest that ERES play important roles in autophagosome formation.
As mentioned above, forming autophagosomes are associated with the ER at two or three sites percell. We also show that the Atg2-Atg18 complex forms two or three dots at the edge of the forming
autophagosome. These observations prompted us to examine the spatial relationship between ERES
and the Atg2-Atg18 complex. By three-dimensional fluorescence microscopy, we found that dots
labeled with Atg18 along the GAC were adjacent to ERES.

Discussion & Conclusion
In this study, taking advantage of yeast genetics, we visualized forming autophagosomes byfluorescence microscopy and finely mapped the localizations of Atg proteins. We found that the
Atg13, Atg17, and PtdIns 3-kinase complex I were localized to the VICS. Atg1, Atg8, and the
Atg16–Atg12–Atg5 complex labeled the VICS and the forming autophagosome. We also found that
the Atg2-Atg18 complex and Atg9 were localized to the edge of the forming autophagosome but not
to the VICS. Moreover, we showed that the forming autophagosome is associated with the ER via- 93 -

ERES. Taken together, our data show that Atg proteins play individual roles at spatially distinct sites
during autophagosome formation. These results will facilitate investigations of each Atg protein’sfunction in the near future.

References
1. Connerly, P. L., Esaki, M., Montegna, E. A., Strongin, D. E., Levi, S., Soderholm, J. and Glick,
B. S. (2005). Sec16 is a determinant of transitional ER organization. Curr. Biol. 15, 1439-1447.
2. Hayashi-Nishino, M., Fujita, N., Noda, T., Yamaguchi, A., Yoshimori, T. and Yamamoto, A.
(2009). A subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation.
Nat. Cell Biol. 11, 1433-1437.
3. Ishihara, N., Hamasaki, M., Yokota, S., Suzuki, K., Kamada, Y., Kihara, A., Yoshimori, T., Noda,
T. and Ohsumi, Y. (2001). Autophagosome requires specific early Sec proteins for its formation
and NSF/SNARE for vacuolar fusion. Mol. Biol. Cell 12, 3690-3702.
4. Kametaka, S., Okano, T., Ohsumi, M. and Ohsumi, Y. (1998). Apg14p and Apg6/Vps30p form aprotein complex essential for autophagy in the yeast, Saccharomyces cerevisiae. J. Biol. Chem.
273, 22284-22291.
5. Kihara, A., Noda, T., Ishihara, N. and Ohsumi, Y. (2001). Two distinct Vps34 phosphatidylinositol
3-kinase complexes function in autophagy and carboxypeptidase Y sorting in Saccharomyces
cerevisiae. J. Cell Biol. 152, 519-530.
6. Mizushima, N., Yoshimori, T. and Ohsumi, Y. (2011). The role of Atg proteins in autophagosome
formation. Annu. Rev. Cell Dev. Biol. 27, 107-132.
7. Nakatogawa, H., Suzuki, K., Kamada, Y. and Ohsumi, Y. (2009). Dynamics and diversity in
autophagy mechanisms: lessons from yeast. Nat. Rev. Mol. Cell. Biol. 10, 458-467.
8. Obara, K., Noda, T., Niimi, K. and Ohsumi, Y. (2008). Transport of phosphatidylinositol
3-phosphate into the vacuole via autophagic membranes in Saccharomyces cerevisiae. Genes
Cells 13, 537-547.
9. Okamoto, M., Yoko-o, T., Umemura, M., Nakayama, K. and Jigami, Y. (2006).
Glycosylphosphatidylinositol-anchored proteins are required for the transport of detergentresistant microdomain-associated membrane proteins Tat2p and Fur4p. J. Biol. Chem. 281, 40134023.
10. Raymond, C. K., Howald-Stevenson, I., Vater, C. A. and Stevens, T. H. (1992). Morphologicalclassification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar
compartment in class E vps mutants. Mol. Biol. Cell 3, 1389-1402.
11. Yla-Anttila, P., Vihinen, H., Jokitalo, E. and Eskelinen, E. L. (2009). 3D tomography reveals
connections between the phagophore and endoplasmic reticulum. Autophagy 5, 1180-1185.

一般の皆様へ
単細胞生物である出芽酵母（パン酵母）も我々哺乳類も、遺伝情報を含む DNA が膜で区画化された真核生物と呼ばれる生き物です。こうした膜区画がどのような分子機構で形成されているかはほ
とんど分かっておらず、我々は栄養飢餓で誘導されるオートファゴソームと呼ばれる膜区画の形成過程に注目して解析を進めています。本研究は形成中のオートファゴソーム上でオートファゴソーム形成に関わるタンパク質の分布を詳細に明らかにした点で斬新であり、今後の個別のタンパク質の機能解析に向けての大きな手がかりとなるものです。

- 94 -

Synthesis of fatty acids having various carbon frameworks
Takanori Iwasaki
Department of Applied Chemistry, Graduate School of Engineering, Osaka University
iwasaki@chem.eng.osaka-u.ac.jp

Abstract
New synthetic method of fatty acids using nickel butadiene system catalyzed cross-coupling
reaction between alkyl groups was developed. Unsaturated fatty acid and its regioisomers can beprepared by iterative coupling using the catalytic system. The Ni catalyst was also effective for the
introduction of secondary alkyl groups into C2 position of benzothiazoles via C-S bond cleavage.
Key Words : fatty acid, cross-coupling, Grignard reagent, alkyl halides, benzothiazole

Introduction
Fatty acids are essential to natural life because they act as key components in our body and havevarious carbon chain frameworks, where unsaturated bond(s) as well as branched structure are often
founds. Therefore, development of efficient synthetic method to construct carbon frameworks ofnatural and artificial fatty acids or their synthetic equivalents is important task.

Results
Transition metal-catalyzed cross-coupling reactions are one of the most important tools toconstruct new carbon skeletons from simple organic molecules in synthetic organic chemistry andnow are widely used in various areas including functional materials, drug synthesis and so on.
However, cross-coupling between two alkyl groups is rarely discussed compared to that of vinyl
and aryl groups partly due to easy β-hydrogen elimination from alkylmetal intermediates.1 We havedeveloped transition metal-catalyzed cross-coupling reaction of alkyl halides with organometallic
reagents in the presence of unsaturated hydrocarbon like 1,3-butadiene as an appropriate additive.2
Because fatty acids consist of long saturated hydrocarbon chain, this C-C bond forming reaction
can be assumed as an appropriate synthetic tool for constructing carbon frameworks of fatty
acids though organometallic reagents as well as transition metal are known to be reactive toward
carboxylic acid moiety in fatty acids, leading unwanted side-reactions. To overcome such drawback,
we initiated our study on the synthesis of fatty acids using cross-coupling reaction by searchingsuitable conditions to achieve selective coupling reaction at C-halogen bond over carboxylic acidmoiety and found that pre-treatment of bromoalkanoic acid with 1 equivalent of t-BuMgCl was
effective to protect carboxylic acid moiety by forming magnesium carboxylate and cross-coupling
reaction smoothly proceeded by catalytic amount of NiCl2 in the presence of 1,3-butadiene as anadditive as shown in eq 1. The present catalyst was effective for various bromoalkanoic acids as well
as alkyl Grignard reagents and fatty acids having various carbon chains were obtained in good to
excellent yields.3- 95 -

By using the Ni-catalyzed cross-coupling reaction, we succeeded to prepare long unsaturated fatty
acid, nervonic acid, from readily available oleic acid by reduction, bromination and cross-coupling
sequence in good yield.
In addition, trans fatty acid, elaidic acid, and its regioisomers could be synthesized by iterative
coupling as shown in scheme 1. It is well known that trans fatty acids increase the risk of coronary
heart disease, however, toxic assessment of their regioisomers is not well explored due to thesynthetic difficulty of such isomers. Therefore, the synthetic method to obtain these regioisomers inselective manner is highly desirable to evaluate their health risk.
For example, 6-bromohexanoic acid coupled with Grignard reagent having trans olefin andsilyl ether moieties to give corresponding 12-bromo-9-dodecenoic acid in 72% yield after direct
bromination of silyl group by Br2PPh3. Subsequent coupling of the 12-bromo-9-dodecenoic acidwith hexyl Grignard reagent gave elaidic acid in 90% yield. Regioisomers of elaidic acid havingtrans olefin moiety at ω-5 to ω-8 positions were prepared by changing length of starting material,
bromoalkanoic acid and Grignard reagent, in good to excellent yields as sole regioisomers.

T h e N i / b u t a d i e n e s y s t e m w a s a l s o g o o d c a t a l y s t fo r t h e c o u pl i n g r e a c t i o n ofmethylthiobenzothiazoles with secondary alkyl Grignard reagents through C-S bond cleavage,
providing alkylated benzothiazoles in good yields (eq 2).4 Interestingly, the cross-coupling did notproceed in the absence of 1,3-butadiene additive. Because thiazole derivatives having alkyl groups
on C2 position are synthetic equivalents of fatty acids and often found in natural and synthetic
compounds, the present catalyst provides a simple and efficient method for introducing an alkyl
group in this position.

- 96 -

Discussion & Conclusion
The present Ni-catalyzed cross-coupling of bromoalkanoic acids and benzothiazoles with alkyl
Grignard reagents provides useful synthetic tools to synthesize carboxylic acids and their synthetic
equivalents having various carbon frameworks and will open up new fields of fatty acid chemistryin both biological and materials disciplines. The iterative coupling using Grignard reagents havingsiloxy group was found to be useful building block to construct various carbon frameworks.
The alkylation of benzothiazoles using Ni-butadiene catalytic system was also developed, wherecongested secondary alkyl groups were efficiently introduced into C2 position of benzothiazoles
through C-S bond cleavage. Further investigations on synthesis of biological active compounds
using transition metal-catalyzed cross-coupling reaction are ongoing in our group.

References
1. N. Kambe, T. Iwasaki, J. Terao Chem. Soc. Rev. 2011, 40, 4937-4947.
2. N. Kambe, J. Terao, T. Iwasaki J. Synth. Org. Chem. Jpn. 2011, 69, 1271-1281.
3. T. Iwasaki, K. Higashikawa, V. P. Reddy, W. W. S. Ho, Y. Fujimoto, K. Fukase, J. Terao, H.
Kuniyasu, N. Kambe Chem. Eur. J. 2013, 19, 2956-2960.
4. A. Ghaderi, T. Iwasaki, A. Fukuoka, J. Terao, N. Kambe Chem. Eur. J. 2013, 19, 2951-2955.

一般の皆様へ脂肪酸は多彩な炭素骨格を有する化合物群であり、生体内をはじめとし幅広く存在しています。この脂肪酸類の炭素骨格を構築する手法の開発は、微量天然脂肪酸や非天然脂肪酸の生理活性等を評価する上で重要な研究課題です。我々は、カルボキシル基を有するアルキルハライド類とアルキル
グリニャール試薬とのニッケル触媒によるクロスカップリング反応を利用し、一段階で様々な炭素骨格を有する脂肪酸類の合成に成功しました。また、炭素̶硫黄結合の切断を伴ったクロスカップリン
グ反応によりベンゾチアゾール類の 2 位に 2 級アルキル基を導入することにも成功した。

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Analyses of structural dynamics of a G protein-coupled receptor usingsite-directed fluorescent labeling.
Hisao Tsukamoto
Institute for Molecular Sciencetsukamoh@ims.ac.jp

Abstract
The effects of constitutively activating mutation on structural dynamics of opsin, a G proteincoupled receptor, were analyzed using site-directed fluorescence labeling techniques. Fluorescence
spectroscopic analyses revealed that the mutation makes the receptor protein more flexible and moreeasily converted to the active state, which couples and activates G protein.
Key Words : GPCR, fluorescence labeling, membrane protein, protein dynamics

Introduction
Constitutively activating mutations (CAMs) have been found in various G protein-coupled
receptors have been identified in various G protein-coupled receptors (GPCRs). These mutations
elevate G protein activation efficiency of ligand-free GPCRs. Also, some of CAMs are known to belinked to genetic diseases. It is still unclear how CAMs makes ligand-free GPCRs can couple andactive G protein in ligand-independent manner. In this study, effects of constitutively activating
mutation on structural dynamics of opsin, the ligand-free form of light sensing GPCR rhodopsin,were analyzed using site-directed fluorescence labeling techniques.

ResultsA f luorescence probe bimane was specifically attached on the cytoplasmic end of sixthtransmembrane helix of opsin with or without a CAM. This is because the sixth transmembrane
helix is known to move outward upon signal-induced activation of various GPCRs including
rhodopsin. Respective labeled opsins were successfully purified in a detergent solution. Using thesebimane labeled opsins, the fluorescence emission spectra were compared in the presence or absenceof the CAM. The fluorescence spectrum was changed by the CAM, indicating that the CAM
induces some conformational changes in the sixth transmembrane helix. Next, G protein mimetic
peptide was added to the labeled opsins and the peptide-induced spectral shift was analyzed. In thecase of normal opsin (without the CAM), addition of the peptide caused no changes, but the peptide
significantly shifted the fluorescence spectra of the labeled opsin with the CAM. These results
suggest that conformation of opsin is highly constrained to the inactive conformation, but the CAM
converts the opsin conformation into the active conformation that can interact with G protein. Also,binding of the G protein mimetic induced additional conformational changes only in opsin with the
CAM. These results indicate that conformational changes in the sixth helix are involved in not only

- 98 -

signal-induced activation but also mutation-induced activation of GPCRs.
In order to resolve the CAM-induced conformational changes in detail, the f luorescencequencher tryptophan was introduced on the fifth transmembrane helix of the labeled opsins. Thechanges in relative arrangement of the fifth and sixth helices are analyzed base on intra-molecularfluorescence quenching profiles. The analyses showed that the CAM caused rearrangement of thesixth transmembrane helix relative to the fifth one. This result confirmed that the CAM induces
rearrangement of the sixth helix like photo-activation of rhodopsin. Next, energetics aspect of the
CAM-induced conformational changes was analyzed. For this purpose, the dynamic quenching
constant was measured at various temperatures. The dynamic quenching constant is calculated as[1/tw – 1/t0], in which tw is a fluorescence lifetime in the presence of quencher tryptophan andto is a fluorescence lifetime without tryptophan. Using the constant values measured at various
temperatures, Arrhenius plot was constructed. The plot clearly showed that introduction of the CAM
decreased the slopes. That means that the activation energy in the formation of dynamic quenchingcomplex is decreased by the CAM. Since fluorophore and quencher were introduced on the sixthand fifth helices, respectively, this result strongly suggests that the CAM decreased energy barrier
between inactive and active conformations of opsin. In other words, the CAM makes conformationalchanges of the helices in opsin more easily. In that way, the CAM affects structural dynamics ofligand-free opsin.
Taken together, the CAM in ligand-free opsin caused some conformational changes and affected
the energetic aspect of conformational changes as well. Further analyses using various GPCRs andthe mutants labeled with a fluorescent probe would reveal how CAMs affect molecular properties
and structural features of GPCRs, resulting in signal-independent G protein activation. Furthermore,such analyses may provide new information to understand how some CAMs cause genetic diseases.

Discussion & Conclusion
This study clearly showed that f luorescence labeling study can assess how a CAM affectconformation(s) of a GPCR and its conformational dynamics. Also, fluorescence labeling methods
used in this study can be easily applied for not only other GPCRs but also other membrane proteins.
Recently, crystallographic studies of a wide variety of GPCRs have been developed dramatically,
providing valuable many static structures of various GPCR conformations. Thus, dynamic aspectof the GPCRs should be studied and revealed. Combination of the crystal structures with structuraldynamics analyses like this study will extend our understanding of structure-function relationshipsof GPCRs. In other words, in order to understand how GPCRs function, both static structures anddynamic aspects of the receptors should be analyzed.

- 99 -

References
1. Homologs of vertebrate Opn3 potentially serve as a light sensor in nonphotoreceptive tissue.
Koyanagi M, Takada E, Nagata T, Tsukamoto H and Terakita A.
Proc Natl Acad Sci USA, 2013, 110, 4998-5003.
2. A constitutively activating mutation alters the dynamics and energetics of a key conformationalchange in a ligand-free GPCR.
Tsukamoto H and Farrens DL.

一般の皆様へヒトなどの動物を構成する細胞は細胞外からのシグナルを、何らかのメカニズムで受容して、そのシグナルをもとに生理活動を調節します。細胞外のシグナルを受け取る役割を果たす主要な部品（タンパク質）の一つが G タンパク質共役受容体 (GPCR と略されます ) です。GPCR をコードする遺伝
子に特定の変異が入ると、シグナルを受けていないのにもかかわらず、細胞内にシグナルを受容したという（間違った）情報を伝達してしまいます。今回私は GPCR に変異が入った時に、タンパク質のかたちが変わりやすくなることで、「間違った」情報伝達をしてしまうことを明らかにしました。

- 100 -

Regulation of the generation and maintenance of memory T cells
in the intestine
Naoto Ishii
Tohoku University Graduate School of Medicine
ishiin@med.tohoku.ac.jp

Abstract
We examined how gut-specific homeostatic proliferation of T cells contributes to mucosal memory
T cells. This study revealed that the homeostatic proliferation of CD4 T cells in the mesentericlymph nodes (MLNs) critically contributes to generating gut-tropic Th17 cells. In addition, Th17
generation in MLNs was markedly suppressed by blockade of OX40 signals in T cells. These results
reveal that MLNs are critical in inducing gut-tropic effector memory Th17 cells through OX40
signaling.
Key Words : Mucosal immunity, homeostatic proliferation, Th17 cell, mesenteric lymph node,

Introduction
Since the systemic homeostatic proliferation of naïve T (T N) cells supplies a memory-phenotype T
cell pool, this system can be used to generate effector memory T (TEM) cells, including Th17 cells.
However, it is unclear whether the homeostatic proliferations may also occur in an “organ-specific”
fashion such as in the intestine and MLNs. Therefore, we examined the homeostatic proliferation in
MLNs and intestinal tissue, and demonstrate that the TEM-phenotypic CD4 T cells accumulate in thehost’s small intestine through homeostatic proliferation.

Results
The small intestine harbors numerous CD4+ effector memory T (TEM) cells
Naive and central memory T cells are reported to circulate through secondary lymphoidtissues, while T EM cells accumulate in extralymphoid organs, including the small intestine (1).
To confirm this, we looked for naïve and memory CD4+ T cell phenotypes. Of the CD4+ T cells
found in secondary lymphoid tissues such as the spleen, inguinal lymph nodes (ILNs), and MLNs,
approximately 70% had a naïve T phenotype and 20–30% had a T EM phenotype. In the lamina
propria of the small intestine, 80–90% of CD4+ T cells had a TEM phenotype. These data confirm
that TEM cells preferentially accumulate in extralymphoid organs, including the small intestine.
Mesenteric lymph nodes (MLNs) but not Peyer’s patches (PPs) are essential for generating
α4β7+ gut-homing T cells
We examined α4β7 levels on donor cells, derived from naïve CD4 T cells, undergoing homeostatic
proliferation. After transfer with naïve CD4 donor T cells, some donor T cells acquired higher levels
α4β7 through homeostatic proliferation. In particular, fast-dividing cell populations from the small- 101 -

intestine and MLNs contained larger proportions of α4β7+ donor cells than did those recovered fromthe spleen. On the other hand, slow-dividing population of the donor cells in these organs retained
their initial, intermediate α4β7 level. These results indicate that the α4β7+ phenotype develops during
fast homeostatic proliferation.
To examine whether PPs and MLNs are required for generating α 4β7+ gut-homing T cells, we
determined the frequencies of α4β7+ donor cells in Peyer’s patch-deficient (PPX) and mesentericlymphadenectomized (MLX) mice. The α 4β7+ proportion was unchanged in fast-dividing cells
recovered from the small intestine or spleen of PPX mice but the proportion was significantly
reduced in MLX mice. These findings suggest that MLNs but not PPs are essential for generating
α4β7+ cells through fast cell division.
OX40 signaling is critical for fast proliferation in MLNs
The in vivo blockade of T cell costimulatory signals mediated through OX40, which belongs
to the TNF receptor superfamily, improves experimental colitis by suppressing the homeostatic
proliferation of pathogenic CD4+ T cells (2). Therefore, we examined OX40 expressed on donor T
cells in MLNs during homeostatic proliferation, and found OX40 on fast- but not slow-proliferating
cells. To clarify the role of OX40 in fast proliferation, we treated recipient mice with an inhibitoryanti-OX40L mAb and monitored donor cell proliferation in MLNs. Treatment with the anti-OX40L
mAb significantly inhibited the generation of fast-proliferating α 4β7+ cells in MLNs, without
affecting slow cell division. Treatment with the anti-OX40L mAb also strongly reduced the total,
fast-proliferating, and α4β7+ donor T cell populations in the small intestine. This indicates that OX40
costimulation is required for generating gut-tropic, fast-dividing cells in MLNs.
Gut-tropic Th17 cells arise in MLNs during homeostatic proliferation
The CD4+ T cell population in the small intestine includes a significant number of Th17 cells (3).
Therefore, we measured IFN-γ and IL-17A cytokine levels in donor cells recovered from the small
intestine, spleen, and MLNs. Neither cytokine was produced by the slow-dividing population, while
the fast-dividing population expressed both. When gating on the fast-dividing population, an IFN-γ
profile was dominant in the spleen, but in the intestine IL-17A-producing cells greatly outnumbered
IFN-γ-producing ones. In MLNs, IFN-γ and IL-17A were detected at equal levels. These results
indicate that Th17 cells are generated through fast proliferation in an organ-specific manner, andthat gut-specific Th17 cells may arise in MLNs or the small intestine. Furthermore, removing MLNs
from recipient mice significantly reduced the IL-17A+ but not the IFN-γ+ population in the small
intestine. Collectively, these results demonstrate that MLNs are essential for the generation of guttropic Th17 cells through fast homeostatic proliferation.

Discussion & Conclusion
Our results clearly demonstrate that gut-specific fast homeostatic proliferation plays a criticalrole in inducing gut-homing α4β7+ IL-17A+ T cells, which is dependent on OX40 signals. Although
the systemic inhibition of OX40 signals, either in OX40L-KO mice or by treatment with an anti-- 102 -OX40L mAb, drastically ameliorates several types of IBDs and GVHD, our study revealed that the
OX40-OX40L interactions necessary for generating intestinal Th17 cells may occur in the MLNs.
Therefore, it may be possible to develop therapeutic strategies for IBDs and GVHD by controlling
the OX40-OX40L interactions in MLNs. In addition, the finding that the role of MLNs in developing
gut-homing TEM cells, including Th17 cells, is distinct from that of the PPs or spleen may contributeto a deeper understanding of intestinal mucosal immunity.

References
1. Sheridan, B.S., and Lefrancois, L. (2011). Regional and mucosal memory T cells. Nat Immunol
12, 485-491.
2. Takeda, I., S. Ine, N. Killeen, L. C. Ndhlovu, K. Murata, S. Satomi, K. Sugamura, and N. Ishii.
2004. Distinct roles for the OX40-OX40 ligand interaction in regulatory and nonregulatory T
cells. J Immunol 172: 3580-3589
3. Ivanov, II, K. Atarashi, N. Manel, E. L. Brodie, T. Shima, U. Karaoz, D. Wei, K. C. Goldfarb, C.
A. Santee, S. V. Lynch, T. Tanoue, A. Imaoka, K. Itoh, K. Takeda, Y. Umesaki, K. Honda, and D.
R. Littman. 2009. Induction of intestinal Th17 cells by segmented filamentous bacteria. Cell 139:
485-498.

一般の皆様へ
免疫反応の司令塔であるヘルパー T 細胞の制御機構の解明は、アレルギーや免疫疾患の原因解明や治療法の開発に直結する重要な研究です。私たちは、腸管粘膜免疫を司る Th17 型と呼ばれるヘルパー T 細胞が作られる場所が腸間膜リンパ節である可能性を見いだしました。さらに、Th17 型
ヘルパー T 細胞が作られる過程で OX40 受容体からのシグナルが必須であることを発見しました。
腸間膜リンパ節の T 細胞やそこに存在する OX40 受容体の働きの仕組みを解明することで、近い将来には、食物アレルギーや炎症性腸疾患の治療法の開発が期待されます。

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Studies on the regulatory mechanisms underlying cell cycle-dependent
centrosome replication
Takaya Satoh
Department of Biological Science, Graduate School of Science, Osaka Prefetkysato@b.s.osakafu-u.ac.jp

cture University

Abstract
The guanine nucleotide exchange factor ARHGEF10 negatively regulates centrosome duplication
through the activation of the small GTPase RhoA. In this study, we investigated the mechanisms
for this novel signalling pathway. Furthermore, the role of ARHGEF10 in malignant progression ofhuman cancer was explored.
Key Words : centrosome; GTP-binding protein; guanine nucleotide exchange factor

Introduction
The centrosome serves as the major microtubule-organizing center in mammalian cells.
Aberrations in centrosome numbers, which frequently occur in aggressive human tumors, represent
a crucial cause of missegregation of chromosomes, leading to a phenotype termed genetic instability.
Therefore, the duplication cycle of the centrosome must be rigorously controlled in the cell. In thisstudy, we attempted to clarify the detailed mechanism through a novel signaling pathway involving
the small GTPase RhoA.

Results
A single centrosome consists of a pair of centrioles surrounded by the amorphous pericentriolar
material. Centrioles are cylindrical structures built of nine sets of triplet microtubules, and lie atright angles to each other and in close proximity at one end. The pericentriolar material harbors a
number of protein complexes, including the γ-tubulin ring complex, which acts as a template formicrotubule nucleation. During M phase of the cell cycle, two centrosomes orchestrate the assemblyof bipolar mitotic spindles, which is prerequisite for accurate chromosome segregation.
The signal transducing small GTPase RhoA has been implicated in a variety of cellular responses
in mammalian cells. Upstream of RhoA, diverse Dbl family guanine nucleotide exchange factors(GEFs) act as signal-specific regulators of RhoA depending on the cellular context. Tandem
catalytic DH and regulatory PH domains are conserved in all Dbl family members. The Dbl family
GEF ARHGEF10 was originally identified as the product of the gene associated with slowed nerveconduction velocities of peripheral nerves. However, underlying molecular mechanisms remain
elusive.
In our effort to clarify the biological function of this poorly characterized GEF, we found a novelsignaling pathway that negatively regulates centrosome duplication during cell cycle progression.

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RhoA, but not other Rho family small GTPases examined, was biochemically identified as a
substrate of ARHGEF10. ARHGEF10 was localized in the centrosome in adenocarcinoma HeLa
cells in both G1/S and M phases. Knockdown of ARHGEF10 expression by RNA interference
resulted in multipolar spindle formation in M phase. An intact centrosome composed of twocentrioles and the pericentriolar material was found in each spindle pole. Partial, but not full,knockdown of RhoA by RNA interference elicited similar phenotypes. Aberrant mitotic spindle
formation following ARHGEF10 knockdown was rescued by ectopic expression of constitutively
activated RhoA. Thus, it is likely that RhoA is a downstream target of ARHGEF10 in this signaling
pathway.
We further isolated the kinesin-like motor protein KIF3B as a binding partner of ARHGEF10.
Knockdown of KIF3B also caused multipolar spindle phenotypes. The supernumerary centrosome
phenotype was also observed in S phase-arrested osteosarcoma U2OS cells when the expression of
ARHGEF10, RhoA or KIF3B was abrogated by RNA interference.
Multinucleated cells were not increased upon ARHGEF10 knockdown. Inhibition of the RhoA
effector kinase ROCK by its specific pharmacological inhibitor Y-27632, in contrast, induced not
only multipolar spindle formation, but also multinucleation. These results suggest that unregulated
centrosome duplication rather than aberration in cytokinesis may be responsible for ARHGEF10
knockdown-dependent multipolar spindle formation.
Given that more than two intact centrosomes appear in G2/M phase, excess duplication may
occur due to aberrant disengagement of the newly synthesized (daughter) centriole in S phase.
To obtain further evidence supporting this possibility, we tried to quantify doublet (engaged) andsinglet (disengaged) centrioles in S phase. Counterstaining of centrioles by anti-centrin and antiC-Nap1 antibodies is expected to give us the engagement status of the centriole (Tsou and Stearns
Nature 442, 947 (2006)). We are already successful in staining centrin in synchronized cells, but
the conditions for the staining of C-Nap remain to be established. We, in fact, tried several different
purchased anti-C-Nap antibodies at various concentrations, and only one antibody is promising.
Currently, we are trying to describe the precise mechanisms for ARHGEF10-RhoA mediatedregulation of centrosome duplication.

Discussion & Conclusion
ARHGEF10 is a RhoA GEF, which is localized in the centrosome in both G1/S and M phases.
ARHGEF10 regulates mitotic spindle formation through the action of RhoA. ARHGEF10-regulaed
RhoA may be implicated in the control of centrosome duplication rather than cytokinesis. The motor
protein KIF3B is a binding partner of ARHGEF10, a subset of which is co-localized with ARHGEF10
in the centrosome. Thus, KIF3B may regulate ARHGEF10 upstream of RhoA, being involved in
the regulation of centrosome duplication. Recently, mutations in the ARHGEF10 gene was found invarious types of human cancer cells. Dysregulated centrosome duplication and cell division in cells
lacking ARHGEF10 activity may be a cause of malignant progression of human cancer.

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References
1. Takuji Aoki, Shuji Ueda, Tohru Kataoka, and Takaya Satoh: Regulation of mitotic spindle
formation by the RhoA guanine nucleotide exchange factor ARHGEF10. BMC Cell Biol. 10, 56(2009)

一般の皆様へ
本研究では、中心体の過剰な複製を抑制することにより、中心体複製と細胞分裂の同調を保証するという、我々が見出した全く新しい制御機構の全容を解明するとともに、その異常による細胞の異数化とヒト癌の悪性化との関連性を明らかにすることを目的としている。本研究において中心体の複製回数を厳密に制御するメカニズムの詳細が明らかにされれば、細胞機能の基本原理の一端が解明されるという意味で学術的にインパクトが大きい。さらに、癌の悪性化を誘導する新規のメカニズムの解明となるとともに、この過程が癌治療の新規の標的となることが期待される。

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microRNA-mediated regulation of vasculogenesis
Yuichiro Mishima
Institute of Molecular and Cellular Biosciences, The University of Tokyomishima@iam.u-tokyo.ac.jp

Abstract
Secreted factors with potent morphogenetic activity are key elements of cellular communication,and precise regulation of their expression is required to elicit appropriate physiological responses.
MicroRNAs (miRNAs) are versatile post-transcriptional modulators of gene expression. We
found that muscle-specific miRNAs miR-1/206 negatively regulate angiogenesis during zebrafish
development. miR-1/206 directly regulate the levels of Vascular endothelial growth factor A (VegfA)in muscle, controlling the strength of angiogenic signaling to the endothelium. Our findings uncovera novel function for miR-1/206 in the control of developmental angiogenesis through the regulationof VegfA, and identify a key role for miRNAs as regulators of cross-tissue signaling.
Key Words : vasculogenesis, zebrafish, microRNA, VEGF

Introduction
Secreted signaling molecules are powerful regulators of development that are able to elicit
behaviors such as proliferation, migration and differentiation in a concentration-dependent
manner. One developmental context in which these signaling molecules play an important roleis the formation of the vascular network. Secreted growth factors such as vascular endothelial
growth factor (VEGF) function at different levels during the establishment and remodeling of thevasculature.

Results
miR-1 and miR-206 are evolutionarily conserved, have common expression patterns in themuscle, and share a large part of their sequence (1). To investigate whether miRNAs regulate thecross-talk between the muscle and surrounding tissues during developmental angiogenesis, weanalyzed the effect of blocking miR-1 and miR- 206 function on the vasculature during zebrafish
embryogenesis. Embryos in which miR-1/206 was inhibited display larger intersegmental vessels(ISVs), accompanied by a significant increase in the number of endothelial cells. Consistent with theexpression of miR-1/206 in the muscle, mosaic analysis supports a non-cell-autonomous effect of
miR-1/206 in the vasculature. These results support a role for miR-1/206 in the regulation of a proangiogenic factor that is non-autonomous to endothelial cells.
Next, we analyzed the 3’UTRs of angiogenic genes for putative miR-1/206 target sites. We foundthat zebrafish vegfaa contains three miR-1/206 target sites in its 3’UTR. To determine whether
miR-1/206 regulate the 3’UTR of vegfaa, we analyzed the expression of a firefly luciferase reporter- 107 -

containing the vegfaa 3’UTR, relative to a Renilla luciferase control, in the presence or absenceof miR-1/206 duplex. The luciferase activity of the wild-type vegfaa reporter was significantly
repressed by miR-1/206, and the regulation of the vegfaa reporter depended on the presence of miR1/206 target sites. Furthermore, we directly visualized the levels of endogenous vegfaa mRNA by insitu hybridization. We found that vegfaa levels in the somites were upregulated in embryos in which
miR-1/206 was inhibited, consistent with the loss of a negative regulatory factor. Together, theseresults indicate that miR-1/206 can regulate vegfaa expression during development. We reasoned
that if the increase in angiogenesis observed in miR-1/206 inhibition is due to VegfAa misregulation,
reducing the levels of VegfAa signaling would rescue this phenotype. To test this possibility, we
first employed SU5416, a selective inhibitor of the VEGF receptor Kdrl (also known as VEGFR2,
Flk1), to block Vegf signaling (2). We found that increasing concentrations of SU5416 rescued theangiogenesis phenotype of miR-1/206 inhibition. Second, reducing the levels of VegfAa with atranslation-blocking antisense oligo nucleotides rescued the miR-1/206 KD vascular phenotype.
Together, these results indicate that the phenotype observed when blocking miR-1/206 function isdependent on VegfAa activity, consistent with the proposed role of miR-1/206 in the regulation ofvegfaa during angiogenesis.
miRNAs are widespread regulators of gene expression, and it is predicted that each miRNA has
the potential to regulate several hundred target mRNAs (3). To directly address the interaction of
miR-1/206 with vegfaa in vivo, we employed target protectors (TPs), which are antisense oligo
nucleotides complementary to the miRNA target sites in a 3’UTR(4). Indeed, protection of the miR1/206 target sites in vegfaa had a pro-angiogenic effect, resulting in ISV with elevated numbers ofendothelial cells compared with controls. These results indicate (1) that despite the large number ofmiR-1/206 targets, the miR-1/206 inhibition phenotype is at least in part a result of altered vegfaa
regulation and (2) that miR-1/206 directly regulate the levels of vegfaa to modulate angiogenesis
during development.

Discussion & Conclusion
Our study shows that miR-1/206 negatively regulate angiogenesis during zebrafish development.
Because miRNAs have hundreds of putative targets, it is challenging to determine the physiological
function of individual miRNA-target interactions. Our findings identify a novel regulatory layerthat modulates the cross-talk between muscle and vasculature during developmental angiogenesis.
VegfA plays a central role in the stimulation and control of angiogenesis, requiring it be tightly
regulated during embryonic development and homeostasis. However, less is known about theregulatory systems that control Vegf production during development. Our findings indicate thatvegfaa is targeted by miR-1/206 in the muscle to modulate the expression of the ligand, allowing theorganism to regulate this signaling pathway.
The identification of miR-1/206-mediated regulation of Vegf might be significant beyond
developmental angiogenesis. As in developmental angiogenesis, VEGF signaling is an essential- 108 -

component of the angiogenic response of a wide variety of tumors, making it a prime clinical target
for the development of therapeutic agents. Future studies will be required to understand whether the
regulation of VEGF by miRNAs plays an important role in angiogenesis during human disease andcancer.

References
1. Wienholds, E., Kloosterman, W. P., Miska, E., Alvarez-Saavedra, E., Berezikov, E., de Bruijn,
E., Horvitz, H. R., Kauppinen, S. and Plasterk, R. H. (2005). MicroRNA expression in zebrafish
embryonic development. Science 309, 310-311
2. Fong, T. A., Shawver, L. K., Sun, L., Tang, C., App, H., Powell, T. J., Kim, Y. H., Schreck, R.,
Wang, X., Risau, W. et al. (1999). SU5416 is a potent and selective inhibitor of the vascularendothelial growth factor receptor (Flk-1/KDR) that inhibits tyrosine kinase catalysis, tumor
vascularization, and growth of multiple tumor types. Cancer Res. 59, 99-106
3. Bartel, D. P. (2009). MicroRNAs: target recognition and regulatory functions. Cell 136, 215-233.
4. Choi, W. Y., Giraldez, A. J. and Schier, A. F. (2007). Target protectors reveal dampening andbalancing of Nodal agonist and antagonist by miR-430. Science 318, 271-274.

一般の皆様へ高度に複雑化した器官の形作りには、細胞同志がコミュニケーションを取り合うことが重要です。
今回私たちは複雑に入り組んだ血管ネットワークの構築に着目し、小型淡水魚ゼブラフィッシュを用いてその制御機構を解析しました。その結果、マイクロ RNA と呼ばれる小さな RNA 分子の一つである miR-1/206 が、骨格筋から発せられる血管内皮細胞増殖因子 A（VegfA）の量を調節する役割をはたしていることを明らかにしました。この知見は基礎生物学のみならず、受傷後の血管網の再構
築やがん細胞による血管誘導現象の解明などの医学分野にも波及効果が期待されます。

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The role of molecular motor for regulating T cell activation
Akiko Hashimoto-Tane
RIKEN Research Center for Allergy and Immunology
akikoh@rcai.riken.jp

Abstract
When T cells are activated, T cell receptors (TCRs) make clusters and move toward center of the
T cell-antigen presenting cell (APC) interface. In this study, I tried to reveal the mechanism of TCR
clustering and movement by protein identification and fluorescence imaging, and found very unique
new structure for T cell receptor clustering. The structure contains general cytoskeletal-regulating
molecules, adhesion molecules and TCR signaling molecules. Analyzing this structure will explain
how the TCR clustering and movement analogously regulate TCR signaling.
Key Words : T cell receptor, cell signaling, dynein, cytoskeleton

Introduction
When T cells recognize a peptide- major histocompatibility complex on APC, dozens of T cell
receptor microclusters (TCR-MCs), which contains several tens of receptors, kinases and adaptors,
are generated and start T cell activation. TCR-MCs move to the center of the T cell-APC interface
to be endocytosed and terminate activation. We have demonstrated that the TCR-MCs movement
depends on dynein motor and microtubules, and the movement negatively regulates T cell activation.
However, we have not revealed molecular mechanism of TCR clustering and movement.

Results
To reveal the molecular mechanism of TCR clustering and movement, I used immuno-precipitaionand protein identification by mass spectroscopy.
Firstly, to know how TCR are connected to dynein motor, I planned double precipitation of taggedTCR and tagged-dynein. Because It has already known that TCR recruit so many molecules, so
I wanted to concentrate the molecules between TCR and dynein by secondary precipitation bydynein. I used His-tag, FLAG-tag, GFP-tag for TCR component CD3ε chain, CD3ζ chain or dynein
light chain. However unfortunately, I failed in double precipitation because every tag had defects inexpression, adsorption or elution.
Next I performed simple dynein light chain (LC-8) -precipitation. GFP-tagged dynein light chain
expressed in T cell hybridoma. The cells were activated by antigen presenting cells or kept restingon ice and lysed with 1% triton-100 solutions and precipitated by GFP antibody labeled with magnet.
The precipitants were examined on two-dimensional protein gel electrophoresis. Comparing thesilver protein staining patterns with or without T cell activation, I fortunately found several spotsthat were detected only on T cell activated sample. Next I identified the spots by mass spectroscopy- 110 -

of out-sourcing service.
The identified molecules were very exciting. Although I cannot declare the molecular names at thismoment, I will report them shortly in my next paper. I confirmed that some of the molecules werealso co-precipitated with TCR component CD3ε chain. I further confirmed the localization of themolecules by using lipid bilayer system. Fortunately, the molecules formed clusters at TCR-MCs andmoved together toward center.
On the other hand, I analyzed TCR and dynein at lower antigen doses. Because, when thestimulation was weak, not only the size and number but also the movement of TCR-MC weredifferent, so I thought the mechanism of TCR-MCs movement is different. I checked the associationand co-localization of TCR-MCs and dynein complexes. T cells were activated by antigen presenting
cells loaded 10nM or 1μM antigen peptide and lysed for anti-CD3ε precipitation. I confirmed
co-precipitation of dynein complex proteins in each dose of stimulation. On the planer bilayer,
co-localization of TCR-MCs and dynein complexes were observed. Therefore even with weakstimulation, dynein was recruited to TCR-MCs. I also checked the association under microtubuleinhibitors, which inhibit tubulin polymerization and disappears tubular structures. The coprecipitation and co-localization still observed under treatment of microtubule-inhibitors. These
results suggested the association of the dynein complex and the TCR complex is independent of
microtubules.
I examined some molecules important for TCR signaling under weak stimulation. I wanted
to know the behavior of signaling molecules. Famous essential kinase ZAP70, which binds to
TCR component CD3ζ chain, makes co-localizing clusters at TCR-MCs, and the number andmovement of ZAP70 clusters were quite similar to TCR-MC. The recruitment and clustering of
ZAP70 will be totally depends on TCR-mediated signals and the movement will depend on dynein.
On the other hand, the adaptor protein SLP76, which is essential for T cell activation, induces
clusters independently of TCR-MCs, particularly by weak stimulation. Further, they seemed to bedissociated from TCR-MCs on the way to leach to cSMAC. Therefore, the regulation of SLP76
clustering and movement appears to be different from TCR-MCs and ZAP70.
Interestingly, I recently found that the SLP76 clusters at lower dose accompany the molecules
identified as T cell activation-dependently dynein-precipitated protein.

Discussion & Conclusion
The molecules identified as T cell activation-dependently dynein binding protein in T cell werevery famous proteins. I think that because they have so well known functions nobody have analyzed
their functions on TCR-MCs. In addition, I found they were recruited at SLP76 clusters under weakstimulation. The SLP76 cluster seems to be independent from TCR clusters. I am thinking about thepossibility that it is a structure to evoke antigen-independent or self-recognizing T cell activation.
Hereafter, I will show the composition of the SLP76 and identified proteins clusters and relationshipbetween dynein complex. I will finally clarify the biological significance of the clusters.

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References
1. Hashimoto-Tane A, Yokosuka T, Sakata-Sogawa K, Sakuma M, Ishihara C, Tokunaga M, Saito T.
Dynein-driven transport of T cell receptor microclusters regulates immune synapse formation
and T cell activation. Immunity 34, p919-31, 2011
2. Yokosuka T, Sakata-Sogawa K, Kobayashi W, Hiroshima M, Hashimoto-Tane A, Tokunaga M,
Dustin ML, Saito T.
Newly generated T cell receptor microclusters initiate and sustain T cell activation by recruitment
of Zap70 and SLP-76. Nature Immunology 6, p1253-62, 2005

一般の皆様へ
本研究では、ワクチンによる免疫記憶形成などを司る T リンパ球が、細胞表面に発現した受容体を介して、体内に侵入した外敵を識別する仕組みを解析しています。蛍光顕微鏡を用いた画像解析で、反応した受容体の集合体がダイナミックに動いて免疫反応の規模を調節する様子を追いながら、その動きに影響を与える分子や因子を特定しています。本研究の結果には、リンパ球が自己に対して免疫反応を開始してしまう自己免疫疾患の発生機序の解明などが期待されます。

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Generation of Batf reporter/conditional knock-out mice forelucidation of Batf function during in vivo immune responses
Wataru Ise
Immunology Frontier Research Center (IFReC), Osaka University
wise@ifrec.osaka-u.ac.jp

Abstract
We have newly generated Batf conditional knock out mice which allow us to analyze cell-type
specific role of Batf. The mice have GFP reporter which is inserted into floxed allele such that wewere able to look at expression of BATF-GFP fusion protein in activated T cells and B cells. By
using this new mouse strain, we are going to investigate the function of Batf in memory T or B cells.
Key Words : T-cell, B-cell, transcription factor, BATF

Introduction
We have previously discovered the transcription factor BATF as a crucial factor for in vivo
antibody response. BATF is essential for not only B cell class-switching but also development offollicular helper T cells which are the helper T cell subsets required for germinal center formation.
Given the multi-function of BATF in lymphocytes, BATF conditional knock-out mice were required
to elucidate cell-type specific function of BATF in vivo.

Results
We generated Batf floxed/GFP Knock-In mice. In these mice, exon 3 of Batf gene is flanked byloxp sequences. Also, GFP is inserted into the stop codon in exon3, so that BATF-GFP fusion
protein should be expressed from the mutated allele. First, we looked at GFP expression in activatedT cells or B cells in vitro. Naïve T cells or B cells did not express GFP. However, upon stimulation
with anti-CD3, purified CD4 or CD8 T cells expressed GFP. Similarly, purified B cells became
GFP positive after culture with LPS or anti-IgM. GFP expression was nicely correlated with Batf
mRNA expression, suggesting that GFP knock-In strategy worked well. Next we looked at GFP
expression in T or B cells in vivo after immunization with soluble protein antigens. At 7 days after
immunization, robust BATF-GFP expression was observed in follicular helper T cells, which are
CXCR5hiPD1hi cells. CXCR5int T cells showed modest levels of BATF-GFP. After 1 month, survived
memory T cells expressed BATF-GFP at low level. By contrast, BATF-GFP expression in B cells at
any time points was quite low. At day7, where most of the antigen-specific B cells showed germinal
center B cell phenotype, BATF-GFP expression was neglectable. Memory B cells did not express
BATF-GFP either. It might be possible that pattern of BATF expression was different between T celland B cells. In T cells, it appeared that BATF was kept expressed from effector to memory response.
In contrast, BATF expression might be very transient in B cells, which made detection of BATF-- 113 -GFP in B cells difficult.
We have crossed Batf floxed/reporter mice with several Cre-expressing strains, such as CD4-cre,
Mb1-cre, or ERT2-cre. We are now able to analyze T- or B cell specific function of BATF in vivo.
Also, we are going to delete BATF in inducible manner at given time point to look at the function of
BATF in memory T cell survival or maintenance.

Discussion & Conclusion
Having established Batf reporter/conditional knock-out mice, we are now able to track BATFGFP expression in T-cell or B cells in vivo during immune responses. Notably, analysis of BATFGFP expression in vivo suggested the difference in regulation of BATF expression between T cells
and B cells. BATF was thought to function in effector T cells. However, our analysis clearly showedthat memory T cells also expressed BATF, suggesting the possibility that BATF regulates survival
or function of memory T cells. We are going to examine the role of BATF in memory T cells using
Batf fl/fl x ERT2-cre mice.

一般の皆様へ
転写因子 BATF は抗体産生応答を制御する重要な因子である。本研究により BATF が抗体産生応答に加えて、記憶免疫応答をも制御する可能性が示唆された。したがって BATF の機能を研究し、人為的にその機能を制御することでより良いワクチン開発の指針が得られる可能性がある。

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A study on the molecular mechanisms of mitochondrial regulationby the genes responsible
for early-onset familial forms of Parkinson’s disease
Yuzuru Imai
Department of Neuroscience for Neurodegenerative Disorders,Juntendo University Graduate School of Medicine
yzimai@juntendo.ac.jp

Abstract
Mitochondrial quality control is an important issue in higher animals that depend on aerobic
respiration. A series of elegant studies has demonstrated that two Parkinson’s disease (PD)associated genes, PINK1 and parkin, are involved in the maintenance of healthy mitochondria.
Parkin, in cooperation with PINK1, specifically recognizes damaged mitochondria with
reduced mitochondrial membrane potential (Δψm), rapidly isolates these mitochondria from themitochondrial network and eliminates these organelles through the ubiquitin-proteasome andautophagy pathways. Our studies, combined with Drosophila genetics and cell biology, contributeto an understanding of the molecular mechanisms of PINK1- and Parkin-mediated mitochondrial
regulation and the pathological mechanisms explaining how defects in the PINK1-Parkin pathway
lead to neurodegeneration in PD.
Key Words : Parkinson’s disease, ubiquitin ligase, kinase

Introduction
Mitochondrial dysregulation is now implicated in various human diseases, including cancer,
diabetes, myopathy and neurodegeneration. The pathological hallmark of the second mostcommon neurodegenerative disorder, Parkinson’s disease (PD), is the progressive degeneration of
dopaminergic neurons in the midbrain. Although mitochondrial dysfunction has long been believed
to be closely associated with the etiology of PD, there was no direct evidence for the association.
A series of studies on two PD-associated genes, PINK1 and parkin, has now shown that these twogenes work in a coordinated manner in mitochondrial maintenance, including the mitophagy ofdamaged mitochondria. This finding strongly implies that the dysregulation of mitochondria is oneof the major causative or contributing factors in the etiology of PD.

Results
The loss of PINK1 or Parkin in Drosophila causes mitochondrial degeneration in the thorax
muscles, which results in abnormal wing posture. Using this wing phenotype, we screened modifier
genes for the PINK1/Parkin phenotype, which revealed two pathways involved in PINK1-Parkinsignaling: namely, a pathway for mitochondrial motility and the target of rapamycin complex 2- 115 -

(TORC2)- nuclear Dbf2-related (NDR1) pathway.
Miro is an outer mitochondrial protein that binds to microtubule-associated transport complexes,
such as the kinesin motor in conjunction with Milton. Knockdown of Miro alleviated themitochondrial degeneration caused by a loss of PINK1. We found that Miro is one of the substratesof the Parkin ubiquitin ligase in mitophagy and that Miro’s ubiquitination and subsequentdegradation by Parkin in damaged mitochondria is an important process that prevents damaged
mitochondria from moving to the axonal terminus (ref. 3). Nearly simultaneously, Wang et al.
reported that Miro directly phosphorylated at Ser156 by PINK1 upon mitophagy is recognized
by Parkin as a degradation substrate (Wang et al., Cell 2011). However, we disproved this idea,
reporting that Miro is not directly phosphorylated by PINK1 in vitro and that the Miro Ser156Ala
phospho-mutant is equally degraded by Parkin in vivo.
TORC2, which is a highly conserved kinase complex, is known to regulate actin cytoskeleton
signaling. We found that the genes that encode TORC2 components, such as rictor and sin1, improve
the PINK1 wing phenotype. Further genetic interaction studies revealed that Tricornered/NDR, akinase downstream of TORC2, behaves as a regulator of the downstream PINK1 pathway, likely
independently of Parkin (ref. 1).
Parkin translocates to the mitochondria upon the loss of mitochondrial membrane potential(ΔΨm) in a PINK1 kinase activity-dependent manner. However, little is known about how PINK1
activation recruits Parkin to damaged mitochondria. We attempted to monitor and compare thephosphorylation status of Parkin in wild-type and PINK1-deficient cells, thereby excluding thepossibility of phosphorylation by kinases other than PINK1. We found that Parkin is phosphorylated
at Ser65 of its ubiquitin-like (Ubl) domain in a PINK1-dependent manner. Furthermore, we showedthat this phosphorylation event is implicated in the regulation of the mitochondrial translocation ofParkin and the subsequent degradation of mitochondrial surface proteins during mitophagy (ref. 2).
However, Parkin mutants lacking mitochondrial translocation activity
were also phosphorylated upon PINK1 activation, suggesting that Ser65
phosphorylation is not sufficient for the mitochondrial translocation ofParkin and raising the possibility that the presence of another cytosolic
kinase(s) regulated by PINK1 (Fig 1).

Discussion & Conclusions
Two gene products implicated in early-onset PD, PINK1 and Parkin,have recently been implicated in mitochondrial quality control. PINK1and Parkin-mediated mitophagy includes two major components:
commitment to elimination by PINK1 and Parkin and the execution ofelimination by autophagy. Although canonical autophagy components
appear to be involved in the autophagy stage, at least under experimental
conditions, the molecular mechanisms by which PINK1 and Parkin signal- 116 -

the commitment to elimination remain largely unclear. This concept is an important issue, and our
future studies may lead to the identification of new molecules associated with neurodegenerative
disorders and an understanding of the etiology of idiopathic PD and PINK1- and parkin-linked PD.

References
1. Wu Z, Sawada T, Shiba K, Liu S, Kanao T, Takahashi R, Hattori N, Imai Y, Lu B: Tricornered/
NDR kinase signaling mediates PINK1-directed mitochondrial quality control and tissue
maintenance. Genes Dev. 27:157-162 (2013)
2. Shiba-Fukushima K, Imai Y, Yoshida S, Ishihama Y, Kanao T, Sato S, Hattori N: PINK1mediated phosphorylation of the Parkin ubiquitin-like domain primes mitochondrial translocationof Parkin and regulates mitophagy. Sci Rep. 2: Article number: 1002 (2012)
3. Liu S, Sawada T, Lee S, Yu W, Silverio G, Alapatt P, Millan I, Shen A, Saxton WM, Kanao T,
Takahashi R, Hattori N, Imai Y, Lu B: Parkinson’s disease-associated kinase PINK1 regulates
Miro protein level and axonal transport of mitochondria. PLoS Genet. 8: e1002537 (2012)
4. Imai Y, Hattori N: Mitophagy Controlled by the PINK1-Parkin Pathway is Associated with
Parkinson’s Disease Pathogenesis. In: Autophagy:
Cancer, Other Pathologies, Inflammation, Immunity, and Infection, Hayat MA ed, Elsevier,
Amsterdam, in press.
5. Imai Y: Mitochondrial regulation by the PINK1-Parkin signaling. ISRN Cell Biol. 2012: Article
ID 926160 (2012).

一般の皆様へ
本研究は、パーキンソン病原因遺伝子の機能を解明し、パーキンソン病の予防や治療に貢献することを究極の目的としています。若年性パーキンソン病の原因遺伝子 Parkin と PINK1 は、細胞活動に必要なエネルギーを産生するミトコンドリアの品質管理に関与することが明らかになってきました。
ミトコンドリアは細胞にとって必須の器官でありながら、一方でエネルギー産生時に加齢や老化につながる活性酸素種を発生させることから、適切な監視下で働かせることが必要です。この監視（品質管理）が Parkin と PINK1 の役割であることが、私たちを含む多くの研究で分かってきました。

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Fundamental study for long intergenic RNA (linc RNA)and epigenetic regulation by tumor suppressor p53
using ES/iPS cells to control cancer stem cells
Tomoaki Tanaka
Chiba University Graduate School of Medicine
tomoaki@restaff.chiba-u.jp

Abstract
The tumor suppressor p53 has been shown to control energy balance and metabolic homeostasis,concerning its role in Warburg effect and cancer stemness. In this context, we have attempted toisolate and characterize p53 chromatin complexes, and to perform genome-wide screening of RNAseq and ChIP-seq using tumor and ES/iPS cells. We found that p53 exerts multiple functions with acomplexity of crosstalk to intra-nuclear structure and transcriptional regulation for a wide variety oftranscripts including linc RNAs in senescent and ES/iPS cells, linking to the molecular mechanismof cancer stemm cells.
Key Words : Tumor suppressor p53, non-coding RNA, epigenetics, stem cell

Introduction
Recent several lines of evidences have indicated that aging process is subjected to regulatory
network and the nuclear reprogramming to generate induced pluripotent stemm cells can beachieved by introducing certain transcriptional factors, OCT3/4, SOX2, KLF4 and Myc so-called“Yamanaka f factors”. It is now well appreciated that these include the nutrient-sencing pathways(such as insulin and AKT/mTOR signals), and transcriptional and chromatin regulations (Sirtuinsetc) with profound consequences. Importantly as well, it is also evident that key molecules including
tumor suppressor p53 that regulate cellular senescence and reprogramming process are criticallyinvolved in the pathogenesis of cancer, cardiovascular disease and metabolic disorders, a testament
to importance and crosstalk of transcriptional and epigenetic regulators in the four inevitables in ourlife. Here, to approach this molecular mechanism, we have performed to isolate and characterize thep53 chromatin complexes using multifaceted biochemical methodologies coupling the ChIP assay,
size-fractionation by sucrose gradient ultracentrifugation and LC-MS/MS analysis and to clarify
the gene expression profile including long intergenic RNA (linc RNA) and epigenetic status using
RNA-sequence and ChIP-sequence in cancer cells and ES/iPS cells.

Results
Tumor suppressor p53 receives multiple forms and diverse range of stress signals such as DNA
damage, oxidative or metabolic stress, and then initiates different cellular outcomes including
cell-cycle arrest, apoptosis, and/or cellular senescence. Recent several lines of evidence have

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suggested that p53 pathway is linked to reprogramming process by pluripotent factors andoncogenic signals and p53 systematically regulates intracellular metabolic pathways to preserveanti-oxidant and bioenergetic function. To elucidate the p53 mechanisms, we have attempted toisolate and characterize p53 chromatin complexes in vivo by manipulating biochemical techniquesand mass-spectrometry, and to perform genome-wide screening of RNA-seq and ChIP-seq using
tumor and non-tumor cells including senescent cells and ES/iPS cells. We found several chromatin
regulators and transcriptional coactivators such as CAS/CSE1 (chromosome segregation factor),
Sp110 (component of PML nuclear body protein forming a multi-protein complex) and zincfinger proteins in association with p53 chromatin complexes. Given that they are components
of intra-nuclear structures and have functional domain such as bromodomain and PHD zincfinger motif that potentially function as “reader” for histone codes in epigenetic and chromatinmediated transcriptional regulation, p53 is bona-fide a epigenetic regulators to be “the guardianof the genome” and “the cellular gatekeeper”. Further, genome-wide analyses revealed that p53
can actively and/or repressively control cell cycle regulators, metabolic regulators, senescence
associated secretary proteins (SASP) and linc RNAs in cancer, senescent and ES/iPS cells. Indeed,
ChIP-sequence and RNA-sequencing revealed that p53 controls glutamine metabolism through
GLS2, a key enzyme to produce glutamate, and thereby a regulator of glutathione synthesis andenergy production via α–ketoglutarate in cancer and iPS/ES cells. The expression of GLS2, mainly
localized in mitochondria, was induced in response to DNA damage in tumor and non-tumor cells.
Biochemical and metabolomics analysis revealed that p53-GLS2 regulates intracellular metabolic
pathways to control ROS and energy supply through glutamine metabolism. Consistently, in vitro
and vivo study including mouse xenograft model displayed that GLS2 overexpression inhibited thetumor formation as well as cancer cell invasion via glutamate-dependent GSH synthetic pathway.
Further, immunohistological analysis displayed that the GLS2 expression significantly increasedin the liver of obesity or NASH patients, whereas it decreased in HCC. Importantly as well, GEO
database analysis showed that GLS2 expression is correlated to the prognosis of breast cancer and
HCC patients, indicating its potential role for tumor suppression. When we examined what kindof intracellular signaling or upstream pathway are significantly regulated in ES/iPS cells using
KEGG pathway analysis, we found that pluripotent markers such as OCT4, NANOG, KLF2 andSOX2, oncogenic genes such as c-Myc and n-Myc, cell proliferation related genes such as E2F1 aresignificantly upregulated as expectedly. Interestingly as well, master regulators such as SREBP1
and 2 or repressor for cellular senescence are elevated. In addition, we found that mitochondria of
ES/iPS cells are immature and TCA cycles are suppressed strongly, suggesting that ES/iPS cells
are like as cancer cells in terms of the aberrant glucose metabolism known as “Warburg effect”.
However, ES/iPS cells are quite different from cancer cells in ROS regulation, in which ES/iPS
cells control the low levels of ROS to maintain the pluripotency and stemmness through p53-GLS2
system. Further, we found the ES/iPS or senescence cell specific linc RNAs are regulated in bothp53-dependent and –independent manner, possibly linking to the epigenetic gene profile.

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Discussion & Conclusion
Thus, our study suggests that p53 exerts multiple functions with a complexity of crosstalk to intranuclear structure and transcriptional regulation for a wide variety of transcripts including lincRNAs in senescent and ES/iPS cells, linking to not only the molecular mechanism of cancer stemm
cells, but also life-style related disease and age-related disease such as diabetes and cardiovasculardisorders.

References
1. Hosokawa H, Tanaka T, Suzuki Y, Iwamura C, Ohkubo S, Endoh K, Kato M, Endo Y, Onodera
A, Tumes DJ, Kanai A, Sugano S, Nakayama T. Functionally distinct Gata3/Chd4 complexes
coordinately establish T helper 2 (Th2) cell identity. Proc Natl Acad Sci U S A. 110(12):46914696, 2013.
2. Toyoda A, Yokota A, Saito T, Kawana H, Higashi M, Suzuki Y, Tanaka T, Kitagawa M, Harigaya
K. Overexpression of human ortholog of mammalian enabled (hMena) is associated with theexpression of mutant p53 protein in human breast cancers. Int J Oncol. 38(1):89-96. (2011)
3. Suzuki S, Tanaka T, Poyurovsky MV, Nagano H, Mayama T, Ohkubo S, Lokshin M, Hosokawa
H, Nakayama T, Suzuki Y, Sugano S, Sato E, Nagao T, Yokote K, Tatsuno I, Prives C. Phosphateactivated glutaminase (GLS2), a p53-inducible regulator of glutamine metabolism and reactive
oxygen species. Proc Natl Acad Sci USA. 107(16);7461-6. (2010)

一般の皆様へ
発がんの鍵を握る「がん幹細胞」という概念が注目を集めたのは、京都大学の山中伸弥博士がiPS 細胞の作製に成功した時期にほぼ近い。がんを治療しても再発や転移を起こしたり、抗がん剤が効かなくなるといった治療抵抗性の原因は、がん幹細胞にあるという概念である。がん幹細胞のメカニズムを遺伝子レベルで解明しようにも数が少なくて難しいことから、がん幹細胞に近い性質をもつ ES/iPS 細胞を用いて共通性と非共通性のメカニズムや分子機構を明らかにすることで、がん幹細胞研究に応用できると着想したのが本研究の目的です。最先端技術を用いた解析から、「ゲノムの守護神」として知られる癌抑制遺伝子 p53 の研究に基づいて、ES/iPS 細胞とがん幹細胞に共通したエ
ピゲノム分子基盤及びがん化メカニズムの一旦が明らかとなり、将来的ながん幹細胞標的治療に応用することが期待される。

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Drugs discovery for CKD patients using transporter manipulation
Takaaki ABE
Tohoku University Graduate School of Meidicine
takaabe@med.tohou.ac.jp

Abstract
The promoter region of SLCO4C1 gene has several GATA motifs, and indoxyl sulfate upregulated GATA3 mRNA and subsequently down-regulated SLCO4C1 mRNA. Overexpression of
GATA3 significantly reduced SLCO4C1 expression, and silencing of GATA3 increased SLCO4C1
expression vice versa. Administration of indoxyl sulfate in rats reduced renal expression of slco4c1
and under this condition, plasma level of guanidinosuccinate, one of the preferable substrates of
slco4c1, was significantly increased without changing plasma creatinine. Furthermore, in 5/6
nephrectomized rats, treatment with oral adsorbent AST-120 significantly decreased plasma indoxyl
sulfate level and conversely increased the expression of slco4c1, following the reduction of plasma
level of guanidinosuccinate. These data suggest that the removal of indoxyl sulfate and blocking
its signal pathway may help to restore the SLCO4C1-mediated renal excretion of uremic toxins in
CKD.
Key Words : SLCO4C1, transporter, GATA, indoxyl sulfate

Introduction
Chronic kidney disease (CKD) is a global health problem that carries a substantial risk for
cardiovascular morbidity and death. With the progression of CKD, various uremic toxins
accumulate, subsequently causing renal damage and hypertension. Recently, we have revealed thathuman kidney-specific organic anion transporter SLCO4C1 excretes uremic toxins, and the upregulation of SLCO4C1 resulted in the reduction of blood pressure and renal inflammation in a
CKD model. However, the expression levels of SLCO4C1 is down-regulated in the renal failure.
The down-regulation mechanism of SLCO4C1 in the renal failure has not been well elucidated.

Results
The accumulated uremic toxins inhibit the expression of various renal transporters and thisinhibition further reduces renal function and subsequently causes the accumulation of uremic
toxins. However, the precise mechanism has been poorly understood. Here we report that indoxyl
sulfate, a uremic toxin, directly suppresses the renal-specific organic anion transporter SLCO4C1
expression through GATA transcription factor. The GATA3 mRNA was increased by indoxyl
sulfate in contrast to SLCO4C1 mRNA. A specific GATA-inhibitor K-7174 completely canceled theinhibitory effects of indoxyl sulfate. In addition, over-expression of GATA3 significantly inhibited
SLCO4C1 and knockdown of GATA3 increased SLCO4C1 conversely. In indoxyl sulfate-treated- 121 -

rats, renal expression of slco4c1 was decreased. Under this condition, plasma guanidinosuccinate,
which is a favorable substrate for slco4c1, was significantly increased without changing plasma
creatinine. In 5/6 nephrectomized rats, treatment with oral absorbent AST-120 significantly
decreased the plasma indoxyl sulfate level and conversely increased the renal expression of slco4c1,
resulting the reduction of plasma guanidinosuccinate concentration.
Furthermore, indoxyl sulfate also suppressed erythropoietin production in erythropoietin
producing cells through induction of GATA2 and GATA3.
These data suggest that indoxyl sulfate enhances the expression of GATA factors and causes theaccumulation of uremic toxins and anemia in CKD status. Therefore, the removal of indoxyl sulfate
may help to restore the SLCO4C1-mediated renal excretion of uremic toxins and anemia.

Discussion & Conclusion
It is suggested that IS plays a key role of the formation of the “vicious cycle” between theaccumulation of uremic toxins and renal damage in CKD. Therefore, the removal of IS andblocking its signaling pathway should be an effective strategy to restore the SLCO4C1-mediatedrenal excretion of uremic toxins.

References
1. Akiyama Y., Kikuchi K., Saigusa D., Suzuki T., Takeuchi Y., Mishima E., Yamamoto Y., IshidaA., Sugawara D., Shima H., Toyohara T., Suzuki C., Souma T., Moriguchi T., Tomioka Y., Ito S.
and AbeT.
Indoxyl sulfate down-regulates SLCO4C1 transporter through up-regulation of GATA. Plos One
in press.
2. Toyohara T., Suzuki T., Morimoto R., Akiyama Y., Souma T., Hiromi O. Shiwaku OH., TakeuchiY., Mishima E., Abe M., Tanemoto M., Masuda S., Kawano H., Maemura K., Nakayama M., Sato
H., Mikkaichi T. Yamaguchi H., Fukui S., Fukumoto Y., Shimokawa H., Inui K., Terasaki T.,
Goto J., Ito S., Hishinuma T., Rubera I., Tauc M., Fujii-Kuriyama Y., Yabuuchi H., Moriyama Y.,
Soga T. and Abe T. SLCO4C1 transporter eliminates uremic toxins preventing hypertension andrenal inflammation. J. Am. Soc. Nephrol. 20: 2546-2555, 2009
3. Toyohara T., Akiyama Y., Suzuki T., Takeuchi Y., Mishima E., Tanemoto M., Momose A., TokiN., Sato H., Nakayama M., Hozawa A., Tsuji I., Ito S., Soga T. and Abe T. Metabolomic profilingof uremic solutes in CKD patients.Hypertens. Res. 33: 944, 2010

一般の皆様へ
慢性腎臓病においてトランスポーターの発現や機能低下が腎不全を発症する契機本研究ではヒトの尿毒症物質を腎臓からくみ出す役割を果たすトランスポーターである SLCO4C1 の発現を増強することで尿毒症物質の排泄が促進されることを明らかにし新たな CKD 治療戦略となる可能性を報告した。

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Molecular mechanism of semaphorin receptor activation
implicated in axon-pathfinding
Terukazu Nogi
Graduate School of Medical Life Science, Yokohama City University
nogi@tsurumi.yokohama-cu.ac.jp

Abstract
Plexin is the cell-surface receptor on neuron and binds with the axon guidance cue semaphorin to
induce axon repulsion. We here established the overproduction system of the extracellular domain ofplexin to analyze its three-dimensional structure.
Key Words : Axon Guidance, Cell-surface receptor, Structural Biology, X-ray Crystallography

Introduction
During development of the central nervous system, neurons extend elongated fibers, called axons,
to form precise connections with their target neurons. Plexin is the cell-surface receptor on neuron
and binds with the axon guidance cue termed as semaphorin to mediate the signal transduction
inducing axon repulsion. To analyze the molecular mechanism by which plexin is activated bysemaphorin, we here attempted to overproduce the extracellular domain of plexin for structure
determination through X-ray crystallography.

Results
Most of the plexin family members contain ten modules, a sema domain, three Plexin-SemaphorinIntegrin (PSI) domains and six immunoglobulin-like domains, in the extracellular region. In thisstudy, we first attempted to produce the extracellular domain of plexin as a soluble protein towards
the structure determination at an atomic level. For that purpose, we designed several kinds ofconstructs of the extracellular domain.
Many of extracellular proteins and extracellular domains of membrane proteins are, in general,
glycosylated and possess disulfide-bonds. Such proteins are difficult to produce in conventional
overproduction systems using E. coli. In this study, we therefore utilized a mammalian expression
system to produce recombinant protein of high quality. Specifically, we used HEK293S GnT1(-), amutant cell line of HEK293 that is defective in glycosylation processing, as the expression host to
reduce the glycosylation heterogeneity. It has been established that proteins produced from this cellline possess high-mannose-type N-glycosylations and are suitable for crystallization. In fact, we
succeeded in determining several crystal structures of the extracellular proteins or the extracellulardomains of cell-surface receptors that are associated with development of the central nervous
system.
For stable expression, we transfected the HEK293S GnT1(-) cell with the plasmids encoding several- 123 -

kinds of the partial fragments of the plexin extracellular domain. The transfectants were plated on
96-well plates and selected for resistance to antibiotics. The clone showing the highest secretionlevel was cultured in the high-density cell-culture system BelloCell® (CESCO). With this system,
we obtained about 2L culture supernatant containing the protein of interest within two weeks.
Since we produced the proteins with a polyhistidine tag, we first apply the culture supernatant to
the ion-chelated chromatography resin for purification. Subsequently, we treated the eluted proteinwith protease to cleave off the tag sequence. Furthermore, we purified the protein by using sizeexclusion chromatography.

Results
Finally, we concentrated the eluate by ultrafiltration and used it for crystallization. With thisprocedure, we successfully purified milligram quantity of the target protein from about 1 L of theculture supernatant.
The initial crystallization screens were conducted using commercial screening kits. 0.1 μL of theprotein solution and the reagents, respectively, were dispensed into 96-well plates using the robotic
crystallization system HYDRA II Plus One (Matrix Technologies Corporation) and equilibrated
against 80 μL of the reservoir at 20°C by the sitting-drop vapor-diffusion method. Optimization of
the crystallization conditions was performed by the hanging-drop vapor-diffusion method using
24-well plate where 0.5 μL of the protein solutions and the reagents, respectively, were mixed
and equilibrated against 500 μL of the reservoir. After we obtained crystals, we performed X-ray
diffraction analysis at the synchrotron radiation facility Photon Factory (KEK, Tsukuba, Japan).
For data collection, crystals were quickly soaked in the cryoprotectant and flash-frozen in liquid
nitrogen. Data were collected with the CCD detector and processed with the program HKL2000.
Since the resolution of X-ray diffraction was very low, we were not able to solve the crystal
structure. We, however, could determine the crystallographic parameters such as the space groupand cell constants. The crystallographic analysis suggested that the protein crystallized in this study
is relatively flexible.

Discussion & Conclusion
We here established the overproduction systems for the extracellular domain of plexin. In
addition, we succeeded in purifying and crystallizing one of the overproduced proteins. However,diffraction quality of the crystal was not sufficient and the structure determination has not been
completed so far. Since the protein that was crystallized is heavily glycosylated, we will probably
have to eliminate the glycosylations by treating with glycosidase or introducing point mutations
to the consensus sequences, which will reduce the flexibility and surface entropy of the protein.
Information about the entire structure of plexin and its mobility will contribute to understanding of
the structural change of plexin during the activation process.

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References
1. Nogi, T., Yasui, N., Mihara, E., Matsunaga, Y., Noda, M., Yamashita, N., Toyofuku, T.,
Uchiyama, S., Goshima, Y., Kumanogoh, A. and Takagi, J. (2010) Structural basis for semaphorin
signalling through the plexin receptor. Nature, 467, 1123-1127.
2. Nogi, T., Sangawa, T., Tabata, S., Nagae, M., Tamura-Kawakami, K., Beppu, A., Hattori, M.,
Yasui, N., and Takagi, J. (2008) Novel affinity tag system using structurally defined antibody-tag
interaction: Application to single-step protein purification. Protein Sci., 17, 2120-2126.
3. Sangawa, T., Nogi, T., and Takagi, J. (2008) A murine monoclonal antibody that binds N-terminal
extracellular segment of human protease activated receptor-4. Hybridoma, 27, 331-335.

一般の皆様へヒトの体を構成する細胞の表面には、外界からのシグナルを受け取るための多種多様な受容体蛋
白質が存在しています。我々は、そのような受容体の中でも、特に神経の発生において重要な役割を担っているプレキシンという受容体に興味を持ち、その立体的な形を X 線結晶解析という手法で明らかにする研究に取り組んでいます。プレキシンがシグナルを受け取る時にどのような形をとっているかが明らかになれば、それを標的とした薬剤のデザインが可能になると期待されます。

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Promoter-based super improvement of gene expression
in an antibody production
Masakiyo Sakaguchi
Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciencesmasa-s@md.okayama-u.ac.jp

Abstract
Recombinant antibodies have become fundamental in biomedical research where they are used innumerous therapeutic and diagnostic applications. For this reason there is an increasing demand foran economical production system for antibodies in mammalian cells. In this study, we performed
to enhance expression of a cargo antibody gene by modifying promoter. A newly designed-plasmid
vector showed 10 to 100 fold higher expression than that attained by commercially available vectors.
This vector certainly improved the antibody production in HEK293 cells. This innovative vector
shows a great promise for productive application of recombinant antibodies.
Key Words : Plasmid, Gene expression, Antibody

Introduction
The production of recombinant antibodies for use in therapeutic and diagnostic applications,attracts growing interest in the development of techniques that allows ever increasing large-scale
production of the antibodies. For an exploitation of the recombinant antibodies, the main problem is
low level of expression in animal cell culture leading to high cost of antibody production. Of course,
during the last decade several approaches have been attempted to optimize these in vitro processes
with the aim of improving production yield. One of the main factors that limit the efficiency ofrecombinant antibody production is insufficient activity of promoters loaded on plasmid vectors.
Therefore, in this study, we attempted to improve vector promoter with structural modification of
the plasmid vector. The exploited vector brought significantly higher expression of the antibody
used in this study than that attained by commercially available vectors.

Results
1. Design of vector: We developed a novel promoter system, designed CMViR-C. The CMViR is
composed of the human cytomegarovirus early promoter (CMV), the same virus-derived intron A(i), and the R segmemnt with part of the U5 sequence of the long terminal repeat of human T-cellleukemia virus type 1 (R), and the connected promoter CMViR is inserted at 5’ head of the inserted
cDNA. On the other hand, C from the CMViR-C shows CMV enhancer (C), which is located to 3’
end of the inserted cDNA-polyA sequence. The designed vector, named pCMViR-C, causes verystrong expression for various kinds of cargo cDNAs in many different mammalian cell types. In
this study, the designed plasmid was employed and tried to express recombinant antibody. Human- 126 -

IgG antibody gene is composed of two subunits, Heavy chain (HC) and Light chain (LC). To force
an expression simultaneously HC and LC genes, we used BiP IRES (internal ribosomal entry site)
sequence as the best one among the examined IRES sequences. As an objective IgG antibody ofthis study, we used the HC and LC genes derived from the anti-myc tag IgG antibody (clone 9E10,ATCC) and inserted them to the designed vector under the BiP IRES-mediated connection.
2. Evaluation of the pCMViR-C vector for antibody expression in transient transfection:
The vector was assessed for the expression of cargo antibody genes, HC and LC, in transienttransfections using HEK293 cells. The vector brought around 10 fold higher expression than thatattained by commercially available vectors loaded CMV and CAG promoter, respectively.
3. Evaluation of the pCMViR-C vector for antibody expression in stable transfection: The
designed vector with antibody genes was then employed for stable transfection. In antibioticsselected pools, the stable transformants in mixed culture unexpectedly gave lower expression for
the antibody production than that induced by the transient transfection. Even after the single cellcloning, however, clonal productivities of the antibody in positive clones searched did not show any
appreciable enhancing-improvement of the expression. These results suggest that the vector favors
as circular structure for the high expression rather than the linear structure of the plasmid after theincorporation into chromosome.
4. Functional improvement of the pCMViR-C vector to stable expression: To make it betterthe plasmid for stable expression, EBNA1-OriP system derived from EBV-based episomal vector
was further employed. The system induces sustained localization of the vector in the nucleus andreplicates it with keeping circular structure in human cells. The newly designed vector, pCMViR-CEBO, was assessed for the antibody expression in HEK293 cells, resulting in similar high expression
in transient transfection as compared with the former pCMViR-C vector. In addition, the expression
was kept even after the establishment of stable clones.
5. Function of the produced antibody: Finally, to determine whether the expressed anti-myc tag
IgG retains the same binding ability as compared with a commercially available one (clone 9E10), a
Western blot of serially diluted purified IgG expressed from HEK293 cell cultures was carried out
against a reference protein, GFP fused with myc-epitope tag. We confirmed that a similar detection
was possible for the antibody preparation, HEK293 expressed IgG, in comparison to the companyderived one.

Results
This study validated a newly designed vector, pCMViR-C, that robustly enhances various kindsof gene expression in mammalian cells. The advanced expression system should be a valuable
tool for recombinant antibody production. We therefore studied whether this system could bealso working on a large scale of antibody production. The antibody IgG gene is composed of two
component genes, HC and LC, so that HC and LC genes were connected by BiP IRES sequence,
which causes simultaneous expression of them in the same vector. The exploited construct actually- 127 -

showed dramatically high expression of the individual two genes, finally leading to produce thematured recombinant IgG protein with high productivity in transiently transfected HEK293 cells.
However, this vector was not suitable for making stable transformants by reason of yet unidentified
mechanism, so that we attempted to improve the vector adapting stable expression. The decreased
expression in stable transformants might be reasoned by vector structure, since the incorporated
vector on chromosome exists as a linear structure. According to the idea, we employed EBV-based
episomal self-replication system. The promoter-EBNA1 and OriP sequence units were inserted
to pCMViR-C, eventually producing the improved vector, pCMViR-C-EBO. When utilized thevector for stable expression, the isolated stable clone did not show any appreciable change about
the production as compared with the former vector in transient expression as well as long-term
stable expression. From these results, we strongly indicate that the innovative construct allows
great promise for economical production of the antibodies utilized in therapeutic and diagnostic
applications.

References
1. Saito K, Sakaguchi M, Iioka H, Matsui M, Nakanishi H, Huh NH, Kondo E. Coxsackie andadenovirus receptor is a critical regulator for the survival and growth of oral squamous carcinoma
cells. Oncogene 2013. doi: 10.1038/onc.2013.66. in press.
2. Yamamoto K, Murata H, Putranto EW, Kataoka K, Motoyama A, Hibino T, Inoue Y, Sakaguchi
M, Huh NH. DOCK7 is a critical regulator of the RAGE-Cdc42 signaling axis that induces
formation of dendritic pseudopodia in human cancer cells. Oncol Rep. 29: 1073-1079, 2013.
3. Hibino T, Sakaguchi M, Miyamoto S, Yamamoto M, Motoyama A, Hosoi J, Shimokata T, Ito T,
Tsuboi R, Huh NH. S100A9 is a novel ligand of EMMPRIN that promotes melanoma metastasis.
Cancer Res. 73: 172-183, 2013.
4. Sugimoto M, Watanabe M, Kaku H, Li SA, Noguchi H, Ueki H, Sakaguchi M, Huh NH, Nasu
Y, Kumon H. Preclinical biodistribution and safety study of reduced expression in immortalized
cells/Dickkopf-3-encoding adenoviral vector for prostate cancer gene therapy. Int J Oncol. 28:
1645-1652, 2012.
5. Hirata T, Watanabe M, Kaku H, Kobayashi Y, Yamada H, Sakaguchi M, Takei K, Huh NH, Nasu
Y, Kumon H. REIC/Dkk-3-encoding adenoviral vector as a potentially effective therapeutic
agent for bladder cancer. Int J Oncol. 41: 559-564, 2012.
6. Kataoka K, Ono T, Murata H, Morishita M, Yamamoto K, Sakaguchi M, Huh NH. S100A7
promotes the migration and invasion of osteosarcoma cells via the receptor for advanced glycation
end products. Oncol Lett. 3: 1149-1153, 2012.
7. Kawauchi K, Watanabe M, Kaku H, Huang P, Sasaki K, Sakaguchi M, Ochiai K, Huh NH, Nasu
Y, Kumon H. Preclinical Safety and Efficacy of in Situ REIC/Dkk-3 Gene Therapy for Prostate
Cancer. Acta Med Okayama. 66: 7-16, 2012.
8. Kataoka K, Du G, Maehara N, Murata H, Sakaguchi M, Huh N. Expression pattern of REIC/
Dkk-3 in mouse squamous epithelia. Clin Exp Dermatol. 37: 428-431. 2012.
9. Jin Y, Murata H, Sakaguchi M, Kataoka K, Watanabe M, Nasu Y, Kumon H, Huh NH. Partial
sensitization of human bladder cancer cells to a gene-therapeutic adenovirus carrying REIC/Dkk3 by downregulation of BRPK/PINK1. Oncol Rep. 27: 695-699, 2012.

- 128 -

一般の皆様へ
近年、がんやリウマチの治療薬として抗体医薬の市場は急速に拡大している。抗体医薬のもっとも大きな課題は、製造コストが高いことにある。コストを下げるために、哺乳動物高発現ベクターの開発等が求められている。抗体医薬の抗体は、目的の抗体遺伝子搭載発現ベクターを導入した動物細胞を大量培養することにより製造されているが、従来の発現ベクターを用いた場合は、導入された抗体遺伝子が抗体を生産しない場合や、培養途中で生産しなくなってしまうことが多く、抗体を安定的に高効率で生産する動物細胞を取得するには多大な時間を要していた。代表者が独自に開発した当
プラスミドベクターは、本研究成果より、上記問題を克服し、抗体医療における様々な抗体や生理活
性タンパク質を大量調製するための哺乳動物産生系に新たな光明をもたらすことが期待された。

- 129 -

Analysis of a novel mechanism of tumorigenesis
involving cMyc proto-oncogene productYoshihiro Kawasaki
Institute of Molecular and Cellular Biosciences, The University of Tokyokawasaki@iam.u-tokyo.ac.jp

Abstract
Aberrant activation of Wnt/β-catenin signaling has been implicated in colon cancer formation(1). The proto-oncogene cMYC has been identified as a target of the Wnt/β-catenin pathway incolorectal cancer cells. Here we report the identification of novel Wnt/cMyc target long non-coding
RNA (lncRNA), termed ncRNA-X. We also show that ncRNA-X is required for the tumorigenicity
of colorectal cancer cells. These findings highlight a key role for ncRNA-X in cMyc-driven
transformation.
Key Words : cMyc, Wnt signal, long non-coding RNA (lncRNA)

Introduction
cMyc is among the most commonly over-expressed genes in many types of human cancers. cMYC
encodes a basic helix–loop–helix/leucine zipper (bHLH/LZ) transcription factor that regulates cell
growth, cell proliferation, cell cycle, and apoptosis (2). Although deregulation of cMYC contributes
to tumorigenesis, the mechanism of cMyc-mediated cell transformation remains elusive.

Results
1) Identification of Wnt/cMyc target lncRNA
To obtain new insights into the function of cMyc, we attempted to identify target genes of cMycand to analyze the role of target genes in cMyc-driven tumorigenesis. In order to identify novel
cMyc transactivated lncRNAs, high throughput RNA sequencing (RNA-seq) was performed on
RNA from HT29 cells following knockdown of cMyc using a lentivirus expressing an shRNAagainst cMyc, and on RNA from cells infected with control lentivirus. We found that 120 lncRNAs
showed a statistically significant down-regulation of their expression levels following knockdownof cMyc in HT29 cells. qRT-PCR analysis revealed that knockdown of β-catenin led to reduce theexpression of 7 (out of 120) candidate target genes. Moreover, to explore that cMyc transactivatesthese genes directly, we performed chromatin immunoprecipitation (ChIP) assays on HT29 cells
using anti-cMyc antibody. We detected cMyc binding to the promoter region of ncRNA-X, whichcontains consensus cMyc/MAX binding sites. Taken together, these results suggest that Wnt/cMycsignaling pathway regulates ncRNA-X expression.
2) Analysis of ncRNA-X expression level in colon cancer tissues
Quantitative analysis of ncRNA-X and cMyc expression in human colon cancerous and- 130 -

corresponding non-cancerous tissues by real-time RT–PCR. ncRNA-X was highly expressed inmost of the colorectal tumors examined, whereas it was expressed at low levels in most of thecorresponding non-cancerous tissues. Furthermore, the expression of ncRNA-X is significantly
correlated with the expression of cMyc and Axin2, which is a well-established general target gene of
Wnt signaling. Thus, ncRNA-X expression is aberrantly enhanced in most human colorectal tumors.
3) ncRNA-X is required for the tumorigenesis of colorectal tumor cells
To clarify the importance of ncRNA-X in colorectal tumorigenesis, we infected colon cancer
HT29 cells with a lentivirus expressing an shRNA against ncRNA-X. When the stably expressing
cells were transplanted into nude mice, cell growth was significantly retarded compared to cells
infected with control lentivirus. Furthermore, the suppressive effects of ncRNA-X knockdown on
tumorigenesis were comparable to those of cMyc or β-catenin knockdown. In addition, knockdownof ncRNA-X in HT29 cells led to significant decrease in cell proliferation under adherent cell
culture conditions. These results indicate that the Wnt/cMyc-ncRNA-X pathway may play animportant role in colorectal tumorigenesis.
4) Identification of proteins that are associated with ncRNA-X
We next wanted to investigate the mechanism by which ncRNA-X facilitates tumor formation.
Thus, we attempted to identify proteins that are associated with ncRNA-X by an RNA-pulldown
experiment. We incubated in vitro-synthesized biotinylated ncRNA-X and antisense ncRNA-X
transcripts (negative control) with whole cell lysates and isolated co-precipitated proteins withstreptavidin beads. We resolved the RNA-associated proteins on a SDS-PAGE gel, cut out two bands
specific to ncRNA-X, and are now subject them to mass spectrometry.

Discussion and Conclusion
It has been shown that cMYC is a target gene of Wnt/β-catenin signaling and plays importantroles in tumor formation and progression. We have demonstrated here that Wnt/cMyc signaling
pathway regulates ncRNA-X expression in colorectal tumor cells. Furthermore, we have shown thatncRNA-X is required for the tumorigenicity of colorectal cancer cells. In addition, we also foundthat ncRNA-X expression is aberrantly enhanced in most human colorectal tumors. Thus, the Wnt/
cMyc-ncRNA-X pathway may be critical for colorectal tumorigenesis. We envision that the cMycncRNA-X pathway could be a promising target for the therapy of colorectal tumors.

References
1. Clevers H and Nusse R: Wnt/β-catenin signaling and disease. Cell, 149, 1192-205 (2012).
2. Meyer N and Penn LZ: Reflecting on 25 years with MYC. Nat Rev Cancer, 8, 976-990 (2008).

- 131 -

一般の皆様へ
Wnt シグナル経路の異常亢進は大腸癌細胞に於いて最も一般的にみられる特徴です。Wnt シグナ
ル経路の標的因子 c-Myc が癌発症に深く関与していることは種々の研究成果から十分に確立しているにも関わらず、c-Myc が介在する癌発症機構は良く分かっていません。さらに、長鎖 ncRNA に関する研究は始まったばかりであり、その生理機能が明らかになっているものは極僅かです。その為、
c-Myc の標的因子として同定した長鎖 ncRNA を手掛かりとして、Wnt シグナル経路の異常亢進による大腸癌発症機構を分子レベルで明らかにしようとする本研究は、新たな創薬ターゲットの提示や癌の病理・病態の理解に貢献できるだけでなく、ncRNA の分野における新たな分子基盤の構築としても意義深いものであると考えています。

- 132 -

III.
Reports from the Recipients of Grants
for International Meetings- 133 -

The 8th Annual Conference of OOTR
1. Representative Person
Masakazu Toi
OOTR (Organisation for Oncology and Translational Research), President
2. Opening period and Place
20th (Fri.) - 21st (Sat.) April, 2012
The Westin Miyako Hotel, Kyoto
3. Number of participants / Number of participating countries and areas
165 people, 22 countries, 5 areas
4. Total cost
JPY16,600,000.5. Main use of subsidy
Programme and Guidance Making expenses
6. Result and Impression
OOTR the 8th Annual Conference concluded successfully. Important lectures were given by
world experts in oncology using leading-edge innovative technologies to progress research into
cancer prevention and treatment. Many current topics regarding cancer treatment were discussed
leading to new ideas for future investigation and as targets for possible clinical trials. Attendees
left feeling they had gained valuable insight into new methods of cancer treatment and theirgeneral impression was that the conference achieved and exceeded all their expectations.
7. Additional description
We would like to take this opportunity to thank Novartis Foundation for their most generous
support of OOTR the 8th Annual Conference. Without their generosity it would not have beenpossible to achieve such a great success.

- 134 -

The 22nd CDB Meeting “RNA Sciences in Cell and Developmental Biology II”
1. Representative Person
Toshifumi Inada
Professor, Tohoku University, Graduate School of Pharmaceutical Sciences
2. Opening period and Place
Jun 11 (Mon) - 13 (Wed), 2012
RIKEN Center for Developmental Biology, Hyogo, Japan
3. Number of participants / Number of participating countries and areas
Number of participants: 149 (abroad 31 / domestic 118)
Number of participating countries and areas: 12 countries (Japan, Taiwan, USA, Singapore,
Australia, China, Canada, Austria, Spain, UK, France, Germany)
4. Total cost
JPY 5,514,119.5. Main use of subsidy
Printing of abstract books (200 copies)
6. Result and Impression
The 22nd CDB Meeting “RNA Sciences in Cell and Developmental Biology II” brought
together nearly 149 scientists in Japan to exchange ideas on how RNAs are transcribed,
processed, localized, exported to cytoplasm, and subjected to quality control, and diseases
caused by defects in mRNA processing. The conference was held in June 2011 in Kobe, andconsisted of 1 keynote, 11 sessions with total of 30 oral presentations, including 18 invited
speakers. In addition, 60 posters were presented and 12 of them were selected for short oralpresentations. The three days conference in which 149 attendants representing 12 countries got
together turned out to be very successful in terms of high quality in science as well as ofdeepness of interaction among young scientists and invited speakers. The conference has greatly
contributed to an advance of RNA research both in Japan and the world.
7. Additional description
The organizing committees greatly appreciate The Novartis Foundation for the support.

- 135 -

20th International Symposium on Molecular Cell Biology of Macrophages
1. Representative Person
Shigeo Koyasu, D.Sc.
Department of Microbiology and Immunology, Keio University School of Medicine
2. Opening period and Place
June 15th and 16th, 2012
Sanjo Conference Hall, The University of Tokyo, Tokyo
3. Number of participants / Number of participating countries and areas
130 participants from 9 countries (Austria, Brazil, China, France, Holland, Japan, UK, USA)
4. Total cost
JPY6,040,000.5. Main use of subsidy
Invited speaker’s airfare, the venue and a meeting program.
6. Result and Impression
Forty papers (19 oral and 21 poster presentations) were presented and discussion was very active.
In particular, a lot of unpublished data were presented in the session for innate lymphocytes,
inducing very active discussions. Recent studies have revealed multiple innate lymphocyte
subsets, which correspond to helper T cell subsets. Those include Th1 cytokine producing NK
and NK-like cells, Th2 cytokine producing natural helper cells and Th17 cytokine producing
lymphoid tissue inducer cells. The interaction between macrophages and these innate
lymphocytes seems to play important roles in controlling macrophage activities.
7. Additional description
Among posters presented by young researchers including graduate students and postdocs, threeposters were chosen for the best poster awards.

- 136 -

Plant and Microbes Adaptations to the Cold 2012: Toward risk assessment andmanagement of sustainable agriculture in the cool and cold regions
1. Representative Person
Naoyuki Matsumoto (Hokkaido University)
Ryozo Imai (NARO Hokkaido Agricultural Research Center)(Co-Chair)
2. Opening period and Place
June 24-28，2012
Conference Center, Hokkaido University (Sapporo, Japan)
3. Number of participants / Number of participating countries and areas
102/14 (Japan, U.K., Sweden, Italy, Hungary, France, Canada, Iceland, Korea, Germany, U.S.A.,
Finland, Russia, Norway)
4. Total cost
JPY5,370,000.5. Main use of subsidy
Travel and accommodation expenses for foreign invited speakers
6. Result and Impression
The conference was started by the Keynote lecture “Global change in winter climate andagricultural sustainability” followed by 7 scientific sessions of 41 oral presentations and 42
poster presentations, a special session for which a farmer from Canada and a representative of
farmers’ association from Tokachi area in Hokkaido of Japan are invited as panelists to report theforefront of winter climate change, and finished by the wrap up discussion. Presentations
indicated the rapid progress in researches on plant-microbe interactions under low temperatureand the future direction: the trend of the understanding the cold acclimation of plants andmicrobes is moving into the “fine tuning” network of cold response genes from major cold
regulated genes; molecular genetical approaches are becoming a powerful tool to accelerate
breeding cold hardy and/or snow mold resistant crops by creating molecular markers and new
breeding strategy; climate changes caused by global warming are requiring new strategies forsustainable agricultural production, such as the control of late frost damage induced by loss of
winter hardiness ascribed to early spring. In general, discussions during formal and informal
sessions were quite fruitful and helpful for future collaborative works.
7. Additional description
The proceedings of the conference will appear as “Plant and Microbe Adaptations to Cold in
Changing World” (Publisher: Springer, NY, USA) in June 2013.

- 137 -

The 18th Young Asian Biochemical Engineers' Community (YABEC)
1. Representative Person
Takeshi Omasa
Chairman, Organizing Committee of YABEC 2012
Professor, The University of Tokushima
Guest Processor, Osaka University
2. Opening period and Place
From 26 th October 2012 to 28th October 2012 at the University of Tokushima, Tokushima,JAPAN
3. Number of participants / Number of participating countries and areas
173 participants including 80 participants from China, Taiwan, and Korea. The participants’nationalities are Japanese, Chinese, Taiwanese, Korean, Vietnamese, and Indian.
4. Total cost
JPY5,500,992.5. Main use of subsidy
Travel and accommodation costs for participants from abroad
6. Result and Impression
Since the first YABEC symposium was held in Seoul in 1995, this is the 18th symposium andthis symposium series has developed into a key academic event among young Asian
Biochemical Engineers. This symposium is 5th YABEC symposium in Japan and first
symposium in local city. Tokushima city is located on eastside of Shikoku Island and delta areaof Yoshino River. This YABEC 2012 is the second YABEC symposium which is held under the
auspices of AFOB. This meeting was very successful with total 4 plenary lectures, 16 oralpresentations and 135 poster presentations. Total 117 Ph.D scientists and 56 students attended.
Most participants had very nice impression on the organization of the meeting. All participantsalso enjoyed food and culture, Awa-odori in Tokushima.
7. Additional description
The particular session, “Biofun” was held at this meeting. The purpose of this session is to enjoy
the biotechnology. The interesting presentation entitled “DNA and music”, “Paintings andbiotechnology” were presented in this section and all participants enjoyed these presentation.

- 138 -

The 8th 3R Symposium(International Symposium on DNA Replication, Recombination and Repair)
1. Representative Person
Kaoru Sugasawa
Professor, Biosignal Research Center, Organization of Advanced Science and Technology, Kobe
University
2. Opening period and Place
November 25th (Sun) - 28th (Wed), 2012
Awaji Yumebutai International Conference Center
3. Number of participants / Number of participating countries and areas
184 participants / 16 countries and areas
4. Total cost
JPY12,350,000.5. Main use of subsidy
Printing - abstract books
Meeting expenses - the equipment fee
6. Result and Impression
The 3R Symposia have been aimed to discuss and better understand basic molecularmechanisms and biological relevance of DNA replication, recombination and repair (collectively
called "3R"), which are fundamental functions for organisms to regulate stability and diversity of
the genetic information.In this 8th Symposium, implications of the 3R in human diseases were a
bit more consciously regarded than the previous ones, thereby subtitled: "Molecular mechanismand pathology of the 3R".
As the leading scientists from the related fields, 19 speakers were invited from abroad and 20
from Japan.In addition, 18 speakers were selected from abstracts submitted to give oralpresentation.In general, scientific quality of their talks was very high, stimulating active
discussion throughout the meeting.In poster sessions, as many as 116 posters were presented,
and it was quite impressive that many participants were absorbed in intense discussion,forgetting about the time.Overall, this meeting was filled with free and pleasant atmosphere, sothat it was very successful.
7. Additional description
Especially to encourage young postdocs and students, 3 or 4 posters were chosen for eachsession and the presenters were invited to short talks.Although this was a new attempt for this
symposium, it was highly evaluated by many participants and thus significant from theviewpoint of nurturing young scientists.

- 139 -

26th Grant Report (FY2012)
The foundation has been conducting public interest activities such as research grant, meeting
grant and international exchange program since its establishment on Sep. 4, 1987 in Japan underauthorization of the Ministry of Education, Science, Sports and Culture. The grants conducted in FY
2012 are as follows.
26th Novartis Research Grant: 40 Researchers (JPY 1 mil.),

Subtotal JPY 40 mil.

Research Meeting Grant:

Subtotal

JPY 2.4 mil.

Total

JPY 42.4 mil.

6 Meetings (JPY 0.4 mil.),		

26th Novartis Research Grant (FY2012)
The grant is to aim supporting creative research in Japan in the field of bio, life science and relevant
chemistry. The 40 grantees are as follows.
#

Name

1 Rikinari Hanayama
2 Satoru Yokoyama
3 Koichiro Nishino
4 Atsushi Kuhara
5 Kohichi Matsunaga
6 Shuji Yamashita
7 Yuji Hara
8 Akira Nakamura
9 Shuh-ichi Nishikawa

10 Katsuaki Hoshino

11 Kyoko Hida

12 Yuko Seko

13 Mamoru Sugita
14 Yutaro Obara
15 Shin-ichi Horike

Institution
Osaka University,
Immunology Frontier
Research Center
Institute of Natural Medicine,
Toyama University
University of Miyazaki,
Veterinary Biochemistry andMolecular Biology

Title

Research Project

Associate
Professor

Inflammation caused by secreted lysosomal
enzymes

Assistant
Professor
Associate
Professor

RAC1 inhibitor is an anti-tumor metastasis
drug?
Analysis of induction mechanism of
human TERT gene expression in humanpluripotent stem cells.

Konan University, Faculty of
Science and Engineering

Junior
Associate
Professor

Molecular mechanism of temperature
response through secretory signaling

Institute for Molecular andCellular Regulation, Gunma
University
Tohoku University, Graduate
School of Science
Tokyo Women's Medical
University, Institute forIntegrated Medical Sciences

Assistant
Professor

Role of Rab GTPases in the intracellularlogistics of insulin granule

Assistant
Professor

Chemical Biology Based on the Unnatural
Steroidal Compounds
mechanisms underlying the
Associate Molecular
repair pathway in skeletal
Professor membranemusclequiescence for the
RIKEN Center for
Laboratory Transcriptional
establishment of germ cell fate in
Developmental Biology
Head
Drosophila
Regulation of plasma membrane receptorNiigata University, Faculty
Professor
like kinase functions by endoplasmicof Science
reticulum quality control in plant cells
Development of a novel therapeutic
Faculty of Medicine, Kagawa Professor approach for the treatment of autoimmune
University
diseases by antibodies against plasmacytoid
dendritic cells.
Hokkaido University,
Associate Prevention tumor metastasis by targeting
Graduate School of Dental
Professor tumor endothelial cells
Medicine
In vitro derivation from humancells to finally differentiatedSection Chief somatic
photoreceptors; generating complete discstructures of rods and cones
Identification of cytidine deaminase activity
Nagoya University, Center
Professor for elucidating the molecular mechanism offor Gene Researchplant organelle RNA editing
Yamagata University, School Associate Clarification of novel mechanism ofof Medicine
Professor catecholamine biosynthesis by ERK5
Kanazawa University,
the biological significance andAssociate Assessing
Advanced Science Researchfunctionofneuron-specific imprinted genes
Professor in brain plasticity
CenterNational Rehabilitation
Center for Persons with
Disabilities- 140 -#Name

16 Masami Yamada

Institution
Osaka-City University,
Graduate School of Medicine

Title
Associate
Professor

17 Takatsune Shimizu

Faculty of Pharmaceutical
Sciences, Hoshi University

Associate
Professor

18 Mitsuhiro Arisawa

Hokkaido University, Facultyof Pharmaceutical Sciences

Associate
Professor

Research Project
Intracellular logistics regulated by LIS1
and lissencephaly
Elucidation of molecular functions of
Imp3 in osteosarcoma progression, anddevelopment of novel anti-Imp3 targeted
therapeutic approach
Development of metal nanoparticles fororganic synthesis aiming a substitution andreduction of rear metals

Professor

Epigenetic regulation by nuclear porecomplex in cancer progression.

Assistant
Professor

Artificial Regulation of Protein Degradationby the Proteasome

Assistant
Professor

Regulatory mechanism of mucosal healing
by DNA methylation
Actin Cytoskeleton Structure Regulation
by TRIOBP, a novel gene responsible tohuman deafness

21 Hiroyuki Tezuka

Institute of Science andEngineering, Kanazawa
University
Frontier Research Core forLife Sciences, University of
ToyamaTokyo Medical and Dental
University

22 Shin-ichiro Kitajiri

Kyoto University Hospital

Assistant
Professor

23 Yuko Ozaki

Research Institute for
Radiation Biology &Medicine, Hiroshima
University

Postdoctoral Study of chromosome instability caused by
Research
Associate the impairment of centrosome maturation

24 Hiroyasu Nakano

Juntendo University,
Graduate School of Medicine

Elucidation of the mechanisms by which
impaired clearance of apoptotic cells results
in severe inflammation
Molecular mechanisms for the production
Section Head and maintenance of proliferative cells inthe subventricular zone

19 Richard Wong
20 Tomonao Inobe

28 Akiko Taguchi

Institute for Developmental
Research, Aichi Human
Service Center
National Institute of
Genetics, Department ofDevelopmental Genetics
Kyoto Prefectural Universityof Medicine, Department ofChemistry
Faculty of Medicine,
University of Miyazaki

29 Kiyotsugu Yoshida

Jikei University, School of
Medicine

25 Hidenori Tabata
26 Kazuhide Asakawa
27 Takayoshi Suzuki

Associate
Professor

Assistant
Professor

Role of the VIPLa-mediated glycoprotein
trafficking in locomotion in zebrafish

Professor

Explorative study on lysine demethylase
inhibitors by rational drug design

Associate
Professor

Molecular mechanism of aging-like effectby diabetes
Comprehensive proteomicanalysis ofmolecules that are targeted for cancer
prognosis or therapy
Molecular mechanism of Derlin familymediated novel endoplasmic reticulum
quality control system
Development of NO donor drug based on
furoxan structure
Development of Novel Genetic Screening
Assay using Next-Generation Sequencer
and Clinical Application of Personalized
Medicine in Inherited Arrhythmia
Syndromes
Research on a novel neuroprotective/
anti-inflammatory prototoxin targeting tonicotinic acetylcholine receptor
Development of an orange-coloredbimolecular fluorescence complementation(BiFC) assay
Regulation of secretion of Wnt proteinsby post-translational modifications inpolarized epithelial cells
Synthetic Studies on the virulent cell wall
lipid of Mycobacterium tuberculosis

Professor

31 Ryosuke Matsubara

Department of Medical
Sciences, University ofMiyazaki
Kobe University, Graduate
School of Science

Associate
Professor

32 Hiroshi Watanabe

Niigata University, Medical
and Dental Hospital

Assistant
Professor

33 Hidemi Misawa

Keio University, Faculty of
Pharmacy

Professor

34 Yutaka Kodama

Center for Bioscience
Research and Education,
Utsunomiya University

Assistant
Professor

35 Akira Kikuchi

Graduate School of
Medicine, Osaka University

Professor

36 Seijiro Hosokawa

Waseda University, Faculty
Associateof Science and Engineering
Professor
Kyushu Institute ofTechnology, Graduate School Associate
Professorof Engineering
Research Center for
Advanced Science andProject
Technology, The University
Researcherof Tokyo

30 Hideki Nishitoh

37 Mitsuru Kitamura

38 Yasuharu Kanki

Professor- 141 -

Synthesis of anticancer anthracycline
antibiotics having aza-spiro structure
Determination about the essential factors invascular endothelial cell differentiation

#

Name

39 Kazuaki Yoshioka
40 Koichi Matsuo

Institution
Kanazawa University, Schoolof Medicine
School of Medicine, Keio
University

Title
Assistant
Professor
Professor

Research Project
Essential roles of class II PI3-kinase C2α invascular formation and homeostasis.
Mechanisms of Eph family receptormediated regulation of bone mineralization

FY 2012 Research Meeting Grant
The grant is to aim supporting international research meetings in Japan in the field of bio, life
science and relevant chemistry. The 6 grantees are as follows.
#

Meeting

8th Annual Conference of OOTR
1 (Organization for Oncology andTranslational Research )Sciences in Cell and Developmental
2 RNA
Biology 2012
International Symposium on
3 20thMolecular Cell Biology of Macrophages
and Microbe Adaptation to the
4 Plant
Cold 2012
18th Symposium of Young Asian
5 The
Biochemical Engineers’ Community
The 8th 3R Symposium (International
6 Symposium on DNA Replication,
Recombination and Repair)

Date(Place)2012.4.20 ～ 21
(Kyoto)Representative
Institution / Title(NPO) OOTR /President

Graduate School of
Pharmaceutical Sciences,
Tohoku University /Professor
2012.6.15 ～ 16 Keio University School(Tokyo)of Medicine / Professor
Agricultural
2012.6.24 ～ 28 Hokkaido
ResearchCenter/(Sapporo)
Research Director
Institute of Technology
2012.10.26 ～ 28 and Science, University(Tokushima)of Tokushima /Professor
Research
2012.11.19 ～ 22 Biosignal
Center,Kobe
University(Awaji)/ Professor
2012.6.11 ～ 13
(Kobe)- 142 -Name

Masakazu Toi

Toshifumi Inada
Shigeo Koyasu
Norio Iriki

Takeshi Omasa

Kaoru Sugasawa

第 26 期（2012 年度）助成事業報告
当財団は、文部大臣の認可を得て 1987 年 9 月 4 日に設立されて以降、研究助成を中心とした公益事業を行っております。2012 年度は、下記の総額 4,240 万円の助成事業を実施しました。
第 26 回ノバルティス研究奨励金
研究集会助成40 件 （1 件 100 万円）4,000 万円
6 件 （1 件 40 万円） 240 万円
総額 4,240 万円

第 26 回ノバルティス研究奨励金 (2012 年度 )この事業は、生物・生命科学および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の助成を目的としています。2012 年度は 40 件の助成を行いました。
（受付順 ・ 敬称略、 所属 ・ 職位は申請時、 贈呈額は 1 件 100 万円）
No
1
2
3
4
5
6
7
8
9
10
11
12

13
14
15

氏名

所属
大阪大学免疫学フロンティア
華山 力成
研究センター

職位
研究課題
特任 リソソーム酵素の放出による炎症の発症機
准教授 構Rac1 阻害剤は “抗がん転移剤” になりう
横 山
悟 富山大学和漢医薬学総合研究所 助教
るのか？
ヒト多能性幹細胞における TERT 遺伝子
西野 光一郎 宮崎大学農学部准教授の発現獲得機構の解析
内分泌シグナルを介した温度応答の分子久 原
篤 甲南大学理工学部
講師
機構
複数の Rab GTPase によるインスリン顆粒
松 永 耕 一 群馬大学生体調節研究所
助教の細胞内物流システム
非天然ステロイドを基盤とする有機分子
山 下 修 治 東北大学大学院理学研究科
助教
ツールの創製と生体機能制御
テニュア
東京女子医科大学統合医科学
骨格筋における形質膜修復機構の分子
原
雄 二
トラック
基盤
研究所
准教授
理化学研究所発生 ・ 再生科学 特別主管 ショウジョウバエ生殖細胞形成におけるゲ中 村輝
研究員 ノムワイドな転写抑制とその意義
総合研究センター
小胞体品質管理による植物細胞膜受容西 川 周 一 新潟大学理学部教授
体様キナーゼの機能発現機構
形質細胞様樹状細胞を標的とした自己免
星 野 克 明 香川大学医学部教授
疫疾患制御手段の開発
特任 腫瘍血管内皮細胞を標的としたがんの転樋 田 京 子 北海道大学大学院歯学研究科
准教授 移制御国立障害者リハビリテーションセ 研究 ヒト体細胞から網膜視細胞への in vitro
世古 裕子
における最終分化―外節のある杆体と錐ンター研究所
室長 体の作製－
植物オルガネラの RNA 編集の分子機構
杉 田
護 名古屋大学遺伝子実験施設
教授 解明に向けたシチジンデアミナーゼ活性の同定
ERK5 による新しいカテコールアミン類の小原 祐太郎 山形大学医学部准教授
生合成促進機構の解明
高次な精神発達制御を司るゲノムインプリ
金沢大学学際科学実験セン
堀家 慎一
准教授 ンティング遺伝子の生物学的意義と機能
ターの解明- 143 -

No

氏名

所属
大阪市立大学大学院
16 山 田 雅 巳
医学研究科

職位

17 清 水 孝 恒 星薬科大学薬学部
18 有 澤 光 弘 北海道大学大学院薬学研究院
ウォング
熊本大学大学院
リチャード 生命科学研究部
富山大学先端ライフサイエンス
20 伊野部 智由
拠点東京医科歯科大学難治疾患
21 手 塚 裕 之
研究所
19

22 北尻 真一郎 京都大学医学部附属病院
23 尾 崎 佑 子

広島大学原爆放射線医科学
研究所

24 中 野 裕 康 順天堂大学医学部
愛知県心身障害者コロニー
発達障害研究所
大学共同利用機関法人情報 ・
26 浅 川 和 秀 システム研究機構国立遺伝学研究所
京都府立医科大学大学院医学27 鈴 木 孝 禎
研究科
25 田 畑 秀 典

28 田 口 明 子 宮崎大学医学部
29 吉 田 清 嗣 東京慈恵会医科大学
30 西 頭 英 起 宮崎大学医学部
31 松 原 亮 介 神戸大学大学院理学研究科
32 渡 部

裕 新潟大学医歯学総合病院

33 三澤 日出巳 慶應義塾大学薬学部
宇都宮大学バイオサイエンス
教育研究センター

34 児 玉豊

35 菊 池章 大阪大学大学院医学系研究科

早稲田大学大学院先進理工学研究科
九州工業大学大学院工学研究
37 北 村充
院
東京大学先端科学技術研究セ
38 神 吉 康 晴
ンター
金沢大学医薬保健研究域医学39 吉 岡 和 晃
系
36 細川 誠二郎

40 松 尾 光 一 慶應義塾大学医学部

研究課題
LIS1 による細胞内ロジスティクスと滑脳症
准教授
発症の関係
骨肉腫悪性化に関わる RNA 結合タンパ
准教授 ク Imp3 の機能解明及び分子標的療法の開発
希少元素代替 ・ 削減型有機合成用金属
准教授
ナノパーティクル触媒の開発
骨格筋由来の新規血管新生因子の同定
助教とその機能解析
特命助 プロテアソームによる蛋白質分解の人工制
御教
DNA メチル化による粘膜治癒機構の解助教
明
ヒト難聴から同定された新規分子 TRIOBP
助教
による、 アクチン束化様式と聴覚制御
博士研 中心体成熟不全によるがん細胞の染色体究員 不安定性惹起メカニズムの解明
死細胞貪食破綻による炎症誘導のメカニ
准教授
ズムの解明
脳室下帯分裂細胞の産生と維持の分子
室長
機構
助教

ロコモーションに必要な糖タンパク質輸送系の解明

合理的設計によるリシン脱メチル化酵素阻害薬の創製研究
糖尿病による老化類似作用の分子機構の准教授
解明
癌の診断 ・ 治療の標的となる分子同定を教授目指した網羅的プロテオーム解析
Derlin ファミリーを介した新規小胞体品質
教授
管理機構の解明
フロキサン構造を有する NO ドナー医薬
准教授
品の開発
次世代シークエンサーを応用した致死性
助教 不整脈症候群の新たな遺伝子検索アッセイの構築と個別化治療の確立
ニコチン受容体をターゲットとする新たな教授
神経保護 ・ 抗炎症プロトトキシンの研究
教授

助教

橙色二分子蛍光補完法の開発

Wnt タンパク質の翻訳後修飾と極性化上皮細胞における分泌制御
化学ワクチンを指向した結核菌の毒性細准教授
胞壁成分の合成研究
アザスピロ構造を有するアントラサイクリン
准教授
系抗がん抗生物質の合成
特任
血管内皮細胞分化決定因子の同定
研究員
クラスⅡ型 PI3 キナーゼ C2 αによる血管
助教
形成 ・ 健全性維持の機能解明
Eph ファミリー受容体による骨ミネラリゼー
教授
ションの制御機構教授- 144 -

2012 年度 研究集会助成
この事業は、生物・生命化学および関連する化学の領域において、我が国で開催される国際色豊かな研究集会の助成を目的としています。2012 年度は 6 件の助成を行いました。
（受付順・敬称略、所属・職位は申請時、贈呈額は 1 件 40 万円）
No

研究集会名

開催日
( 開催地 )
2012.4.20-21
（京都）

代表者
所属・職位

氏名

特定非営利活動法人
OOTR ・ 理事長

戸井

雅和

1

第 8 回 OOTR 年次学会

2

細胞と分化生物学における RNA 2012.6.11-13
研究会
（神戸）

東北大学大学院
薬学研究科 ・ 教授

稲田

利文

3

第 20 回マクロファージ分子細胞 2012.6.15-16
生物学国際シンポジウム（東京）

慶應義塾大学医学部 ・ 教授

小安

重夫

4

植物と微生物の低温適応 20122012.6.24-28（札幌）

農業 ･ 食品産業技術総合研究機構 北海道農業研究 入 来センター ・ 研究領域長規雄

5

第 18 回生物化学工学アジア
2012.10.26-28
若手研究者の集い YABEC2012 （徳島）

徳島大学大学院 ソシオテク
大政
ノサイエンス研究部 ・ 教授健史

6

第 8 回国際 3R シンポジウム

2012.11.19-22（淡路）- 145 -

神戸大学 バイオシグナル
研究センター ・ 教授

菅

澤

薫

26th Financial Report

Balance Sheet

As of March 31, 2013
Account
I Assets
1. Current Assets
Total Current Assets
2. Fixed Assets (Long-term)(1) Basic Fund
Basic Fund Total(2) Other Long-term Assets
Other Long-term Assets Total
Long-term Assets Total
Assets Total
II Liabilities
1. Current Liabilities
Current Liabilities Total
Liabilities Total
III Equity (Net Assets)
1. Designated Net Assets
Designated Net Assets Total(Amount appropriating to Basic Fund)
2. General Net Assets(Amount appropriating to Basic Fund)
Equity Total (Net Assets)
Liabilities & Equity Total

Movement of Net Assets(Unit: JP Yen)
Amount49,067,338

1,100,000,000
59,861,958
1,159,861,958
1,208,929,296

40,054,374
40,054,374

1,000,000,000(1,000,000,000)
168,874,922(100,000,000)
1,168,874,922
1,208,929,296- 146 -

April 1st, 2012 - March 31, 2013
(Unit: JP Yen)

Account
I General Net Assets Changes
1. Ordinary Income & Expenditure(1) Ordinary Income
Interest from Basic Fund
Donation
Other Income
Ordinary Income Total(2) Ordinary Expenditure
Project Expenses
Grant Expense
Novartis Research Grant
Research Meeting Grant
Administrative Expense
Ordinary Expenditure Total
Ordinary Balance of Current Period
2. Nonrecurring Profit & Loss
Nonrecurring Balance of Current Period
General Net Assets Balance of Current Period
General Net Assets Ending Balance

Amount20,583,328
40,050,000
334,524
60,967,852
52,966,501
42,400,000
40,000,000
2,400,000
4,351,506
57,318,007
3,649,845
0
3,649,845
168,874,922

II Designated Net Assets Changes
Designated Net Assets Change0
Designated Net Assets Ending Balance 1,000,000,000
III Net Assets Balance Ending Balance
1,168,874,922

第 26 期（2012 年度）財務報告

貸借対照表

正味財産増減計算書

2013 年 3 月 31 日現在（単位：円）

科目
Ⅰ資産の部
１． 流動資産
流動資産合計
２． 固定資産(1) 基本財産
基本財産合計(2) その他固定資産
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
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（うち基本財産への充当額）
２．一般正味財産（うち基本財産への充当額）
正味財産合計
負債及び正味財産合計

金額49,067,338

1,100,000,000
59,861,958
1,159,861,958
1,208,929,296

40,054,374
40,054,374

1,000,000,000(1,000,000,000)
168,874,922(100,000,000)
1,168,874,922
1,208,929,296- 147 -

2012 年 4 月 1 日～ 2013 年 3 月 31 日
（単位：円）

科
目
Ⅰ一般正味財産増減の部
１． 経常増減の部(1) 経常収益
基本財産運用益
受取寄付金
雑収益
経常収益計
(2) 経常費用
事業費
支払助成金
ノバルティス研究奨励金
研究集会助成金
管理費
経常費用計
当期経常増減額
2． 経常外増減の部(1) 経常外収益
経常外収益計(2) 経常外費用
経常外費用計
当期経常外増減額
当期一般正味財産増減額
一般正味財産期末残高
Ⅱ指定正味財産増減の部
当期指定正味財産増減額
指定正味財産期末残高
Ⅲ正味財産期末残高

決算額

20,583,328
40,050,000

334,524
60,967,852
52,966,501
42,400,000
40,000,000

2,400,000
4,351,506
57,318,007
3,649,8450003,649,845
168,874,922
01,000,000,000
1,168,874,922

List of Board Members[Board of Trustees]

As of Sep. 1, 2013

Post

Name

Chairman

Akimichi KANEKO

Dean, Prof., MD, Grad. School of Health Science, Kio Univ.,
Emer. Prof., Keio Univ.

Shigetaka ASANO

Visiting Prof., MD, School of Medicine, Kobe Univ.,
Emer. Prof., Univ. of Tokyo

Masao ENDOH

Emer. Prof., MD, Yamagata Univ.

Hiroko ISHIKAWA

President, Novartis Holding Japan K.K.,
Vice-president, Novartis Pharma K.K.

Toshio SUDA

Prof., MD, Keio Univ., School of Medicine

Tokuzo NAKAJIMA

Certified Public Accountant

Masanori FUSE

Head, Accounting Dept., Novartis Pharma K.K.

Trustee

Auditor

Title[Board of Councilors]
Post

Name

Chairman Tsuneyoshi KUROIWA

Title
Member of the Japan Academy,
Emer. Prof., Univ. of Tokyo

Norio AKAIKE

Director, Kumamoto Health Science Univ.,
Emer. Prof., Kyushu Univ.

Max M. BURGER

Novartis Science Board
Prof., MD, Univ. of Basel

Hiroyuki KAWASHIMA

Former Prof., Grad. School of Medical & Dental Sciences,
Niigata Univ.

Shigeo KOYASU

Acting Director, RIKEN Center for Integrative Medical Sciences

Tadanori MAYUMIEmer. Prof., Osaka Univ.

Councilor Miwako MORI

Prof., Health Sciences Univ. of Hokkaido,
Emer. Prof., Hokkaido Univ.

Akihiko NAKANO

Prof., Univ. of Tokyo, Science Dept., Team Leader, RIKEN (Inst.
of Physical & Chemical Research)

Yoshiyasu NINOMIYA

President, Novartis Pharma K.K.

Masakatsu SHIBASAKI

Director, Microbial Chemistry Research Center, Microbial
Chemistry Research Foundation

Takao SHIMIZU

Director-General, MD, Research Institute, National Center for
Global Health and Medicine

Toshifumi WATANABE

Corporate Officer, Senior Science Advisor, Novartis Pharma K.K.

- 148 -

[Grantee Selection Committee]
Post

Name

Chairman Hiroaki SASAI

MemberTitle
Prof., Inst. of Scientific & Industrial Research, Osaka Univ.

Hidenori ICHIJO

Prof., Dentist, Grad. School of Pharmaceutical Sciences,
Univ. of Tokyo

Nobuya INAGAKI

Prof., MD, Grad. School of Medicine, Kyoto Univ.

Akihiro UMEZAWA

Director, MD, National Inst. for Child Health & Development

Shinichi OKA

Director, MD, Aids Clinical Center, National Center for Global
Health & Medicine

Shigeyuki KAWANO

Prof., Grad. School of Frontier Sciences, Univ. of Tokyo

Takeo KISHIMOTO

Visiting Prof., Science & Education Center, Ochanomizu Univ.

Makoto SASAKI

Prof., Grad. School of Life Sciences, Tohoku Univ.

Toshiharu SHIKANAI

Prof., Grad. School of Science, Kyoto Univ.

Hiroshi TAKAYANAGI

Prof., MD, Grad. School of Medicine, Univ. of Tokyo

Hiroyuki TSUTSUIProf., MD, School of Medicine, Hokkaido Univ.

Hiroyuki NAKAMURA

Prof., Faculty of Science, Gakushuin Univ.

Junichi NABEKURA

Prof., MD, National Inst. for Physiological Sciences

Masanori HATAKEYAMA Prof., MD, Grad. School of Medicine, Univ. of Tokyo
Tomoko BETSUYAKUProf., MD, School of Medicine, Keio Univ.

Tetsuji MIURA

Prof., MD, Sapporo Medical Univ.

Masabumi MINAMI

Prof., Grad. School of Pharmaceutical Sciences, Hokkaido Univ.

Takashi MINEGISHI

Prof., MD, Grad. School of Medicine, Gunma Univ.

Toyoaki MUROHARA

Prof., MD, Grad. School of Medicine, Nagoya Univ.

Masato YASUIProf., MD, School of Medicine, Keio Univ.

- 149 -

公益財団法人ノバルティス科学振興財団

役 員 名 簿2013 年 9 月 1 日現在 ( 順不同、 敬称略 )
職

名

氏

名

現

職

就任年月日

常勤･非常勤代表理事 金 子 章 道

畿央大学大学院健康科学研究科長･教授
慶應義塾大学名誉教授

2012 年 4 月 1 日

非常勤理

神戸大学大学院医学系研究科客員教授
東京大学名誉教授

2012 年 4 月 1 日

非常勤

ノバルティスホールディングジャパン(株)代表
石 川 裕 子 取締役社長、ノバルティス ファーマ(株)取締役 2012 年 4 月 1 日
副社長

非常勤

遠 藤 政 夫 山形大学名誉教授

2012 年 4 月 1 日

非常勤

須 田 年 生 慶應義塾大学医学部教授

2012 年 4 月 1 日

非常勤中嶋德三公認会計士事務所
公認会計士

2012 年 4 月 1 日

非常勤

ノバルティス ファーマ株式会社
経理部長

2012 年 4 月 1 日

非常勤

監

事 浅野 茂隆

事 中嶋 德三

布施

正則

評議員名簿
2013 年 9 月 1 日現在 ( 順不同、敬称略 )
職

名

氏

名

現

職

就任年月日

常勤･非常勤評議員長 黒岩 常祥

日本学士院会員
東京大学名誉教授

2012年 4月1日

非常勤

評 議 員 赤池 紀扶

熊本保健科学大学理事 ・ 研究顧問
九州大学名誉教授

2012年 4月1日

非常勤

2012年 4月1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
小安 重夫

理化学研究所
統合生命医科学研究センター長代行

2012年 4月1日

非常勤

柴崎 正勝

公益財団法人微生物化学研究会
微生物化学研究所長

2012年 4月1日

非常勤

清水 孝雄 国立国際医療センター研究所長

2012年 4月1日

非常勤

中野 明彦 東京大学大学院理学系研究科教授

2012年 4月1日

非常勤

マックス ・ ノバルティス サイエンスボード
ブルガー バーゼル大学教授

2012年 4月1日

非常勤

眞弓 忠範 大阪大学名誉教授

2012年 4月1日

非常勤

二之宮 義泰 ノバルティス ファーマ㈱代表取締役社長

2013 年7月4日

非常勤- 150 -

職

名

氏

名

現

評 議 員 森 美和子
渡邉 敏文

職

就任年月日

常勤･非常勤北海道医療大学客員教授
北海道大学名誉教授

2012年 4月1日

非常勤

ノバルティス ファーマ㈱執行役員シニアサイエンスアドバイザー

2012年 4月1日

非常勤

選考委員名簿2013 年 9 月 1 日現在 ( 順不同、敬称略 )
職 名

氏 名

現

職

就任年月日

常勤･非常勤選考委員長 笹 井 宏 明 大阪大学産業科学研究所教授

2010 年 6月18日

非常勤

選考委員 一 條 秀 憲 東京大学大学院薬学系研究科教授

2011年 6月17日

非常勤

2012年 6月15日

非常勤

稲 垣 暢 也 京都大学大学院医学研究科教授
梅澤 明弘

国立成育医療研究センター
再生医療センター長

2012年 6月15日

非常勤

岡国立国際医療センター
エイズ治療 ・ 研究開発センター長

2011年 6月17日

非常勤

河 野 重 行 東京大学大学院新領域創成科学研究科教授 2013 年 6月14日

非常勤

お茶の水女子大学
2011年 6月17日
サイエンス & エデュケーションセンター客員教授

非常勤

慎 一

岸本 健雄

佐 々 木 誠 東北大学大学院生命科学研究科教授

2012年 6月15日

非常勤

鹿 内 利 治 京都大学大学院理学研究科教授

2011年 6月17日

非常勤

高 柳

広 東京大学大学院医学系研究科教授

2011年 6月17日

非常勤

筒 井 裕 之 北海道大学大学院医学研究科教授

2012年 6月15日

非常勤

中 村 浩 之 学習院大学理学部教授

2013 年 6月14日

非常勤

鍋 倉 淳 一 自然科学研究機構生理学研究所教授

2013 年 6月14日

非常勤

畠 山 昌 則 東京大学大学院医学系研究科教授

2013 年 6月14日

非常勤

別 役 智 子 慶應義塾大学医学部教授

2013 年 6月14日

非常勤

三 浦 哲 嗣 札幌医科大学医学部教授

2013 年 6月14日

非常勤

南2011年 6月17日

非常勤2010 年 6月18日

非常勤

室 原 豊 明 名古屋大学大学院医学系研究科教授

2011年 6月17日

非常勤

安 井 正 人 慶應大学医学部教授

2013 年 6月14日

非常勤

雅 文 北海道大学大学院薬学研究院教授

峯 岸

敬 群馬大学大学院医学系研究科教授- 151 -

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