1

Foreword
Printed media or electronic media
In the late 1960s, the time when I started my research career, I used to expect a large
number of reprint requests by colleagues for my publication in leading international
journals. It was my happiest moment if the reprint request cards accumulated more than300. Of course I had to bear considerable amount of charge from the publisher. In
today's academic society, even if you publish a superb paper in a leading journal withhigh impact factor, you never receive any reprint request. Also most of us place noreprint order to the publisher.
Many academic publications are moving from paper to electronic media. Referees,
reviewers, and editors sitting in front of computer displays work together by exchanging
texts and pictures in an electronic format. Use of electronic media reduces time forediting and printing as well as postage. Journals on medical and biological sciences aresearched and retrieved easily by using PubMed and many other databases. It becomes
important for university libraries to offer a good electronic access to magazines andjournals rather than a vast selection of printed books or journals.
Recently NIH requests all NIH-funded investigators to make their peer-reviewed
author's final manuscripts available to other researchers and the public at the NIH
National Library of Medicine's PubMed Central (PMC) immediately after the final dateof journal publication. Many medical journal publishers are against this NIH policy
because it makes their publishing difficult. Researchers are generally in favor of the
NIH policy because they naturally want to let the world know their work as soon as
possible. NIH seems to reach a compromise that it waits for one year after journal
publication.
NOVARTIS Foundation (Japan) introduced HTML version of Annual Report on website this year and ceased the publication of printed version. If Annual Reports are
published on our web page anyone interested in the research topic can read it and at thesame time we can reduce the cost for publication. We may keep this policy if noproblems arise.
However I recently realized that keeping printed matter as a part of archives is still
very important. When I tried to find some materials describing NOVARTIS Foundation(Japan) at the time of its establishment, the first issue of the Annual Reports told me
what I was wanting to know and I could read it without any difficulty of its retrieval.

2Electronic media is useful only when retrieval means are available. Printed media has its
history for more than a thousand years and is of vital importance. We NOVARTISFoundation (Japan) are seriously reviewing how to store irreplaceable knowledge ofhuman being.

Akimichi Kaneko, MD, PhD
Chairman, Board of Trustees
The NOVARTIS Foundation (Japan)

3Contents
Part Ⅰ:
Reports from the Recipients of Novartis Research Grants(Fiscal Year 2003)
1) Biological Sciences
1-1)

Molecular Biology
The mechanism of p16INK4a gene induction by stress response
Naoko Ohtani
Division of Protein Information, Institute for Genome Research, The University ofTokushimaohtani@genome.tokushima-u.ac.jp

Regulation of NF-κB activation by PYPAF1/Cryopyrin
Takashi Suda
Kanazawa University, Cancer Research Institutesudat@kenroku.kanazawa-u.ac.jp

Identification of genes involved in neurodegenerative disorders and ER-stress byusing a library of randomized ribozymes
Reiko Onuki
Graduate School of Pharmaceutical Sciences, the University of Tokyo
onuki@mol.f.u-tokyo.ac.jp

Role of small GTPase Rac in formation of neural circuit and synaptic plasticity inthe central nervous system
Atsu Aiba
Kobe University Graduate School of Medicine
aiba@med.kobe-u.ac.jp
Molecular Analysis of Salivary Gland Branching Morphogenesis
Takayoshi Sakai
Osaka University Dental Hospitalsakai@dent.osaka-u.ac.jp

Structural manipulation of integrin ligand binding affinity and its use in thedevelopment of novel class of inhibitors
Junichi Takagi
Institute for Protein Research, Osaka University
takagi@protein.osaka-u.ac.jp

4

Molecular mechanism of transcriptional regulation controlled by histone
methylation
Yoichi Shinkai
Institute for Virus Research, Kyoto University
yshinkai@virus.kyoto-u.ac.jp

1-3） Cell Biology
Analysis of the functional role of vacuolar protein sorting (Vps) protein, Hrs/Hgs
in the regulation of TGFb-induced signal transduction
HIRONOBU ASAO
Yamagata University faculty of Medicine
asao-h@med.id.yamagata-u.ac.jp

Comprehensive
studiesofgene
regulationbyposttranslational
processing-mediated carboxy-terminal fragments of the EGF family members.
Shigeki Higashiyama Ph.D.
Ehime University School of Medicine
Shigeki@m.ehime-u.ac.jp

Molecular Network Underlying Sperm Motility
Kazuo Inaba
University of Tsukuba
inaba@kurofune.shimoda.tsukuba.ac.jp

Isolation of ES-like cells and production of cloned animals from geneticallymodified blastocysts derived from equine or bovine
Kazunari K. Yokoyama
Gene Engineering Division, BioResouce Center, RIKEN
yokoyama@brc.riken.jp

Physiological functions and regulation mechanisms for tankyrase and TAB182
Hiroyuki Seimiya
Division of Molecular Biotherapy, Cancer Chemotherapy Center,
Japanese Foundation for Cancer Researchhseimiya@jfcr.or.jp

Receptor protein tyrosine phosphatase ζ (Ptprz) signaling in gastric ulcer induction
Masaharu Noda
National Institute for Basic Biology
madon@nibb.ac.jp

51-4） Developmental Biology
Regulation of spermatogenesis by cytoplasmic polyadenylation of mRNAs in male
haploid germ cells
Shin-ichi Kashiwabara
Graduate School of Life and Environmental Sciences, University of Tsukuba
kashiwa@sakura.cc.tsukuba.ac.jp

Mechanism of the functional neuronal histogenesis in the developing brain
Kazunori Nakajima
Department of Anatomy, Keio University School of Medicine
kazunori@sc.itc.keio.ac.jp

Development of the neural network and the blood vessels
Wataru ShojiInstitute of Development, Aging, and Cancer, Tohoku University
wshoji@idac.tohoku.ac.jp

1-5) Physiology
Involvement of Na,K-ATPase in Respiratory Rhythm Generation during Perinatal
Period.
Keiko Ikeda
Jichi Medical School, Center for Molecular Medicine, Division of .Biology
kikeda@jichi.ac.jp

1-6） Pharmacology
Studies on a Role for PRIP in the Function of GABAA Receptor
Masato Hirata
Kyushu University, Faculty of Dental Scie
Hirata1@dent.kyushu-u.ac.jp

1-7)

Plant Biology
Molecular Genetic Studies on Maternal Inheritance of Chloroplasts Using a Model
Plant System
Wataru Sakamoto
Research Institute for Bioresources, OkayamaUniversity
saka@rib.okayama-u.ac.jp

Molecular genetic analysis of light signal transduction from the N-terminal domainof phytochrome B
Tomonao Matsushita
Department of Botany, Graduate School of Science, Kyoto University
matsushita@physiol.bot.kyoto-u.ac.jp

62) Medical Sciences2-1) Immune System
Molecular Mechanisms of immune defenses against infection by IgM
Shibuya Akira
Department of Immunology, Institute of Basic Medical Sciences, University of Tsukuba
ashibuya@md.tsukuba.ac.jp

Physiological roles of the cannabinoid receptors
Takayuki Sugiura
Faculty of Pharmaceutical Sciences, Teikyo University
sugiurat@pharm.teikyo-u.ac.jp

Regulation of Inflammation and Obesity by MAP kinases
Masato OGATA
Mie university graduate school of medicine
ogata@doc.medic.mie-u.ac.jp

2-2) Central Nervous System
Analyses of molecular mechanisms of endoplasmic reticulum-localized Bcl-xL to
ER stress induced cell death
Toshiyuki Nakagawa
Gifu University Graduate School of Medicine, Department of Neurobiology
tnakagaw@cc.gifu-u.ac.jp

Molecular mechanisms of inflamed pain hypersensitivity in spinal cord neurons
Tatsuro Kohno
Division of Anesthesiology, Niigata University Graduate School of Medical and Dental Sciencekohno@med.niigata-u.ac.jp

2-5)

Cardiovascular Metabolic Endocrine
The role of IL-6/gp130 signaling and its negative regulator SOCS3 in viral
myocarditis
Hideo Yasukawa
Cardiovascular Research Institute, Kurume University, Fukuoka Japanyahideo@med.kurume-u.ac.jp

2-7)

Infection
Role of lipid rafts on the infection of intracellular bacteria into macrophages
Masahisa Watarai
Obihiro University of Agriculture and Veterinary Medicine
watarai@obihiro.ac.jp

7

Identification of new disease genes and development of new gene therapy protocol
for primary immunodeficiency
Yoshiyuki Minegishi
Tokyo Medical and Dental University
Yminegishi.mbch@tmd.ac.jp2-8) Oncology
Molecular cloning of the gene for mitotic spindle checkpoint deficiency
Shinya MATSUURA
Research Institute for Radiation Biology and Medicine, Hiroshima University
shinya@hiroshima-u.ac.jp

Biochemical and molecular mechanisms of drug resistance of gastrointestinal
stromal tumors against Imanitib.
Toshirou Nishida
Osaka University Graduate School of Medicine
toshin@surg1.med.osaka-u.ac.jp

2-12) Hematology
A role for transcription factors in hematopoiesis and leukemogenesis
MINEO KUROKAWA
Department of Hematology and Oncology, Graduate School of Medicine, University ofTokyokurokawa-tky@umin.ac.jp

3) Chemical Sciences3-1) Organic Chemistry
Development of Stereoselective Synthetic method of Procyanidin Oligomers
and Their Activities
Noriyuki Nakajima
Biotechnology Research Center, Toyama Prefectural University
nori@pu-toyama.ac.jp

Synthetic studies on insect anifeedants
Shigefumi Kuwahara
Graduate School of Agricultural Science, Tohoku University
skuwahar@biochem.tohoku.ac.jp

8

Global identification of Akt substrates by a novel phosphoproteome analysis.
Seisuke Hattori, Dsc.
Institute of Medical Scinece, The University of Tokyo
hattoris@ims.u-tokyo.ac.jp

Development and Application of Chiral Ligand based on N-Ar Chiral Mimetic
Kazuhiro Kondo
Graduate School of Pharmaceutical Sciences, Nagoya City University
kazuk@phar.nagoya-cu.ac.jp

Synthetic Studies on Marine Cytotoxic Halogen-Containing Oxasqualenoids
Yoshiki Morimoto
Graduate School of Science, Osaka City University
morimoto@sci.osaka-cu.ac.jp

Studies on the structure and the chemical synthesis of marine toxin Azaspiracid
Masato Oikawa
Graduate School of Life Sciences, Tohoku University
mao@bios.tohoku.ac.jp

Synthesis of optically active a-aryl carboxylic acids by catalytic asymmetricprotonation
Akira YANAGISAWA
Chiba University
ayanagi@scichem.s.chiba-u.ac.jp

Synthetic Study on Glycopeptide Using Glycosyl Amino Acid Having Natural
Oligosaccharide
Toshiyuki INAZU
Department of Applied Chemistry, School of Engineering, Tokai University
inz@keyaki.cc.u-tokai.ac.jp

4) Other
Identification of new peptide agonists specific for circulatory orphan receptors
using internalization assay
Kenji Kuwasako
First Department of Internal Medicine, Miyazaki Medical College, University of Miyazaki
kuwasako@fc.miyazaki-med.ac.jp

9Part Ⅱ:
Reports from the Recipients of Garants for International Meeting(Fiscal Year 2003)
11th International Conference on the Cell and Molecular Biology ofChlamydomonas (Chlamy2004)
Report of 21st International Lectin Meeting (Interlec 21)
International Narcotics Research Conference 2004/Kyoto
The 4th Tateshina Conference on Organic Chemistry

10

PartⅠ
Reports from the Recipients of Novartis
Research Grants (Fiscal Year 2003)

111) Biological Sciences1-1) Molecular Biology

The mechanism of p16INK4a gene induction by stress response
Naoko Ohtani
Division of Protein Information, Institute for Genome Research, The University of Tokushimaohtani@genome.tokushima-u.ac.jp

Introduction
Oncogenic Ras/MAP kinase signalling provokes irreversible growth arrest in cultured humanprimary cells through the induced expression of p16INK4a tumour suppressor gene, which is an
inhibitor of cyclin-dependent kinases (CDKs). This phenomenon is termed “premature senescence”,
and is proposed to be a fail-safe mechanism, which protects normal cells from uncontrolledcell-proliferation and tumour formation.

We have previously demonstrated that the activation of

Ets2, which is a downstream mediator of Ras/MEK signalling cascade, is responsible for the
up-regulation of p16INK4a in Ras-induced premature senescence. This finding was published in
Nature (Ohtani et al. Nature 409, 1067-1070, 2001).
The phosphorylation of Ets2 takes place within several hours upon expression of oncogenic Ras.
However, it takes about four to five days for p16INK4a induction. This time gap suggests that someother factors induced by Ets2 are necessary for p16INK4a induction.

Results
1 Ets2 induces JunB expression in human diploid fibrlblasts
Therefore, we searched for such factors by using micro-array technique. We found that atranscription factor JunB is induced by Ets2. Recently it was reported that the cells from JunBtransgenic mice express higher level of p16INK4a.
2 Ets2 and JunB cooperatively stimulate p16INK4a gene promoter activity
Therefore, we checked the possibility that Ets2 might cooperate with JunB to induce p16INK4agene by reporter assays. As we anticipated, Ets2 and JunB cooperatively stimulated p16INK4a genepromoter activity by 16-fold in the presence of activated Ras/MEK signalling.
3 Ras/MEK signalling facilitates binding between Ets2 and JunB
Moreover, we found that the binding between Ets2 and JunB is strongly facilitated by activatedRas/MEK signalling in 293T cells.

Also we were able to confirm that activated Ras/MEK

signalling facilitates the binding between endogenous Ets2 and JunB in Ras infected human diploidfibroblasts.

12

4 Ets2 and JunB expression induces endogenous p16INK4a gene expression
We confirmed the induction of endogenous p16INK4a gene by Ets2 and JunB.

Transfection into

primary human fibroblasts is usually very difficult. Therefore, we used Nucleofector transfection
system to transfect primary cells with Ets2 and JunB expression vectors, which reveals 95%
transfection efficiency.
5 Endogenous Ets2 and JunB bind to the p16INK4a gene promoter
We were also able to confirm that endogenous Ets2 and JunB bind to the p16INK4a gene promoter
in Ras-expressing primary humanfibroblasts by chromatin immunoprecipitation (ChIP) assay

using Ets2 and JunB antibodies.

Discussion & Summary
Oncogenic stress, such as activated Ras/MEK signalling, provokes irreversible growth arrest in
cultured human primary cells through the induction of p16INK4a tumour suppressor gene expression.
We previously identified that one of the key transcription factor to induce p16INK4a tumour
suppressor gene upon oncogenic stress is Ets2 (Ohtani et al. Nature 409, 1067-1070, 2001). However,
our previous results suggested that Ets2 needs other co-factors to transactivate p16INK4a genepromoter.
Our current study suggests that one of the key factor to cooperate with Ets2 is JunB. Our results
suggests that activated Ras/MEK signalling activates Ets2 activity and induces transcription factor
JunB. These two transcription factors bind to p16INK4a gene promoter and cooperatively induces
p16INK4a gene expression.

Figures & Tables

Figure 1.

Ets2 induces p16INK4a expression cooperatively with JunB

A hormone (Muristerone A) -inducible expression vector encoding JunB(PIND-JunB) was13co-transfected with or without Ets2 expression vector into early passage HDFs using “Nucleofectortransfection system” (Amaxa biosystems, Germany).

Judging from the GFP signals driven by thetransfected plasmids, nearly 100% of cells were successfully transfected.

Although we wereunable to see any significant increase of p16INK4a level by overexpression of either Ets2 alone or
JunB alone, significant induction of p16INK4a was observed within 2 days if both Ets2 and JunB
were coexpressed ( lane6).

As shown previously(Ohtani et al. Nature 2001), p16INK4a

expression can be induced by Ets2 alone, 3 days after transfection (lanes 8 and 11), although thelevels are still further enhanced by coexpression with JunB (lane 12).

References
Ohtani N, Zebedee Z, Huot TJ, Stinson JA, Sugimoto M, Ohhashi Y, Sharrocks AD,

Peters G andHara E.
Opposing effects of Ets and Id proteins on p16INK4a expression during cellular senescence.
Nature. 409, 1067-70, 2001

141) Biological Sciences1-1) Molecular Biology

Regulation of NF-κB activation by PYPAF1/Cryopyrin
Takashi Suda
Kanazawa University, Cancer Research Institutesudat@kenroku.kanazawa-u.ac.jp

Introduction
Cryopyrin/PYPAF1/NALP3 is an Apaf-1-like protein that induces activation of NF-κB andcaspase-1 through an adaptor molecule ASC. The gene encoding cryopyrin has been identified as thegene mutated in the familial cold autoinflammatory syndrome and Muckle-Wells syndrome.
Recently, we identified molecules (IPNs) that inhibit NF-κB activation induced by cryopyrin. IPN2
was a novel Apaf-1 like protein, which we named PYNOD (1). PYNOD also inhibited the
cryopyrin-induced IL-1β secretion. Here, we investigated the inhibitory mechanism of PYNOD
against NF-κB activation and IL-1β secretion. In addition, we have explored the mechanism ofASC-mediated NF-κB activaton.

Results
1) Inhibitory mechanism of PYNOD against crypyrin-induced NF-κB
Expression of cryopyrin in combination with ASC (cryopyrin + ASC) induces NF-κB activation inhuman embryonic kidney (HEK) 293 cells. In this experimental system, co-expression of PYNOD
inhibited the cryopyrin + ASC-induced NF-κB activation. PYNOD also inhibited NF-κB activationinduced by coexpression of CARD12 (another Apaf-1-like molecule that induces NF-κB activation
using ASC, like cryopyrin) and ASC, or overexpression of ASC alone. In contrast, PYNOD failed toinhibit MEKK1-, Myd88- or TNF-α-induced NF-κB activation. In addition, PYNOD inhibited
ASC-induced apoptosis without any effects on Fas- or Bid-induced apoptosis. PYNOD and ASC
were coprecipitated with each other when either of them was immunoprecipitated from cell extracts
containing both of them. There results indicate that PYNOD inhibits ASC-mediated NF-κB
activation and apoptosis by physically interacting with ASC.
2) Inhibitory mechanism of PYNOD against crypyrin-induced IL-1β secretion.
Crypyrin induced caspase-1-mediated IL-1β processing and secretion when cryopyrin was
coexpressed with ASC, caspase-1 and IL-1β in HEK293 cells. In this experimental system,
coexpression of PYNOD inhibited the crypyrin-induced IL-1β secretion. PYNOD also inhibited

15

IL-1β secretion induced by caspase-1 overexpression in the absence of cryopyrin and ASC. WhenPYNOD was coexpressed with caspase-1, PYNOD inhibited autoprocessing of caspase-1. PYNOD
and caspase-1 were co-precipitated with each other when either of them was immunoprecipitated.
There results indicate that PYNOD physically interacts with caspase-1 and inhibits autoprocessingof caspase-1.
3) Establishment of an experimental system for the ASC-mediated NF-κB activation.
To characterize the signaling pathway mediated by ASC under physiologically relevant conditions,
we generated a cDNA encoding a chimeric protein consisting of the signal transducing region of
cryopyrin and the ligand binding domain of Nod2, whish is an Apaf-1-like molecule that recognizes
muramyl dipeptide as a ligand and induces NF-κB activation using RICK as a downstream signaling
molecule. We also constructed a cDNA encoding a similar chimeric protein from CARD12 andNod2 (Fig. 1). Although both chimeric proteins induced NF-κB activation in response to muramyl
dipeptide stimulation when they were expressed with ASC in cells, the CARD12-Nod2 chimera
protein induced stronger response than the cryopyrin-Nod2 chimera protein. Therefore, weestablished stable cell lines (MAIL8 cells) expressing both the CARD12-Nod2 chimera protein andASC. Successfully, stimulation by muramyl dipeptide induced an interaction between the
CARD12-Nod2 chimera protein and ASC, and elicited cell-autonomous NF-κB activation in MAIL8cells (3).

MDP

A
Nod2

RICK

B

?
CARD12

ASC

NF-κB

MDP

C
CARD12-Nod2

ASC

?

?

NF-κB
apoptosis

NF-κB
apoptosis

Fig. 1. Construction and function of a CARD12-Nod2 chimera protein
A.Nod 2 recognizes muramyl dipeptide (MDP) and activates NF-κB using RICK. B.
CARD12 induces NF-κB activation and apoptosis using ASC, but its ligand is unknown. C. Our
CARD12-Nod2 chimera protein induces NF-κB activation and apoptosis upon MDP stimulation.

164) Mechanism of ASC-induced NF-κB activation
Recently, it was reported that casapse-8 is involved in the ASC-induced apoptosis. On the other hand,
we found that caspae-8 is involved in not only apoptosis but also NF-κB activation induced by Fas
ligand (2). There results prompted us to investigate whether casapse-8 is involved in the
ASC-mediated NF-κB activation (3). Small interfering RNA (siRNA) that attenuated caspase-8
expression inhibited the MDP-induced NF-κB activation in MAIL8 cells, whereas siRNAs against
RICK, which mediates Nod2-induced NF-κB activation, did not do so. CLARP, a cellular caspase-8
inhibitor, as well as pan-caspase inhibitor z-VAD-fmk and caspase-8-specific inhibitor z-IETD-fmk
but not granzyme B inhibitor z-AAD-fmk inhibited this response. These results indicate that
caspase-8 and its catalytic activity is required for the ASC-mediated NF-κB activation. In contrast,
siRNA against FADD and a dominant negative mutant of FADD did not influence the ASC-mediated
NF-κB activation. ASC forms large aggregates called “speck” when it is activated. Consistent withabove conclusions, ASC recruited caspase-8 and CLARP but not FADD and Nod2 to its speck-likeaggregates when these proteins were overexpressed in cells. Furthermore, muramyl dipeptide
stimulation induced formation of ASC-specks in MAIL8 cells and endogenous caspase-8 wascolocalized with the specks. Finally, muramyl dipeptide induced IL-8 production in MAIL8 cells.
These results are the first to indicate that caspase-8 plays an important role in the ASC-mediated
NF-κB activation, and that the ASC-mediated NF-κB activation actually induces physiologically
relevant gene expression..

Discussion & Summary
Mutation in cryopyrin causes systemic autoinflammatory syndromes. So far, pyrin, which is not amember of Apaf-1-like proteins, is known to inhibit cryopyrin-induced NF-κB activation and IL-1β
secretion by interacting with ASC (Fig. 2). Importantly, mutation of the pyrin gene causes foranother hereditary inflammatory disease, familial Mediterranean fever. Our results indicated that
PYNOD has similar function with pyrin, suggesting that PYNOD may modulate the severity of thesehereditary diseases. In addition, PYNOD directly interacts with caspase-1 and inhibits its activity(Fig. 2). We recently investigated the functions of two other Apaf-1-like proteins, PYPAF2 andPYPAF3, and demonstrated that PYPAF2 inhibits ASC and PYPAF3 inhibits caspase-1 (4). In
addition PYPAF4 was reported to interact with IKKα and inhibits cytokine induced NF-κB
activation (5). These results suggest that there is an anti-inflammatory subfamily among Apaf-1-likeproteins. We demonstrated that caspase-8 is involved in the ASC-mediated NF-κB activation.
Consistently, caspase-8 inhibitor CLARP inhibits ASC mediated NF-κB activation (Fig. 2). In
contrast, CLARP does not inhibit ASC-induced IL-1β secretion, indicating that CLARP inhibits apart of cryopyrin function with a mode of action different form those of pyrin and PYNOD. Taken

17

together, our study revealed novel molecular mechanisms for the signal transduction and regulationof cryopyrin.
Cryopyrin

PYNOD

Caspase-1IL-1β
secretion

ASC

Pyrin

Caspase-8

NF-κB
activation

CLARP

Apoptosis

Fig. 2. Signal transduction and regulation of cryopyrin

References
1. Wang, Y., M. Hasegawa, R. Imamura, T. Kinoshita, C. Kondo, K. Konaka, and T. Suda. 2004.
PYNOD, a novel Apaf-1/CED4-like protein is an inhibitor of ASC and caspase-1. Int. Immunol.
16:777-786.
2. Imamura, R., K. Konaka, N. Matsumoto, M. Hasegawa, M. Fukui, N. Mukaida, T. Kinoshita, andT. Suda. 2004. Fas ligand induces cell-autonomous NF-kappa B activation and IL-8 production bya mechanism distinct from that of TNF-alpha. J. Biol. Chem. 279:46415-46423.
3. Hasegawa, M., R. Imamura, T. Kinoshita, N. Matsumoto, J. Masumoto, N. Inohara, and T. Suda.
2005. ASC-mediated NF-kappa B activation leading to IL-8 production requires caspase-8 and isinhibited by CLARP. J. Biol. Chem. 280:15122-15130.
4. Kinoshita, T., Y. Wang, M. Hasegawa, R. Imamura, and T. Suda. 2005. PYPAF3, a
PYRIN-containing APAF-1-like protein, is a feedback regulator of caspase-1-dependentinterleukin-1beta secretion. J Biol Chem, in press.
5. Fiorentino, L., C. Stehlik, V. Oliveira, M.E. Ariza, A. Godzik, and J.C. Reed. 2002. A novel
PAAD-containing protein that modulates NF-kappa B induction by cytokines tumor necrosis
factor-alpha and interleukin-1beta. J. Biol. Chem. 277:35333-35340.

181) Biological Sciences1-1) Molecular Biology

Identification of genes involved in neurodegenerative disorders andER-stress by using a library of randomized ribozymes
Reiko Onuki
Graduate School of Pharmaceutical Sciences, the University of Tokyo
onuki@mol.f.u-tokyo.ac.jp

Introduction
Various types of stress, such as disruption of calcium homeostasis, inhibition ofprotein glycosylation and reduction of disulfide bonds, result in accumulation of
misfolded proteins in the endoplasmic reticulum (ER).

The initial cellular response

involves removal of such proteins by the ER, but excessive and/or long-term stressresults in apoptosis.
associated with

Furthermore, recent studies indicate that this apoptosis isneurodigenerative disorder.

In this study, we used a randomized

ribozyme (Rz) library and ER stress-mediated apoptosis (tunicamycin (Tm) -induced
apoptosis) in SK-N-SH human neuroblastoma cells as a selective phenotype to identify
factors involved in this process

Results
We isolated the candidate Rzs using three types of screening and performed the homology
searching to identify the Rz-targeted proteins. Double-stranded RNA-dependent protein kinase(PKR) and SREBP cleavage-associated protein (SCAP) were suspected to be involved in thisprocess. To confirm the effect of isolated Rzs on PKR, ribozyme stably expressing cells, named
EB1-47 and EB2-68, were constructed.

We examined the levels of Rz 47 and 68 in EB1-47 andEB2-68 cells and performed an analysis by RT–PCR with Rz-specific primers.

The expression ofeach Rz was detectable in Rz-transfected cells. To confirm the activities of Rz 47 and Rz 68
against the PKR transcript, we also performed analysis by RT–PCR using primers specific for genes
for PKR and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

The level of PKR mRNA in

EB1-47 cells was 53% lower than that in empty vector-transfected cells and similar results wereobtained in the case of Rz 68 (37% reduction).

We also performed Western blotting and found thatthe level of PKR was reduced in EB1-47 and EB2-68.

Thus, Rz 47 and Rz 68 specificallysuppressed the expression of PKR at the post-transcriptional level.
To identify the role of PKR in vulnerability to ER stress, we prepared cytoplasmic and nuclear
fractions from Tm-treated and untreated SK-N-SH cells and subjected them to Western blotting with
anti-actin and anti-KDEL antibodies (this antibody interacts with GRP78 and 94).

Levels of actin

19remained unchanged but levels of GRP94 and GRP78/Bip in the cytoplasmic fraction increased afteran 8-h incubation with Tm.

Neither of these chaperones was detected in the nuclear fraction,suggesting that the nuclear fraction was not contaminated by cytoplasmic material.

Westernblotting analysis with anti-PKR and anti-phospho-PKR antibodies revealed that, when cells were
stimulated with Tm for 8 h, the level of PKR rose in the nuclei (Figure 1) while the level ofcytoplasmic PKR barely changed.

Phosphorylation or cleavage of PKR is required for kinase

activity. Using an anti-phospho-PKR antibody (which recognizes phosphorylation of Thr446), we
detected phosphorylated PKR in the nuclear fraction, and the level of this form of PKR depended on
the incubation time. The maximum level of phosphorylated PKR was detected 24 h after the start oftreatment (Figure 1).

When we examined the subcellular localization of PKR by

immunofluorescence microscopy after SK-N-SH cells had been treated with Tm, we found most of
the PKR in the cytoplasm of the untreated control. . After cells had been treated with Tm, levels of
nuclear PKR increased and PKR was clearly localized in the nuclei, while levels of GRP 78 and 94
increased in the ER of treated cells.

These results suggest that Tm-mediated ER stress promotes

the expression of PKR in SK-N-SH cells.
We wanted to investigate whether phosphorylated PKR might affect Tm-induced cell death and so
we constructed dominant negative (DN) PKR expressing cells and incubated them with Tm for 30 h.
The level of cleaved caspase-3 (activated caspase-3) was examined and small amounts of cleaved
caspase-3 were detected in these cells, while a large amount of cleaved caspase-3 was detected in thecontrol cells.

This result suggests that the expression of DN-PKR inhibits apoptosis.

Considerable evidence suggests that ER stress is closely related to genetic and/or neuronaldegenerative diseases, such as Alzheimer’s disease. Therefore, we examined the levels of PKR in
the brains of sporadic AD patients obtained post-mortem.

We prepared crude nuclear andcytoplasmic fractions (for seven disease-free controls and 17 patients with AD) for Western blottingwith anti-phospho-PKR and anti-actin antibodies.

Levels of phosphorylated PKR weresignificantly higher in crude nuclear fractions of AD tissues compared with controls (Figure 2).
Figure 1

Figure 2

20

Discussion & Summary
In summary, we have identified PKR as a proapoptotic protein in Tm-induced apoptosis. During
apoptosis, phosphorylation of PKR is increased and phosphorylated PKR is localized in the nuclei of
SK-N-SH cells. In the nuclei in AD brains, similar results underscore the potential importance ofphosphorylated PKR.
brains.

Furthermore, we found aggregated PKR in the nuclei of samples from AD

Although many cells exhibited aggregation of PKR in their nuclei, histochemical

investigations showed that these cells had not, apparently, undergone apoptosis.

It is possible thatthe aggregation of PKR in the nuclei occurs at an early phase in the pathogenesis of AD.

We alsodetected elevated levels of PKR in the brains of patients, at autopsy, who had died of Huntington's
disease or Parkinson's disease.

It is possible that PKR may be widely involved in a number ofneuronal degenerative diseases.

References
Bando Y, Onuki R, Katayama T, Manabe T, Kudo T, Taira K, Tohyama M.
Double-strand RNA dependent protein kinase (PKR) is involved in the extrastriatal degeneration in
Parkinson's disease and Huntington's disease.
Neurochem Int. 2005 Jan;46(1):11-8.
Onuki R, Bando Y, Suyama E, Katayama T, Kawasaki H, Baba T, Tohyama M, Taira K.
An RNA-dependent protein kinase is involved in tunicamycin-induced apoptosis and Alzheimer's
disease.
EMBO J. 2004 Feb 25;23(4):959-968.

211) Biological Sciences1-1) Molecular Biology

Role of small GTPase Rac in formation of neural circuit and synaptic
plasticity in the central nervous system
Atsu Aiba
Kobe University Graduate School of Medicine
aiba@med.kobe-u.ac.jp

Introduction
The Rac is a member of the Rho family GTPases regulating actin reorganization. During
development, Rho family GTPases integrate many extracellular signals to direct the outgrowth ofaxons and dendrites and the formation and dynamics of dendritic spines. To examine the roles of Rac
in formation of neural circuit and synaptic plasticity in the central nervous system (CNS), we
generated Rac conditional knockout mice.

Results
Of three mammalian Rac isoforms, Rac1 and Rac3 are expressed in the CNS. To gain an insight
into a possible role of Rac in the CNS, we first examined the expression patterns of rac1 and rac3
mRNAs in the developing mouse brain. At embryonic day 15 (E15), postnatal day 7 (P7) and adult,
mouse brain sections were prepared and analyzed by in situ hybridization. Rac1 mRNA isubiquitously expressed in the mouse brain at E15, P7 and adult. On the other hand, rac3 mRNA isdetected mainly hindbrain neurons at E15. At P7, rac3 mRNAs are prominently expressed in wholebrain regions and rac3 mRNA expression declines in adult mouse brain. Overlapping but distinct
expression pattern of rac1 and rac3 mRNAs in the CNS suggests distinct roles of the two Rac
proteins in the CNS.
We previously showed that rac1 (-/-) embryos die before E9.5. Thus, we planed to generate Rac1
and Rac3 conditional knockout mice which lack Rac proteins in the specific neurons in order toexamine the role of the Rac proteins in the CNS. We generated rac1 (flox/flox) mice in which theexon1 containing the initiation codon is flanked by two loxP sites in rac1 allele by gene targeting
using embryonic stem (ES) cells. We also generated rac3 (+/flox-HcRed) mice in which loxP is
inserted in the noncoding region upstream of the initiation codon of the exon1 and a loxP-HcRed,
far-red fluorescent protein gene, is inserted downstream of the poly A addition signal in rac3 allele.
rac1 (flox/flox) mice and L7 (+/cre) mice expressing Cre recombinase only on cerebellar Purkinjecells in the brain were crossed to generate rac1 (flox/flox) L7 (+/cre) mice. We confirmed the exon1of the rac1 was deleted in the cerebellum of the rac1 (flox/flox) L7 (+/cre) mice (Purkinje

22cell-specific Rac1 knockout mice) by PCR analysis of genomic DNAs. Purkinje cell-specific Rac1
knockout mice appeared normal and did not display ataxia. To further examine motor coordination,
we used the rotating rod task. Purkinje cell-specific Rac1 knockout mice quickly learned how tokeep themselves on the rod. There was no significant difference between the knockout and control
mice, suggesting motor coordination of the Rac1 conditional knockout mice is normal. Next, we
examined the structure of the cerebellum in the Purkinje cell-specific Rac1 knockout mice. Nissl
staining showed that size and overall structure of the cerebellum of the knockout mice were normal
as compared with controls. We further examined the dendrite structures of the Rac1-deficient
Purkinje cells by immunohistochemical analysis using anti-calbindin antibody. We found that theRac1-deficient Purkinje cells have more abnormal dendrites than those of wild-type mice. The
mutant mice which lack GluR delta2, Purkinje specific ionotropic glutamate receptor, were reported
to have reduced density of parallel fiber-Purkinje cell synapse. Thus we examined the synapse
density of parallel fiber-Purkinje cell using an electron microscope and found that synapse density issignificantly decreased in the Purkinje cells lacking Rac1. This result suggests that Rac1 is involved
in synapse formation or synapse maintenance on the parallel fiber-Purkinje synapses.

Discussion & Summary
We generated the mice which have rac1 flox allele or rac3 flox allele by gene targeting using ES
cells. Analysis of Purkinje cell-specific Rac1 knockout mice reveals that Rac1 plays an importantrole in formation or/and maintenance of parallel fiber-Purkinje cell synapses. Actin cytoskeleton
whose reorganization is known to be regulated by Rac protein is implicated in regulation of synaptictransmission and plasticity. Further electrophysiological study using these knockout mice will show
the role of Rac in these functions. Purkinje cell-specific Rac1 knockout mice showed apparent
normal motor coordination in spite of reduced parallel fiber synapse density on the Purkinje cells.
Associative leaning using Purkinje cell function such as eye-blink conditioning task may reveal thefunctional significance of the synapse density in the motor learning.

23

Figures & Tables

241) Biological Sciences1-1) Molecular Biology

Molecular Analysis of Salivary Gland Branching Morphogenesis
Takayoshi Sakai
Osaka University Dental Hospitalsakai@dent.osaka-u.ac.jp

Introduction
Epithelial branching morphogenesis is essential for forming many organs such as exocrine glands,
salivary gland, lung, and kidney. Repetitive epithelial cleft and endbud formation create thethree-dimensional branching structures characteristic of many organs, yet the mechanisms are poorly
understood. The purpose of this project is to identify molecules that are necessary for branchingmorphogenesis and to apply these molecules for regeneration of salivary gland. To identify
molecules that are necessary for branching morphogenesis, laser microdissection (LMD) andanalysis of gene expression by T7-SAGE (reference 1) were performed.

Results
1. Embryonic 12-day salivary glands were extracted from mouse embryonic body, andwere cultured on filter membrane and cleft and endbud epithelia of frozen-sectioned salivary gland
were laser-microdissected as previously described using LMD (laser microdissection) microscope
system (reference 2). We realized that twelve micron frozen section was suited for our experiment.
Figure 1 indicated the design for LMD and strategy for microanalysis of gene expression. Red areashows cleft epithelium and green area does endbud epithelium. Both of their tissues ware used forthe production of T7-SAGE (serial analysis of gene expression, http://www.sagenet.org) libraries.
2. Conventional plastic slide was changed to new glass slide to avoid any static power on themembrane for LMD. Because previously plastic ones caused trouble to catch up salivary tissue
using the cap of centrifuge tubes. Toluidine blue staining was performed to clearly identify
epithelial cleft in salivary gland. Each 500 cells were collected to extract RNA.
3. Using column technique, total RNA was extracted from both cleft and end bud
epithelia, and the cDNAs were synthesized after the RNAs were treated with DNase.

25

4. T7-SAGE was our modified technique of conventional SAGE that we previously established thatwas utilized T7-based RNA amplification method (reference 1). We have started the T7-SAGE
method to make the gene expression profile from limited small amount of salivary epithelial cells.
5. Initial profiles of micro gene expression in salivary epithelium were identified.
Our T7-SAGE data showed that salivary gland epithelium expressed Fibronectin
and E-cadherin. Epithelial cleft expressed Fibronectin higher than epithelial end bud,and epithelial cleft decreased E-cadherin.
6. T7-SAGE tags were quantitated using SAGE software (http://www.sagenet.org), and the patternof gene expression including were determined. (Figure 1).
We started identifying the gene using NCBI database and SAGEmap database to identify someinteresting gene expression.

We initially started confirming some data from our T7-SAGE database using quantitative real-time
RT-PCR and in situ hybridization technique.
After we estimated the candidate genes that regulate the branching morphogenesis, we will performfunctional knockdown technique, small interfering RNA (siRNA) to identify the role in organ
development. Full length cDNA will be cloned using RACE technique for new or rare gene study.
Further study is necessary to complete our project after we complete the gene expression profile inembryonic epithelial cells of mouse salivary gland.
Finally we will identify the candidate gene which is required for glandular branchingmorphogenesis, and apply the information to organ development and regeneration research. These26

data will provide new possibilities for the guide of regeneration therapy in human organ, such as
kidney and lung, salivary gland.

Discussion & Summary
T7-SAGE was performed to identify the profile of gene expression. Initial T7-SAGE data showedthat salivary gland epithelium expressed upregulated fibronectin and downregulated E-cadherin. Our
preliminary data was confirming our previous work (reference 2). Epithelial branchingmorphogenesis could be control by the interaction between cell-cell adhesion and cell-ECM (extra
cellular matrix) adhesion.
Our analysis of gene expression using T7-SAGE method was very useful for microanalysis fromlimited small amount of sample such as laser microdissected tissue. Future works will be continued
to identify the genes that regulate branching morphogenesis of some organs to regenerate importantorgan such as kidney and lung, prostate, pancreas, salivary gland.

References
1. Sakai, T., Larsen M., and Yamada K.M. Microanalysis of Gene Expression in Tissues Using
T7-SAGE: Serial Analysis of Gene Expression After High-fidelity T7-based RNA Amplification. in
Current Protocols in Cell Biology 19.3.1-19.3.30 (John Wiley & Sons, New York, 2002)
2. Sakai, T., Larsen M., and Yamada K.M. Essential Role for Fibronectin in Branching Morphogenesis.
Nature 423, 876-881, 2003
3. Sakai, T., Kogo, M., Larsen M. and Yamada, K.M. Review: Molecular Analysis of Salivary Gland
Branching Morphogenesis. Oral Sci. Int.1, 16-21, 2004

27

1) Biological Sciences1-1) Molecular Biology

Structural manipulation of integrin ligand binding affinity and its use
in the development of novel class of inhibitors
Junichi Takagi
Institute for Protein Research, Osaka University
takagi@protein.osaka-u.ac.jp

Introduction
Integrins are major metazoan adhesion receptors that play a fundamental role in cellular organization.
One unique aspect of integrins is that the affinity of their extracellular domain for biological ligands
can be rapidly upregulated by signals from within the cell(1).

Rapid and precise control of integrin

activation is particularly important for leukocytes and platelets. In order for screening anti-integrin
drugs with minimum side-effects, we try to design integrin receptor which is fixed in its low-affinity
or high-affinity state.

Results
Mutant αIIbβ3 integrin that has constitutively high affinity toward ligand has been designed(2).
The design is based on the hypothesis that the angle between I-like domain and hybrid domain of
integrin b subunit controls ligand affinity. The “glycan wedge” introduced at specific domainboundary activated integrin expressed on cell surface(3). We have proposed that the change inaffinity at the ligand binding site in the I-like domain around its metal ion-dependent adhesion site
(MIDAS) is communicated to the interface with the hybrid domain on the opposite end of the I-likedomain by axial displacement in the C-terminal direction of the I-like domain α7-helix(4). We
hypothesized that in both the unliganded and liganded aVβ3 structures the C-terminal α7 helix of theβ3 I-like domain is in a position stabilizing a closed, low affinity conformation; therefore, thesestructures were used to design low affinity mutants. An open, high affinity conformation wasmodeled assuming that the α7-helix was displaced in the C-terminal axial direction by two α-helicalturns.

The distance between Cβ atoms of T329 and A347 in the unliganded and RGD-liganded

αVβ3 structures is 4.4 Å and 4.9 Å, respectively, whereas it is 9.6 Å in the hypothesized high affinity
model.

Therefore, the mutant b3T329C/A347C was expected to form a disulfide bond in the low but not

the high affinity conformation, and to be stabilized in the low affinity, closed conformation.

On theother hand, the distance between the Cβ atoms of V332 and M335 in the unliganded and
RGD-liganded αVβ3 structures is 10.0 Å and 8.3 Å, respectively, whereas it is 3.7 Å in the
hypothesized high affinity model.

Therefore,

the mutant b3V332C/M335C was expected to form a28

disulfide bond in the high but not the low affinity conformation, and to be stabilized in the highaffinity, open conformation.

Wild type and mutant β3 subunits were co-transfected with wild typeaIIb in 293T cells, and subjected to immunostaining flow cytometry.
35

Non-reducing SDS-PAGE of

S-labeled, immunoprecipitated receptors showed that the wild type and mutant αIIb subunits

migrated similarly, whereas the mutant b3T329C/A347C and b3V332C/M335C subunits migrated slightly
faster than wild type b3 . By contrast, all β3 single cysteine mutants migrated similarly to wild-typeβ3 .

In general, disulfide bonds increase the mobility of proteins in SDS-PAGE, and these results

suggest that the cysteines introduced into the β3T329C/A347C and β3V332C/M335C mutants form a disulfide
bond. Disulfide bond formation was confirmed by labeling free sulfhydryls with themaleimide-containing reagent, biotin-BMCC.
Binding to soluble fibrinogen was first examined using two-color flow cytometry in transiently
transfected 293T cells, in which wild type aIIbb3 basally has low affinity for ligand.

Wild typeαIIbβ3 bound fibrinogen when stimulated with the activating mAb PT25-2, but not basally in Ca2+
(Fig. 1).

Each of the four single cysteine mutants behaved similarly to the wild type receptor1). By contrast, the putative locked closed, double cysteine mutant aIIbb3
soluble fibrinogen even in the presence of PT25-2 (Fig. 1).
mutant aIIbb3

V332C/M335C

T329C/A347C(Fig.

did not bind

Furthermore, the putative locked open2+

bound soluble fibrinogen even in Ca , and addition of PT25-2 mAb did notfurther increase binding (Fig. 1). Constitutive binding in Ca2+ by the aIIbb3V332C/M335C mutant was
abolished by two blocking αIIb mAbs HA5 and 10E5, but neither blocked nor further activated by theactivating β3 mAb AP5 (Fig. 2), confirming that the high affinity fibrinogen binding of thetransfected cells was specific.

Figure 1

Figure 229

Discussion & Summary
The results with the αIIbβ3

T329C/A347C

and αIIbβ3V332C/M335C mutations strongly support theimportance of β I-like domain α7-helix movement in integrin affinity regulation.

Bothhypothesis-driven mutations in the β I-like domain had the predicted effect. One, designed todisplace the β6-α7 loop downward, indeed activated ligand binding.

The other, designed to holdthe β6-strand and α7-helix together near the end of the α7-helix, indeed maintained αIIbβ3 in the low
affinity state and demonstrated that this relative arrangement of these two secondary structureelements, visualized in crystal structures, corresponds to the low affinity state.
The rational design of mutations that allosterically stabilize high affinity or low affinity
conformations of integrins demonstrates marked advances in our understanding of the molecularbasis of affinity regulation. These “hyperactive” or “inactive” mutant integrins can be used in the
future as a tool for screening novel class of small molecule integrin inhibitors and/or monoclonal
antibodies.

References(1)R. O. Hynes: "Integrins: bi-directional, allosteric, signalling machines", Cell, 110, 673-687 (2002)(2)B.-H. Luo, J. Takagi and T. A. Springer: " Locking the beta3 integrin I-like domain into high andlow affinity conformations with disulfides. ", J Biol Chem, 279, 10215-10221 (2004)(3)B.-H. Luo, T. A. Springer and J. Takagi: "Stabilizing the open conformation of the integrin
headpiece with a glycan wedge increases affinity for ligand", PNAS, 100, 2403-2408 (2003)(4)J. Takagi and T. A. Springer: "Integrin activation and structural rearrangement", ImmunologicalRev., 186, 141-163 (2002)

30

1) Biological Sciences1-1) Molecular Biology

Molecular mechanism of transcriptional regulation controlled byhistone methylation
Yoichi Shinkai
Institute for Virus Research, Kyoto University
yshinkai@virus.kyoto-u.ac.jp

Introduction
Histone H3 Lys 9 (H3-K9) methylation is a crucial epigenetic mark for transcriptional silencing.
G9a is the major mammalian H3-K9 methyltransferase that targets euchromatic regions and isessential for murine embryogenesis. There is a single G9a-related methyltransferase in mammals,
called GLP/Eu-HMTase1. To elucidate in vivo function of GLP and functional interaction between
G9a and GLP, we generated and characterized GLP deficient mouse and ES cells.

Results
The human counterpart of GLP, Eu-HMTase1, has been shown to express H3-K9 HMTase activity
in vitro. To investigate the substrate specificity and the enzymatic activity of GLP versus G9a, we
generated GST-fusion proteins carrying their respective SET domains. The GST-fusion proteins
were incubated with S-adenosyl methionine and several recombinant GST-histone H3 tail substrates.
As reported previously, the GST-G9a SET protein preferentially methylated K9 and less efficiently
K27 in our in vitro assay system. The GST-GLP SET protein also methylated K9 preferentially andK27 weakly Thus, the enzymatic activities of GLP were quite similar to those of G9a. To further
explore the biological function(s) of GLP, we examined the intracellular localization profile of GLP
as compared with G9a. Newly generated monoclonal antibodies specific for G9a or GLP were used
for detection of the corresponding endogenous proteins using immunohistochemistry. G9a and GLP
displayed similar intracellular localization profiles in mouse ES cells and embryonic fibroblasts.
Both enzymes were detected exclusively in nuclei, but did not accumulate at DAPI-dense
heterochromatin, and were excluded from nucleoli. We also examined RNA expression profiles of
G9a and GLP by multi-tissue Northern blot hybridization in adult mice. G9a and GLP wereubiquitously expressed with some variations at steady-state mRNA levels when comparing different
tissues. It was notable that the relative levels of GLP transcripts among the examined tissues werequite proportionate to those of G9a, except in testis, where G9a transcripts were elevated.
Collectively, these results suggest that G9a and GLP may possess similar functions as H3-K9

31

HMTases in vivo. Next, to generate GLP-/- mice and cells by genetic mutation, we designed atargeting construct for replacement of exon 25 and a portion of exon 26 with the neomycin gene
cassette. The replaced sequences encode the catalytic core region of the GLP SET domain. The
targeting construct was introduced into TT2 mouse ES cells and selected by G418/Gnc. From thethree hundred clones screened, we identified eight that carried a correctly targeted GLP allele and no
extra copy of the construct. Three clones were injected into the morula stage of mouse embryos togenerate chimeric animals. To establish GLP deficient ES cells, at first, we introduced a Flag-tagged
GLP cDNA flanked with two loxP sites into GLP+/- ES cells and then generated GLP-/- ES cells
expressing exogenous Flag-tagged GLP molecules. Finally, using Cre-recombinase treatment, we
generated GLP-deficient ES cells. Analyses of GLP deficient mice and ES cells showed that GLP is
also important for H3-K9 methylation of mouse euchromatin and murine embryogenesis.
GLP-deficiency led to embryonic lethality around E9.5 (Figure 1), a severe reduction of H3-K9
mono- and dimethylation (Figure 2), the induction of Mage-a gene expression, and HP1 (HP1α, βand γ) relocalization in embryonic stem cells, all of which were phenotypes of G9a-deficiency.
Interestingly, GLP-deficiency caused a reduction in the steady-state levels of G9a protein toapproximately ~1/3 of those observed in wild-type ES cell, TT2. RNA hybridization analysis of
GLP-deficient ES cells demonstrated no decrease in G9a-transcript levels, indicating that the
reduction in G9a protein occurred post-transcriptionally. Importantly, all phenotypes observed for
the GLP deficiency, including G9a protein instability, loss of
H3-K9 dimethylation, and de-repression of Mage-a transcripts,

Figure1

were rescued by the wild-type GLP expression, indicating that the

GLP‑/‑ embryos die around E9.5

observed GLP-/- phenotypes were intrinsic. Furthermore, we
observed that G9a and GLP tightly formed a stoichiometricheteromeric complex in a wide variety of cell types. Biochemical
analyses revealed that full-length G9a, GLP and their SET domains
but not SET-domain deletion mutants formed complexes indicating
the formation of the G9a/GLP complex is dependent on their
enzymatic SET domains.

32

Discussion & Summary
In this study using G9a and GLP deficient mice and ES cells, we showed that GLP is also importantfor H3-K9 methylation of mouse euchromatin. Furthermore, the phenotypes observed for germline
mutations of the G9a and GLP genes were identical in many respects, including embryonic lethality,
drastic reduction of H3-K9 mono- and di-methylation, induction of Mage-a genes, and HP1
re-localization in ES cells. Moreover, we showed that G9a and GLP formed a stoichiometricheteromeric complex in a wide variety of cell types. Biochemical analyses revealed that formation of
the G9a/GLP complex was dependent on their enzymatic SET domains. Taken together, our new
findings revealed that G9a and GLP cooperatively exert H3-K9 methyltransferase function in vivo,
likely through the formation of higher-order heteromeric complexes.

References
Tachibana, M. Ueda, J. Fukuda, M, Takeda, N. Ohta, T. Iwanari, H. Sakihama, T. Kodama, T.
Hamakubo, T. & Shinkai, Y. Histone methyltransferases G9a and GLP form heteromeric complexes
and are both crucial for methylation of euchromatin at H3-K9. 2005. Gene Dev. 19:815-826.

331) Biological Sciences
1-3） Cell Biology

Analysis of the functional role of vacuolar protein sorting (Vps)protein, Hrs/Hgs in the regulation of TGFβ-induced signaltransduction
HIRONOBU ASAO
Yamagata University faculty of Medicine
asao-h@med.id.yamagata-u.ac.jp

Introduction
We identified an IL-2-induced tyrosine-phosphorylated protein, Hrs/Hgs and STAMs in T cell,isolated a cDNA clone and then reported that Hrs and STAMs are associated to each other. Some
reports showed that Hrs and STAMs were important molecules for vesicular transport from early tolate endosomes.
TGFβ is a multifunctional cytokine activating Smad2/3 and has suppressive functions on immune
system. We previously reported that Hrs formed a complex with Smad2/3 and TGFβ receptor andthat the expression level of Hrs was diminished accompanied with T cell activation and then TGFβ
signal was enhanced. In this study, we are going to ascertain the regulatory mechanism of the
TGFβ signaling by Hrs.

Results
Recently we established cells that defect Hrs gene expression1. In the Hrs-deficient cells establishedfrom mouse embryonic fibroblast (MEF), TGFβ signal was tested. In the Hrs-deficient cells (HRSd),
Smad2, which is a major signaling molecule of TGFβ signal transduction, was phosphorylated
before TGFβ stimulation and Smad2 activation was enhanced and prolonged than that of wild typeMEF (MEFw) (Figure 1). Translocation of Smad2 was studied by using transiently expressed
GFP-Smad2 in these cells. In Hrs-deficient cells, Smad2 was located in the cytoplasmic region andalso in the nucleus before TGFβ stimulation and then nuclear Smad2 was enhanced after
TGFβ stimulation. On the other hand, in the wild type Hrs-introduced cells, Smad2 was located in
the cytoplasmic and translocated into nucleus only after TGFβ stimulation. Introduction of wild-type
Hrs (HRSw) or Hrs mutant with an intact STAM binding domain (Hrs-dFYVE) into theHrs-deficient cells completely recuperated normal Smad2 activity while introduction of mutant Hrs
with no STAM binding ability (Hrs-dC2, Hrs-dM) did not.
Then we investigated STAM that is an association molecule with Hrs in the Hrs-deficient cell lines.
Surprisingly in the Hrs-deficient cells only trace amounts of STAM1 and STAM2 were detected34

comparing with wild type MEF. While wild-type Hrs or Hrs-dFYVE mutant-introduced
Hrs-deficient cells fully restored STAM1 and STAM2 expression, whereas Hrs-dC2, Hrs-dM
mutants with no STAM binding ability failed to restored the expression of the STAMs2.
This regulated control of STAM expression by Hrs was independent of transcription. Interestingly,
STAM1 degradation was mediated by proteasomes and was partially dependent on theubiquitin-interacting motif of STAM1. Revertant Hrs expression in the Hrs-deficient cells not only
led to the accumulation of ubiquitinated proteins, including intracytoplasmic vesicles, but also
restored STAM1 levels in early endosomes and eliminated the enlarged endosome phenotype causedby the absence of Hrs. These results suggest that Hrs is a master molecule that controls in part thedegradation of STAM1 and the accumulation of ubiquitinated proteins.

Discussion & Summary
In this study, we showed Hrs has an important role in regulating TGFβ signaling. Without Hrs,
TGFβ-induced major signal transducing molecule, Smad2 is constitutively activated even without
TGFβ. For the regulatory function of TGFβ signaling, STAM-binding domain of Hrs wasindispensable suggesting that this regulatory mechanism required also STAM. We previously
reported that epidermal growth factor receptor degradation is partially regulated by the Hrs-STAM
complex. Degradation of TGFβ receptor, which is important negative regulatory mechanism, mayalso be regulated by the Hrs-STAM complex that are important molecules in the mammalianendosomal/vacuolar protein sorting pathway.

35

References
1.

Kanazawa C, Morita E, Yamada M, Ishii N, Miura S, Asao H, Yoshimori T and Sugamura K;

Effects of deficiencies of STAMs and Hrs, mammalian class E Vps proteins, on receptor
downregulation. Biochem. Biophys. Res. Commun. 309, 848-856, 2003.
2.

Kobayashi H, Tanaka N, Asao H, Miura S, Kyuuma M, Semura K, Ishii N, Sugamura K.; Hrs, amammalian master molecule in vesicular transport and protein-sorting, suppresses the degradation of
ESCRT proteins STAM1 and STAM2. J. Biol. Chem. 280, 10468-10477, 2005.

361) Biological Sciences
1-3） Cell Biology

Comprehensive studies of gene regulation by posttranslational
processing-mediated carboxy-terminal fragments of the EGF family
members.
Shigeki Higashiyama Ph.D.
Ehime University School of Medicine
Shigeki@m.ehime-u.ac.jp

Introduction
Cleavage of membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF) via
metalloprotease activation yields amino- and carboxy-terminal regions (HB-EGF and HB-EGF-C),
with HB-EGF widely recognized as a key element of epidermal growth factor receptor
transactivation in G-protein coupled receptor signaling.

Currently we have shown that subsequent

to proteolytic cleavage of proHB-EGF, HB-EGF-C translocated from the plasma membrane into the
nucleus.

This translocation triggered nuclear export of the transcriptional repressor PLZF, which

we identify as a HB-EGF-C binding protein. In this study, we focused on the functional analyses of
C-terminal fragments of the EGF family members including HB-EGF as well.

Results
Based on the analysis of the HB-EGF-C binding domain of PLZF, we screened candidates as abinding partner of HB-EGF-C, and listed up 15 members of the PLZF family consisted of 75
members.

Pull-down assay using GST-HB-EGF-C revealed at least additional 4 members, BCL-6,

BAZF, Myoneulin and KIAA0441, of the PLZF family were able to interact with HB-EGF-C. In
order to analyze the localization of C-terminal fragment of the EGF family members after their
shedding, we carried out immunostining by their C-terminal fragment specific antibody, revealingthat the C-terminal fragments of TGF-α, amphiregulin, epiregulin and epigen were tranlocated into
nucleus. Their nuclear translocation effectively blocked by the addtion of a metalloproteaseinhinibtor KB-R8301.

We further confirmed that their C-terminal fragments were able to bind to

PLZF. On the other hand, we have screened the genes which transcription are regulated by PLZF
using DNA microarray, and identified Cycline A2 Pbx-1, tzfp and c-myc.

We are currently

investigating the target genes which regulates by BCL-6, BAZF, Myoneulin and KIAA0441.

37

Discussion & Summary
The C-terminal fragments on the EGF family members as well as HB-EGF-C translocate into
nucleus after ectodomain shedding on cell surface. The The C-terminal fragments interact severaltranscriptional repressors including PLZF.

Since the ectodomain shedding of the EGF family

members is well known to be a crucial event of EGFR transactivation induced by various
extracellular stimuli, the C-terminal fragment signaling of the EGF family members might be afundamental event in cell growth and differentiation.

Figures & Tables
C-terminal fragment signaling of
membrane-anchored growth factor HB-EGF
HB-EGF
Wounding

GFR

CR
Ca2+
GPCR

proHB-EGF
ADAMs

HB-EGFEGFR
HB-EGF-C

SH3 Pro.
PKC,etc.

repressors

MAPK

CRM1

repressors

Regulated geneTaregeted repressors
PLZF, Bcl-6, BAZF, Myuneulin, KIAA0041
Regulated gene
Cyclin A2, Pbx-1, tzfp, c-myc

References
1.Higashiyama, S. and Nanba, D. (2005) ADAM mediated ectodomain shedding of the EGFR
ligands in receptor cross talk. Biochemica. Biophysica. Acta. in press.
2. Toki, F., Nanba, D., Matsuura, N., and Higashiyama, S. (2005) Ectodomain shedding of
membrane-anchored heparin-binding EGF like growth factor and subcellular localization of the
C-terminal fragment in a cell cycle J.Cell. Physiol. 202, 839-848.
3. Tanaka, M., Nanba, D., Mori, S., Shiba F., Ishiguro, H., Yoshino, K., Matsuura,N., and Higashiyama, S. (2004) ADAM‑binding protein Eve‑1 is required for ectodomain
shedding of EGF receptor ligands J. Biol. Chem. 279, 41950‑41959, 2004

381) Biological Sciences
1-3） Cell Biology

Molecular Network Underlying Sperm Motility
Kazuo Inaba
University of Tsukuba
inaba@kurofune.shimoda.tsukuba.ac.jp

Introduction
Activation of sperm motility is one of the dynamic changes after completion of spermatogenesis.
This is a sophisticated process that many organisms have acquired during evolution and theabnormality in this process results in serious deficiency in fertilization, such as male infertility. This
research was aimed to reveal the molecular interaction underlying the reception of extracellularsignal, intracellular signal transduction and activation of motile apparatus of sperm flagella.

Results
To reveal the molecular network underlying the activation of sperm motility, the ascidian Ciona
intestinalis was used as an experimental organism. Ciona sperm are immotile when they were
spawned into seawater, but the motility was initiated and activated by an egg-derived substance
SAAF (Yoshida et al., 2002). The activation process is easily observed in vitro. This animal provides
not only a simple system for analysis of sperm activation but also genomic information that havebeen recently established: We have analyzed approximately 10,000 ESTs of testis-expressed genes in
C. intestinalis (Inaba et al., 2002) and genome draft sequence has been determined (Dehal et al.,2002).
The research was started from the establishment of the analysis system for sperm proteins using
peptide mass fingerprinting (PMF). The open reading frames of approximately 4,000 genes
expressed in C. intestinalis testis were determined from EST data complemented with genome draft
sequence. The accurate mass values of tryptic fragments derived from these testis-expressed genes
were in silico calculated by the help of the program MS-Fit. These data was summarized as“MSCITS”, a database comprising peptide masses of 4,000 proteins expressed in C. intestinalis
testis. Then a search program was devised for PMF analysis using Perl and was named as “PerMS”.
To test the efficiency for identification of proteins by this system, we have randomly selected 50
proteins from Ciona sperm proteins separated by two-dimensional gel electrophoresis, treated withtrypsin and analyzed by a matrix-assisted laser desorption ionization/time of flight/mass
spectrometry (MALDI-TOF/MS). Approximately 80% of the proteins could be correctly identified,39suggesting that the MSCITS/PerMS is a efficient system for identification of Ciona sperm proteins(Hozumi et al., 2004).For comprehensive understanding of the signaling network for the activationof sperm motility, we carried out the analysis on 1) changes in proteins at activation of sperm
proteins using two-dimensional gel electrophoresis, 2) molecular construction of outer and inner arm
dyniens, 3) isolation of radial spokes and its molecular architecture, and 4) proteomic analysis ofmembrane/matrix fraction of sperm.
During the course of these study using MSCITS/PerMS system, we have identified a heat shockprotein 40 in the radial spoke of flagellar axonemes, which is thought to be quite important in thesignal transduction for the activation of dyneins (Satouh et al., 2005). Members of the heat-shockprotein (HSP) 40 regulate the protein folding activity of HSP70 proteins and help the functional
specialization of this molecular chaperone system in various types of cellular events, suggesting acorrelation between Hsp40 related chaperone system and flagellar function. Furthermore we havefound that Ciona 37-kDa Hsp40 is extracted from KCl-treated axonemes with 0.5 M KI solution andcomigrates with radial spoke protein (RSP) 3 along with several proteins as a complex through gel
filtration and ion exchange columns. PMF with MALDI-TOF/MS revealed that other proteins in thecomplex include a homolog of sea urchin spokehead protein (homolog of RSP4/6), a membrane
occupation and recognition nexus repeat protein with sequence similarity with meichroacidin, and afunctionally unknown 33-kDa protein. A spoke head protein, LRR37 (Padma et al., 2003), is notincluded in the complex, suggesting that the complex constructs the stalk of radial spoke.
Immunoelectron microscopy indicates that Hsp40 is localized in the distal portion of spoke stalk,possibly at the junction between spoke head and the stalk (Figure 2).

Figure 1. Two-dimentional gel electrophoresis of Ciona axonemal proteins (left) and
MALDI-TOF/MS analysis of an axonemal proteins.

40

Figure2.

Immunologicalanalyses

of

Ciona

axonemal

HSP40.A,

westernblot;

B,

immunofluorescence; C, immunogold localization.

Discussion & Summary
In order to perform a proteomic study on the molecular mechanism of sperm activation, we haveconstructed a MS database from Ciona testis ESTs and the genome draft sequence, along with a
newly devised, perl-based search program PerMS for peptide mass fingerprinting. This system could
identify more than 80% of Ciona sperm proteins, suggesting that it could be widely applied forproteomic analysis for a limited tissue with less genomic information. By using this system, we
could identify HSP40, a homolog of sea urchin spokehead protein, a membrane occupation andrecognition nexus repeat protein with sequence similarity with meichroacidin, and a functionally
unknown 33-kDa protein in the radial spoke complex of flagellar axonemes.

References
Dehal, P. et al.,(2002) The draft genome of Ciona intestinalis: insights into chordate and vertebrate
origins. Science, 298, 2157-2167
Hozumi, A., Satouh, Y., Ishibe, D., Kaizu, M., Konn,o A., Ushimaru, Y., Toda, T. and Inaba, K.
(2004) Local database and the search program for proteomic analysis of sperm proteins in the

41

ascidian Ciona intestinalis. Biochem. Biophys. Res. Commun., 319:1241-1246.
Inaba K, Padma P, Satouh Y, Shin-I T, Kohara Y, Satoh N, Satou Y.(2002) EST analysis of gene
expression in testis of the ascidian Ciona intestinalis. Mol Reprod Dev.,62, 431-445.
Satouh, Y., Padma, P., Toda, T., Satoh, N., Ide, H. and Inaba, K. (2005)
Molecular characterization of radial spoke subcomplex containing radial spoke protein 3 and heat
Shock protein 40 in sperm flagella of the ascidian Ciona intestinalis. Mol. Biol. Cell, 16: 626-636.
Padma, P., Satouh, Y., Wakabayashi, K., Hozumi, A., Ushimaru, Y., Kamiya, R., and Inaba, K.
(2003). Identification of a novel leucine-rich repeat protein as a component of flagellar radial spokein the ascidian Ciona intestinalis Mol. Biol. Cell 14, 774-785.
Yoshida M, Murata M, Inaba K, Morisawa M. (2002) A chemoattractant for ascidian spermatozoa isa sulfated steroid. Proc Natl Acad Sci USA 99, 14831-14836.

421) Biological Sciences
1-3） Cell Biology

Isolation of ES-like cells and production of cloned animals fromgenetically modified blastocysts derived from equine or bovine
Kazunari K. Yokoyama
Gene Engineering Division, BioResouce Center, RIKEN
yokoyama@brc.riken.jp

Introduction
One of the goals of animal biotechnology is the application of current techniques of genetic
engineering to the production of farm animals with desired traits. At present, foreign genes can be
induced into the murine genome via embryonic stem (ES) cell and the use of homologous
recombination techniques.

In this system, cultured murine ES cells can be used as a tool for the

addition or deletion of genes at specific sites in the genome.

With respect to species other thanmice, human, sheep, pig, bovine, mice, mink, hamster, rat, monkey and equine have all beenreported as source of putative ES cells.

However, neither production of chimeric animals withtraits that are transmitted through the germ line nor satisfactory demonstration of the essential traitsof ES cells has yet been reported.
In attempt to generate that genetically modified ES (like) cells and cloned animals, we haveexamine gene.
The possibility of the generation of transgenic calves and equines from ES cells introduced byfollowing genes,(A)

Japanese specific HLA class I and β2m genes;(B)

Human telomease genes.

Results
Capacity for self-renewal and pluripotency
Mouse ES cells can be maintained indefinitely in the presence of Leukemia inhibitory factor (LIF),
and they express markers of the undifferentiated state that indicate the capacity for self-renewal andpluripotency, such as transcription factors Oct4, STAT-3, stage-specific embryonic antigen (SSEA-1),
and alkaline phosphatase (AP).

Upon removal of LIF, the cells cease to express markers such as

Oct 4, rapidly losing the capacity for self-renewal and differentiating into a variety of cell types.
In addition to the mouse and man, the sheep, pig, rabbit, bovine, mink, hamster, rat and monkey
have all been reported as sources of putative ES cells. However, in these otter animals, neither the43

production of chimeric animals with traits that are transmitted through the germ line nor satisfactory
clarification of the essential characteristics of ES cells has yet been reported.
Recently, we demonstrated the successful isolation of immortalized equine and bovine ES cellswith a normal karyotype. We have cultured such cells for more than 50 passages in vitro with aminimum of 250 cell divisions. Strelchenko et al.
cells is approximately 60 divisions.

reported that the limit for most non-immortalized

Therefore, we can distinguish immortalized cell lines from

non-immortalized cell lines operationally on the basis of the ability of cells to undergo more than 60
divisions. Both our equine and our bovine ES cells exhibit the essential features of ES cells,
expressing Oct4, STAT-3, SSEA-1 and AP (Fig. 1). We confirmed the pluripotency of these cellswith respect to somatic differentiation in vitro to neural progenitor cells and to endothelial orhematopoietic lineages.

Fig.1:Morphology of equine (A) and bovine (B) ES cells.
Phase-contrast image of colonies of ES cells cultured for5days in the absence of a feeder layer but in the presenceof human LIF.
Diferentiated eqithelia-like,rounded and bi-polar shaped
cells are visible among other cells in culture.
Magnification: A and B,×200;

44

By contrast to murine ES cells, equine ES cells differentiated to epithelial-like cells in the absenceof a feeder layer, provided the medium was supplemented with LIF.

Equine and bovine ES cells

both required the support of a feeder layer for serial cultivation in the undifferentiated state.
Furthermore, LIF was unable, by itself, to maintain self-renewal of murine ES, cells in serum-free
medium.

Hence, some other growth factors must be necessary, in combination with LIF to support

the self-renewal capacity of ES cells.
Ying and colleagues showed recently that bone morphogenetic protein (BMP) can replace serum,
defining the molecular requirements for the self-renewal of murine ES cells. These researchers
demonstrated that, when ES cells were grown in serum-free medium, BMP4 and LIF acted
synergistically to support the derivation and maintenance of ES cells, which are driven by thecoordinated actions of BMP and STAT-3 signaling pathways, even when the staｒting material issingle cells. Thus, it appears necessary to include BMP in the culture medium in the absence of afeeder layer for the consistent generation of ES cells, in which the full detail of the molecular basisof self-renewal remains to be clarified.
While it has proved difficult to identify the factors that are required for the maintenance of ES
cells in the absence of a feeder layer, progress in establishing methods for inducing thedifferentiation of such cells.

We developed a method for the induction of differentiation to neuralprecursors of equine and bovine ES cells, using a combination of basic fibroblast growth factor(bFGF; 10 ng/ml; Sigma), epidermal growth factor (EGF; 10 ng/ml; Sigma) and platelet-derived
growth factor (PDGF; 10 ng/ml; Sigma) in serum-free MEMα medium..

We also developed aprotocol for the differentiation of equine ES cells in vitro to hematopoietic and endothelial celllineages in response to bFGF, stem cell factor (SCF) and oncostatin M.
Equine ES cells that were proliferating in two dimensions were able to differentiate tohematopoietic cells and, thus, it appeared that neither a three-dimensional ES structure nor a layer of
feeder cells was required for this process, as was also the case for the differentiation of equine andbovine ES cells to neural precursors cells.

Now, we need to develop methods that will allow us topathways of differentiation of ES cells and the formation of tissues and organs from them in vitro.

Transgenesis of animal ES cells
Other research group, working on the isolation of porcine ES cells, determined conditions formaintaining such ES cells in an undifferentiated state for short periods of time. However, they failed
to maintain the cell lines in an undifferentiated state for a long enough time to allow attempts at
genetic modification. By contrast, our bovine ES cell lines can be maintained indefinitely in vitro
and retain characteristics of pluripotency, as do mouse ES cells. Then, we have been able to achieve
genetic transformation and the selection of transformants.
Bovine ES cells were transfected by a lipid-based reagent (Effectene, Qiagen, Tokyo, Japan) with

45

DNA fragments derived from pCX-Neo-EGFP and incubated for 3 days. Then, we selected colonies
that expressed fluorescent GFP protein by exposing of cells to the antibodies G418 (200-400 ug/mg).
Recipient embryos produced by in vitro fertilization (IVF; day 4 embryos) were injected with 10 to
15 transgenic ES cells per embryo.
After 5 days in culture, more than 40% of the chimeric embryos (17 out of 41) included
contributions due to the injected GFP-expressing stem cells. Fluorescence due to GFP was detectable
both in the ICM and in trophectodermal cells of the injected blastocysts. Our data showed clearly
that the transfected bovine ES cells were able to participate in the formation of chimeras after
injection into embryos at the 8- to 16- cell stage, while similarly transfected fibroblast did not havethis ability. Our observations indicated that the procedures for genetic transformation and selection
had not interfered with the ability of the ES cells to participate in the formation of chimeras.
Human leukocyte antigen-β(HLA-B) are useful, therapeutic agents but, because they are available
only from human donors, their supply and, thus, their applications are limited.

Transgenic animals

carrying a gene for human leukocyte antigen (HLA) might be a good source of HLA-B. Therefore,
we recently established lined of bovine ES cells that express HLA plus GFP (HLA-B-GFP) or
human telomerase reverse transcriptase plus GFP (HTERT-GFP).

These genes were efficiently

introduced using FuGene 6 (Roche, Tokyo, Japan) or FluoroGene (ImaRx, Tucson, AR, USA ) in
combination with an Enhancer A (Nepagene, Chiba, Japan) as transfection reagents.

The efficiencyof transfection using these reagents was apprpximately 50%, as determined 72h after the start of thetransfection. Consequently, the expression of GFP in bovine ES cells was 5 to 10 times higher thanthat obtained with other reagents such as LipofectAMINE (Life Technologies, Rockville, USA),
Effectene (Quiagen) and Escort (Sigma).
Nuclear transfer (NT) from murine ES cells allows generation of mice with DNA insertions
without necessity of generating chimeric mice, as is required when convential gene-targeting
techniques are used because animals produced by NT are entirely transgenic.

Thus, with improved

efficiency, NT with ES cells should shorten the time required for the generation of transgenic
animals.

Furthermore, it might be possible to insert genes at a single site in multiple lines ofanimals to generate homozygous lines of animals rapidly while avoiding inbreeding.

The realistic

prospect of gene targeting in large domestic animals opens up a vast range of novel applications andresearch possibilities.

Nuclear transfer of animal ES cells
Nuclear transfer (NT) was accomplished first in frogs to determine whether nuclei fromdifferentiated cells remain genetically totipotent. Unsuccessful results from cloning experimentswith adult cells suggested that the nuclei from differentiated frog cells could not support thedevelopment of an entire frog.

However, the generation of Dolly, the sheep from an adult

46

mammary gland cell suggested the nuclear totipotency of at least some cells within an adult animal.
It remains to be determined whether cloned animals are derived selectively and for the most part,from the nuclei of less-differentiated adult cells, such as adult stem cells, rather than from the nuclei
of fully differentiated adult cells.

The generation of cloned mice, sheep and bovine derived frompluripotent blastomeres or ES cells suggests that the nuclei of less-differentiated cells are more
useful.
For example, mouse blastocysts derived from 2- to 8- cell blastomeres and from morula stage nuclei
developed to term with an efficiency of 20-40%. Similarly, Willadsen et al. reported a success rateof 36% for live cloned calves derived from morula-stage nuclei by NT in cattle. Furthermore, inmice blastocysts cloned from nuclei of cultured ES cell developed to term at an efficiency (10-30%)
that was 3 to 10 times greater than in the case of blastocysts cloned from the nuclei of somatic cells(1-3%).
We performed NT experiments using bovine ES cells, that had been purified by flow cytometry ina cell sorter (FACScan, Becton Dickinson, Mountain View, CA, USA) with SSEA-1- specificantibodies. Cloned blastocysts derived from cultured ES cells yielded a pregnancy rate of 71% byultrasonography 50 days after transfer, and calves developed to term at a frequency of 43%. These
values are higher than those for early pregnancies (55%) and term pregnancies (8-14%) that havebeen reported after transfer of nuclei from differentiated somatic cells.

Wakayama et al.

demonstrated that live offspring cannot be generated after direct transfer of nuclei from terminallydifferentiated lymphocytes to early embryos.

Their observations suggest that reprogramming of aterminally differentiated cell might be extremely inefficient and, thus, the development of cloned
embryos is likely to fail. Indeed, the genome might undergo not only epigenetic but also genetic
changes during differentiations to, for example, terminally differentiated lymphocytes.
Compared to differentiated somatic cells, embryonic cells require less-reprogramming of theirgenome, perhaps because many genes critical for early development are already active in the donor
cells and do not need to be reactivated.

Thus, it might prove possible to generate cloned animals

from the nuclei of adult stem cells but not from those of terminally differentiated cells. Adult stem
cells and ES cells might exist in a similar epigenetic state that allows them to differentiate tomultiple lineages.
As a next step towards our goal of applicable ES cell technologies, we are now attempting thegeneration of transgenic clonal calves that harbor an HLA gene from cultured ES cells to produce
human leukocyte antibodies against HLA.

47

Discussion & Summary
It is important to note that NT often results in problems, such as respiratory distress, kidney and liverdefects, and abnormal growth of the placenta and/or fetus. In our experiments, one of cloned calvesdied two days after birth as a result of abdominal bleeding. Experiments on mice indicate that, mostcloned animals suffer from abnormalities that range from, subtle to severe, as confirmed by aberrant
patterns of gene expression.

Many of the observed abnormalities in cloned animals might becaused by the inefficient and incomplete reprogramming of post-zygotic epigenetic modifications
that were imposed on the genome during differentiation.

Such epigenetic modifications, include

DNA methylation, histone modifications and the expression of development-related genes.
We understand very little about reprogramming and ways in which it can be achieved. A better
understanding of the molecular basis of reprogramming might allow us to improve the efficiency ofNT and to facilitate its application to gene and cell therapy.
Studies of NT may help us to clarify the contribution of epigenetic and genetic modification tocellular differentiation and transformation.

Figures & Tables

48

References
・ Saito, S., Ugai, H., Sawai, K., Yamamoto, Y., Minamihashi, A., Kurosawa, A., Kobayashi, Y.,
Murata, T., Obata, Y. and Yokoyama, K.: Isolation of embryonic stem-like cells from equine
blastocystes and their differentiation in vitro. FEBS Letters, 531，389-396 (2002).
・ Saito, S., Sawai, K., Ugai, H., Moriyasu, S., Minamihashi, A., Yamamoto, Y., Hiroyama, H.,
Kageyama, S., Pan, J., Murata, T., Kobayashi, Y., Obata, Y. and Yokoyama, K.: Generation ofcloned calves and transgenic chimeric embryos from bovine embryonic stem-like cells.
Biochem. Biophys. Res. Communi. 309, 104-113 (2003).
・ Saito, S., Liu, B. and Yokoyama, K.: Animal embryonic stem (ES) cells: Self-renewal,
pluripotency, transgenesis and nuclear transfer.

Human Cell, 17, 107-115 (2004).

・ Yokoyama, K., Murata, T., Ugai, H., Suzuki, E., Terashima, M., Kujime, Y., Inamoto, S., Hirose,M., Inabe, K. and Yamasaki, T.: Recombinant Virus BANK for gene delivery. Science 307, 1722
(2005).
Saito, S., Sawai, K., Minamihashi, A., Ugai, H., Murata. T. and Yokoyama, K.: Derivation,
maintenance and differentiation of equine ES cells. Methods in Molecular Biology, in press(2005).

49

1) Biological Sciences

1-3） Cell Biology

Physiological functions and regulation mechanisms for tankyrase and
TAB182
Hiroyuki Seimiya
Division of Molecular Biotherapy, Cancer Chemotherapy Center,
Japanese Foundation for Cancer Researchhseimiya@jfcr.or.jp

Introduction
Tankyrase 1, a member of poly(ADP-ribose) polymerases, binds to a telomeric protein, TRF1, andpoly(ADP-ribosyl)ates it. This post-translational modification inhibits the ability of TRF1 to bind
telomere

DNA

and

the

TRF1-unboundtelomeres

allow

accessoftelomerase,atelomere-synthesizing enzyme. Thus, tankyrase 1 is a positive regulator for telomere length (Refs. 1& 2). TAB182 is another tankyrase 1-binding protein that is poly(ADP-ribosyl)ated by tankyrase 1(Ref. 3). Tankyrase 1 is present not only at telomeres but also at Golgi apparatus and mitotic
centrosomes, suggesting that this enzyme has additional non-telomeric functions. In this study, we
focused on physiological significance of the tankyrase 1-TAB182 interaction (especially, functionsof TAB182).

Results(1) Generation of TAB182 knockout mice.
After cloning mouse TAB182 genomic DNA clones by the lambda phage screening, we constructed
two types of the TAB182 gene targeting vectors (conventional and conditional). These vector
plasmids were linearlized by Not I digestion and then transfected into mouse ES cells byelectroporation. As for conventional targeting, 9 homologous recombinants (TAB182+/-) weresuccessfully isolated from 215 G418-resistant ES cell clones (Figure 1). In the targeted allele,
TAB182 exon 2 was disrupted by in-frame insertion of a lacZ gene. The downstream portion of theexon 2 and whole exon 3 were also deleted in the targeted allele, which would abolish the
production of TAB182 transcripts. Patterns for genomic integration of the vector were verified bySouthern blot analysis with four different probes and PCR. Representative ES cell clones have justbeen injected into blastocysts to obtain chimeric mice. As for the Cre recombinase/loxP-based
conditional targeting of the gene, 5 homologous recombinants (TAB182+/flox) were isolated from 190
G418-resistant ES cell clones. However, none of these clones retained the critical 3rd loxP sequence

50

because one side of homologous recombination occurred at the locus between the 3rd loxP sequenceand the neo gene cassette of the vector. Thus, for conditional targeting of the gene, we still need to
add the 3rd loxP sequence to the floxed allele of the recombinants in the future.

(2) Functional analysis of TAB182 by using RNA interference.
Human fibrosacoma HTC75 cells were transfected with several kinds of small interfering RNA(siRNA) to knockdown TAB182 protein expression in the cells. After verifying the effect of eachsiRNA on TAB182 protein level by western blot analysis, retroviruses that produced respective short
hairpin RNA (shRNA) were constructed with the same target sequences. Then, the cells were
infected with the viruses to establish cell lines in which the amount of TAB182 protein was stably
down-regulated. Of note, the pilot study with siRNAs did not predict the extent of knockdown effectof shRNAs even with the same target sequences. The cell lines with decreased TAB182 expression
grew at comparable rates to that of parental cells. After prolonged cultivation for 100 population
doublings, these cells did not exhibit any change in mean telomere length as compared to that ofparental cells.
(3) Estimation of the tankyrase 1-TRF1 interaction in mouse cells.
Human TRF1 has a tankyrase 1-binding motif, RGCADG, at the amino terminal acidic domain

51

whereas mouse TRF1 does not have such a motif. By using a pull-down assay with recombinant
glutathione-S-transferase fusion proteins and in vitro translated proteins, we confirmed that neither
human or mouse tankyrase 1 bound mouse TRF1 in vitro. On the other hand, both human and mousetankyrase 1 bound human TRF1 in vitro (Figure 2). Consistently, transient overexpression of mousetankyrase 1 in human HeLaI.2.11 cells released TRF1 from telomeres whereas that of humantankyrase 1 in mouse NIH3T3 cells had no effect on telomeric localization of TRF1, which wasassessed by indirect immunofluorescence staining. Meanwhile, both human and mouse tankyrase 1bound TAB182, which had a well-conserved tankyrase 1-binding motif, RPQPDG. Chromatin
immunoprecipitation assay showed that telomere DNA was pulled down by anti-TRF1 but not byanti-TAB182 antisera. Consistent with this observation, affinity-purified anti-TAB182 antibody did
not stain telomeres.

Discussion & Summary
Inability of tankyrase 1 to recognize TRF1 in mouse cells strongly suggests that telomere length
regulation by tankyrase 1 could be a species-restricted phenomenon. This also implies that tankyrase
1, especially in mice, would play a role for non-telomeric events through interaction with otherbinding partners, such as TAB182. In this study, so far, our observations have not shown any linkage
between TAB182 and telomere dynamics. We have generated TAB182+/- ES cells and are expecting
the chimeric mice. In the near future, we would like to confirm germ line transmission of thetargeted allele and obtain TAB182+/- F1 and TAB182-/- F2 mice. We expect that these knockout micewould tell us the physiological importance of TAB182. Recently, we have reported thatpharmacological targeting of tankyrase 1 enhances the anti-tumor impact of a telomerase inhibitor onhuman cancer cells (Ref. 1). Before assuming that tankyrase 1 is a good anticancer molecular target,

52

we still need to investigate whether tankyrase 1 inhibition disrupts its crucial physiological functionsat other loci than telomeres. Functional analyses on TAB182 would give further insight into
expanding network of tankyrase 1-mediated biological events.

References
1.

Seimiya, H., Muramatsu, Y., Ohishi, T., and Tsuruo, T.: Tankyrase 1 as a target for

telomere-directed molecular cancer therapeutics. Cancer Cell, 7:25-37, 2005.
2.

Seimiya, H., Muramatsu, Y., Smith, S., and Tsuruo, T.: Functional subdomain in the ankyrindomain of tankyrase 1 required for poly(ADP-ribosyl)ation of TRF1 and telomere elongation. Mol.
Cell. Biol., 24:1944-1955, 2004.
3.

Seimiya, H., and Smith, S.: The telomeric poly(ADP-ribose) polymerase, tankyrase 1,

contains multiple binding sites for telomeric repeat binding factor 1 (TRF1) and a novel acceptor,
182-kDa tankyrase-binding protein (TAB182). J. Biol. Chem., 277:14116-14126, 2002.

531) Biological Sciences
1-3） Cell Biology

Receptor protein tyrosine phosphatase ζ (Ptprz) signaling in gastriculcer induction
Masaharu Noda
National Institute for Basic Biology
madon@nibb.ac.jp

Introduction
Protein tyrosine phosphorylation plays crucial roles in various aspects of cellular function.

Thelevel of tyrosine phosphorylation is determined by the balance between the activities of protein
tyrosine kinases (PTK) and protein tyrosine phosphatases (PTP).

Ptprz (PTPz/RPTPb) is a memberof the receptor-like PTP family and predominantly expressed in the brain.

Recently, we found that

Ptprz is expressed in the stomach, and that the mice deficient in Ptprz gene do not show mucosal
damage by vacuolating cytotoxin, VacA, secreted from Helicobacter pylori.
behaves as a ligand for Ptprz.

We revealed that VacA

In this study, we aimed to elucidate Ptprz-signaling cascade

underlying the gastric ulcer induction.

Results
We here developed a genetic method to screen for PTP substrates which we have named the“yeast substrate-trapping system” (Fig. 1).

This method is based on the yeast two-hybrid systemwith two essential modifications: the conditional expression of a PTK to tyrosine-phosphorylate theprey protein, and screening using a substrate-trap PTP mutant as bait.

In our screening of

substrates for Ptprz, we isolated four substrate candidates and 11 putative continuously interacting
molecules (Fig. 2).
The four substrate candidates are Git1, p190, PIST and MAGI-1. Git1 is a GTPase-activating
protein (GAP) for ADP-ribosylation factor (ARF) family of GTPases and believed to function in thewide range of cellular events including vesicle trafficking, cell adhesion, and cytoskeletalorganization. p190 is a GAP for the Rho family of GTPases and known to participate in thecytoskeletal organization through the regulation of Rho activity.

Rho family GTPases act as themain regulator of actin cytoskeletal dynamics in a variety of situations including neurite extension,
cell migration, cell adhesion, and cell morphological changes.

PIST was first identified as a PDZ

domain-contining protein which interacts with TC10, a member of the Rho family of GTPases and54

most closely related to cdc42.

MAGI-1 contains a guanylate kinase domain, two WW motifs, andfive PDZ domains, and is believed to function as a molecular scaffold in the formation ofmultiprotein complexes on the cytoplasmic surface of the cell membrane as is PSD-95.

Thus, Ptprz

likely regulates the cytoskeletal dynamics and cell adhesion through the control of Rho family
GTPase activity.
Furthermore, we demonstrated that all of the four candidates can be tyrosine phosphorylated by
v-src in mammalian cells, and dephosphorylated by GST-Ptprz in vitro.

As the next step, we needto analyse the tyrosine phosphorylation of these molecules upon VacA treatments to know whichmolecule is involved in gastric ulcer induction.
common functional features as described above.

This is highly intriguing because they are given

55

Discussion & Summary
In contrast to PTKs, our understanding of the biological functions of PTPs has been limited due to alack of information concerning the physiological substrates for most PTPs.

To fully understand thebiological functions of PTPs, the identification of each of their substrates is essential.

The yeast

substrate-trapping system can be applied to all members of the PTP family and identify substratecandidates together with continuously interacting molecules.

The identification of PTP substrates

and elucidation of cellular phenotypes correlated with the tyrosine phosphorylation levels of
substrates will lead to a full understanding of the physiological roles of individual PTPs.

We

believe that the clones isolated by the yeast substrate-trapping system will be promising candidates
for substrates and that they will shed light on the Ptprz-signaling cascade in gastric cells underlying
the gastric ulcer induction.

56

References
1. Shintani, T., Watanabe, E., Maeda, N. and Noda, M. (1998) Neurons as well as astrocytes express
proteoglycan-type protein tyrosine phosphatase ζ/RPTPb: Analysis of mice in which the
PTPζ/RPTPβ gene was replaced with the LacZ gene.

Neurosci. Lett. 247, 135-138.

2. Kawachi, H., Fujikawa, A., Maeda, N. and Noda, M. (2001) Identification of GIT1/Cat-1 as a
substrate molecule of protein tyrosine phosphatase ζ/β by the yeast substrate-trapping system.
Proc. Natl. Acad. Sci. USA 98, 6593-6598.
3. Fujikawa, A., Shirasaka, D., Yamamoto, S., Ota, H., Yahiro, K., Fukada, M., Shintani, T., Wada, A.,
Aoyama, N., Hirayama, T., Fukamachi, H. and Noda, M. (2003) Mice deficient in protein tyrosine
phosphatase receptor type Z are resistant to gastric ulcer induction by VacA of Helicobacter pylori.
Nature Genetics 33, 375-381.
4. Fukada, M., Kawachi, H., Fujikawa, A. and Noda, M. (2005) Yeast substrate-trapping system forisolating substrates of protein tyrosine phosphatases: Isolation of substrates for protein tyrosine
phosphatase receptor type z.

Methods 35, 54-63,

571) Biological Sciences1-4） Developmental Biology

Regulation of spermatogenesis by cytoplasmic polyadenylation ofmRNAs in male haploid germ cells
Shin-ichi Kashiwabara
Graduate School of Life and Environmental Sciences, University of Tsukuba
kashiwa@sakura.cc.tsukuba.ac.jp

Introduction
A testis-specific cytoplasmic poly(A) polymerase, TPAP, is involved in poly(A) tail extension of
specific mRNAs, including TRF2, TFIIAγ, and TAF10, in the cytoplasm of haploid, roundspermatids. Mice lacking TPAP resulted in spermatogenesis arrest at the round spermatid stage dueto the severely reduced expression of haploid-specific genes required for morphogenesis of germ
cells1). The purpose of this study is to elucidate the molecular basis of spermatogenesis arrest
caused by TPAP deficiency.

Results
1. Transgenic expression of TPAP in the wild-type and TPAP-deficient mice2) --- Transgenic miceoverexpressing TPAP showed normal spermatogenesis and fertility.
substrate mRNAs was also unaffected.

The poly(A) tail length ofIntroduction of TPAP transgenes in the TPAP-deficient

mice complemented both the incomplete elongation of the poly(A) tails of substrate mRNAs, andreduced expression of haploid-specific genes (Fig. 1A), resulting in the resumption of normal
spermatogenesis and fertility (Fig. 1B).

Fig. 1. Transgenic expression of TPAP in TPAP-deficient mice.

58

2. Mechanism of poly(A) tail extension of specific mRNAs in the cytoplasm of round spermatids --CPEB2, a paralogue of CPEB1, which is implicated in cytoplasmic polyadenylation of maternal
mRNAs during oocyte maturation, and CPSF160 showed similar expression patterns with TPAP
during spermatogenesis.

In addition, GST-pull down assay revealed that TPAP interacts withCPEB2 through CPSF160.
3. Nuclear transport of TAF10 in round spermatids --- In testes lacking TPAP, TAF10 was
insufficiently transported into the nucleus of germ cells.
protein of TAF10 may be a true substrate of TPAP.

Thus, mRNA encoding a transporter

Immunoprecipitation of testicular extracts byanti-TAF10 antibodies identified KIF2β and KIF2γ, which are splicing isoforms of motor protein
KIF2A.

The mRNAs encoding KIF2β and KIF2γ were overexpressed in testis. Although theexpression levels of these two mRNAs in mice lacking TPAP were the same as those in the wild-type
mice, the poly(A) tail extension of the mRNAs was incomplete, and translational efficiency was
reduced.
4. Profiling of proteins affected by TPAP deficiency --- When protein extracts of round spermatids ofthe wild-type and TPAP-deficient mice were compared by two dimentional gel electrophoresis, atleast two proteins, sorbitol dehydrogenase and adenylate kinase, were reduced in round spermatidsof mice lacking TPAP.

However, the mRNA levels of the two proteins were the same between thewild-type and mutant mice, suggesting that poly(A) tail extension of some mRNAs in roundspermatids is required for normal translation.

Discussion & Summary
Transgenic expression of TPAP in the wild-type testes implys the presence of a regulatory
mechanism(s) defining the extent and specificity of the cytoplasmic mRNA polyadenylation.
Resumption of normal spermatogenesis and fertility in TPAP-deficient mice with TPAP transgenes
conclusively demonstrates the essential role of TPAP in spermatogegesis.

The mechanism ofpoly(A) tail extension by TPAP is suggested to be similar to that in oocyte maturation.

The reduced

expression of haploid-specific genes due to inefficient nuclear transport of TAF10 in the mutant mice
may be attributed to incomplete poly(A) tail extension of mRNAs for KIF2A splicing isoforms.

By

proteomic approach, it is also suggested that proper translation of some mRNAs in round spermatids
requires poly(A) tail elongation.

References
1. Kashiwabara, S., Noguchi, J., Zhuang, T., Ohmura, K., Honda, A., Sugiura, S., Miyamoto, K.,
Takahashi, S., Inoue, K., Ogura, A., and Baba, T. Science 298, 1999-2002 (2002)
2.

Zhuang, T., Kashiwabara, S., Noguchi, J., and Baba, T.

J. Reprod. Dev. 50, 207-213 (2004)

591) Biological Sciences1-4） Developmental Biology

Mechanism of the functional neuronal histogenesis in the developing
brain
Kazunori Nakajima
Department of Anatomy, Keio University School of Medicine
kazunori@sc.itc.keio.ac.jp

Introduction
In the developing mouse cerebral cortex, gamma-aminobutyric acid (GABA)ergic neurons andnon-GABAergic neurons arise in distinct places and migrate into the cortical plate via different
pathways.

Although the “inside-out” alignment of projection neurons in the cortex has beenthoroughly analyzed, the pattern of interneuron alignment is not well understood.

Results
To investigate the birth-date dependent alignment of GABAergic neurons in the mouse visualcortex, we initially analyzed the birth-date dependent alignment of GABA+/BrdU+ cells
quantitatively and compared the distributions of GABA+/BrdU+ cells to those of GABA-/BrdU+
cells (projection neurons).
The distributions of GABA+/BrdU+ cells and GABA-/BrdU+ cells were analyzed by dividing
the cortex into 10 bins positioned parallel to the brain surface. The correspondence between each bin
and the cortical layers was determined by staining the sections with anti-NF and anti-NeuN. Bin 10
nearly corresponded to layer I, while bin 1 contained the lowest part of layer VI and the whitematter.
The distribution of E12.5 GABA+/BrdU+ cells (n=2321 cells, 5 animals) showed two peaks
around bins 4 and 9; the following significant differences were obtained between the bins: 1-2(p=0.0007), 2-3 (p=0.011), 3-4 (p=0.02), 4-5 (p=0.0099), 5-6 (p=0.001), 7-8 (p=0.03), 8-9 (p=0.006),
and 9-10 (p=0.008). The distribution of E12.5 GABA-/BrdU+ cells (n=1229 cells, 5 animals)
showed only one peak around bin 2; the following significant differences were obtained between thebins: 1-2 (p=0.0000007), 2-3 (p=0.02), 5-6 (p=0.006), 6-7 (p=0.02), 7-8 (p=0.01), and 8-9 (p=0.02).
Thus, in P9.5 mice, GABAergic neurons born on E12.5 were distributed in the cortex around two
peak locations, a major peak near layer V and a minor peak near layer II/III. On the other hand,
non-GABAergic neurons from the same day were only distributed around one peak location, in layer
VI. Interestingly, the major peak location of the GABA+/BrdU+ cells was always slightly above the

60peak location of the GABA-/BrdU+ cells in all 5 animals analyzed in this study. In the mostsuperficial quarter of the cortex, the proportion of GABA+/BrdU+ cells among all BrdU-positivecells

was 98.9±0.68 % (n=537 cells, 5 animals). This result indicates that most of the

BrdU-positive cells in this area are GABAergic neurons.
In contrast, the E15.5 GABA+/BrdU+ cells (n=1526 cells, 3 animals) exhibited only one peaklocation around bin 9, with the following significant differences between bins: 6-7 (p=0.04), 7-8(p=0.03), and 9-10 (p=0.003). E15.5 GABA-/BrdU+ cells (n=595 cells, 3 animals) also showed only
one peak location around bin 9, with the following significant differences obtained between bins: 6-7(p=0.03), 7-8 (p=0.03), and 9-10 (p=0.002). These results indicate that both GABAergic andnon-GABAergic neurons born on E15.5 are aligned around a common, single peak location nearlayer II/III in the cortex.
To investigate whether the two peak locations of GABA+/BrdU+ cells born on E12.5 still exist
in the mature visual cortex, we performed double-immunostaining with anti-GABA and anti-BrdU in
P30 mice and displayed the results as a histogram. The distributions of E12.5 GABA+/BrdU+ cells
in P30 mice (n=478 cells, 3 animals) showed a two-peak pattern, which was very similar to thatobserved in P9.5 mice
To investigate whether the two peak locations of E12.5 GABAergic neurons differ according totheir GABAergic neuron subtypes, we then analyzed the subtypes of E12.5 GABAergic neurons.
The calretinin-positive cells were located preferentially at the minor peak location near layer II/III,
whereas somatostatin-positive cells were located preferentially at the major peak location near layer
V.

Discussion & Summary
In conclusion, the present results show that, in the mouse visual cortex, 1) E12.5
GABAergic neurons are distributed around two peak locations, while non-GABAergic
E12.5 neurons are distributed around only one peak location, 2) The major peak locationof GABAergic E12.5 neurons was always slightly above the peak location of the
non-GABAergic E12.5 neurons, 3) Calretinin-positive E12.5 cells did not distribute
similarly to the projection neurons with the same time of birth. These results suggestthat the birth-date dependent alignment of GABAergic neurons occurs in a different
pattern from that of non-GABAergic neurons in the mouse visual cortex. Further
analysis of how the termination of interneuron migration is regulated is essential tounderstanding cortical histogenesis.

61

Figures & Tables

References
Birth-date dependent alignment of GABAergic neurons occurs in a different pattern from that of
non-GABAergic neurons in the developing mouse visual cortex. Masato Yozu, Hidenori Tabata, andKazunori Nakajima. Neurosci. Res. 49 (4), 395-403 (2004).

621) Biological Sciences1-4） Developmental Biology

Development of the neural network and the blood vessels
Wataru ShojiInstitute of Development, Aging, and Cancer, Tohoku University
wshoji@idac.tohoku.ac.jp

Introduction
During early embryogenesis, development of nervous system and blood vessels suggest a variety ofinteraction. In some cases, they run together with close contact, whereas in other cases, they
develop exclusively.

By chemical mutagenesis for zebrafish and following F3 screening, we

obtained a mutant that exhibits defect in both nervous and blood vessel network.

Results
Zebrafish mutant mujina was initially identified with missing posterior lateral line ganglion at 3 dpf(days post-fertilization).

The lateral line nerve plays an important role in the escape reflex circuitby transmitting sensory information from the neuromasts on the surface of the fish larva. The
neuromasts are equipped with hair cells to detect water vibration that implies approaching predators.
Interestingly, development of the posterior lateral line nerve and neuromasts are intimately
associated (Figure).

They were born together as cranial ganglia just posterior to the otocyst, anddifferentiate as neurons and neuromast primordium.

When peripheral axons begin extension fromthe neurons of the ganglion, the neuromast primordium migrates with the growth cones, the leadingedge of the axons.

The primordium migrates from the head to the tail and periodically deposits

group of cells that give rise to the neuromasts.

In corresponding to the deposition, some axonscease extension to innervate the neuromasts, which ensures rigid association between axons and theirtargets.
In mujina mutant, the lateral line neurons are missing at 3 dpf.
process of the neurons and the primordium in the mutant.

We examined developmental

The course described above, from cranial

ganglia to deposition of the neuromast occurred normally.

The lateral line axons extended in anormal pattern until at least 1.5 dpf. Defect was seen first on 2 dpf when hair cells should
differentiate in the center of the neuromast.

In the mutant embryos, the lateral line neurons begin todegenerate and the hair cells do not appear in the neuromast.

We are now investigating relation

between the hair cell defect and the neuronal degeneration by cellular and molecular analysis.
In addition to the defect in the nervous system, we found that blood vessel system also decay in the

63mutant embryos.

The blood circulation starts on 1 dpf and appeared normal by 2 dpf. Subsequently

at 3 dpf, the mutant suffers with brain infarction by emborization at the primordial midbrain channel
and middle cerebral vein.

We are now investigating more closely for the blood vessel defects.

Thus, identification of mutated gene for mujina may elucidate new developmental process governing
both neural and blood vessel development.

Our intentional effort for positional cloning recently

picked up closest microsatellite marker within 0.4 cM.

Discussion & Summary
We characterized a new zebrafish mutant, mujina, which exhibits defects in neurons and bloodvessels.
In mujina embryos, lateral line neuron develops once normally but dies when its target hair cells
failed to differentiate.

In other vertebrates, it is known that peripheral sensory axon requires

trophic support from its target organs. Based upon this idea, we are hypothesizing and examining if
hair cells may support lateral line axons or vice versa.
are once established in normal, then decays later.
blood vessel development in the mutant.
this study.

Interestingly, blood vessels and circulation

This may imply a common defect in neurons andCloning of the responsible gene should be next step of64

References
Liu, Y., Berndt, J., Su, F., Tawarayama, H., Shoji, W., Kuwada, J. Y. and Halloran, M. C.,
Semaphorin3D guides retinal axons along the dorsal-ventral axis of the tectum., J Neurosci., 24
p310-318, 2004
Ober EA, Olofsson B, Makinen T, Jin SW, Shoji W, Koh GY, Alitalo K, Stainier DY., Vegfc isrequired for vascular development and endoderm morphogenesis in zebrafish., EMBO Rep. 1
p78-84, 2004
Yoshida, M., Inoue, T., Shoji, W., Ikawa, S., and Obinata, M., Reporter gene stimulation by MIDA1
through its DnaJ homology region, Biochem Biophys Res Commun., 324 p326-332, 2004
Wolman, M. A., Liu, Y., Tawarayama, H., Shoji, W., and Halloran, M. C. Repulsion and Attraction ofAxons by Sema3D are Mediated by Different Neuropilins in vivo, J Neurosci, 24 p8428-8435, 2004

651) Biological Sciences
1-5） Physioiogy

Involvement of Na,K-ATPase in Respiratory Rhythm Generation
during Perinatal Period.
Keiko Ikeda
Jichi Medical School, Center for Molecular Medicine, Division of .Biology
kikeda@jichi.ac.jp

Introduction
The sodium pump is the enzyme responsible for the maintenance of Na+ and K+ gradient across thecell membrane. Among four isoforms of the catalytic a subunits, α2 subunit gene (Atp1a2) is
specifically expressed in excitable tissues. Mice homozygous for the α2 mutation survive when they
are born under spontaneous labor, but died just after birth when they are born through cesarean
section. The aim of this research is to clarify why Atp1a2 knockout homozygotes survive when bornunder spontaneous labor, by comparing respiratory neural network activities between the wild-type
and the cesarean delivered neonatal mice.

Results
The α2 subunit is abundantly expressed in neurons in the brainstem, including respiratory center.
The neural network for respiratory rhythm is genetically programmed and developed in utero, andnewborn mammals breathe immediately after birth. This neural circuit is located rostrocaudally inthe ventrolateral medulla (VLM) of the lower brainstem. It produces rhythmic activity, which istransmitted to spinal motoneurons to generate a periodic contraction of respiratory muscles, such as
diaphragm. As a measure of respiratory output, we recorded the respiratory activity from the C4, oneof phrenic nerve motoneurons, in an en bloc brainstem-spinal cord preparation (figure 1) isolated
from E18.5-E19 mice through cesarean section. During the recording, the brainstem-spinal cord
preparation was superfused with oxygenated modified Krebs' solution at 26°C. Spontaneous
rhythmic discharges related to inspiration were recorded in the wild-type and heterozygous mice (n =5), but were completely absent in the homozygous mice (n = 3). We also recorded C4 in thebrainstem-spinal cord preparation from fetuses, which were born under spontaneous labor (within 9hours after birth). In this case, we observed C4 spontaneous discharges not only in the wild -type andheterozygous mice (n = 3), but also in the homozygous mice (n = 3). These results were consistent
with the observation that the homozygous mice spontaneously breathe under spontaneous labor, but
completely lacked breathing through cesarean section, leading their immediate death after birth.

66

It has been known that some neuromodulators are released in the fetal brain during passing throughthe mother’s birth canal. Therefore, we applied various neuromodulators to the brainstem-spinal cord
preparation by adding them in the superfusate. We found that adrenaline and substance-P induced
more regular and strong respiratory rhythmic discharges in the case of the wild-type/heterozygous
mice. These chemicals also induced regular inspiratory bursts in the homozygous mice. The results
strongly indicated that exposure of neuromodulators to the respiratory rhythm generator neurons
during spontaneous delivery are important (but not essential in the wild-type/heterozygous mice) forthe establishment and maturation of autonomous respiratory rhythm generation in the central
nervous system.
To obtain insights into the underlying defects in the homozygous rhythm generator neurons, we
performed optical recordings to visualize respiratory neural activity from the ventral surface using
voltage-sensitive dye (50 μg/ml Di-2-ANEPEQ; Molecular Probes, Eugene, OR) and the system of
MiCAM01 (Brain Vision Inc.)(Ref.1). We compared the optical signals during adrenaline application
between the wild-type and homozygous mice born through cesarean section. Strong fluorescencechanges at two regions of medulla (one is rhythm generator neuron complex and the other is patterngenerator neuron complex=preBoetinger. complex), that indicate electrical burst, were detected close
to the peak of C4 inspiratory activity. The fluorescence decreased (i.e., depolarization) in thewild-type (n = 3), heterozygous (n = 6), and homozygous (n = 5) mice in the pattern generator
neuron complex. We observed an increase in the fluorescence (i.e., hyperpolarization) in thewild-type and heterozygous rhythm generator neuron complex. In contrast, we observed a decreasein the fluorescence (i.e., depolarization) in the homozygous mice. These results suggest impairmentof the inhibitory neural activity in the ventral medulla and its transformation to excitatory activity inthe homozygous mice. To get insight into the molecular basis for the transformation in thehomozygous mice, we measured intracellular chloride ion concentration in the rhythm generator
neurons using gramicidin patch clamp techniques. We found that higher intracellular chloride ion
concentration in the homozygous mice neurons when born through cesarean section than in thewild-type mice.

Discussion & Summary
Using an en bloc brainstem-spinal cord preparation, we observed that Atp1a2 knockout homozygous
mice showed spontaneous respiratory rhythm neural activity when they are born under spontaneous
labor, but lacked through cesarean section. We found that adrenaline and substance-P, that are
released in the fetus brain during passing through the mother’s birth canal, induced regularinspiratory bursts in the homozygous preparation. We suggest that the neuromodulators such as
adrenaline and substance-P are important for the establishment and maturation of respiratory rhythm
activity during perinatal period. The impairment of the inhibitory neural activity in the ventral

67

medulla and its transformation to excitatory activity in the homozygotes was due to the higher
intracellular chloride ion concentration, resulting the respiratory defect in the homozygous mice
when born through cesarean section.

Figures & Tables

References
1. Ikeda K, Onimaru H, Kawakami K. Novel function of sodium pump. Tanpakushitsu Kakusan
Koso. 2005 May;50(5):434-439.
2. Ikeda K, Onimaru H, Yamada J, Inoue K, Ueno S, Onaka T, Toyoda H, Arata A, Ishikawa TO,
Taketo MM, Fukuda A, Kawakami K. Malfunction of respiratory-related neuronal activity in Na+,
K+-ATPase alpha2 subunit-deficient mice is attributable to abnormal Cl- homeostasis in brainstem
neurons. J Neurosci. 2004;24(47):10693-10701.

681) Biological Sciences
1-6） Pharmacology

Studies on a Role for PRIP in the Function of GABAA Receptor
Masato Hirata
Kyushu University, Faculty of Dental Scie
Hirata1@dent.kyushu-u.ac.jp

Introduction
PRIP (PLC-related inactive protein) was first identified as an inositol 1,4,5-trisphosphate binding
protein. An attempt to explore interacting molecule(s) with PRIP revealed GABARAP (GABA
receptor associated protein) and PP1 (protein phosphatsae-1), which prompted us to examine thepossible involvement of PRIP in GABA(A) receptor signaling at several aspects.

Results
Biochemical and Behavioral Analysis of PRIP Double Knock-Out Mouse:
Our recent studies revealed that a novel Ins(1,4,5)P3 binding protein, PRIP-1, regulates the assembly
and/or function of GABAA receptors as assessed by analyzing the PRIP-1 knockout (KO) mice.
There are two PRIP genes in mammals, thus the roles of PRIP-1 might partly be substituted by
PRIP-2 in the PRIP-1 KO mice. In the present study, we produced the PRIP-1 and -2 double
knockout (DKO) mice and examined the roles for PRIP molecules in regulating the assembly of
GABAA receptors. The expression of α, β, γ and d subunits of GABAA receptors in the brain of
DKO mice were slightly higher than those of wild type (WT) mice at both protein and gene levels,
as analyzed by western blots and semi-quantitative RT-PCR. Ligand binding assays using[3H]muscimol, a GABA agonist, and [3H]Ro15-1788, a diazepam antagonist, showed that the cellsurface expression levels of GABA binding site (α/β subunit interface) were increased in DKO, but
the numbers of diazepam binding site (α/γ2 subunit interface) were reduced in the DKO mice,compared to WT mice. Diazepam sensitivity whose target is α/γ2 subunit interface, was reduced in
DKO mice in electrophysiological and behavioral analysis. These findings indicate that PRIP are
involved in trafficking of GABAA receptors to cell surface membrane.
Possible Involvement of PRIP in the Regulation of β-subunit Phosphorylation of GABA(A)
Receptors:
We have further examined the role for PRIP-1, which binds and inactivates protein phosphatase 1α(PP1α), in facilitating GABAA receptor phospho-dependent regulation, using PRIP-1 knock-out(PRIP-1-/-) mice. In wild-type animals, robust phosphorylation and functional modulation of GABAA

69

receptors containing theβ3 subunits by cAMP-dependent protein kinase (PKA) was evident,

which was greatly diminished in PRIP-1-/- mice. PRIP-1-/- mice exhibited equivalent levels of PKA
activity compared to controls, and equivalent levels of PP1α at the protein level. However, the PP1αactivity in PRIP-1-/- mice was enhanced compared to controls. Inhibiting phosphatase activity in
PRIP-1-/- mice restored PKA mediated phosphorylation of GABAA receptor β3 subunit to levels seenin control animals. In addition, PRIP-1 was able to interact directly with GABAA receptor β subunits,
and moreover, these proteins were found to be PP1a substrates. Finally, PRIP-1 was phosphorylatedon threonine 94 and serine 96, and the phosphorylation of threonine 94 facilitated the dissociation of
PP1α/PRIP-1 complexes, providing a local mechanism for the activation PP1a. Together, theseresults suggest an essential role for PRIP-1 in controlling GABAA receptor activity, via regulating
subunit phosphorylation and thereby the efficacy of neuronal inhibition mediated by these receptors.

Discussion & Summary
All these results strongly indicate the involvement of PRIP in GABA(A) receptor signaling at many
aspects including the transcription and expression of several subunit components of the receptors,
the cell-surface expression of pentameric GABA(A) receptors, the phospho-dependent modulationof the receptors, and the internalization of the receptors once worked. Therefore, several
impairments regarding GABA(A) receptor function were observed in PRIP-DKO mice compared to
WT mice by electrophysiological and behavioral analysis. However, the molecular basis explaining
these impairments are largely unknown, which is our future projects

Figures & Tables

701) Biological Sciences
1-7） Plant Biology

Molecular Genetic Studies on Maternal Inheritance of Chloroplasts
Using a Model Plant System
Wataru Sakamoto
Research Institute for Bioresources, OkayamaUniversity
saka@rib.okayama-u.ac.jp

Introduction
Plastid and mitochondrion are the organelles that contain their own genomes. These “organelle
genomes” are different from the nuclear genome in that they are 1) multiplicated in plant cells and 2)
inherited from either parent. On fertilization, nuclear fusion takes place and one set of chromosomesfrom each parent is inherited in a zygote. However, it is not the case in chloroplasts andmitochondria: an organelle genome from either parent tends to be excluded, and the genome fromthe other parent is uniformally maintained. Recent survey implicated that about 20 % of flowering
plants inherits plastid genome paternally (i.e. from pollen), while 80% inherits it maternally (i.e.
through egg cell) (Zhang et al., 2003). This phenomenon, referred to as uniparental inheritance, hasbeen revealed by genetic and cytological analyses, but little is understood on its molecularmechanism in higher plants. This study is to establish the experimental system that could allow us to
study maternal inheritance at the molecular level.

Results
At the beginning of this research project, I proposed to conduct the following three experiments.
1) Transformation of the chloroplast genome by introducing a foreign gene (aadA) that confers
resistance to an antibiotics spectinomycin (spec)2) Spec resistance as a selection marker for investigating uniparental inheritance of plastid DNA
3) Isolation of a mutant in meternal inheritance in Arabidopsis
The first two projects concern the introduction of foreign genes into Arabidopsis chloroplasts inorder to give chloroplast a genetically scorable marker. So far, transformation of chloroplast
genomes has been reported only in limited plant species. We reasoned that the longer flanking region
included in the transformation construct may improve the transformation efficiency in Arabidopsis.
As an effort to design the DNA construct for chloroplast transformation, using the region between

71

rbcL and atpE, is still underway, we will test these construct for transformation in the future.
In the third project of screening mutants in maternal inheritance, we examined approximately
4,000 EMS-mutagenized M2 individual plants. We established a ‘pollen-squash’ method, by which
we were able to fix pollens and simultaneously stain DNA by fluorescent dye DAPI (Sodmergen etal., 2002). In the wild-type pollens, one relatively large vegetative nucleus and two tightly packed
sperm nuclei can be stained. In this method, however, DAPI stains corresponding to organelle DNAs
are not detectable, suggesting that organelle DNAs diminish during pollen development.
In our screening, we were able to isolate putative mutants in which organelle DNAs could be
detected by DAPI. So far, we isolated five mutants and classified into three categories. The firstcategory included #1411 and #2214, and these mutants showed substantial intensities of fluorescentgranules in the pollen cytoplasm (Figure 1). Although our initial genetic analysis (in progress)
indicates that it is heritable, appearance of this phenotype is not 100% and variable in individuals.
The mutant occasionally showed abnormal morphology in both vegetative and reproductive tissues.
The second category included #2084, in which the fluorescence is not as strong as #1411 but most of
the pollens show this phenotype (Figure 1). The third category included #2213 and #2041, in which
the DAPI fluorescence is similar to #1411, but the pollens looked immature and therefore we
considered that pollen development is defective in this category. Because DAPI stains can besometimes observed in the wild type at the younger stage of pollen development, it was assumed thatpollen development itself might be deficient.
Given that the mutations obtained by EMS mutagenesis are likely recessive ones, our genetic
screening suggests that a positive mechanism to eliminate organelle DNAs during pollen
development exists. Further characterization of the granule bodies detected by DAPI stain may benecessary to confirm the containment of organelle DNAs in these mutants. Our genetic analysis isunder progress to identify the genetic factors affecting maternal inheritance in higher plants.
In addition to the afore mentioned, we also isolated a mutant that has altered nucleoploidity. Thismutant, #1415, segregated normal tri-nuclear pollen grains and abnormal di-nuclear pollen grains to
1:1. Our genetic analysis showed that the di-nuclear phenotype was transmitted only maternally, andthe transmission rate was always 1:1. These results suggest that pollen mitosis II is impaired in#1415. Further characterization indicated that #1415 is an allele of duo mutant, which has been
recently identified by Dr. Twell’s group in U.K.

72

Figure1.
A r a b i d o p s i s m u t a n t s o f a l t e r e d o rg a n e l l e D N A c o n t a i n m e n t i n p o l l e n s . S h o w n
are the squashed pollens simultaneously fixed and stained by DAPI (seeResults).

Discussion & Summary
This work has been conducted in order to establish the experimental system that could allow us to
study maternal inheritance at the molecular level. To this end, we utilize the model plant Arabidopsis
thaliana to study and identify factors involved in maternal inheritance molecular genetically.
Previous studies suggest that amount of organelle DNAs in pollens decreases during pollen
development. Such diminish of organelle DNA could follow a regulatory mechanism that plays animportant role in maternal inheritance in higher plants. We reasoned that some mutants can beobtained by EMS mutagenesis, based on these assumptions. Screening of the possible mutants byour pollen squash method indeed led us to isolate some mutants in which the organelle DNA
containment was altered. Further characterization of these mutant, in terms of genetic analysis andsubsequent map-based cloning, may enable to understand maternal inheritance in more detail.

References
Zhang Q, Liu Y, Sodmergen (2003) Examination of the cytoplasmic DNA in male reproductive cells
to determine the potential for cytoplasmic inheritance in 295 angiosperm species. Plant Cell Physiol
44: 941-951.
Sodmergen, Zhang Q, Zhang YT, Sakamoto W, Kuroiwa T (2002) Reduction in amounts ofmitochondrial DNA in the sperm cells as a mechanism for maternal inheritance in Hordeum vulgare
Planta 216:235-244.

731) Biological Sciences

1-7） Plant Biology

Molecular genetic analysis of light signal transduction from the
N-terminal domain of phytochrome B
Tomonao Matsushita
Department of Botany, Graduate School of Science, Kyoto University
matsushita@physiol.bot.kyoto-u.ac.jp

Introduction
Phytochrome is a major informational photoreceptor in plants. The phytochrome molecule has
two domains: the N-terminal photosensory domain and the C-terminal domain which contains
signaling motifs such as a kinase domain. The latter has been widely believed to transduce the signal
to downstream components. Our recent study clearly demonstrated that the N-terminal domain, but
not the C-terminal domain, of Arabidopsis phytochrome B (phyB) transduces the signal (1, 2), which
forces us to reconsider our view on phytochrome. However, nothing has been known about themolecular mechanism of this phenomenon. In this study, we took a genetic screening strategy toaddress the following two points: 1) which amino acid residue within the N-terminal domain ofphyB is directly involved in signal transfer? and 2) what is the downstream signaling component
responsible for it?

Results & Discussion
In order to elucidate the molecular mechanism of the N-terminal domain signaling of phyB, wemutagenized transgenic Arabidopsis expressing the N-terminal domain of phyB and screened forlong hypocotyl mutants in red light. We have so far screened at least 1,000,000 M2 seedlings which
is derived from approximately 200,000 M1 plants. Then, we analyzed intragenic missense mutations
and extragenic mutations to identify the amino acid residues critical for signaling within phyB
N-terminal domain and novel downstream components mediating phyB signaling, respectively.

1) Analysis of intragenic missense mutations within the N-terminal domain of phyB
We identified 16 missense mutations within phyB N-terminal domain through the genetic screen
described above. Although these mutations distributed throughout the N-terminal domain of phyB,
some of them should affect spectral properties of phyB molecule instead of being directly involved
in signaling, because it has been established that the N-terminal domain of phytochrome is required
and sufficient for light perception. Therefore, we examined in detail the effects of these missense

74

mutations on the spectral properties of phyB N-terminal domain fragment. As a result, we identified
6 missense mutations that exhibited normal photochemical properties, which means these mutations
abolished signaling competence of phyB without affecting its spectral nature. Interestingly, they fellwithin a relatively small region with significant homology to PAS domain, suggesting that this site is
directly involved in phyB signal transduction. More experiments are needed to confirm thispossibility, and they are currently in progress.

2) Analysis of extragenic mutations
We isolated 16 extragenic mutant lines to date and designated them as pns (phyB N-terminal
domain signaling) mutants. We first examined their responses to various wavelengths of light, andthe results of the photophysiological study indicated that pns mutants can be classified roughly into
two categories: phyB pathway-specific ones that exhibit long hypocotyl only under red light, andphyB pathway-nonspecific ones with long hypocotyl also under other wavelengths of light than red
light. Since our interest focuses on phyB signal transduction, we decided to further analyze only theformer class of pns mutants. Then we performed rough mapping of three such pns mutants, and at
least one of them was mapped in a region not known to contain any other loci involved inphotomorphogenesis. We plan to continue rough mapping of more pns mutants and then to begin
map-based cloning of the responsible genes for those pns mutants which are more likely to represent
novel photomorphogenic loci.
We believe these two different types of analyses from inside and outside of the phyB molecule
will give us a clue to unravel the long-standing mystery of phytochrome signal transduction.

References
1. Matsushita T, Mochizuki N, Nagatani A (2003) Dimers of the N-terminal domain ofphytochrome B are functional in the nucleus. Nature 424, 571-574.
2. Oka Y, Matsushita T, Mochizuki N, Suzuki T, Tokutomi S, Nagatani A (2004) Functional
Analysis of a 450-Amino Acid N-Terminal Fragment of Phytochrome B in Arabidopsis. Plant Cell
16, 2104-2116.

752) Medical Sciences2-1) Immune System

Molecular Mechanisms of immune defenses against infection by IgM
Shibuya Akira
Department of Immunology, Institute of Basic Medical Sciences, University of Tsukuba
ashibuya@md.tsukuba.ac.jp

Introduction
IgM is the first antibody to be produced in a humoral immune response and plays
an important role in the primary stage of immunity.

In addition, IgM is a major

iostype of natural antibodies, which already exist in innate animal and are involved in
the prevention of pathogen dissemination to vital organs. However, how this “innatetype” of antibody are involved in immunity has not necessarily been elucidated.

Results
We previously identified an Fc receptor for IgA and IgM, designated, Fcα/µR which is a
~70 kDa monomeric protein containing a immunoglobulin-like domain in the extracellular region(Fig 1).

No tyrosines are present in the cytoplasmic domain, but there is a di-leucine motif in themouse Fcα/µR that may be involved in receptor internalization.

These genes were mapped tosyntenic regions of mouse chromosome 1 (1F) and human chromosome 1 (1q32.3), near several
other Fc receptors, including Fcγ receptors I, II, and III, Fcε receptor, and the polymeric Ig
receptor (Fig 1).
The Fcα/µR is constitutively expressedon the majority of B lymphocytes, including
follicular, marginal and B1 B cells.

The

Fcα/µR mediates endocytosis Staphylococcus
aureus /anti-S. aureus IgM antibody immune
complexes by primary B-lymphocytes.

We

have recently found that the Fcα/µR is also
expressed on follicular dendritic cells (FDC).
To analyze in vivo role of the Fcα/µR, we haverecently generated Fcα/µR -deficient andtransgenic mice.

These mice exhibited

normal level of antibody subclasses, except of

Fig 1

76

IgA that is higher than that in control wild type mice.
While immunization with T cell-dependent (TD) antigen NP-CG did not affect antibody
production, enhanced production of IgM and IgG3 antibodies against T-independent (TID) antigen
such as NP-Ficoll and NP-LPS was observed in Fcα/µR-/- mice.

Moreover, we also observed thatgerminal center formation was augmented in response to TID, but not TD, antigen in Fcα/µR-/mice.

On the other hand, the Fcα/µR transgenic mice produced significantly lower amount of

IgM and IgG3 than wild type mice in response to TID antigen.

Discussion & Summary
In the present study, we have demonstrated that Fcα/µR plays an important role for antibody
production against TID antigen, suggesting that Fcα/µR is involved in innate immunity.

However,it has remained undetermined how Fcα/µR mediates such an immune response. We have also shown
an interesting result that FDC substantially express Fcα/µR.

Because FDC plays an important role

for antigen retention and presentation to B cells in germinal center and involved in germinal center
formation and antibody production and maturation, Fcα/µR may regulate FDC function.
studies should be required to clarify the in vivo rele of the Fcα/µR.

Further

772) Medical Sciences2-1) Immune System

Physiological roles of the cannabinoid receptors
Takayuki Sugiura
Faculty of Pharmaceutical Sciences, Teikyo University
sugiurat@pharm.teikyo-u.ac.jp

Introduction
2-Arachidonoylglycerol (2-AG) is an endogenous cannabinoid receptor ligand. Evidence isaccumulating which shows that 2-AG is the true natural ligand for the cannabinoid receptors.
Two types of cannabinoid receptors have been identified to date. The CB1 receptor is abundantly
expressed in the brain and assumed to be involved in the attenuation of neurotransmission. On theother hand, the physiological roles of the CB2 receptor, mainly expressed in several types ofinflammatory cells and immunocompetent cells, have not yet been fully elucidated. In this study, weinvestigated possible pathophysiological roles of the CB2 receptor and 2-arachidonoylglycerol inacute inflammation in mouse ear.

Results
Precursors and the metabolic pathways for 2-AG.
In this study, we examined the effects of several metabolic inhibitors such aphosphatidylcholine-phospholipase C inhibitor, a diacylglycerol-lipase inhibitor, a phospholipase D
inhibitor, a phosphatidic acid phosphohydrolase inhibitor, phosphatidylinositol- phospholipase C
inhibitor

on

the

level

of

2-AG

in

inflamed

mouse

ear.

We

found

that

a

phosphatidylcholine-phospholipase C inhibitor, a diacylglycerol-lipase inhibitor, a phospholipase D
inhibitor and a phosphatidic acid phosphohydrolase inhibitor reduced both 2-AG formation and ear
swelling. These results suggest that phosphatidylcholine is an important precursor molecule for
2-AG in inflamed tissues and that phosphatidylcholine-phospholipase C, diacylglycerol lipase,
phospholipase D and phosphatidic acid phosphohydrolase are involved in the synthesis of 2-AG. We
also found that the generation of various species of 2-monoacylglycerols such as 2-oleoylglyceroland 2-linoleoylglycerol in addition to 2-AG were generated in TPA-treated mouse ear. Theseobservation support our hypothesis that 2-AG was generated from phosphatidylcholine. This is quite
different from the case of the nervous system where 2-AG is generated mainly from inositol
phospholipids and selective formation of 2-AG is observed.
Pathophysiological roles of 2-AG in inflammation.

78

We found that TPA-treatment of mouse ear induced the generation of 2-AG. The level of 2-AG
was elevated by 4-fold. We also found that SR144528, a CB2 receptor antagonist, markedly reduced
ear swelling induced by TPA. In contrast to SR144528, AM251, a CB1 receptor antagonist did notaffect ear swelling. We also found that the level of LTB4 in mouse ear was markedly elevatedfollowing the application of TPA. Notably, treatment of the ear with SR144528 blocked LTB4
production triggered by TPA. Treatment of the ear with TPA also induced the infiltration ofneutrophils. The TPA-induced infiltration of neutrophils was diminished by treatment of the ear with
SR144528. Interestingly, the application of 2-AG or 2-AG ether, s stable analog of 2-AG, to mouseear evoked ear swelling. On the other hand, anandamide, another endogenous cannabinoid receptor
ligand did not induce ear swelling. These results strongly suggest that 2-AG rather than anandamide
is crucially involved in acute inflammation induced by TPA. We also found that human eosinophils
express high levels of the CB2 receptor. Notably, 2-AG induced the migration of human eosinophis.
The migration evoked by 2-AG was abolished in the presence of SR144528, a CB2 receptor
antagonist, or by pretreatment of the cells with pertussis toxin, suggesting that the CB2 receptor andGi/o are involved in the 2-AG-induced migration. The migration of EoL-1 cells induced by 2-AG
was suggested to be due to chemotaxis. In contrast to 2-AG, neither anandamide nor free arachidonic
acid elicited the migration. These results suggest that the CB2 receptor and 2-AG are involved inallergic inflammation as well.
Effects of 2-AG on the vascular system.
We found that treatment of mouse ear with 2-AG induced the generation of nitric oxide. We also
found that treatment of mouse ear with nitric oxide synthase inhibitors such as L-NMMA and7-nitroindazole reduced ear swelling induced by 2-AG. L-NMMA and 7-nitroindazole also reduced
TPA-induced ear swelling. These results strongly suggest that ear swelling induced by 2-AG or TPA
is mainly due to the generation of nitric oxide. The mechanism by which 2-AG stimulated nitricoxide formation is not known. It is possible that p42/44 MAP kinase and p38 MAP kinase are
involved in the production of nitric oxide in 2-AG- and TPA-treated mouse ear, because we foundthat U0126, an inhibitor of MEK that is upstream of p42/44 MAP kinase, and SB239063, a p38
MAP kinase inhibitor, markedly suppressed 2-AG-induced ear swelling (93 % inhibition and 87 %inhibition, respectively) and TPA-induced ear swelling (57 % inhibition and 81 % inhibition,
respectively).

Discussion & Summary
The CB2 receptor is abundantly expressed in several types of inflammatory cells andimmunocompetent cells and has been assumed to play an essential role in inflammatory reactions
and immune responses. Nevertheless, its physiological function in vivo still remains an enigma.
Based on our recent experimental results, we assume that the physiological roles of 2-AG in

79

inflammatory reactions are as follows: Various types of inflammatory cells and immunocompetentcells, such as macrophages, generate 2-AG when these cells are stimulated, and release 2-AG into
the extracellular fluid. The released 2-AG then binds to the CB2 receptor expressed on otherinflammatory cells or immunocompetent cells, induces the augmented production of chemokines
such as IL-8 that induce the infiltration of neutrophils, and triggers direct migration of macrophages,
eosinophils and natural killer cells, thereby stimulating inflammatory reactions. On the other hand,∆9-THC and SR144528, CB2 receptor antagonists, interfere with the actions of the CB2 receptor andits physiological ligand 2-AG, thereby causing suppression of inflammatory reactions. Further
detailed studies on the CB2 receptor and 2-AG are necessary for a thorough elucidation of the
precise regulatory mechanisms of various inflammatory reactions.
Finally, 2-AG synthesis inhibitors and the CB2 receptor antagonists would be quite valuable asanti-inflammation drugs and immune suppressive agents.

References
1. Kishimoto, S., Gokoh, M., Oka, S., Muramatsu, M., Kajiwara, T., Waku, K. and Sugiura, T. (2003) J.
Biol. Chem. 278: 24469-24475.
2. Kishimoto, S., Kobayashi, Y., Oka, S., Gokoh, M., Waku, K. and Sugiura, T. (2004) J. Biochem. 135:
517-524.
3. Gokoh, M., Kishimoto, S., Oka, S., Yanagimoto, S., Ikeda, S., Waku, K. and Sugiura, T. (2005)
Biochem. J. 386: 583-589.
4.Kishimoto, S., Muramatsu, M., Gokoh, M., Oka, S., Waku, K. and Sugiura, T. (2005) J. Biochem. 137:
217 – 223.
5.Oka, S., Yanagimoto, S., Ikeda, S., Gokoh, M., Kishimoto, S., Waku, K., Ishima, Y. and Sugiura, T.
(2005) J. Biol. Chem. 280:18488-18497.

802) Medical Sciences2-1) Immune System

Regulation of Inflammation and Obesity by MAP kinases
Masato OGATA
Mie university graduate school of medicine
ogata@doc.medic.mie-u.ac.jp

Introduction
It is well demonstrated that inflammation plays a critical role in the development of atherosclerosis.
It is also reported that increased levels of CRP is a risk factor of Type 2 diabetes. These suggest thepresence of common molecular mechanisms that link inflammation and metabolic syndrome.
Mitogen-activated protein kinases (MAPKs) are critical components of signal transduction pathways.
They are involved in the regulation of various cytokine productions and signal transductions ofcytokine receptors. Cytokines and adipocytokines are regulators of inflammation and obesity. Thus,
it is possible that MAPK is a critical regulator that is involved both in inflammation and obesity.

Results
There are three major subgroups of MAPK, namely, ERK, p38 and JNK. p38 can regulate its
substrates, such as various transcription factors and kinases, through phosphorylation. p38 binds tosubstrates through the CD (common docking) site and the binding is very important for thephosphorylation and regulation of the substrates. We introduced mutations in the CD region of p38
and found that the sevenmaker (sem) type mutant of p38 (p38sem) failed to associate with several
substrates.
We have established p38sem knockin mice to study the physiological role of p38. It was reported that
p38-deficient mice were embryonic lethal due to the defect in the placental development. In contrast,
p38sem knockin mice are viable and fertile, demonstrating that the signaling pathway required for theplacental development is not affected by the p38sem mutation. Since p38sem can still associate withlimited number of substrates, activation of these substrates seems to be sufficient for the normal
placental development. Though the p38sem knockin mice looked apparently normal, we found a
profound defect in the production of TNF-a. When LPS was injected i.p. in the wild type mice,
serum TNF-a level was increased transiently while little increase was observed in the p38sem knockin
mice. Moreover, p38sem knockin mice were more resistant to LPS toxicity than wild type mice.
These results clearly demonstrate that p38 is important for the LPS-induced TNF-a production in
vivo. Furthermore, it is also shown that some signaling molecule(s) downstream of p38 is essential

81

for LPS-induced TNF-a production but not for the placental development.
Unexpectedly, the body weight of p38sem knockin mice was found to be lighter than that of wild typemice. p38 MAPK has been implicated in the differentiation of white adipose tissues and could be amodulator of the signaling cascade that promotes thermogenesis in brown adipose tissues. However,in vivo function of p38 remains to be determined since p38 knockout mice are embryonic lethal. We
thus used p38sem knockin mice to delineate a potential role of p38 in adipose tissue metabolism
and/or differentiation. As described above, p38sem knockin mice is a partial loss of function mutant
and they are viable. Interestingly, low body weight and the reduction in white adipose tissue weight
were observed in the p38sem knockin mice. In addition, the adipose tissue weight did not change verymuch with a high-fat diet. These results show that the p38 signaling pathway is involved in theregulation of adipose tissue weight probably through regulation of lipid metabolism and/or
differentiation of adipocytes.
These observations demonstrate that p38 is involved in inflammation and obesity, suggesting p38 isa key molecule that links inflammation and metabolic syndrome.

Discussion & Summary
We have developed mutant mice of p38 MAPK and found that p38sem mutation results in the strong
inhibition of proinflammatory cytokine productions as well as obesity. Thus, p38 could be involvedin the regulation of inflammation and obesity as a key-signaling molecule connecting these
conditions.
p38 is activated by LPS and cytokines such as IL-1, IL-6 and IL-10. p38 activation results in the
production of proinflammatory cytokines such as TNF-a. Thus, the role of p38 in the regulation of
inflammation is relatively well understood. In contrast, the mechanism that regulates obesity throughp38 remains to be clarified. TNF-a is produced by not only inflammatory cells but also adipocytes
and it induces insulin resistance. Thus, one possible mechanism could be regulation of soluble
factors by p38. Our study suggests that inhibitors of p38 cascade could be candidate drugs to treat or
prevent metabolic syndrome.

82

References
Takahiko Fujikawa, Yoshiko Mori, and Masato Ogata. Mutation of p38 MAPK in mice reduces
adipose tissue weight and protects from high fat diet-induced obesity. (manuscript in preparation)

832) Medical Sciences2-2) Central Nervous System

Analyses of molecular mechanisms of endoplasmic
reticulum-localized Bcl-xL to ER stress induced cell death
Toshiyuki Nakagawa
Gifu University Graduate School of Medicine, Department of Neurobiology
tnakagaw@cc.gifu-u.ac.jp

Introduction
In response to functional disturbance, the ER sends distinct signals to the nucleus and cytosol
resulting in the survival or demise of the cell. Such a response is indicative of ER stress, indicating
the fundamental mechanisms of several disease, such as stroke, Alzheimer’s disease, Diabetic
Mellitus.

Bcl-xL prevents apoptosis caused by proapoptotic Bcl-2 family proteins, such as Bax,

Bak, and Bid.
unknown.

However, the function of antiapoptotic Bcl-2 family proteins at ER remains to be

The objectives are to understand the molecular mechanisms and regulations of Bcl-xL

against ER stress induced cell death and develop its therapeutic intervention.

Results
In C. elegans, the Apaf-1-like cell death activator CED-4 translocation from mitochondria to nuclear
envelope may activate CED-3 procaspase to cause program cell death (Chen et al, 2000).

However,

it remains to be unknown the function at nuclear membrane and endoplasmic reticulum (ER) to
activate caspases in mammal.

To address this cascade, we examined the subcellular localization of

caspases by western blotting using subcellular fractionation of PC12 cells.

ER localizing proteins,

such as caspase-12 (Nakagawa et al, 2000), p28Bap31, and grp78 (BiP), were detected membrane
fractions.

Cytosol localizing caspase, such as caspase-1, was detected in S100 fraction, but not inmembranes fraction. Caspase-7 was detected in cytosolic fraction and membranes fraction.

Next,

We generated the stable PC12 cells constitutively expressed of mutant Bcl-xL. C-terminaltransmembrane domain of membrane bound cytochrome b5 was substituted for that of Bcl-xL,
which made Bcl-xL specifically translocate to ER (Figure).

Immunostain indicated that ER-Bcl-xL

specifically localized at ER, not mitochondria. To study the role of ER-Bcl-xL at ER during cell
death, ER-Bcl-xL and control retroviruses were infected PC12 cells, which were treated by various
drugs to induce cell death. The cell death of ER-Bcl-xL constitutively expressing PC12 cells werepartially inhibited after non-ER stress inducible reagents: etoposide, staurosporine as well as ER
stress treatment: thapsigargin, which was assayed by MTS.

To examine the regulation of caspases

84

activation by ER-Bcl-xL, we carried out the western blotting of several caspases (caspase-12, -7, and–3) using ER-Bcl-xL expressing PC12 cells after thapsigargin treatment.

2 mM of thapsigargin

was challenged into culture medium and incubated for six hours. After treatment, cells were
washed twice with fresh medium and cultured in normal medium without reagent. Caspase-12 and-3 were activated even in control and ER-Bcl-xL expressing PC12 cells, whereas, interestingly,
caspase-7 activation was specifically inhibited by the expression of ER-Bcl-xL.

ER stress

responses, such as, PERK phosphorylation (P-PERK), unfolded protein response (UPR) indicated by
grp78 (BiP) induction, were similarly detected in both cells in time dependent manner.

The

splicing product of Xbp-1 mRNA, which was carried out by active Ire1a, was not discriminated inboth PC12 cells by RT-PCR.

ER stress stimulates JNK through Ire1a-TRAF2-ASK1.

activity was detected in ER-Bcl-xL expressing PC12 cells.
did not have an effect on ER stress responses.

JNK

These data indicated that ER-Bcl-xL

Next, we investigated whether disease related

mutation of proteins, such as a-synuclain (Parkinson’s disease), SOD1 (Amyotrophic lateralschrelosis), and polyglutamine (polyQ78) (Huntington’s disease), induce caspase-7.
plasmids into 293T cells to examine caspases activation.

We transfected

Western blotting indicated that polyQ78

induced caspase-7 activation as well as caspase-8, but not caspase-3 in this experiment.

Since

polyQ induces ER stress through the dysfunction of ERAD-proteasome system, polyQ78 induced
caspase-7 activation may be achieved at ER.

To explore the mechanisms of caspase-7 activation,

we examined the caspase-7 cleavage in several mutant MEF.

Caspase-7 was cleaved in caspase-1

and caspase-12 knockout MEF cells after thapsigargin treatment like in wild type MEF cells.

In

contrast, caspase-7 activation was completely inhibited in bax;bak double knockout MEF cells.
These data implied that caspase-7 activation might be required for Bax and Bak at ER during
apoptosis.

Figure. The mutant Bcl-xL (ER-Bcl-xL), C-terminal transmembrane domain of membrane boundcytochrome b5 was substituted for that of Bcl-xL, resulting in translocation of Bcl-xL specifically to
ER. Western bloting showed the ER-Bcl-xL (ER) is slightly bigger than wild type Bcl-xL (W).
Immunostain by anti-FLAG antibody showed that ER-Bcl-xL is specifically localized at ER.

85

Discussion & Summary
The Bcl-2 protein plays an important role in apoptosis by antagonizing Bax and Bak function at
mitochondria and regulating initiator caspase activation independently of the apoptosome(cytochrome c/Apaf-1/caspase-9).

In this study, we showed that ER targeting BH3 proteins,polyglutamine (pQ), and ER stress caused caspase-7 activation, which was specifically inhibited by
Bcl-xL at ER.

These data indicated that the Bcl-2/Bcl-xL proteins function to regulate the releaseof proapoptotic molecules from mitochondria as well as the caspase-7 activation at ER by competingwith BH3 proapoptotic proteins.

References
Chen F., B.M. Hersh, B. Conradt, Z. Zhou, D. Riemer, Y. Gruenbaum, and H.R. Horvitz. 2000.
Translocation of C. elegans CED-4 to nuclear membranes during programmed cell death. Science
287:1485-1489
Nakagawa T., h. Zhu, N. Morishima, E. Li, J. Xu, B.A. Yankner, and J. Yuan. 2000.

Caspase-12

mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-b. Nature 403:
98-103

862) Medical Sciences2-2) Central Nervous System

Molecular mechanisms of inflamed pain hypersensitivity in spinal
cord neurons
Tatsuro Kohno
Division of Anesthesiology, Niigata University Graduate School of Medical and Dental Sciencekohno@med.niigata-u.ac.jp

Introduction
Bradykinin has long been established to be a key inflammatory mediator playing an integral role in
the pathophysiological processes that produce acute and chronic inflammatory pain. For decades,
most studies on the algesic (pain-promoting) effects of bradykinin have been focused principally on
the periphery. Besides its peripheral effects, bradykinin may also modulate pain processing in the
spinal cord. The major plan here is to decipher the role of bradykinin and its receptors in paintransmission in the spinal cord.

Results
We show that central sensitization, an activity-dependent increase in synaptic efficacy in the spinal
cord, requires bradykinin B2 receptor activation. B2 receptors are expressed in dorsal horn as well as
dorsal root ganglion neurons and B2 agonists substantially augment AMPA and NMDA
receptor-mediated currents in lamina II neurons of spinal dorsal horn, recorded in adult rat slices,
acting pre- and post-synaptically. Augmentation of these currents by bradykinin requires
co-activation of protein kinase C (PKC) and A (PKA), the latter downstream of cyclooxygenase-1(COX1) and extracellular signal-regulated kinase (ERK). Administration of bradykinin to the spinal
cord in vivo induces an NMDA- and ERK-dependent hyperalgesia, while both an intrathecal
B2-selective antagonist suppress behavioral manifestations of central sensitization. Bradykinin is asynaptic neuromodulator that potentiates glutamatergic synaptic transmission in the spinal cord by
activating multiple kinases, producing pain hypersensitivity.

Discussion & Summary
The major form of therapy targeted at acute central sensitization has been NMDA receptor
antagonists. These antagonists are effective for reducing central sensitization-mediated pain inlaboratory animals but their use in patients is severely limited by their psychotomimetic side effects.
A better target, based on our findings, may well be bradykinin as an initiator both of NMDA and87

AMPA use-dependent synaptic potentiation. The action of bradykinin upstream of multiple signaling
pathways that modify neuronal excitability in the peripheral nervous system to produce peripheral
sensitization, and in the central sensitization, indicate that preventing its production (using kallikreininhibitors) or action (with B2 receptor antagonists) may provide useful means for controlling acutepain hypersensitivity.

References
Kawasaki Y, Kohno T, Zhuang ZY, Brenner GJ, Wang H, Van Der Meer C, Befort K, Woolf CJ, Ji
RR.

Activation of ionotropic and metabotropic receptors, PKA, PKC, and Src contributes to

C-fiber-induced ERK activation in dorsal horn neurons leading to CREB phosphorylation andcentral sensitization. Journal of Neuroscience, 24, 8310-8321, 2004.
Kohno T, Moore KA, Baba H, Woolf CJ.

Peripheral nerve injury alters excitatory synaptictransmission in lamina II of the rat dorsal horn.

Journal of Physiology, 548:131-13, 2003.

882) Medical Sciences2-5) Cardiovascular /Metabolic/Endocrine

The role of IL-6/gp130 signaling and its negative regulator SOCS3 inviral myocarditis
Hideo Yasukawa
Cardiovascular Research Institute, Kurume University, Fukuoka Japanyahideo@med.kurume-u.ac.jp

Introduction
Enterovirus infection can cause acute myocarditis, a debilitating and potentially lethal disease in
adults and children, and has been implicated in the development of chronic dilated cardiomyopathy,
one of the main indications for cardiac transplantation. Interleukin-6 (IL-6) family cytokines
including leukemia inhibitory factor (LIF) and cardiotrophin1 (CT-1) activate JAK/STAT pathway.
IL-6 transduces signals through gp130, the common receptor component of IL-6 family cytokines.
IL-6 family cytokines have multi-functions in cells, however, little is known regarding the role ofIL-6 family cytokines in anti-viral defense in viral myocarditis.

Results
The purpose of this research project is to investigate the role of IL-6/gp130 signal and its negative
regulator SOCS3 in viral myocarditis. We generated cardiac-specific SOCS3 transgenic mice. These
mice and cardiac specific gp130 knockout mice were backcrossed into a susceptible, Balb/c, strain
and infected with coxsackievirus B3 (CVB3). JAK-STAT signaling and antiviral effects were
evaluated in isolated cardiac myocytes. Cardiac-specific SOCS3 expression resulted in a marked
increase in the percent of infected myocytes, virus titer (nearly 100 fold) and cardiac cell damage
from Hematoxylin-Eosin staining. There was also a loss of cardiac function by echocardiogram andearly mortality (80% vs. 5%) compared to wild-type, inbred littermates. In isolated adult cardiac
myocytes, SOCS3 expression had no effect on IFN-mediated JAK-STAT signaling or anti-viraleffects. However, gp130 signaling measured by cardiotrophin-1-stimulated STAT3 phosphorylation
was completely inhibited. Furthermore, CVB3 infection of cardiac-specific gp130 knockout miceincreased the percent of infected myocytes and cardiac cell damage (Figure). Interestingly, gp130
stimulation with cardiotrophin-1 has no effect on the viral replication in isolated myocytes, but
increases cardiac cell survival even with CVB3 infection. Adenoviral gene transfer ofdominant-negative STAT3 but not PI3-kinase inhibitor nor MEK1 inhibitor treatment abolished thecytoprotective effect of gp130 cytokines on CVB3 infected myocytes

89

Figurelegend
In vivo significance of cardiac gp130 signaling. Knockout efficiency of gp130 in the adultventricular myocytes from the knockout mice was confirmed by immunoblotting with anti-gp130
antibody. Evans blue dye uptake in the cardiac cells was significantly increased in gp130 KO on
the fifth day after the virus inoculation.

Discussion & Summary
1) There are important susceptibility factors within the cardiac myocyte that can be inhibited by
SOCS3 expression. 2) These factors are independent of IFN signaling. And 3) gp130 signaling could
account for a portion of the protective effect provided by JAK-STAT signaling. 4) STAT3-dependent
signaling pathway is essential for the antiviral effect of gp130 cytokines during CVB3 infection.
These data demonstrate that a cardiac myocytic innate immune response has a powerful effect on
viral infection in the cardiac myocyte, and gp130 signaling plays important roles in this innate
immune response during viral myocarditis.

References
1) Yasukawa H, Sasaki A, Yoshimura A: Negative regulation of the cytokine signaling pathways.
Annu Rev Immunol 18:143-164, 2000

2) Yasukawa H, Yajima T, Duplain H, Iwatate M, Kido M, Hoshijima M, Weitzman W, Nakamura T,
Woodard S, Xiong D, Yoshimura A, Chien KR, Knowlton K: The suppressors of cytokine
signaling-1 (SOCS1) is a novel therapeutic target for enterovirus-induced cardiac injury.
Invest 111:469-478, 2003

J Clin

902) Medical Sciences2-7) Infection

Role of lipid rafts on the infection of intracellular bacteria into
macrophages
Masahisa Watarai
Obihiro University of Agriculture and Veterinary Medicine
watarai@obihiro.ac.jp

Introduction
Brucella species are facultative intracellular pathogens that survive in a variety of cells, including
macrophages, and their virulence and chronic infections are thought to be due to their ability to
avoid the killing mechanisms within macrophages (1, 2).

The molecular mechanisms of theirvirulence and chronic infections are incompletely understood. Brucella internalizes into
macrophages by swimming on the cell surface and then the bacteria are enclosed bymacropinosomes.

Lipid

raft-associatedmacropinosomes containing B. abortus (3-6).

moleculesareselectively

incorporated

into

In this study, we investigated receptor for thebacteria on macrophage surface and roles of lipid rafts on signal transduction through the receptor.

Results1)SR-A promotes internalization and intracellular replication of Brucella.
To investigate the role of class A scavenger receptor (SR-A) in Brucella infection, several
phenotypes of B. abortus virulence were tested by using bone marrow-derived macrophages from
SR-A deficient mice.

Macrophages from parent mice supported internalization of the wild type B.

abortus strain, but macrophages from SR-A-deficient mice inhibited the internalization.
Macrophages from SR-A-deficient mice also showed inhibition of internalization of the virB4
mutant, suggesting that the type IV secretion system does not contribute to bacterial internalization
dependent on SR-A.

The wild type B. abortus strain replicated slightly in macrophages from

SR-A-deficient mice between 24 h and 48 h after infection, but the intracellular growth rate wasstill lower than those macrophages from parent mice.
2)Roles of LPS in the SR-A dependent internalization of Brucella.
LPS from smooth and rough B. abortus inhibited internalization of B. abortus, but not LPS from
E. coli.

Interestingly, LPS from Salmonella enterica serovar Typhimutium also inhibitedinternalization of B. abortus into macrophages, suggesting that soluble LPS from B. abortus and S.

91

enterica serovar Typhimurium was competing with LPS on the surface of B. abortus for receptors
on the macrophage plasma membrane. In addition to LPS, internalization of B. abortus wasinhibited by another SR-A ligand, polyinosinic acid, whereas a control reagent, polycytidylic acidthat does not block the SR-A receptor had no effect. Furthermore, 2F8, a monoclonal antibody
that specifically recognizes mouse SR-A and that blocks phagocytosis of apoptotic thymocytes
mediated by SR-A, inhibited internalization of B. abortus.

These inhibitors did not affectinternalization of opsonized bacteria.
3)Decreased proliferation of Brucella in SR-A deficient mice.
Many bacteria were recovered from the spleen of parent mice infected with the wild type B.
abortus strain at 10 and 20 days after infection, but markedly fewer bacteria were recovered from
SR-A-deficient mice, judged from the number of CFU in each spleen. These results indicated thatbacterial internalization mediated by SR-A contributed to B. abortus proliferation in mice.
4)Association between SR-A and lipid rafts during bacterial internalization
Our previous studies showed that lipid raft-associated molecules were selectively incorporated into
macropinosomes containing B. abortus to induce signal transduction into macrophages and to form
replicative phagosomes (3-6).

If SR-A has an important role in the signal transduction into hostcells, it may be contained in lipid rafts on the macrophage plasma membrane infected with B.
abortus. To ensure that SR-A localizes in lipid rafts, we prepared detergent-resistant membranes(DRMs) fraction from macrophages infected with wild type B. abortus strain or virB4 mutant.
Lipid rafts can be isolated as DRMs from cells.

To isolate DRMs from infected macrophages, we

extracted them in cold 1 % Triton X-100 lysis buffer, and analyzed them on sucrose gradients for
DRMs that float in light membrane fractions away from bulk cellular proteins.

SR-A wascontained in detergent soluble fractions isolated from the uninfected control or macrophages infectedwith the virB4 mutant and it was not detected in detergent insoluble fractions, which were fractions
1-3 of the gradient (15-30% sucrose).

In macrophages infected with the wild type strain, in contrast,

most SR-A was contained in detergent insoluble fractions.

These results suggest that SR-A shifted

into detergent insoluble fractions, lipid rafts, from detergent soluble fractions by bacterialinternalization dependent on the type IV secretion system and that SR-A may participate in thesignaling pathway in B. abortus infection into macrophages (7).

Discussion & Summary
Brucella abortus is a facultative intracellular bacterium that can survive inside macrophages (1).
Intracellular replication of B. abortus requires the VirB complex, which is highly similar to the

92

conjugative DNA transfer system (2).

In this study, we showed that a class A scavenger receptor(SR-A) of macrophages is required to internalize B. abortus and contributes to the establishment ofbacterial infection in mice.

Macrophages from SR-A-deficient mice inhibited internalization andintracellular replication of both wild type strain and the virB4 mutant, and that bacterial proliferation
was inhibited in SR-A-deficient mice.

Adding lipopolysaccharide from B. abortus and Salmonella

enterica serovar Typhimurium, but not from Escherichia coli, to macrophages inhibited bacterialinternalization.

VirB-dependent bacterial internalization induced localization of SR-A into

detergent-resistant membrane lipid rafts.

These results indicate that B. abortus internalizes into

macrophages by using SR-A as a receptor and that the VirB type IV secretion system of B. abortus
regulates signal transduction dependent on SR-A to form replicative phagosomes, and which ismediated by lipid rafts (7).

Figures & Tables

93

References
1. Watarai M, Makino S-I, Shirahata T. An essential virulence protein of Brucella abortus, VirB4,
requires an intact nucleoside triphosphate-binding domain. Microbiology 2002; 148: 1439-1446.
2. Kim S, Watarai M, Kondo Y, Erdenebaatar J, Makino S-I, Shirahata T. Isolation andcharacterization of mini-Tn5Km2 insertion mutants of Brucella abortus deficient ininternalization and intracellular growth in HeLa cells. Infect Immun 2003; 71: 3020-3027.
3. Watarai M, Makino S-I, Fujii Y, Okamoto K, Shirahata T. Modulation of Brucella-induced
macropinocytosis by lipid rafts mediates intracellular replication. Cell Microbiol 2002; 4:
341-356.
4. Watarai M, Makino S-I, Michikawa M, Yanagisawa K, Murakami S, Shirahata T. 2002.
Macrophage plasma membrane cholesterol contributes to Brucella abortus infection of mice.
Infect Immun 2002; 70: 4818-4825.
5. Watarai M, Kim S, Erdenebaatar J, Makino S-I, Horiuchi M, Shirahata T, Sakaguchi S, Katamine
S. Cellular prion protein promotes Brucella infection into macrophages. J Exp Med 2003; 198:
5-17.
6. Kim S, Watarai, M, Makino S, Shirahata T. Membrane sorting during swimming internalization of
Brucella is required for phagosome trafficking decisions. Microb Pathog 2002; 32: 1-13.
7. Kim S, Watarai M, Suzuki H, Makino S, Kodama, T, Shirahata T. Lipid raft microdomains
mediate class A scavenger receptor-dependent infection of Brucella abortus. Microb Pathog
2004; 37: 11-19.

942) Medical Sciences2-7) Infection

Identification of new disease genes and development of new genetherapy protocol for primary immunodeficiency
Yoshiyuki Minegishi
Tokyo Medical and Dental University
yminegishi.mbch@tmd.ac.jp

Introduction
Patients with genetic defects in B-cell development suffer from severe recurrent infections including
pneumonia and sepsis, resulting in poor prognosis in the absence of appropriate diagnosis andtreatment.

All of the known genetic defects that result in a failure of B-cell development involve

signaling through the pre-B-cell receptor. Most of the male patients having X-linked inheritancepossess mutations in the X chromosome-encoded cytoplasmic tyrosine kinase Btk. Some sporadic
patients having autosomal recessive inheritance possess defects in immunoglobulin m heavy chain,Igα, λ5 or B-cell linker protein gene. These findings demonstrated that the components of pre-BCR
and its downstream molecule are critical for normal human B cell development.

Results
Although we have identified 90% of genetic origins of human B cell deficiency, genetic basis of
10 % of the patients are still unknown.

To determine the genetic origin of these patients, we

decided to screen genomic DNA samples from the patients without mutations in Btk, µ heavy chain,Igα, λ5, or BLNK using SSCP analysis. To determine if mutations in Bam32 or LAB could give riseto human B cell immunodeficiency, we designed to amplify individual exons of these two genes by
PCR for analysis by single-strand conformation polymorphism (SSCP). Genomic DNA samples
were analyzed from 25 patients with a Btk-deficient phenotype, in whom we had not identified
mutations in Btk, µ heavy chain, Igα, Igβ, BLNK or the surrogate light chain.
The representative results of SSCP were shown in Figure 1. Left panel showed the SSCP result ofexon 2 of LAB gene. Patient 9 demonstrated altered migration pattern in this analysis, indicating thepresence of heterozygous sequence alteration in this patient (C: control). Right panel showed thenormal pattern of SSCP, indicating all the patients and a control revealed identical migration pattern,indicating no sequence alteration in all the individuals evaluated. We also identified homozygous
sequence alteration on exon 6 of Bam32. The sequence analysis revealed that the patients had
homozygous sequence alteration at the –3 splice acceptor site. We also sequence entire Bam32

95

cDNA in eight patients and found the insertion of111 bp aberrant intronic sequences between exon 7
and exon 8, which resulted in truncation of the PH domain of Bam32.

The third alteration was a

ATC to ATG substitution, occurred at the third base-pair position in codon 39 of LAB gene, which
resulted in missense mutation from isoleucine to methionine.

These three alterations were locatedat the site, which is important for normal function of the genes and were not present in 100 normal
individuals.
In order to evaluate the functional consequences of the expression of this mutant, we expressed
I39M mutant of LAB protein using pMX-IRES-GFP retroviral vector into B cell line WEHI-231(Figure 2). WEHI-231 can induce apoptosis by B cell receptor cross-linking. Under our experimental
condition, approximately 20 % of WEHI-231 cells expressed GFP. By staining with apoptosis
marker annexin V and 7AAD, we evaluated the apoptosis of WEHI-231 in GFP negative control
population and GFP positive population separately. Anti-µ treatment increased the apoptotic cells
from 0.6% to 6.4 % in GFP negative cells. In GFP positive fraction the increase is more drastic, from
0.4% to 13%, suggesting the expression of the mutant protein might have negative effect for thesurvival of B cells in the patient.

These studies suggested the presence of new genetic origin ofhuman B cell deficiency and should help to diagnose early in childhood for prompt treatment of thepatients.

Figure 1

96

Figure 2

Discussion & Summary
The identification of genes that cause human agammaglobulinemia has both clinical and biologic
implications. Early diagnosis and therapy is essential to prevent morbidity due to the recurrent
pulmonary infections in childhood. Further, as improved therapies for immunodeficiencies becomes
available, it should be critical to know the exact nature of the genetic defect. Mutations in thespecific genes provide new intellectual framework to the normal development of the human B-celllineage. The findings in our patients support the hypothesis that signaling from pre-BCR and BCR isessential for the progression of B-cell differentiation beyond the pre-B-cell stage.

References
1. Yel, L., Minegishi, Y., Coustan-Smith, E., Buckley, R. H., Trubel, H., Pachman, L. M.,
Kitchingman, G. R., Campana, D., Rohrer, J., Conley, M. E. Mutations in the mu heavy-chain gene inpatients with agammaglobulinemia. New Engl. J. Med. 335, 1486-1493, 1996
2. Minegishi, Y., Coustan-Smith, E., Wang, Y.H., Cooper, M.D., Campana, D., Conley, M.E.
Mutations in the human λ5/14.1 gene result in B cell deficiency and agammaglobulinemia. J. Exp.
Med. 187, 71-77, 1998
3. Conley, M.E., Mathias, D., Treadaway, J., Minegishi, Y., Rohrer, J. Mutations in Btk in patients

97with presumed X-linked agammaglobulinemia. Am. J. Hum. Genet. 62, 1034-1043, 1998
4. Minegishi, Y., Hendershot, L. M., Conley, M. E. Novel mechanisms control the folding andassembly of λ5/14.1 and VpreB to produce an intact surrogate light chain. Proc. Natl. Acad. Sci.
U.S.A. 96. 3041-3046. 1999.
5. Minegishi, Y., Coustan-Smith, E., Rapalus, L. Ersoy, F., Campana, D., Conley, M.E. Mutations in
Igα (CD79a) result in a complete block in B-cell development. J. Clin. Invest. 104, 1115-1121, 1999
6. Minegishi, Y., Rohrer, J., Coustan-Smith, E.,

Lederman, H.M., Pappu, R., Campana, D., Chan,

A.C., Conley, M.E. An essential role for BLNK in human B cell development. Science, 286,
1954-1957, 1999
7. Minegishi, Y., Rohrer, J., Conley, M.E. Defects in B cell development-Recent progress in diagnosis
and therapy. Curr. Opin. Pediatr. 11, 528-532, 1999
8. Minegishi, Y., Conley, M.E. Negative selection at the pre-BCR checkpoint elicited by human µheavy chain with unusual CDR3 regions. Immunity 14, 631-641, 2001
9. Tomizawa D, Imai K, Ito S, Kajiwara M, Minegishi Y, Nagasawa M, Morio T, Nonoyama S,
Mizutani S. Allogeneic hematopoietic stem cell transplantation for seven children with X-linked
hyper-IgM syndrome: a single center experience. Am. J. Hematol. 76, 33-39, 2004
10. Chen L, Morio T, Minegishi Y, Nakada S, Nagasawa M, Komatsu K, Chessa L, Villa A, Lecis D,
Delia D, Mizutani S. Ataxia-telangiectasia-mutated dependent phosphorylation of Artemis in response
to DNA damage. Cancer Sci. 2005 96, 134-141
11. Kawano Y, Yoshikawa S, Minegishi Y, Karasuyama H. Selection of stereotyped
VH81X-{micro}H chains via pre-B cell receptor early in ontogeny and their conservation in adults
by marginal zone B cells. Int Immunol. 2005

(in press)

982) Medical Sciences2-8) Oncology

Molecular cloning of the gene for mitotic spindle checkpoint
deficiency
Shinya MATSUURA
Research Institute for Radiation Biology and Medicine, Hiroshima University
shinya@hiroshima-u.ac.jp

Introduction
Cancer-prone syndrome of premature chromatid separation with mosaic variegated aneuploidy(PCS syndrome) is a rare autosomal recessive disorder characterized by growth retardation,
microcephaly, childhood cancer, premature chromatid separation of all chromosomes and mosaicism
for various trisomies and monosomies.

Biallelic BUB1B mutations were recently reported in fiveof eight families with MVA syndrome (which we assume is identical with PCS syndrome).

Wecarried out molecular and functional analysis of BUB1B (encoding BubR1) in seven Japanesefamilies with PCS syndrome.

Results
BUB1B mutations were found in all seven families studied: a single base deletion (1833delT) in
four families; and a splice site mutation (IVS10-5A>G), a nonsense mutation (670C>T), and a
missense mutation (107G>A) in one family each. Transcripts derived from the patients with the1833delTand

IVS10-5A>G

mutations

weresignificantly

reduced,possiblydue

tononsense-mediated mRNA decay, and hence these alleles were effectively null. No mutation was
found in the second allele in each of the five patients studied, including the full coding regions,intro-exon boundaries, the promoter region 5-kb upstream of the translation initiation site, and the3’-untranslated region.

cDNA sequencing indicated that the second allele expressed intact mRNAtranscripts.
We analyzed the seven families for microsatellite marker 26020GT situated 54-kb upstream of thegene; SNP 1046G/A at exon 8 of BUB1B; and microsatellite marker D15S994, 69 kb downstream of
the gene.

The 26020GT-1046G/A-D15S994 haplotype of 2A7 was linked to the 1833delT mutationin three of the four families analyzed, a finding that suggested a common origin of the mutation.
The haplotype in the second allele in which no mutation was detected was 6G3 in five of the sevenfamilies.
Western blotting analysis of BubR1 expression was performed on immortalized fibroblast cell

99

lines using anti-BubR1 polyclonal antibody.

Consistent with the mutation analysis, BubR1 bandsof normal size but reduced intensities were detected in these cell lines.

We also found that theexpression of BubR1 from the 6G3 haplotype was moderately reduced.
We examined the effect of colcemid treatment on the mitotic index of immortalized fibroblast
cells.

Control cells showed an increase of mitotic index with a peak at 24 hr, whereas the cells

from the patients showed abnormal response with no clear peak in mitotic index with an increasing
accumulation of micronucleated cells.

Introduction through microcell-mediated chromosometransfer of human chromosome 15 containing the BUB1B locus into the cells restored theaccumulation of mitotic cells and abolished the accumulation of micronucleated cells.
Immortalized fibroblast cells were analyzed for mitotic checkpoint proteins, with HeLa cells
serving as a normal control.

In the patients’ cells, normal levels of Mad2, Bub1, and Bub3 proteins

were detected with western blotting analysis, and Mad2, Bub1, Bub3, and CENP-B, -C, -E, and –F
proteins localized to the kinetochores efficiently.
Western blot analysis of the patients’ cells demonstrated reduced expression of BubR1 but normal
levels of p55cdc protein (data not shown).

Kinetochore signals of BubR1 were faint, andkinetochore p55cdc signals were markedly reduced.

Transfer of chromosome 15 (containing

BUB1B) into the patients’ cells restored kinetochore association of both BubR1 and p55cdc.

These

results suggest that in these patients reduced expression of BubR1 caused abolished kinetochore
localization of 55cdc and abnormal checkpoint signaling.

Discussion & Summary
BUB1B mutations were found in all seven families studied: a single base deletion (1833delT) in
four families; and a splice site mutation, a nonsense mutation, and a missense mutation in one family
each.

Transcripts derived from the patients with the 1833delT mutation and the splice site mutation

were significantly reduced due to nonsense-mediated mRNA decay.

No mutation was found in thesecond alleles in the seven families studied, but RT-PCR, western blot, and haplotype analysis of
BubR1 indicated a modest decrease of their transcripts.

BubR1 showed both reduced protein

expression and diminished kinetochore localization in cells from two patients.

The expression levelof p55cdc, a specific activator of anaphase-promoting complex, was normal but its kinetochoreassociation was abolished. Microcell-mediated transfer of chromosome 15 (containing BUB1B)
into the cells restored normal BubR1 levels, kinetochore localization of p55cdc, and the normal
responses to colcemid treatment.

These findings emphasize the importance of BubR1 in

p55cdc-mediated mitotic checkpoint signaling, and indicate that PCS syndrome results from >50%
decrease in expression (or activity) of BubR1.

100

Figures & Tables

101

References
Kajii, T., Ikeuchi, T., Yang, Z.Q., Nakamura, Y., Tsuji, Y., Yokomori, K., Kawamura, M., Fukuda, S.,
Horita, S. and Asamoto, A. (2001) Cancer-prone syndrome of mosaic variegated aneuploidy andtotal premature chromatid separation: report of five infants. Am J Med Genet, 104, 57-64.
Kajii, T., Kawai, T., Takumi, T., Misu, H., Mabuchi, O., Takahashi, Y., Tachino, M., Nihei, F. andIkeuchi, T. (1998) Mosaic variegated aneuploidy with multiple congenital abnormalities:
homozygosity for total premature chromatid separation trait. Am J Med Genet, 78, 245-9.
Matsuura, S., Ito, E., Tauchi, H., Komatsu, K., Ikeuchi, T. and Kajii, T. (2000) Chromosomal
instability syndrome of total premature chromatid separation with mosaic variegated aneuploidy is
defective in mitotic-spindle checkpoint. Am J Hum Genet, 67, 483-6.
Hanks, S., Coleman, K., Reid, S., Plaja, A., Firth, H., Fitzpatrick, D., Kidd, A., Mehes, K., Nash, R.,
Robin, N. et al. (2004) Constitutional aneuploidy and cancer predisposition caused by biallelicmutations in BUB1B. Nat Genet, 36, 1159-61.
Matsuura, S., Matsumoto, Y., Morishima, K., Izumi, H., Matsumoto, H., Ito, E., Tsutsui, K.,
Kobayashi, J., Tauchi, H., Komatsu, K., et al.

BUB1B mutations and defective mitotic-spindle

checkpoint in seven Japanese families with premature chromatid separation (PCS) syndrome (MVA
syndrome).

Submitted.

1022) Medical Sciences2-8) Oncology

Biochemical and molecular mechanisms of drug resistance ofgastrointestinal stromal tumors against Imanitib.
Toshirou Nishida
Osaka University Graduate School of Medicine
toshin@surg1.med.osaka-u.ac.jp

Introduction
Gastrointestinal Stromal Tumor (GIST) has been demonstrated to be caused by gain-of-function
mutations in the KIT or PDGFRalpha gene. Mutations in the KIT or PDGFRalpha gene are found in
regulatory domains (Exon 9 and 11 for KIT and Exon 12 for PDGFRalpha) and kinase domains(Exon 13 and 17 for KIT and Exon 18 for PDGFRalpha). Downstream kinases including
RAS-GAP-MAP kinases, PI3K- AKT kinases also play an important role in progression andneoplastic proliferation of GIST. Imatinib mesylate, a selective inhibitor for KIT and PDGFRalpha,
are active for advanced GISTs. However, primary or secondary resistance is going to appear after
long time-use of imatinib. The purpose of this project is to elucidate molecular mechanisms of
secondary resistance to imatinib. In this project, we have evaluated the target gene mutations andactivation of its downstream kinases in resistance clones.

Results
In GISTs, we and others have shown that mutations in the kinase domains of the KIT orPDGFRalpha gene are involved in the primary resistance for imatinib. GIST without any mutationsin the KIT and PDGFRalpha genes also shows primary resistance to the drug.

In CML, The

mechanisms of acquired drug resistance of GIST are shown to be newly generated target genemutation in the activation loop. Similar mechanisms are postulated to be in secondary resistance of
GIST. In the present study, we have examined newly developed KIT and PDGFRalpha mutations,over-expression of KIT and PDGFRalpha, and mutations in downstream kinases in 9 secondary
resistant GISTs.
All resistant GISTs are viable, express strongly KIT protein in the immunohistochemical
examinations, and also show activation of downstream kinases including RAS-GAP-MAP kinases
and PI3K- AKT kinases. Full sequencing of the KIT and PDGFRalpha genes revealed that threeresistance GISTs have newly generated mutations in the kinase domains of the KIT gene. The new
mutations are occurred in the same allele to the primary mutation. Kinetic analysis indicated that

103

these newly generated KIT mutations showed resistance to imatinib. Three GISTs with primary KITgene mutations have no additional mutation in the KIT and PDGFRalpha genes.
We have also investigated primary resistance mechanisms of GISTs without any mutations in
the KIT and PDGFRalpha genes for imatinib. At present , we observed that these GISTs also express
phosphorylated KIT protein, that downstream kinases of the KIT protein are activated, and that the
KIT protein in these GISTs are smaller in the molecular weight than the wild type and mutated KIT
protein. Now, we are investigating activation mechanisms of these KIT proteins using proteome
analysis and differential display methods.

Discussion & Summary
In summary, we have examined resistance mechanisms of GISTs which showed secondary resistanceto imatinib. Although we have now analyzed the limited number of patients with resistant GISTs,
almost half of GISTs show newly generated c-kit gene mutations in the kinase domain. Although theother GISTs show strong expression of activated KIT protein and its downstream kinases, precise
mechanisms of KIT protein activation under imatinib are still investigated.

Figures & Tables
Table 1. Analyzed cases
PrimarylesionImatinibBestprimary

response

c-kit

resistanceexon11(delCase 1stomach

600

PR

Alive

Case 2intestine

600

PR

Alive

c-kit exon11 (Lys558Asn)

same to the primary

Case 3duodenum

400

PR

Alive

c-kit exon11(Del:559-574 )same to the primary

Case 4stomach

600

PR

Alive

c-kit exon11(Del:557-558

c-kit exon17 (c-kit Trp 823

TrpLys)Asp)

Case 5intestine

400

SD

Alive

c-kit exon11(Del:557-558

c-kit exon17 (c-kit Asp 816

TrpLys)

His)

Case 6

duodenum

600

PR

Alive

c-kit exon11(Del:560-572)

Case 7stomach

400

SD

Death

wild type KIT

wild type KIT

Case 8intestine

400

PR

Alive

c-kit exon11 (Val560Asp)

same to the primary

Case 9stomach

600

PR

Alive

Unknown at present

Unknown at present

codon557-558)

Unknown at present

c-kit

exon17

Ala829-Pro )(c-kit104

References
1. Ohashi A, Kinoshita K, Isozaki K, Nishida T, Shinomura Y, Kitamura Y, Hirota S: Different
inhibitory effect of Imatinib on phosphorylation of MAPK and Akt, and cell proliferation in cells
expressing different types of mutant PDGF receptor alpha. Int J Cancer. 2004:111:317-321.
2. Kinoshita K, Hirota S, Isozaki K, Ohashi A, Nishida T, Kitamura Y, Shinomura Y, Matsuzawa Y.
Absence of c-kit gene mutations in gastrointestinal stromal tumours of neurofibromatosis type I
patients. J Pathol 2004; 202:80-85.
3. Hirota S, Ohashi A, Nishida T, Isozaki K, Kinoshita K, Shinomura Y, Kitamura Y.
Gain-of-function mutations of platelet-derived growth factor receptor alpha gene in gastrointestinal
stromal tumors. Gastroenterology 2003; 125:660-667.
4. Wakai T, Kanda T, Hirota S, et al.: Late resistance to imatinib therapy in a metastatic
gastrointestinal stromal tumour is associated with a second KIT mutation. Br J Cancer
2004;90:2059-2061.
5. Chen L.L., Trent J.C., Wu E.F., et al.: A missense mutation in KIT kinase domain 1 correlateswith imatinib resistance in gastrointestinal stromal tumors. Cancer Res. 2004;64:5913-5919.
6. Wardelmann E, Thomas N, Merkelbach-Bruse S, Pauls K, Speidel N, Buttner R, Bihl H, Leutner
CC, Heinicke T, Hohenberger P. Acquired resistance to imatinib in gastrointestinal stromal tumours
caused by multiple KIT mutations. Lancet Oncol. 2005;6:249-251.
7. Debiec-Rychter M, Cools J, Dumez H, Sciot R, Stul M, Mentens N, Vranckx H, Wasag B, Prenen
H, Roesel J, Hagemeijer A, Van Oosterom A, Marynen P. Mechanisms of resistance to imatinib
mesylate in gastrointestinal stromal tumors and activity of the PKC412 inhibitor against
imatinib-resistant mutants. Gastroenterology. 2005 Feb;128:270-9.

1052) Medical Sciences2-12） Hematology

A role for transcription factors in hematopoiesis and leukemogenesis
MINEO KUROKAWA
Department of Hematology and Oncology, Graduate School of Medicine, University of Tokyo
kurokawa-tky@umin.ac.jp

Introduction
Embryonic development of multilineage hematopoiesis requires the precisely regulated expressionof lineage-specific transcription factors including AML1/Runx1 (also CBFA2 or PEBP2αB).

In

vitro studies and findings in human diseases including leukemias, myelodysplastic syndromes(MDS) and familial platelet disorder with predisposition to acute myelogenous leukemia alsosuggest it plays a pivotal role in adult hematopoiesis. However, this role in vivo has not been fully
disclosed due to the embryonic lethality of AML1-targeted mice.

Results
We assessed the requirement of AML1 in adult hematopoiesis by an inducible gene targeting method.
Using the Cre-loxP sequence-specific recombination system, we generated mutant mice in which
exon 5 of the AML1 gene can be selectively deleted by the expression of Cre recombinase. We
bred the mutant mice with Mx-cre transgenic mice to generate mice, in which deletion of AML1
allele could be evoked in hematopoietic progenitors by inducible expression of Cre reconbinase by
the injection of polyinosinic-polycytidylic acid (pIpC). Injection of pIpC did not cause significant
differences in neutrophil counts or hemoglobin levels between AML1-deficient and control mice.
However, platelet counts for AML1-deficient mice declined to one third to one sixth of those for thecontrol mice, immediately after pIpC administration. Lymphocyte counts were slightly depressed
after more than four weeks in AML1-deficient mice. These results suggest that AML1 is required
for the maintenance of platelets and lymphocytes, but not for the sustained production oferythrocytes and neutrophils.

AML1-deficient bone marrow revealed the absence of normal

megakaryocytes with abundant cytoplasm and lobulated nuclei.

Instead, it contained a small

number of immature megakaryocyte-like cells with occasionally separated round nuclei resemblingabnormal “micromegakaryocytes” observed in human MDS.

Given the prominent immaturity ofmegakaryocytes in AML1-deficient mice, we examined the ultrastructure of the megakaryocytes.
In addition to the smaller size, AML1-deficient megakaryocytes showed poorly developed
demarcation membranes compared to those of the control.

During the maturation process,106

megakaryocytes acquire high DNA ploidy (>4 n) through a cell cycle process known as endomitosis.
As expected from the smaller size of nuclei, CD41+ megakaryocytes from AML1-deficient mice
showed a remarkably lower polyploidy level (≤8 n) than those from the control mice (modal ploidy,
16 n) as revealed by the flow cytometric measurement of DNA content. This demonstrates that
AML-1 deletion also causes defective polyploidization in megakaryocytes.

Subsequently, we

performed in vitro colony forming assays to evaluate the frequency of hematopoietic progenitors.
AML1-deficient bone marrow cells formed more megakaryocytic colonies (CFU-Meg) than control
bone marrow cells in semisolid media.

The numbers of myeloid and mixed (myeloid anderythroid) colonies were also elevated in AML1-deficient bone marrow.

By single colony PCR

genotyping, AML1‐/– genotype was detected in 149 of 150 myeloid colonies, 48 of 50 erythroid
colonies, 35 of 36 mixed colonies and 14 of 14 megakaryocytic colonies from pIpC-treated
AML1-deficient bone marrow cells, thus excluding the possibility that those hematopoietic cells
might be predominantly derived from progenitor cells still expressing intact AML1.

Using flow

cytometry, we observed an increased cell number in the Lin− c-Kithi CD41hi fraction, which waspresumed to contain CFU-Meg, in AML1-deficient bone marrow.

Similarly, The number of cells

defined by CD34− Lin−/lo c-Kithi Sca-1hi, which represent the most immature hematopoietic
progenitor cell population was elevated in AML1-deficient bone marrow.

Since immature

hematopoietic progenitor cell fractions were elevated in AML1-deficient bone marrow, we assessed
their ability to reconstitute adult hematopoiesis using a competitive repopulation assay.

We

employed isotypes of the pan-hematopoietic marker CD45 (Ly5) to distinguish the origins of therepopulating cells.

Sublethally irradiated Ly5.1 recipient mice (Ly5.1+Ly5.2−) were intravenously

injected with a mixture of bone marrow cells from Ly5.1–Ly5.2 competitor mice (Ly5.1+Ly5.2+) andAML1-deficient or control mice (Ly5.1−Ly5.2+) previously injected with pIpC. Subsequently, we
analyzed Ly5 isotypes on the repopulating cells by flow cytometry.
+

Notably, the floxed bone+marrow cells reconstituted neither peripheral T (Thy-1.2 ) nor B (B220 ) cells, while there were no
significant differences in the mature neutrophil (Gr-1hi Mac-1+) and monocyte populations (Gr-1lo
Mac-1+).

Discussion & Summary
In the absence of AML1, hematopoietic progenitors are fully maintained with normal myeloid cell
development.

However, AML1 deficient bone marrow showed megakaryocytic maturation block,increased hematopoietic progenitor cells and defective T and B lymphocyte development.

AML1

is thus required for the maturation of megakaryocytes and differentiation of T and B cells, but not forhematopoietic stem cell maintenance in adult hematopoiesis.

Our present data also demonstratethat AML-1 is essential for the terminal differentiation of hematopoietic progenitors ofmegakaryocytic and lymphocytic lineages, which establishes AML-1 as a multi-role regulator in the107

maintenance of lineage-committed cells in adult hematopoiesis.

However, our results also suggestthat the maintenance of hematopoietic stem cells and their commitment to more mature progenitors
in adult hematopoiesis does not always require a transcription factor essential for hematopoietic
ontogeny during embryogenesis, and can be induced by other hematopoietic genes.

Figures & Tables

Megakaryocytic maturation arrest of AML1-deficient mice. In contrast to control mice, small anddysplastic megakaryocytes were increased in AML1-deficient mice (Floxed). Bone marrow (BM)sections stained with hematoxyllin and eosin (H-E), cytocentrifuged specimens of the BM cells
stained with Wright-Giemsa (W-G), and cytochemically stained for acetylcholinesterase (AchE).
BM was collected from mice 2 months after pIpC injection. Arrows : megakaryocytes, bars : 50
µm (H-E, W-G, and AchE-right) ; 250 µm (AchE-left).

AML1-deficient BM cells show maturation block in T and B cells. BM cells from AML1-deficient(floxed) or control (“test”) mice (Ly-5.1-5.2+) 1 – 3 months after pIpC injection were injected
simultaneously with “competitor” Ly-5.1+5.2+ BM cells, and the contribution to peripheral blood
cells was analysed by FACS. AMl1-dificient BM cells contribute to granulocytes and monocytes,
but not to B cell or T cells. Data are shown as mean (AML1-deficient : squares, control : closed
circles) + S.D. (error bars) of Ly-5.1−Ly-5.2+ objective cell number / Ly-5.1+Ly-5.2+ competitive cellnumber ratios of each point. * : P < 0.05, ** : P < 0.01 (Wilcoxon rank sums test).

108

References
Ichikawa M, Asai T, Saito T, Yamamoto G, Seo S, Yamazaki I, Yamagata T, Mitani K, Chiba S,
Ogawa S, Kurokawa M, and Hirai H. AML-1 is required for megakaryocytic maturation andlymphocytic differentiation, but not for maintenance of hematopoietic stem cells in adulthematopoiesis.

Nature Medicine 10: 299-304, 2004.

1093) Chemical Sciences3-1) Organic Chemistry

Development of Stereoselective Synthetic method of Procyanidin
Oligomers and Their Activities
Noriyuki Nakajima
Biotechnology Research Center, Toyama Prefectural University
nori@pu-toyama.ac.jp

Introduction
Proanthocyanidins are known as condensed tannins and/or oligomeric flavonoids. The
investigation of proanthocyanidins is now increasingly important because of their various strong
bioactivities. However, the structure-activity relationship of proanthocyanidin oligomers has notbeen proved yet, because a large number of isomers in the plants make it very difficult to purify andsupply each pure compound for assay. So we have undertook a stereoselective synthesis ofprocyanidin oligomers, and further investigated their bioactivities.

Results
We applied the intermolecular TMSOTf-catalyzed condensation to the synthesis of procyanidin
trimers

andgalloyl-substitutedprocyanidin

dimers.

A stereoselective condensationof(2R,3R,4S)-5,7,3’4’-tetra-O-benzyl-4-(2’’-ethoxyethyloxy)flavan (epicatechin electrophile) withfour dimeric nucleophiles in CH2Cl2 at –40°C to –20°C gave the corresponding four trimers in
excellentyields(Table1,entries

1-4).

On(2R,3S,4S)-5,7,3’4’-tetra-O-benzyl-4-(2’’-ethoxyethyloxy)flavan

the

other

(catechin

hand,

when

electrophile)wascondensed with four dimeric nucleophiles in CH2Cl2 at –78°C, only procyanidin C2 (catechintrimer) was obtained in excellent yield (entry 5). The condensation yields of other two trimers were
only in 47% and 44 % yields yield (entries 7 and 8) and catechin-catechin-epicatechin trimer was
not obtained yield (entry 9). The structure of condensed product was confirmed by comparing the1

H-NMR spectra of products, that was synthesized by two different condensation approaches(entries 5 and 6). Finally, deprotection of (+)-catechin and (-)-epicatechin trimers derivatives gave
seven natural procyanidin trimers in good yields.1 Twelve galloyl-substituted procyanidin B1, B2,2
B33 and B44 3-O-gallate, 3''-O-gallate and 3,3''-di-O-gallate, were also synthesized under ourcondensation conditions using TMSOTf as a catalyst.
Their bioactivities, antioxidant activities on UV-induced lipid peroxide formation using the TBA
method, DPPH radical scavenging activity and inhibitory activity of DNA polymerases, of all110synthesized compounds were investigated (Fig 1).

Figures & Tables
Table 1Synthetic studies of procyanidin trimers
Method 1

Method 2

OBn
BnO

A-unit

O
OR1

OBn

OBn
BnO

electrophile

OBn

OBn
OR2

OBn

OBn
BnO

electrophile

B-unitO

O

OBn

nucleophile
C-unitnucleophile

OR3
OBn

Temp,

Yield,

°C

%

1

-20

70

1

-20

97

Epicatechin - Catechin

1

-40

100

Epicatechin

Epicatechin - Epicatechin

1

-40

91

Catechin

Catechin - Catechin

1

-78

100

Catechin

2

-78

44

Entry

A-unit1

Epicatechin

Catechin - Catechin2

Epicatechin

Catechin - Epicatechin

3

Epicatechin

4
5
6

B-unitCatechin -Catechin

C-unit

Method7

Catechin

Epicatechin - Catechin

1

-78

47

8

Catechin

Epicatechin - Epicatechin

1

-78

44

9

Catechin

Catechin - Epicatechin

1

-78

0

111

OH
HO

O

HO

OH
OR

OH
HO

OH
O
OR

OH

O

OH

OH
HO

OH

O

OR'
OH

R=R'=H; 1.3 µM (25.4 µM)
R=galloyl, R'=H; 0.8 µM (0.72 µM)
R=H, R'=galloyl; 0.7 µM (0.89 µM)
R=R'=galloyl; 0.6 µM (0.23 µM)

R=R'=H; 1.2 µM (24.0 µM)
R=galloyl, R'=H; 0.8 µM (0.70 µM)
R=H, R'=galloyl; 0.6 µM (0.85 µM)
R=R'=galloyl; 0.6 µM (0.24 µM)

OH
O

OH
HO

OH
OR

OH
HO

OH
OR'

OH

HO

OH

O

O
OR

OH
OH
HO

OH

OR'
OH
R=R'=H; 1.3 µM (36.0 µM)
R=galloyl, R'=H; 3.2 µM (0.26 µM)
R=H, R'=galloyl; 1.1 µM (0.96 µM)
R=R'=galloyl; 1.1 µM (0.12 µM)

OH

O

OH
OH

OR'
OH
R=R'=H; 2.0 µM (24.6 µM)
R=galloyl, R'=H; 0.9 µM (0.66 µM)
R=H, R'=galloyl; 0.8 µM (0. 70 µM)
R=R'=galloyl; 0.9 µM (0.09 µM)

Fig. 1 IC 50 Values of DPPH radical scavenging activity and enzyme inhibition
against mammalian DNA polymerase α in parenthesis for procyanidin dimers.

Discussion & Summary
The TMSOTf-catalyzed intermolecular condensation method was effective for the synthesis of
3,4-trans procyanidin trimers. Seven procyanidin trimers and twelve galloyl-substituted procyanidin
dimers were systematically synthesized. From the results of the activity tests, the galloyl group of isvery important for activities. 3,3"-Di-O-gallate dimers acted as strong inhibitor against DNA
polymerase α and β, whereas the desgalloyl and monogalloyl compounds did not exhibit any
appreciable inhibitory activity against the DNA polymerase β.

References
1. Saito A.; Tanaka, A.; Ubukata, M.;Nakajima, N. Synlett, 2004,1069-1073.
2. Saito A.; Mizushina, Y.; Ikawa, H.; Yoshida, H.; Doi, Y.; Tanaka, A.; Nakajima, N., Bioorg. Med.

Chem., 2005, 13, 2759-2771.
3. Saito A.; Emoto M.; Tanaka, A.; Doi, Y.; Shoji, K.; Mizushina, Y.; Ikawa, H.; Yoshida, H.;

Matsuura, N.;

Nakajima, N. Tetrahedron, 2004, 60, 12043-12049.

4. Sakuda, H.; Saito, A.; Mizushina, Y.; Ikawa, H.; Yoshida, H.; Tanaka, A.; Nakajima, N.,

Heterocycles, 2006, 67, submitted.

1123) Chemical Sciences3-1) Organic Chemistry

Synthetic studies on insect anifeedants
Shigefumi Kuwahara
Graduate School of Agricultural Science, Tohoku University
skuwahar@biochem.tohoku.ac.jp

Introduction
The use of antifeedants as eco-friendly agrochemicals to reduce crop damage by insect pests has
recently been attracting increasing attention among academic circles as well as manufacturers ofagrochemicals. Among antifeedants so far reported, which are produced by plants and prevent insect
pests from eating the plants, azadirachtin (1), and clavigerins A and B (2 and 3, respectively) are
known for their extremely potent antifeedant activity. In this report, our studies directed toward thetotal synthesis of these substances are described.

Results
As a basic synthetic strategy, we envisaged installing the highly sterically congested C8–C14 bondof 1 by dissecting the C ring of pseudo-steroidal compound 4. Along this basic plan, enone 6,
obtained readily from the Wieland-Miescher ketone, was converted into 7 through oxidation andacetylation. The enone (7) was subjected to Diels-Alder reaction conditions with cyclopentadiene in
the presence of methyl aluminum dichloride, affording quantitatively the corresponding adduct,
which was then reduced to give 8. Epoxidation of 8 with mCPBA brought about cyclization of theresulting epoxy alcohol to furnish an pentacyclic alcohol, which was then oxidized to give 9.
Reductive cleavage of the a-C–O bond of 9 gave 10, which could be convertible to an azadirachtin
model compound (12), although lacking the A-ring of azadirachtin, via oxidative cleavage of 11.
For the synthesis of clavigerins A and B, compound 16, obtained from benzyloxyacetaldehyde in 2
steps, was exposed to the Carroll rearrangement conditions to give 17, which was converted intoacid chloride 18 by a three-step sequence including a Wittig reaction. Heating of 18 in xylene in thepresence of tributylamine afforded the desired bicyclo[3.1.1]heptane derivative (19) via a [2 + 2]
cycloadditon reaction of a ketene intermediate. This bicyclic compound is considered to beconvertible to 1 and 2 via alkylation of the ester with an equivalent of the side chain.

113

Discussion & Summary
Based on a proposed biosynthetic pathway for azadirachtin, a new strategy for its total synthesis,featuring the construction of its C8–C14 moiety by cleaving the C-ring of a fused polycyclic
intermediate, was examined. Starting from the Wieland-Mischer ketone, a substrate for the cleavage
reaction was prepared. Since the cleavage proceeded smoothly with some analogous compounds,
application of the conditions to intermediate 11 would produce the corresponding C-ring cleaved
intermediate, which would probably lead to our initial goal, an azadirachtin model compound
lacking only its A-ring portion.

References
For a recent synthetic study of azadirachtin, see: K. C. Nicolaou et al., Angew. Chem. Int. Ed., 44,3447 (2005).
For structural determination of clavigerins, see: N. B. Perry et al., Tetrahedron Lett., 44,
1651 (2005).

1143) Chemical Sciences3-1) Organic Chemistry

Global identification of Akt substrates by a novel phosphoproteome
analysis.
Seisuke Hattori, Dsc.
Institute of Medical Scinece, The University of Tokyo
hattoris@ims.u-tokyo.ac.jp

Introduction
Akt is a serine/threonine kinase the abnormal regulation of which is implicated in the
carcinogenesis of several types ofcancer. Akt is also a major signal transducer for insulin signaling

pathways. Therefore, it is important to globally identify Akt substrates and characterize theirfunctions. We addressed this issue by a novel phosphoproteome strategy.

Results
We established conditions to purify phosphoproteins by immobilized metal affinity
chromatography (IMAC) by optimizing pH and ionic strength. Under the established conditions, thepurity of phosphoryated Akt substrates increased about ten-folds by the chromatography.
Next, we analyzed the phosphoprotein profiles from Akt-activated (NIH3T3 cells treated withPDGF) and Akt-suppressed cells (NIH3T3 cells treated with PDGF in the presence of wortmannin,
an inhibitor for PI3-kinase, an upstream activator for Akt).

The proteins were labeled with Cy5-

(Akt-activated cells) and Cy3-derivatives (Akt-suppressed cells), and the combined sample wasanalyzed on 2-D differential fluorescence gel electrophoresis (Fig.1). Proteins specific to
Akt-activated cells give red spots, while proteins specific to Akt-suppressed cells give green spots.
Proteins with similar amounts between two samples give yellow spots (as an overlap of red andgreen). As shown in the figure, multiple red or orange spots were observed, among the many spots
being yellow. Since, phosphoprotein-enriched fraction was used as a sample for this analysis, theresult suggests that these spots are Akt substrates. Currently, we are trying to identify the proteins inthese spots and characterized the function of these proteins.

Discussion & Summary
This system could be applied to other kinases if the activation conditions would be established and if
the specific inhibitor would be available. Therefore, the system may be very useful to analyze thesignaling pathways of a given kinase.

115

Summary:
We established the conditions to purify phosphoproteins from total cell lysate by using immobilized
metal affinity chromatography. The phosphoproteins

were analyzed by 2-D fluorescencedifferential gel electrophoresis where multiple spots unique to Akt-activated cells were identified.

References
1. Ueda, K., Kosako, H., Fukui, Y., and Hattori, S. Proteomic identification of Bcl2-associatedathanogene 2 as a novel MAPK-activated protein kinase 2 substrate. J. Biol. Chem. 279,
41815-41821, 2004.
2. Tsuruta, F., et al. JNK promotes Bax translocation to mitochondria throughphosphorylation of 14-3-3 proteins.

EMBO J. 23, 1889-1899, 2004.

1163) Chemical Sciences3-1) Organic Chemistry

Development and Application of Chiral Ligand based on N-Ar Chiral
Mimetic
Kazuhiro Kondo
Graduate School of Pharmaceutical Sciences, Nagoya City University
kazuk@phar.nagoya-cu.ac.jp

Introduction
We designed the ligand 1 on the following concept: the ligand 1 may have the two diasteromers
due to the N-Ar axis and the chiral carbon in solution.

But, if the N-Ar axis in 1 isconfigurationally flexible, and the complexation of 1 and a metal is largely reflected by theasymmetric center in 1, one of the two diastereomer complexes due to the N-Ar axis, is expected to
be selectively formed (2a), leading to the creation of favorable asymmetric environment.

Results
We synthesized the ligand 1a,b.
carbon, were not observed.

The diastereomers of 1a,b due to the N-Ar axis and the chiralWith the ligands 1a, we first concentrated on a palladium-catalyzed

asymmetric allylic alkylation, using the allylic acetate 3 as the substrate and dimethyl malonate as
the nucleophile.

The ligand 1a was found to be effective (98% yield and 99% ee).

NMR studies on the complex 2a.
one isomer complex.

1

The H-and31

We carried out

P-NMR spectra of the complex 2a showed only

Since it is important to develop versatile chiral ligands, which afford goodenantioselectivity in a variety of reactions, we next were interested in the asymmetric Grignard
cross-coupling reaction of 1-phenylethylmagnesium chloride with alkenyl halides.

One type of

substrate with which it has been more difficult to achieve high selectivity is E-bromostyrene
derivatives. We performed in the asymmetric cross-coupling reaction with the ligand 1b, and1-phenylethylmagnesium chloride (4), affording enantioselectivity of up to 80%.

The product 5

was transformed into lipoxygenase inhibitor 6.
Allylic Alkylation
OAc

X

N

PPh 2

1a: X= NPh
1b: X= NPh
3
Grignard Cross-CouplingX
Stable Chiral
N-Ar Axis

N

X

Pd
PPh 2

+ Br
N

Pd
PPh2

M = metal

Stable complex 2a

CH2 (CO2Me)2
Pd-1a (cat)Unstable complex 2 b

Ph

MgCl

MeO2C

CO2Me

Ph

Ph 99% ee

Pd-1b (cat)Ph *Ar
5: up to 80% ee

Ar

4
Ph *
6

OH

117

Discussion & Summary
We have shown that an N-Ar axially chiral mimetic-type ligand 1a,b are efficient in the standard
asymmetric allylic alkylation and the asymmetric Grignsrd cross-coupling reaction of
1-phenylethylmagnesium chloride with E-bromostyrene derivatives.

Our ligand 1a can beconsidered to be one of the best ligands for the standard asymmetric allylic alkylation described in
the literature to date, and our ligand 1b are, to the best of our knowledge, the third bestenantioselectivity in the reported literature for the asymmetric Grignsrd cross-coupling reaction of
1-phenylethylmagnesium chloride with E-bromostyrene derivatives.

Further application to othercatalytic asymmetric reactions is now in progress.

References
1) Kondo, K.; Kazuta, K.; Fujita, H.; Sakamoto, Y.; Murakami, Y. Tetrahedron 2002, 58, 5209.
2) Horibe, H.; Kazuta, K.; Kotoku, M.; Kondo, K.; Okuno, H.; Murakami, Y.; Aoyama, T. Synlett
2003, 2047.
3) Horibe, H.; Fukuda, Y.; Kondo, K.; Okuno, H.; Murakami, Y.; Aoyama, T. Tetrahedron 2004, 60,
10701.
4) Horibe, H.; Kondo, K.; Okuno, H.; Aoyama, T. Synthesis 2004, 986-988.

1183) Chemical Sciences3-1) Organic Chemistry

Synthetic Studies on Marine Cytotoxic Halogen-Containing
Oxasqualenoids
Yoshiki Morimoto
Graduate School of Science, Osaka City University
morimoto@sci.osaka-cu.ac.jp

Introduction
The plane structure and partial stereochemistry of a bromotriterpene polyether (+)-aurilol (1), a
member of marine cytotoxic halogen-containing oxasqualenoids, were mainly elucidated by NMR
methods; however, determination of the entire stereochemistry has not been reached.1 Although there
have also been many other types of oxasqualenoids, it is often difficult to determine theirstereostructures even by the current highly advanced spectroscopic methods, especially in acyclic
systems

includingquaternary carbon centerssuch as C10–C11, C14–C15,
and C18–C19 in 1. The
purpose of this research is
the total assignment of theincomplete

stereostructureof 1 by chemical synthesis(Chart 1).

Results
In the course of our structural studies on oxasqualenoids, which could biogenetically be derived by
epoxide-opening cyclizations of squalene polyepoxides, we predicted the unknown stereochemistry
to be erythro configuration as shown in 2.
The synthesis of target molecule 2 began with SEM protection of the hydroxy group in the known
optically active epoxy alcohol 3 (98% ee) (Scheme 1). Selective deprotection of the TBDMS ether in
4 and Sharpless’ epoxidation of the allylic alcohol 5 using L-(+)-DET afforded diepoxide 6. The C

119

ring formation from 6 under the alkaline condition stereoselectively proceeded via a regioselective
5-exo-tet epoxide opening of the intermediate A to provide triol 7 in 88% yield. The following
sequence, (1) mesylation; (2) epoxidation; (3) chain extension with the C10 unit 8; and (4)desulfurization, uneventfully yielded diol 9 from 7. Shi’s epoxidation of 9 catalyzed by chiral ketone
10 diastereoselectively gave labile bishomoepoxy alcohol 11. Treatment of 11 with a protic acidunderwent a regioselective 5-exo-tet cyclization to produce diol 12 in 98% yield.
Deprotection of the SEM ether in 12 afforded triol 13, from which diene 15 was derived by the same
sequence as that of 7 to 9 except for employing sulfide 14. Shi’s epoxidation of the diene 15 with
ent-10 proceeded in a regioselective manner to provide monoepoxide 16 with the terminal alkene
intact. The construction of the desired B ring was regioselectively carried out by treating thebishomoepoxy alcohol 16 with TIPSOTf and 2,6-lutidine in CH3NO2 at 0 °C for 20 min.
After removal of the TIPS group in 17, cross metathesis of olefin 18 with 2-methyl-2-butene using
Grubbs’ catalyst 19 produced alkene 20 in 90% yield. The regio- and stereoselective 7-endo-trig
bromoetherification of 20 was brought about under the optimal condition to give the A,B-ringsystem 21, wherein removal of the acetonide, finally furnished the target molecule 2. The spectral
characteristics (1H and 13C NMR) of the synthetic 2 were identical to those reported for the natural
product. Thus, it has been found that the hitherto unknown relative configuration of (+)-aurilol (1) iserythro as indicated in 2.

120

Discussion & Summary
The unprecedented 6-endo-tet cyclization promoted by a silyl triflate against Baldwin’s rule would
be very stimulating because Brønsted acid-catalyzed cyclizations for these types of bishomoepoxy
alcohols, such as 11, generally afford 5-exo-tet regioselectivity, regardless of the relative
configurations of the tertiary alcohol-epoxide substrates. It has been found that (CF3)2CHOH withhigh polarity and non-nucleophilicity is the solvent of choice to successfully form the A ring.
In conclusion, we have accomplished the first asymmetric total synthesis of a cytotoxic
bromotriterpene polyether (+)-aurilol (4.74% overall yield in 21 steps from 3) featuring the highly
regio- and stereocontrolled biogenetic-like A–D ether ring formations. The total synthesis hasrealized the total assignment of the incomplete stereostructure of aurilol (1), which is difficult todetermine the stereochemistry otherwise.2

References(1) Suenaga, K.; Shibata, T.; Takada, N.; Kigoshi, H.; Yamada, K. J. Nat. Prod. 1998, 61, 515–518.
(2) Morimoto, Y.; Nishikawa, Y.; Takaishi, M. J. Am. Chem. Soc. 2005, 127, 5806–5807.

1213) Chemical Sciences3-1) Organic Chemistry

Studies on the structure and the chemical synthesis of marine toxin
Azaspiracid
Masato Oikawa
Graduate School of Life Sciences, Tohoku University
mao@bios.tohoku.ac.jp

Introduction
Azaspiracids are family of polyether toxins isolated first from Irish mussels as causative organic
compounds for Azaspiracid poisoning prevailed in Europe [1].

The novel structure as well as its

unique biological activity, the mode of which is supposed to be much different from those for otherknown marine polyether toxins, prompted us to develop studies on the chemical structure and thesynthesis.

Results
Though we have already successfully achieved the chemical synthesis of the FGHI-ring domain of
Azaspiracid [2], the domain can not be used for further homologation of the E-ring moiety toward
the total synthesis because of the lack of a suitable functional group.

In addition, we have noticed

in the previous work that the Boc group used for protection of C40-amino group was not robust
enough under even mild acidic conditions used for construction of the FGHI-ring domain.

To dealwith these issues we have studied the possibility for Alloc and Ns protecting groups for the aminogroup.
The precursors 4 and 5 were independently synthesized by the modified manner used before [2], andsubmitted to cyclization reaction using Yb(OTf)3 in CH2Cl2.

It was found that natural-type andunnatural-type aminal diastereomers 6 and 7 were selectively obtained from 4 and 5, respectively inreasonable yields.

In both cases undesirable decomposition of these protecting groups was notobserved.
The synthesis of the E-ring moiety was also studied.

In the present study, we have developed a new

strategy using intramolecular cyclization starting from α-halosulfide 9 by the successive treatment
with NCS and then with AgOTf.
but with high stereoselectivity.

By this reaction, the thiopyranoside 10 was obtained in low yield

122

Discussion & Summary
The development of high-yield and stereoselective synthesis of the FGHI domain is essential for the
total synthesis of Azaspiracid-1.

In the present study, we have explored amenable protecting

groups for C40-amino functionality stable under conditions for the construction of the FG-ringmoiety.

In the event, we have found Ns and Alloc protecting groups were suitable for this purpose

and, quite interestingly, provided unnatural and natural diastereomers, respectively, stereoselectively
in good yield.

The efficient method for construction of the E-ring moiety was also developed andthe conditions are now being under optimization to improve the yield.
Our efforts are continued to synthesize the EFGHI-ring moiety toward the total synthesis of
Azaspiracid on the basis of the knowledge found in these studies.

Figures & Tables
fragment 1(C1-C12)
HOA

B
O 10 OH

6

1

fragment 2(C13-C20)

12

O

O

H

19 H
D
OH
O 20

C

Me 14

H

HO

azaspiracid-1
40

Me

NH
I

35

H

H

O 28

H GO
F
O
Me H

O

21

Me

E

25

Me
30 Me

fragment 3(C21-C40)

X

X

HN
MeO
I

Me

H

OR

H GOR
F
O
H
Me

OR

N

Yb(OTf)3Me
CH2Cl2Me

4 (X = Alloc, R = Bn)

H

OR OR
H GOR
F
O
Me
Me H

I 36

(36S)-6: 45% desired(from 4)(36R)-7: 89% undesired (from 5)

5 (X = Ns, R = Ac)
AgOTf
OH
Me

SPh
Me

NCS
CCl4

SPh

2,6-di-tert-butyl
methylpyridine

X

Me

MS4A, CH2Cl2

8 (X = H)

25% (2 steps)

9 (X = Cl)

O
Me

Me
E
Me

10

123

References[1] M. Satake, K. Ofuji, H. Naoki, K. J. James, A. Furrey, T. McMahon, J. Silke, and T. Yasumoto, J.

Am. Chem. Soc., 120, 9967-9968 (1998).
[2] M. Sasaki, Y. Iwamuro, J. Nemoto and M. Oikawa, Tetrahedron Lett., 44, 6199-6201 (2003).

1243) Chemical Sciences3-1) Organic Chemistry

Synthesis of optically active a-aryl carboxylic acids by catalytic
asymmetric protonation
Akira YANAGISAWA
Chiba University
ayanagi@scichem.s.chiba-u.ac.jp

Introduction
Asymmetric protonation of enolates is one of the most effective methods for preparing optically
active α-substituted carbonyl compounds. Main objective of this research is to develop a truly
practical catalytic asymmetric protonation of prochiral metal enolates or silyl enolates.

Especially,

we focus on enolates derived from α-aryl carboxylic acids, some of which are known to havebiological activity, and aim to develop chiral proton sources or chiral catalysts, which can provide
both enantiomers of α-aryl carboxylic acids with high optical purity.

Results
BINAP·AgF is an efficient chiral catalyst for asymmetric aldol reaction of silyl enolates1 and also forasymmetric allylation of aldehydes with allylsilane.2 Because the activation of a trimethoxysilylgroup by the fluoride ion is remarkable, we envisaged that the silver fluoride complex would behave
as a chiral catalyst also for asymmetric protonation of silyl enolates with an appropriate achiral
Brønsted acid such as methanol.

We first studied the protonation of 2-methyl-1-tetralone–derived

trimethylsilyl enolate 1 (shown in entry 1 of Table 1) with methanol to find the optimum reaction
conditions. We attempted the reaction using various ratios of a mixture of BINAP and AgF at –20
˚C and found that a 0.6:1 mixture was more effective in obtaining a nonracemic product with higher
enantioselectivity than a 1:1 mixture.
We then examined solvent effects on the chemical yield and enantioselectivity.

Among the solvents

tested, THF or chlorinated hydrocarbons indicated better ee than did methanol, and dichloromethane
was the solvent of choice. In fact, when the protonation was carried out in a 20:1 mixture ofdichloromethane and methanol, (S)-enriched 2-methyl-1-tetralone was obtained with 56% ee.
Further improvement in the enantiomeric ratio was attained when a twofold volume of methanol anddichloromethane was employed. Additionally, with other achiral alcohols, we investigated theenantioselectivity of this catalytic protonation and as a consequence, methanol was found to be themost effective.

125

This asymmetric protonation was applied to diverse trimethylsilyl enolates and the results are
summarized in Table 1. Not only the 5-MeO derivative, but also the 2-ethyl derivative afforded
good optical purities similar to that of 2-methyl-1-tetralone (entries 1-3). Although simple ketones
possessing no aromatic group at the α-carbon are known to give unsatisfactory results in the
catalytic

asymmetricprotonation

underacidicconditions,employmentof

2,2,6-trimethylcyclohexanone-derived silyl enolate in the present protonation resulted in highenantioselectivity of more than 80% ee (entry 4).

A very high enantiomeric ratio was obtained withthe silyl enolate of 2-phenylcyclohexanone (entry 5). 2-Arylcycloalkanones are the best substrates
for

the

present

asymmetricprotonation

and,

for

example,trimethylsilyl

enolate2-phenylcycloheptanone also showed high enantioselectivity and reactivity (entry 7).

ofAs forp-methoxyphenyl, p-tolyl, and 2-naphthyl derivatives, almost perfect asymmetric induction wasobserved and (R)-enriched products were formed nearly quantitatively in every case (entries 8-10).3
On the basis of these results, we further attempted the catalytic asymmetric protonation of α-arylcarboxylic acid enolates under the optimized reaction conditions. When ketene bis(trimethylsilyl)
acetal of Naproxen 2 was treated with a catalytic amount of (R)-BINAP·AgF complex in a 20:1
mixture of dichloromethane and methanol, (S)-enriched Naproxen (3) was obtained in 93% yieldwith 8% ee (eq 1). Although we tried several other α-aryl carboxylic acids, satisfactory results
were not obtained at all.

Further studies on the reaction conditions including reaction temperatureand solvent are necessary to improve the enantioselectivity.

Discussion & Summary
The reaction mechanism was not fully elucidated; however, two mechanisms can be considered forthe catalytic asymmetric protonation. As a probable catalytic mechanism, a BINAP·AgF complex
is regenerated from an initial transition-state structure accompanied by the formation of the
protonated product and methoxytrimethylsilane.

In contrast, another mechanism generates

BINAP·AgOMe and fluorotrimethylsilane from the transition-state structure via a transmetallation
step.

In the second cycle and thereafter, BINAP·AgOMe is recycled and acts as a chiral catalyst in

the second transition-state structure.
In summary, we have developed a novel catalytic asymmetric protonation system.

By using a

BINAP·AgF complex as a chiral catalyst and MeOH as an achiral proton source, various nonracemic
ketones were prepared with enantioselectivity up to 99% ee.

Further studies on the protonation ofsilyl enolates of α-aryl carboxylic acids and other carbonyl compounds are currently underway.

126

127

References(1)(a) A. Yanagisawa, Y. Nakatsuka, K. Asakawa, H. Kageyama, H. Yamamoto, Synlett 2001,
69; (b) A. Yanagisawa, Y. Nakatsuka, K. Asakawa, M. Wadamoto, H. Kageyama, H.
Yamamoto, Bull. Chem. Soc. Jpn. 2001, 74, 1477.

(2)

A. Yanagisawa, H. Kageyama, Y. Nakatsuka, K. Asakawa, Y. Matsumoto, H. Yamamoto,Angew. Chem. Int. Ed. 1999, 38, 3701.

(3)

A. Yanagisawa, T. Touge, T. Arai, Angew. Chem. Int. Ed. 2005, 44, 1546.

1283) Chemical Sciences3-1) Organic Chemistry

Synthetic Study on Glycopeptide Using Glycosyl Amino Acid Having
Natural Oligosaccharide
Toshiyuki INAZU
Department of Applied Chemistry, School of Engineering, Tokai University
inz@keyaki.cc.u-tokai.ac.jp

Introduction
Recently we have reported the coupling efficiency of Fmoc-Asn(GlcNAc)-OH during solid phase
synthesis of glycopeptide.

In order to confirm the coupling efficiency of glycosyl amino acidhaving natural oligosaccharide for solid phase synthesis of glycopeptide, we prepared the Fmoc
derivatives of amino acids having natural oligosaccharides from natural glycoproteins, such as
ovalbumin, ovomucin, and so on.
Additionally we found the method for recovery of glycosylated amino acid derivatives, which wereusually expensive, and valuable derivatives.

Results
1) Preparation of glycosylated amino acids
First we prepared ovalbumin from hen eggs.
chicken yard.

We are provided fresh eggs by the collaborated

These eggs were laid at the same day by the same aged hens and were available

within 2 days.
Ovalbumin was prepared by the combined method using saturation of ammonium sulfate andisoelectric point of the corresponding protein.

We got ovalbumin in good yield under theconditions of a half saturation of ammonium sulfate at pH 4.6 from egg white.
ovalbumin was detected by gel electrophoresis.

The purity of

We have prepared pure ovalbumin byreprecipitation method, which step was repeated 3 times.
Next the limited hydrolysis of ovalbumin using a-chymotrypsin was carried out. After several
purification steps, such as ion-exchange chromatography and gel chromatography, we obtained
glycosylated peptides.
Hydrolysis of glycosylated peptides by pronase, which step was repeated twice times, gave themixture of glycosylated asparagines having several kinds of natural N-linked oligosaccharides.
Finallythereaction

of

these

glycosylated

asparagines

andFmoc-OSu(N-9-fluorenylmethyloxycarbonyloxy-succiniimide) gave the corresponding Fmoc-Asn(Sugar)-OH,

129

which were isolated by HPLC.
In this study we also attempted to prepare ovomucin to obtain mucin-type (O-linked) glycosylatedamino acids, such as Fmoc-Ser(Sugar)-OH and Fmoc-Thr(Sugar)-OH.

It was found ovomucin was

prepared easily from egg white in good yield by the direct centrifugation.
stored as lyophilized powder.

Pure ovomucin was

The preparation of glycosylated amino acid having O-linked natural

oligosaccharide is now in progress.
2) Solid phase synthesis of glycpeptides
The solid phase synthesis of glycopeptides by dimethylphosphinothic mixed anhydride (Mpt-MA)
method has been studied. No protective group for hydroxyl function of sugar moiety was necessaryduring the coupling reaction using Mot-MA method.
Usually solid phase synthesis of glycopeptide was carried out using a large excess of glycosylatedamino acid derivatives.

The recovery of glycosylated amino acid derivative has been expected.

Mpt-MAs were expected to decompose to Fmoc-amino acid and dimethylphosphinothic acid by only
addition of water because they are classified into acid anhydride. Coupling reaction of Mpt-MA
proceeded in the presence of tertiary amine, such as triethylamine, N, N-diidopropylethylamine(DIEA), and so on.

When the reaction mixture after coupling reaction by Mpt-MA method waspoured into water, glycosylated amino acid would be regenerated.

We tried the recovery of

Fmoc-Thr(Man)-OH during the solid phase synthesis of mannosyl peptide by Mpt-MA method.
After the solid phase coupling reaction of Fmoc-Thr(Man)-OMpt, which was derived from
Fmoc-Thr(Man)-OH and Mpt-Cl, in the presence of DIEA in N, N-dimethylformamide (DMF), all of
DMF filtrate and washings were poured into water.
by TLC.

The decomposition of Mpt-MA was detected

A large excess of water was added and the aqueous solution was charged on a polymericadsorbent, such as Dia-ion HP-20 and Amberlite XAD-2 resin.
with water and eluted with methanol.
reduced pressure.

The resin was washed sufficiently

The methanol solution was corrected and evaporated under

We obtained Fmoc-Thr(Man)-OH in 98 % recovery yield.

Recoveredglycosylated amino acid was able to use for the synthesis of the corresponding Mpt-MA.

This

simple recovery system was applicable for only Mpt-MA method because there are Mpt-MA, tertiaryamine, and DMF in the reaction medium by Mpt-MA.

Because of this simplicity, this recovery

step would be applied for the automated synthesizer.
3) The coupling efficiency of glycosylated amino acid derivative
The solid phase syntheses of several kinds of H-AA-Asn(Sugar)-Ala-Leu-Ala-resin and theevaluation of their coupling efficiency are now in progress.

130

Discussion & Summary
We prepared Fmoc-Asn(Sugar)-OH having natural N-linked oligosaccharide from ovalbumin.
Similary we attempted to prepare ovomucin in order to obtain Fmoc-Ser(Sugar)-OH and
Fmoc-Thr(Sugar)-OH having natural O-linked oligosaccharide.

It was found the preparation of

ovomucin was easily by only centrifugation of egg white.
Syntheses and evaluation of glycopeptide using these glycosylated amino acid derivatives are now inprogress.
Usually a large excess of glycosylated amino acid was used for the synthesis of glycopeptide. We
tried the recovery of glycosylated amino acid and found a simple method for Mpt-MA method.
This recovery method consisted 2 steps, 1) decomposition of mixed anhydride to acid; 2) recovery of
the Fmoc glycosylated amino acid by polymeric adsorbent.

We are now studying the optimum

conditions for this recovery system.

References
1) T. Inazu, M. Mizuno, T. Yamazaki, and K. Haneda, “Peptide Science 1998”, ed by M. Kondo,
Protein Research Foundation (Osaka) 1999, pp.153-156.
2) M. Mizuno, M. Noguchi, M. Imai, T. Motoyoshi, and T. Inazu, Bioorg. Med. Chem. Lett., 2004,
14, 485-490.

1314) Others

Identification of new peptide agonists specific for circulatory orphan
receptors using internalization assay
Kenji Kuwasako
First Department of Internal Medicine, Miyazaki Medical College, University of Miyazaki
kuwasako@fc.miyazaki-med.ac.jp

Introduction
It is well known that almost G protein-coupled receptor molecules are rapidly internalized after
agonist exposure to inhibit over signaling in vivo.

Nothing is reported about new peptide isolation

using internalization assays of orphan receptors.

In this study, we will establish stable transfectantsfor orphan receptors that abundantly expressed in circulatory systems and identify their specificpeptides from extracts of human pheochromocytoma or normal porcine adrenal medulla using
receptor internalization assay.

Results
We initially selected orphan receptors that would need peptide agonists by computer research andlimited the two ones (GPRX and CGRY) that fully expressed in the circulatory organs andup-regulated in cardio-vascular diseases. We next amplified the two targeted orphan receptor genes
by PCR methods using appropriate primers sets. To quantify the receptor internalization using flow
cytometry, double V5 epitope tags were used in-frame to the 5’ end of cDNAs encoding V5-GPRX
and

V5-GPRY,

andthenativesignalsequenceswereremovedand

MKTILALSTYIFCLVFA (Guon et a. J Biol Chem. 1992; 267: 21995-21998).

replacedwith

In addition, EGFP

genes were ligated in-frame to the 3’ end of the V5 epitope-tagged gene constructs to directly
observe receptor internalization using fluorescence microscopy.

Both sided, tagged receptor genes(V5-GPRX-EGFP and V5-GPRY-EGFP) were then cloned into the mammalian expression vector
pCAGGS/Neo using the 5’ Xho I and 3’ Not I sites (1), termed pCAGGS/Neo-V5-GPRX-EGFP and-V5-GPRY-EGFP.

We selected human embryonic kidney (HEK)-293 cells for transfections

because they possess adequate components required for receptor internalization, such as G
protein-coupled receptor kinases (GRKs).

Cells grown to 70% confluence were transfected with

pCAGGS/Neo-V5-GPRX-EGFP and -V5-GPRY-EGFP using LipofectAMINE transfection reagent.
Two days after transfection, cells were plated in selective medium containing 1 mg/ml G418.

A

pool of G418-resistant colonies was trypsinized followed by limited dilution and single cell plating
in to 96-well plates. Three weeks after transfection, EGFP-expressing clones were all screened for132

expression of V5 epitope tags by flow cytometry.
internalization assay.
mg/ml G418.

We selected the most suitable clones for

These two transfectants were maintained in the same buffer containing 0.25

On the other hand, bovine and porcine adrenal medullae and porcine atria, ventricles

and lungs were extracted by our previous methods by Kitamura, K., et al (2).

Discussion & Summary
We have succeeded to establish the transfectant cell lines stably expressing V5-GPRX-EGFP and
V5-GPRY-EGFP and to fully obtain the aforementioned tissue extracts.
experimental procedures will be performed from now on:

The following

Unknown peptides are purified using gel

filtration, ion-exchange chromatography, various reversed-phase HPLC, etc.

Following 30-min

sample treatment at 37°C, cells are stained by rhodamine-labeled antibody at 4°C and the loss of cellsurface receptor expression are quantified using flow cytometry.

The fractions that induced fullinternalization are collected and used for the next step of peptide purification.
purifications are analyzed by protein sequencer.

The final

The tissue distribution and functional properties of

the unknown peptides are further examined.

Figures & Tables

133

References
1. Kuwasako, K., et al. J Biol Chem. 2000; 275: 29602-29609
2. Kitamura, K., et al. Biochem Biophys Res Commun. 1993; 192: 553-560

134Part Ⅱ
Reports from the Recipients of Garants
for International Meetings(Fiscal Year 2003)

135

11th International Conference on the Cell and Molecular Biology ofChlamydomonas (Chlamy2004)
1. Representative
Yoshihiro Matsuda, the chairman of organizing committee
2. Opening period and Place
May 11-15, 2004, International Conference Center Kobe, Kobe, Japan
3. Number of Participants
167 persons (89 persons from 10 foreign countries)
4. The total costs
Eleven million yen
5. Main use of the subsidy
Costs for setting of poster panels and for equipment rent at the opening place
6. Result and Impression
The Chlamydomonas conference has been held every 2 years since1982 – seven times in USA,
twice in Europe, and once in Canada. The present conference was held in Japan for the first time.
Three organizers (Y. Matsuda, R. Kamiya and H. Fukuzawa) hoped to have participants frommany countries who use Chlamydomonas and related organisms, especially to have Japanesephycologists who have been doing excellent works on the fields of molecular biology andbiotechnology, in order to enhance scientific exchange. For this purpose, Chlamy2004 was
supported by 6 foundations and backed by 6 Societies in Japan, related to plant and algal
researches. We are pleased to report that these financial aids were able to support a part of travel
expenses of 55 young scientists and 18 invited persons.
This conference aimed at providing a platform on which world-wide researchers using
Chlamydomonas and related organisms can present their new findings, and thereby promoting
development of the cell and molecular biology of this organism. Since the value ofChlamydomonas as an excellent model organism has recently been further raised, owing to thedevelopment of new biotechnology as well as completion of its genome project, this meeting wasparticularly timely for constructing a strong international network of researchers in theforthcoming post-genome era.
During 5 days of this meeting, we had 72 oral presentations and 85 poster presentations, dividing
into 11 sessions: (1) Photo- and Chemosensory Processes, (2) Structure and Function of the
Photosynthetic Apparatus, (3) Genomics and Proteomics, (4) Basal Body and Flagellar Assembly,(5) Acclimation to Environmental Stress, (6) Genomic Techniques, (7) Flagellar Motility:
Regulation and Mechanism, (8) Organelle Biogenesis, (9) Mating and Cell Division, (10) Little

136

Green Cells and Major Evolutionary Questions, and (11) Discussion on Genome Annotation. Allsessions were held at the same one big room from 9 am to 9 pm everyday so that all participants
could listen to all sessions of different research fields. Furthermore, we reserved a room at the
Portopia Hotel for further discussion after the night sessions. Especially, the participation of many
Japanese scientists with different research careers and interests other than Chlamydomonas to thismeeting provided a proper jolt to their biological knowledge of Chlamydomonas biologists.
7. Additional description
At the business meeting, it was decided that the meeting report on the Chlamy2004, Kobe, will be
published soon in Protist and that the next Chlamydomonas conference will be held at Oregon(USA) in 2006.

137

Report of 21st International Lectin Meeting (Interlec 21)
1.

Representative
Kenichi Kasai, Chairman of the Organizing Committee of 21st International Lectin Meeting

2.

Opening Period and Place
May 23 - 28, 2004.
Shonan Village Center and Hotel Okuyumoto in Hakone

3.

Number of Participants
182 persons (52 persons from foreign countries)

4.

Total Cost
18,190 thousand yen

5.

Main use of the subsidy
Support for travel expenses of invited speakers

6.

Result and Impression
Importance of research on lectins, natural molecules having the role of deciphering glycocodes,

has been growing day by day, in concordance with the significant development of glycobiology.
Glycomics should be the most important field of life science after the era of genomics andproteomics, and lectinology will certainly take an essential part in it.

Interlec meeting, only one

series of international scientific forum focused on lectin research, have been held every two years
around the world, and made a great contribution to the progress of life science, especiallyglycobiology.

It was extremely important to organize the Interlec meeting for the first time inJapan at such a significant moment.
Thanks to many scientists who have submitted excellent abstracts, we were able to make asplendid program.

A number of papers on the latest results in a variety of aspects of lectins were

presented by both established and young researchers. Principal topics were as follows: 1) Structure,function and specificity of lectins.

2) Regulation of cells by lectins. 3) Mechanism of sugarrecognition. 4) Characterization of newly identified lectins.

5) Lectins as tools for life science.

6) Modification of lectin function by genetic engineering. 7)

Medical application of lectins.

8)

Application of lectins to agricultural and technological sciences. 9) Molecular evolution of lectin.
10) New methodology for lectin research.
During the meeting, 61 papers were presented orally and 70 papers were presented as posters.

138

Participants could gain a comprehensive view on current state of lectin research and enjoy thescientific program.

For all oral presentations, there were active questions, comments anddiscussions between the presenters and audience.

Although the poster sessions were assigned

after dinner, discussions by enthusiastic participants did not end until late hours in the evening.
The meeting seems to be extremely fruitful especially for young researchers because it was avaluable experience for them to have been able to meet for the first time many established andfamous lectin researchers whom they knew only by name, listen to their lectures, and make personal
contact.

Such an experience should certainly have greatly stimulated and encouraged these young

researchers. It seems also very fruitful for them to get aquainted with many other young lectin
researchers because they will be able to collaborate each other and contribute to further developmentof lectin research in the future.
During the course of the meeting, many items were planned to entertain participants bothscientifically and socially.

We especially wished to offer participants from foreign countries

opportunities to experience Japan's unique culture, so different from that of the west, and gain a newperspective on human activities, which may help to produce new ideas for scientific research.

We

have already received letters from many participants which are kindly enough to estimate that themeeting was the most exciting and fruitful one ever held.
by the organizing committee.

They also acknowledge all efforts madeTherefore, we now feel very happy to be albe to finish our task

successfully, and would like to express our sincere gratitude for the support from your foundation.
7.

Additional description
Nationality of participants from foreign countries: USA, Canada, Argentin, Brazil, Urguai,

Australia, Taiwan,
China, Thai, India, Israel, Germany, The Netherland, Switzerland, Belgium, France, United
Kingdom, Sweden,
Denmark.

139

International Narcotics Research Conference 2004/Kyoto
1. Representative
Masamichi Satoh
2. Opening period and Place
July 18- July 23, 2004
3. Number of participants
291 persons (including 140 persons from 18 foreign countries and areas)
4. The total cost
¥29,663,5215. Main use of the subsidy
Support for travel expenses of an invited speaker and 4 young scientists.
6. Result and impression
The International Narcotics Research Conference (INRC) is held annually at the middle of July,where a lot of scientists from diverse places on earth and various study fields discuss the novel
findings regarding Opioids.

The first international conference was held in a classroom of the

University of Basel (Switzerland) as a satellite session of narcotics of International Union of
Pharmacology (IUPHAR) on July 19, 1969.
Kosterlitz and Harry Collier.

The meeting was organized by the Late Drs. Hans

After that, the conference has been held every year.

In 1981, the12th meeting of INRC was held in Kyoto, Japan, organized by Dr. Hiroshi Takagi, as a satellite
meeting of IUPHAR held in Tokyo.

And 23 years later, we had this present conference again in

Japan and it was the 35th meeting of INRC, held in Kyoto from July 18-23, 2004, and named“INRC2004/Kyoto”.
INRC2004/Kyoto was jointly sponsored by the Japan Foundation for Neuroscience and Mental
Health, the Japanese Narcotics Research Conference and the National Institute on Drug Abuse(NIDA), and supported by 5 societies and associations in Japan (including the JapanesePharmacological Society, the Pharmaceutical Society of Japan, Japan Neuroscience Society,Japanese Association for the Study of Pain, and Japan Society of Pain Clinicians).

There were

financial aids, the subsidies by NIDA and 6 foundations, including yours, and the donation from the
Osaka Pharmaceutical Manufacturers Association, the Pharmaceutical Manufacturers’ Association ofTokyo, and 20 enterprises.

140

In this conference, two plenary lectures were presented as follow; “Psychological Basis ofCompulsive Drug-Seeking: Implications for Treatment” by Dr. Barry J Everitt (University ofCambridge, UK), and “Regulatory Mechanisms of Synaptic Transmission in the Neural Circuit” by
Dr. Shigetada Nakanishi (Kyoto University, Japan).

A Founders’ Lecture, “Acupuncture,

Endogenous Opioids and Anti-opioids” was presented by Dr. Ji-Sheng Han (Peking University,
China).

It showed us the surprising effectiveness of acupuncture for the pain control.

In 10

symposia, (I) Intractable Pain and Analgesia, (II) New Molecules in Receptor Trafficking/
Signaling-I, (III) Gene Regulation and Human Genetics, (IV) Neurobiology and Opioids, (V) New
Compounds/Molecules in Opioid World, (VI) New Trends in Opioid-related Peptide Research, (VII)New Molecules in Receptor Trafficking/ Signaling-II, (VIII) Molecular Psychiatry, (IX)
CB1-Opioid-Glutamate Cross-talk in Reward and Addiction, and (X) New Frontier in Addiction
Research, 29 symposiasts and 21 regular speakers selected from regular paper gave us lectures. In
the poster session, 129 posters remained on display throughout the conference, and were excitingly
discussed for three hours on Monday 19 and Thursday 22.

In addition, two luncheon lectures were

presented as follow; “Clinical Utility of Oxycodone in the Management of Pain” by Dr. Barry A.
Eagel (Purdue Pharma L.P.), and “Locus-specific Rescue of Opioid and Anti-opioid genes into
Knock-out Mice for the Study of Opioid Tolerance and Dependence” by Dr. Hiroshi Ueda (Nagasaki
University, Japan). Total 184 papers were fully presented for 5 days.
every 8:30 am, and there was no break except for lunchtime.
throughout the conference.

The sessions started at

We all had a solid time for discussion

In addition, because all oral presentations were held in the same room,

the participants were able to fully listen to all lectures.
Although opioids have been widely used in the clinical management of pain including chronic
cancer pain, their liability to develop tolerance and dependence often restricts the range ofapplication. The mechanisms of the development of opioid tolerance and dependence have been
vigorously investigated for a long time, and many recent researchers have tried to elucidate them at
the molecular level.

In this conference, there were a lot of advanced findings about the molecularmechanisms from various points of view.

These studies proposed novel points of view and new

methods in succession, indicating the high activities of opioid researchers.

Furthermore, it wasreported that opioidergic system plays an important role in the dependence on other drugs of abusesuch as cannabinoids and nicotine.

Subsequently more researchers in the field of other substancedependence are thought to take part in the field of opioid research. Opioids and their receptors are
the issue of discussion for not only their side effects such as tolerance and dependence, but also their
possible application for depression, anxiety, learning and memory, itch, and immune suppression.
There is a body of evidence about the relationship between opioids and glial cells, reported in some
symposia, “Neurobiology and Opioids” and so on.

All together, the field of opioid research is still

deriving a large amount of interest from many scientists in the world.

141

It was decided that the next 36th meeting of INRC were going to be held at Annapolis, Maryland in
the U.S.A. from July 10-15, 2005.

142

The 4th Tateshina Conference on Organic Chemistry
1. Representative
Eiichi Nakamura, the chairman of organizing committee
Keisuke Suzuki, the general secretary of organizing committee
Takuzo Aida, Makoto Fujita, Terunori Fujita, Takehiko Iida, and Keiji Maruoka, the members oforganizing committee
2. Opening period and place
November 12-14, 2004 at Lodge Mitsui-no-Mori and Tateshina Forum in the city of Chino,
Nagano prefecture, Japan
3. Number of participants
73 persons (16 persons from foreign countries)
4. The total cost
2.5 million yen
5. Main use of the subsidy
Support for travel expenses of invited foreign speakers
6. Results and impression
The Conference was hold at Tateshina Mitsui-no-Mori Lodge, located at an altitude 1,100 m in thecity of Chino, Nagano prefecture between November 12 (Fraiday) and November 14 (Sunday). We
had seven scientific sessions, including six lectures and one poster session.

Six invited speakers

presented general description of their subject area as well as the most recent discovery from the
speaker’s laboratories.

The session included 50-min talk and 30-min discussion so that participants

could fully appreciate the present status and the future development of the field where you areactive.
The organizing committee gave one-paragraph explanation of the Conference for the information.
For sometime, relatively young generations of Japanese organic chemists and myself have been
pondering on a possibility of having a conference for Asian chemists.

The organizing committee

therefore consulted some eminent chemists in Japan, China, Korea, Taiwan, and Hong Kong about a
new style conference to be held in our country.

Professors Mukaiyama and Noyori were among

those who strongly supported this idea. We therefore gave a trial meeting in November 2000 withindustrial and academic people from Japan, Korea, and Thailand including topics of synthetic,

143

supramolecular, natural product chemistry and molecular biology.

There, all participants agreed to

put forward the idea of East Asian conference. Professor Eiichi Nakamura was elected to become
Chairman of the 4th Conference.
One of the most attractive features of the Conference was high-standard speakers, Professor C.-H.
Wong of The Scripps Research Institute, Professor K. Nakanishi of Columbia Univ., Dr. Y. Hsiao of
Merck, Mr. T. Arimoto of MEXT, Professor R. F. Ismagilov of The Univ. of Chicago as well as
Professor A. Fujishima of KAST.

The participants are as important as speakers, and about

sixty-five of them included people of all area related to organic chemistry, from industry andacademia, and from various parts of East Asia.
The organizing committee and Asian liaison members are confident that the Conference is uniquein its style, and effective in generating a friendly, cooperative and creative atmosphere among
participants, providing Asian chemists with a forum for the advancement of organic chemistry in thispart of the world.