1

Foreword
To create good research environment
Recently deterioration of the moral of Japanese is questioned. In terms of “moral”, thetarget of criticism had been always young people, but now, it shifted to adults.
Newspapers showed us examples such as an architect’s fabrication of structural
calculation sheets for earthquake-resistance strength of buildings, and next, fraudulent
acts carried out by new-riches called “Hills zoku” as they live in a high-rise
condominium “Roppongi Hills” - the symbol of wealth and success. Also in our daily
life, the bad-mannered are mostly adults. People who are using their mobile phones in a
train or who throw private garbage into a trash box in a park are rather senior people.
This means, adults, who normally should lead young generation to a right way, arepractically to be blamed by them. Similarly, some symbolic events of moral corruption
occurred in the academic research field. A fabricated report about cloning technology
was uncovered in Korea, and also a certain famous Japanese university faked researchdata and their report. According to news reports, the Ministry of Education, Culture,
Sports, Science and Technology announced that researchers who committed serious
unethical acts such as falsification of report or data will not allowed to apply for
research grants up to 10 years from next year. If a researcher lost his research fund evenfor a few years, it could be the end of his research life. Tough punishment!
In our childhood, our parents told us a saying “dishonesty does not pay”. This saying is
made based on self-control. If people are self-controlled, they are all good-natured andthe world is peaceful. If you assume that all the people are not good-natured, the world
turns cold and cunning people gain power. Unfortunately it seems like the world we live
in is rather the latter. Why did the world become so? I think the way people assessthings has changed from the analog method to the digital one, namely, they doquantitatively rather than doing qualitatively. Moreover, the cycle of the assessment gets
shorter. In the business world, annual sales and profits are compared with those of lastterm and forced to be increased unremittedly. In the academic world too, impact factors
in science journals have wings and replace those in each report. Can the academic valueof a report be truly quantified? As for personnel affairs in universities, they are also
overwhelmed by figures like research expenditure.

2

In a university whose wrongful use of research fund was recently brought to the light,
a delivery check center was newly set up to check if the content of purchasing slips anddelivery slips match with the actually purchased materials. This way, however, seriously
disturbs a smooth achievement of research in the name of “check”. Academic people
now must control themselves to bring back the world for good-natured.

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 2004)
1) Biological Sciences
1-1)

Molecular Biology
Molecular Design for Bioactive Small Compounds based on Phage-displayed
Combinatorial Peptide Libraries
Ikuo Fujii
Department of Biological Science, Osaka Prefecture University
fujii@b.s.osakafu-u.ac.jp

Molecular mechanism of class switch recombination and somatic hypermutationinduced by AID
Reiko Shinkura
Immunology and Genomic Medicine, Kyoto University, Graduate School of Medicine
shinkura@mfour.med.kyoto-u.ac.jp

Mechanisms of transcriptional and posttranscriptional gene silencing by small
RNA molecules
Mikiko C. Siomi
Institute for Genome Research, University of Tokushimasiomim@genome.tokushima-u.ac.jp

Analysis of HSF1-mediated cell death and alive signaling pathways
Akira Nakai
Yamaguchi University School of Medicine
anakai@yamaguchi-u.ac.jp

Visualization of demyelinating neuropathic pain by lysophosphatidic acid
MAKOTO INOUE
Nagasaki University
minoue@net.nagasaki-u.ac.jp

4

Molecular mechanism of axonal guidance of the injured
central nervous system
Toshihide Yamashita
Chiba University
t-yamashita@faculty.chiba-u.jp

Analysis of the new death receptor signal for the treatmentof cancers and immune diseases
Tadaaki Miyazaki
Department of Bioresources,Hokkaido University Research Center for Zoonosis Control
miyazaki@czc.hokudai.ac.jp

Neuronal cell death and malfunction of intracellular-trafficking
Shinji Hadano
Department of Molecular Neuroscience, The Institute of Medical Sciences,
Tokai University
shinji@is.icc.u-tokai.ac.jp

Proteomic Studies of Hepatitis C Virus
Tohru Ichimura
Tokyo Metropolitan University
ichimura@mail.comp.metro-u.ac.jp

Analysis of physiological roles of catecholamines and vasopressin on fat andcarbohydrate metabolism in insulin resistance, obesity and diabetes.
Akito Tanoue
National Research Institute for Child Health and Development
atanoue@nch.go.jp

1-3） Cell Biology
The molecular mechanism of activity-dependent vesicle transport regulated bysynaptotagmin IV
Mitsunori Fukuda
Fukuda Initiative Research Unit, RIKEN
mnfukuda@brain.riken.go.jp

Relationship between carcinogenesis and the control of the ribosome andtranslation
Daita Nadano
Nagoya University
nadano@agr.nagoya-u.ac.jp

5Studies of the regulatory mechanism in EMT mediated by the Zn signaling
Susumu Yamashita
Laboratory of Developmental Immunology, Graduate School of Frontier Biosciences,and Graduate School of Medicine, Osaka University, Suita, Osaka, Japansyamasi@molonc.med.osaka-u.ac.jp

The function of human BIG family proteins in the membrane trafficking between
TGN and endosomes
Hye-Won Shin
Graduate school of pharmaceutical sciences, Kyoto University
shin@pharm.kyoto-u.ac.jp

1-4） Developmental Biology
Studies on human hereditary disease using medaka as a model
An analysis of pc medaka mutant showing a phenotype of polycystic kidneydisease.
Hisashi Hashimoto
Gene Engineering Division, BioResouce Center, RIKEN
yokoyama@brc.riken.jp

The role of Notch ligand for organogenesis and regeneration of various tissues.
Katsuto Hozumi
Department of Immunology, Tokai University School of Medicine
hozumi@is.icc.u-tokai.ac.jp

Regulation of mesenchymal stem cell differentiation by phosphoinositidemetabolism
Kiyoko Fukami
Tokyo University of Pharmacy and Life Sciencekfukami@ls.toyaku.ac.jp

Molecular genetic analysis of the signaling pathway of the DIF, an anti-tumor
substance involved in the development of a model organism, Dictyostelium
discoideum.
Masashi Fukuzawa
Faculty of Agriculture and Life Science, Hirosaki University
fukuzawa@cc.hirosaki-u.ac.jp

61-5) Physiology
Analysis of primary afferent-evoked synaptic plasticity in spinal dorsal horn bysimultaneous imaging of nitric oxide and presynaptic neuronal excitation.
Hiroshi Ikeda
University of Fukui
ikeda@synapse.his.fukui-u.ac.jp

Roles of presynaptic calcium store in synaptic plasticity
Haruyuki Kamiya
Hokkaido University School of Medicine
kamiya@med.hokudai.ac.jp

1-6） Pharmacology
Photodynamic therapy for cancer using photoreactive nano-micelles with thefavorable property of accumulation in a tumor
Yuji Morimoto
Department of Medical Engineering, National Defense Medical College
moyan@ndmc.ac.jp“Studies on the molecular mechanisms for physiological regulation of urinary
bladder smooth muscle and etiology of overactive bladder Analysis from theviewpoint of large-conductance,Ca2+-sensitive K+ (MaxiK) channel role”
Yoshio Tanaka, Ph.D.
Toho University School of Pharmaceutical Sciences
yotanaka@phar.toho-u.ac.jp

Molecular dissection of the role of CLICK-III/CaMKIγ in fear- andeating-related behavior
Haruhiko Bito
University of Tokyo Graduate School of Medicine
hbito@m.u-tokyo.ac.jp

1-7)

Plant Biology
Molecular mechanism of stem cell maintenance in the plant root meristem
Keiji Nakajima
Nara Institute of Science and Technology
k-nakaji@bs.naist.jp

7

Molecular and genetic studies on the function of polyaminesin plant development
Taku Takahashi
Graduate School of Bioscience and Technology, Okayama University
perfect@cc.okayama-u.ac.jp

Molecular mechanism of phototropin kinase regulation by blue light
Satoru Tokutomi
Osaka Prefecture University
toxan@b.s.osakafu-u.ac.jp

Plant-plant communication mediated by volatile compounds.
〜Elucidation of its molecular mechanisms〜
Kenji MATSUI
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University
matsui@yamaguchi-u.ac.jp

Non-Cell-autonomous flowering signal of FT is conferred by its protein trafficking.
Koji Goto
Research Institute for Biological Sciences, Okayama
kgoto@v004.vaio.ne.jp

2) Medical Sciences2-1) Immune System
Role of negative regulators of cytokine signaling in atopic disorders
Hiromasa Inoue, MD
Research Institute for Diseases of the Chest, Graduate School of Medical Sciences,
Kyushu University
inoue@kokyu.med.kyushu-u.ac.jp

Identification of novel immune adjuvants from parasites or fungi
Tsuneyasu Kaisho
RIKEN Research Center for Allergy and Immunology
tkaisho@rcai.riken.jp

Molecular mechanism of the immune regulation maintained by CD8+CD122+
immuno-regulatory T cells.
Haruhiko Suzuki
Nagoya University, Graduate School of Medicine
K46200a@nucc.cc.nagoya-u.ac.jp

8Studies on Immune Regulation by Interleukin (IL)-27, A New Member of The
IL-12 Cytokine Family
Takayuki Yoshimoto
Intractable Immune System Disease Research Center, Tokyo Medical University
yoshimot@tokyo-med.ac.jp

2-2) Central Nervous System
The analysis of molecular mechanismｓ of schizophrenia and bipolar disordercaused by 22q11.2 deletion
Kenji Tanigaki
Research Institute, Shiga Medical Center
tanigaki@shigamed.jp

Role of forkhead transcription factor Foxo1 in feeding behavior
Jun Nakae
Department of Clinical Molecular Medicine, Division of Diabetes, Digestive and Kidney
Disease, Kobe University Graduate School of Medicine
nakaej@med.kobe-u.ac.jp

2-4) Arthritis/Bone Metabolism
Cell cycle regulation therapy to treat rhueumatoid arthritis
Hitoshi Kohsaka,M.D.
Tokyo medical and Dental University
Kohsaka.rheu@tmd.ac.jp
2-5)

Cardiovascular Metabolic Endocrine
Creation of knock-down transgenic rabbits using RNAi technology
for the study of atherosclerosis
Jianglin Fan
Department of Molecular Pathology, Interdisciplinary Graduate School of Medicine
and Engineering, University of Yamanashi
Jianglin@yamanashi.ac.jp

2-6)

Gastro-intestinal
Regulation of macrophage responses to commensal flora by the colonic mucosal
microenvironment
Taeko Dohi
Research Institute, International Medical Center of Japan
dohi@ri.imcj.go.jp

92-10) Dermatology
A study of metastatic trait of malignant melanoma with expression of
MCM3-binding β-primase GANP correlated with highly metastatic clinical cases
Kazuhiko Kuwahara
Department pf Immunology, Graduate School of Medical Sciences, Kumamoto University
kazukuwa@kaiju.medic.kumamoto-u.ac.jp

2-12) Hematology
Role of Polycomb-group complexes in Geminin degradation which is crucial forhematopoietic stem cell regulation
Yoshihiro Takihara
Research Institute for Radiation Biology and Medicine
takihara@hiroshima-u.ac.jp

3) Chemical Sciences3-1) Organic Chemistry
Design and Synthesis of Optically Active Supramolecular Capsules in Aqueous
Solution and Their Application to Asymmetric Recognition.
Shin Aoki
Faculty of Pharmaceutical Sciences, Tokyo University of Scienceshinaoki@rs.noda.tus.ac.jp

Development of New Oxidation Reactions and Synthesis
of Bio-active Compounds
Jun-ichi Matsuo
Kanazawa University
jimatsuo@p.kanazawa-u.ac.jp

Development of enantioselective reactions based on organocatalysis directing at the
total synthesis of biologically active compounds
Makoto Nakajima
Kumamoto University
nakajima@gpo.kumamoto-u.ac.jp

103-3) Protein Chemistry
Development of real-time sensors to simultaneously assess in vivo productions of
two different second messengers
TAKASHI MORII
Institute of Advanced Energy, Kyoto University
t-morii@iae.kyoto-u.ac.jp

3-4) Analytical Chemistry
Role of chymase in the development of aortic aneurysm
Shinji Takai
Osaka Medical College
Pha010@art.osaka-med.ac.jp

Part Ⅱ:
Reports from the Recipients of Garants for International Meeting(Fiscal Year 2004)
4th European-Japanese Bioorganic Conference (EJBC-4)
6th Asian-Pacific Conference on Medical and Biological Engineering(APCMBE 2005)
The 58th Meeting of the Japan Society for Cell Biology
International Symposium on Ran and the Cell Cycle
International Interdisciplinary Conference on Vitamins, Coenzymes,
and Biofactors 2005

11

PartⅠ
Reports from the Recipients of Novartis
Research Grants (Fiscal Year 2004)

121) Biological Sciences1-1) Molecular Biology

Molecular Design for Bioactive Small Compounds based on
Phage-displayed Combinatorial Peptide Libraries
Ikuo Fujii
Department of Biological Science, Osaka Prefecture University
fujii@b.s.osakafu-u.ac.jp

Introduction
Combinatorial peptide libraries are widely recognized as useful sources for screening bioactive
ligands that bind to receptors and enzymes. However, the selected small peptides generally possess
considerable conformational flexibility, so the entropic costs of binding to target molecules are high.
In addition, the flexibility does not allow one to determine the precise three-dimensional orientationof the side-chains, which impedes the structure-based design of the mimetic. A solution to these
problems is to construct conformationally constrained peptide libraries. Therefore, we propose
designing structured peptides as the library scaffolds and incorporating randomized peptide
sequences into them.

Results
In this work, we attempt to construct a peptide library on a scaffold of a de novo designed
helix-loop-helix (AELHALEHELAALE-(G7)-KLAALKAKLAALKA:1)1). Since the scaffold folds
by virtue of the interactions between the amino acid residues positioned inside the helix-loop-helix,
the solvent-exposed, outside residues (X24, X25, X28, X31, and X32) would be randomized to give alibrary of helix-loop-helix peptides2). A peptide library of helix-loop-helix peptides was displayedon the major coat protein of M13 filamentous phage by a modification of the pComb8 system. We
obtained a phage library with the size of 1.5 X 106, which was screened against the CRH region of
G-CSF receptor.
After ten rounds of biopanning, sixteen clones were randomly selected and subjected tonucleotide sequencing. Of the sixteen, nine clones were found to posses the same sequence. The
obtained peptide (2) was prepared as a soluble form by the solid-phase synthetic method andexamined for its structural and binding properties. In surface plasmon resonance (SPR) assay,
peptide 2 was found to bind to the CRH region with a Kd of 150 mM and the CD spectra suggested
α-helical potency of peptide 2. To define the correlation between the folding and the binding affinity,
we examined point-mutations of the peptide to improve the α-helical potential. Since proline wasconsidered to destroy α-helical structures, a Pro at the randomized position 31 in peptide 2 was13replaced with alanine to give peptide 4. In addition to the Pro31, two histidine residues (His4 andHis8) on the N-terminal helix were substituted with alanine to afford peptide 5. In the CD spectra,
peptide 4 exhibited an increasing α-helical content and peptide 5 showed higher α-helical content
than that of the peptide 4. As expected, peptides 4 and 5 showed Kd values of 28.0 µM and 3.8 µM,
respectively. Thus, there was a parallel correlation between folding of the peptides into thehelix-loop-helix and binding affinity against the CRH region.
In addition, the ability of the peptides to inhibit G-CSF-dependent cell proliferation was
examined with NFS-60 cell lines. Cells were incubated with various concentrations of peptide in thepresence of 17 pM of G-CSF; native G-CSF stimulates this cell line to proliferate with half maximal
response occurring at 5 to 10 pM. The cell proliferation rate was monitored with MTT assay.
Scaffold 1 was inactive at concentrations up to 100 mM, whereas peptide 4 and 5 showed inhibitory
activity in a dose-dependent manner (4: ED50 = 79.1 mM, 5: ED50 = 25.0 mM).
The constrained conformation of the peptides suggested a precise three-dimensional orientation of
the pharmacophore (Arg, Glu, Lys, and Leu) in the interaction between the peptides and the receptor.
Base on 3D information, we designed biphenyi sulfonamide derivatives, which possess thefunctional groups corresponding to the side chains of the active amino acids. In the SPR assay, thesmall compounds were found to be active against G-CSF receptor.

Discussion & Summary
In this work, we have attempted to construct a library of helix-loop-helix peptides

on M13filamentous phage. The library was screened against mouse G-CSF receptor [4], and a phage clonethat bound to the target was selected. Mutations of the selected peptide to increase the a-helicalcontent reflecting increased the stability of the two-helix template led to concomitant increases inbinding affinity for the receptor, providing variants with Kds ranging from 150 to 3.8 µM. The
receptor-binding assay with NMR showed the competitive binding of the peptide against the native
G-CSF, consisting with the antagonist activity observed in MTT assay. These studies can be used toshrink the cytokine to moieties more amenable to small molecule drug design.

14

Figures&Tables
Molecular Design for Bioactive Small Compounds based on Phage-displayed Combinatorial
Peptied Libraies

References
1) Suzuki, N & Fujii, I., (1999) Tetrahedron Lett. 40, 6013-6017
2) Fujii, I., Takaoka, Y., Suzuki, K. & Tanaka, T., (2001)Tetrahedron Lett. 42, 3323-3325

151) Biological Sciences1-1) Molecular Biology

Molecular mechanism of class switch recombination and somatic
hypermutation induced by AID
Reiko Shinkura
Immunology and Genomic Medicine, Kyoto University, Graduate School of Medicine
shinkura@mfour.med.kyoto-u.ac.jp

Introduction
Activation-induced cytidine deaminase (AID) is the essential molecule for two distinct events on
immunoglobulin genes, class switch recombination (CSR) and somatic hypermutation (SHM).
CSR is a region-specific recombination that takes place between switch (S) regions.
introduces point mutations at high frequency in the variable (V) region.

SHM

We are asking twoquestions; what is the target of AID, and how AID regulates differentially CSR and SHM.

Results
1. What is the target of AID?
AID is important for the initiation of DNA cleavage in S region.
DNA cleavage is still controversial.

However, how AID initiates

All other researchers except for us believe that AID

deaminates cytocine on S region DNA directly, resulting in DNA double strand breakage incollaboration with UNG and mismatch repair enzymes.

On the other hand, we think that AID is an

RNA editing enzyme like Apobec-1, which edits mRNA to make the transcript encoding theactivated endonuclease, resulting in DNA cleavage.

To check whether AID has cytidine

deaminase activity on RNA or DNA, we first purified the recombinant AID from E. coli and tried
cytidine deaminase assay on single-stranded DNA in vitro. However, purified AID from E. coli
did not show the cytidine deaminase activity on single-stranded DNA although crude cell lysate
always showed cytidine deaminase activity.

Furthermore, we observed that cytidine deaminase

activity on single-stranded DNA with crude cell extract even from so-called cytidine
deaminase-deficient strain. We conclude that E. coli system is not good for deaminase assay
because of contaminated activity from E coli itself.

Then we tried the in vitrotranscription/translation synthesis of AID protein using wheat-germ extract and purified AID byaffinity purification. In vitro synthesized AID showed the cytidine deaminase activity on
single-stranded DNA, whereas the catalytic domain-deficient AID mutant did not. It indicates thatin vitro synthesized AID has cytidine deaminase activity on DNA. Next, using this AID protein, we16

are going to set up RNA editing assay.
2. How does AID regulate differentially CSR and SHM?
We have shown that mutants with alterations in the C-terminal region of AID retain normal SHM
activity but almost completely lose CSR activity, indicating an essential function for the C-terminal
region of AID in CSR.

In addition, we also found that mutations in the N-terminal region of AID

resulted in specific loss of SHM activity but retained CSR activity. Those mutations do not locate
in the cytidine deaminase motif of AID. From these results, we hypothesize that the CSR and SHM
activities of AID are regulated differentially through the interaction of CSR- or SHM-specificcofactors with C-terminus or N-terminus of AID, respectively.

These mutants are useful toinvestigate the molecular mechanism of CSR and SHM not only in vitro but also in vivo. In
physiological condition, upon antigen stimulation, B cells usually undergo both CSR and SHM.
Therefore it is difficult to observe the effect of CSR and SHM separately in view of immune
surveillance for whole body.

For in vivo study, we will generate the AID knock-in mice thoseexpress the N- or C-terminal AID mutants instead of wild-type AID.
been established.

Knocked-in ES cells haveThose ES cells will be injected into blastcyst to make chimera.

Discussion & Summary
Several groups have reported that AID purified from E. coli showed cytidine deaminase activity
on single-stranded DNA. However, we speculate that they might observe the contaminated activity
from

host

E.

coli.

Thereforetranscription/translation system.

wesynthesizedandpurified

AID

using

in

vitro

In vitro synthesized AID showed cytidine deaminase activity on

single-stranded DNA, whereas deaminase-deficient mutant did not.

We are going to check the

RNA editing activity of AID using in vitro synthesized protein.
We have shown that C-terminal mutations of AID cause loss of CSR specifically, whereas
N-terminal mutations of AID show SHM-specific defect.

We propose that CSR and SHM activities

of AID are regulated differentially through the interaction of CSR- or SHM-specific cofactors with
C-terminus or N-terminus of AID, respectively.
phenotype in vivo, we will make knocked-in mice.

To check if these mutants show any pathological17

Figures&Tables

References
Shinkura, R. et al., Separate domains of AID are required for somatic hypermutation andclass-switch recombination.

Nature Immunology, 5, 707-712, 2004.

Honjo, T. et al., AID to overcome the limitations of genomic information.
Nature Immunology, 6,655-661, 2005.
Begum, NA. et al., Uracil DNA glycosylase activity is dispensable for immunoglobulin class-switch.
Science, 305, 1160-1163, 2004.

181) Biological Sciences1-1) Molecular Biology

Mechanisms of transcriptional and posttranscriptional gene silencing
by small RNA molecules
Mikiko C. Siomi
Institute for Genome Research, University of Tokushimasiomim@genome.tokushima-u.ac.jp

Introduction
RNAi triggers cleavage of complementary mRNAs, which in turn results in gene silencing at the
posttranscriptional level. Small dsRNA functioning in the RNAi pathway is known as siRNA. Workover the past several years has revealed that several factors such as AGO2 are necessary for RNAiand that small RNAs called miRNA also trigger posttranscriptional gene silencing. However, all thedetails of the RNAi/miRNA pathways are still not well understood. One of our ultimate goals is tounderstand how gene silencing via small RNAs (siRNA, miRNA and others) occurs at the molecularlevels.

Results
Functional analyses of AGO family members in fly
We showed that in Drosophila both AGO1 and AGO2 possess the Slicer activity (the activity tocleave the target mRNA). Slicer activity was reconstituted with recombinant full-length AGO1.
Further, reconstitution of Slicer activity with recombinant PIWI domains of AGO1 and AGO2
demonstrates that other regions in the Argonautes are not strictly necessary for siRNA-binding andcleavage activities. In circumstances with AGO2-lacking, the siRNA duplex is not unwound andconsequently RNA-induced silencing complex (RISC) is not formed. We show that upon addition ofsiRNA duplex in S2 lysate, the passenger strand is cleaved in an AGO2-dependent manner and that
nuclease-resistant modification of the passenger strand impairs RISC formation. These findings giverise to a new model in which AGO2 is directly involved in RISC formation as “Slicer” of thepassenger strand of the siRNA duplex.
In Drosophila, Piwi encodes a protein of the Argonaute family, which is essential for the germ
stem cell self-renewal. We show that Piwi immunopurified from ovary specifically associates withsmall RNAs (25 to 29 nt in length). It was found by sequencing that the Piwi-associated small RNAs
are rasiRNAs (repeat-associated siRNAs), mainly derived from the retrotransposon transcripts. We
are currently investigating how Piwi-associated with rasiRNA mechanistically regulates genesilencing of the target RNA.

19

Revealing the molecular mechanism of the miRNA processing pathway
miRNAs are a large family of 21 to 23 nt non-coding RNAs that interact with target mRNAs at
specific sites to induce cleavage of the targets or inhibit the translation. miRNAs are excised in a
stepwise process from pri-miRNA transcripts. The Drosha-Pasha complex cleaves pri-miRNA torelease hairpin-shaped pre-miRNA. The pre-miRNAs are further processed by Dicer to mature
miRNAs. We showed that Drosophila Dicer1 interacts with Loquacious, a dsRNA-binding protein.
This data and others support a model in which Loquacious with Dicer1 mediates miRNA biogenesis
and thus the expression of gene regulation by miRNAs. Lately, we found that in the miRNAbiogenesis pahway, ATP hydrolysis is required. It might be that the unwinding process ofmiRNA/miRNA* duplex requires the reaction. We are currently investigating what factor(s) are
involved particularly in the unwinding process.

Discussion & Summary
Once we could identify the miRNA unwinding factor(s), it means that we do understand throughout
the miRNA biogenesis pathway starting from the pri-miRNA synthesis to mature miRNA loading
onto AGO1. We are hoping that it happens soon as we expect. Regarding AGO family members infly, we still do not know much about functional contributions of Aubergine and AGO3 to genesilencing mechanisms. Hopefully soon, though, we could identify small RNAs associated with themas we have done for Piwi; then, we will be able to get some idea of how they are mechanisticallyinvolved in the gene silencing pathways in Drosophila.

20

References
Miyoshi K, Tsukumo H, Nagami T, Siomi H, Siomi MC.
Slicer function of Drosophila Argonautes and its involvement in RISC formation. Genes Dev. 2005.
19:2837-2848.
Saito K, Ishizuka A, Siomi H, Siomi MC.
Processing of pre-microRNAs by the Dicer-1-Loquacious complex in Drosophila cells. PLoS Biol.
2005. 3(7): e235.

211) Biological Sciences1-1) Molecular Biology

Analysis of HSF1-mediated cell death and alive signaling pathways
Akira Nakai
Yamaguchi University School of Medicine
anakai@yamaguchi-u.ac.jp

Introduction
Heat shock response is an adoptive response to proteotoxic stress, and a major transcription factor
1 (HSF1) has been believed to protect cells from cell death by inducing heat shock proteins (Hsps)
that assist protein folding and prevent protein denaturation. However, it revealed recently that HSF1
also promotes cell death of male germ cells exposed to thermal stress. Recently, we found aproapoptotic Tdag51 (T-cell death associated gene 51) gene as a direct target gene of HSF1.
Therefore, here, we will reveal a novel HSF1-mediated death pathway.

Results(1) HSF1 activates a pro-apoptotic geneWe found that a profile of time-dependent accumulation of Tgad51 mRNA was similar to thoseof Hsp70 when MEF cells were heat-shocked at 42oC. Reporter activity of Tdag51 gene was
induced in the presence of the putative HSE element, but was never induced when the HSE element
was deleted or mutated. Furthermore, gel shift assay showed that an activated HSF1 boundspecifically to the HSE element, but not to a mutated HSE. Moreover, ChIP analysis showed that
HSF1 binds to the Tdag51 gene. These results indicate that HSF1 directly binds to and activatesTdag51 gene in response to heat shock.
(2) Hsps inhibit Tdag51-mediated cell deathOverexpression of GFP-Tdag51 into HeLa cells caused detachment-induced cell death attime-dependent manner. We firstly examined effects of Hsp40 on Tdag51-mediated cell death. We
found that HeLa/Hsp40 cells were more resistant to low levels of Tdag51 than control HeLa cells.
Furthermore, cells expressing high levels of Hsp40 were more resistant to Tdag51 than cells
expressing low levels of Hsp40. HeLa cells overexpressing Hsp70 and Hsp110, but not cells
overexpressing Hsp27, were also resistant to Tdag51.
(3) Hsps bind directly to a PHL domain of Tdag51
We examined direct interaction of Hsps with Tdag51. We found that Hsp110, Hsp70, and Hsp40
bind directly to Tdag51 in vivo, whereas Hsp27 does not. Tdag51 is a member of the PHL domain
family that has a conserved motif of ~100 amino acids, and its C-terminal domain is composed of

22long proline/glutamine- repetitive and proline/histidine-repetitive tracts. We found that Hsp40 binds
to the N-terminal region containing the PHL domain. Furthermore, Hsp70 and Hsp110 also boundto the same regions.
(4) A PHL domain is required for inhibiting cell death activity of C-terminal regions
We determined which regions are responsible for its ability to induce cell death. We found thatcells expressing C-terminal proline/glutamine-rich and proline/histidine- rich tracts died.
Interestingly, cell death induced by the C-terminal region was not inhibited by overexpression of
Hsp40, Hsp70, and Hsp110. These data indicate that the PHL domain negatively regulates cell death
activity of the C-terminal regions through interaction with Hsps.
(5) Tdag51 is uniquely induced by heat shock
To reveal functional relevance of HSF1-Tdag51 pathway in vivo, we focused on male germ cells
that are susceptible to high temperatures. When the testicle was exposed to hot water at 43oC for 15
min, Tdag51 mRNA as well as Hsp70-1 mRNA was induced in wild-type mice. As we reported
previously, Hsp70-1, Hsp110, and Hsp27 mRNAs were hardly detectable in unstressed testes. In
contrast, Tdag51 mRNA was expressed at substantial levels in unstressed germ cells, especiallypachytene spermatocytes. Surprisingly, we found marked induction of Tdag51 mRNA in wild-type
germ cells including pachytene spermatocytes in response to heat shock. In HSF1-null mice,
however, constitutive expression of Tdag51 mRNA was decreased, and its heat shock-induced
expression was significantly impaired.
(6) Tdag51 promotes heat-induced cell death in vivo
We next analyzed the testis of Tdag51-null mice. Tdag51-null mice were fertile and the testis wasmorphologically normal. We examined apoptotic cells when the testicles were heat-shocked at 43oC
for 15 min, and found less numbers of apoptotic germ cells in Tdag51-null mice than that inwild-type mice. These data demonstrated an important role of HSF1-Tdag51 pathway on germ cell
death in vivo, and suggest that a proapoptotic Tdag51 functionally dominates antiapoptotic geneproducts such as Hsps in heat-shocked germ cells.

Discussion & Summary
These observations revealed a novel mechanism how HSF1 manages against proteotoxic stress.
Cell fate on proteotoxic condition is determined at least by balance between Hsp and Tdag51 levels,
which are differently regulated by HSF1 (Figure 1). HSF1 not only protect cell death, but also
promotes it depending on cell types.

Analogous to p53, HSF1 is a major factor that senses

proteotixic stress and transduces it to expression of proapoptotic and antiapoptotic genes depending
on cell types (Figure 2). Thus, the HSF1-mediated pathway may be one of quality control
mechanisms not only in male germ cells, but also in other somatic cells.

23

24

References
1) M. Fujimoto, E. Takaki, T. Hayashi, Y. Kitaura, Y. Tanaka, S. Inouye, and A. Nakai. Active HSF1
significantly suppresses polyglutamine aggregate formation in cellular and mouse models. J. Biol.
Chem. 280: 34908-34916, 2005.
2) T. Mikuriya, K. Sugahara, T. Takemoto, K. Tanaka, K. Takeno, H. Shimogori, A. Nakai, and H.
Yamashita. Geranylgeranylacetone, a heat shock protein inducer, prevents acoustic injury in theguinea pig. Brain Res. 1065: 107-114, 2005.
3) V. Hietakangas, J. Anckar, H. Blomster, M. Fujimoto, J. J. Palvimo, A. Nakai, and L. Sistonen.
PDSM, a motif for phosphorylation-dependent SUMO modification. Proc. Natl. Acad. Sci. USA
103: 45-50, 2006.
4) E. Takaki, M. Fujimoto, K. Sugahara, T. Nakahari, S. Yonemura, Y. Tanaka, N. Hayashida, S.
Inouye, T. Takemoto, H. Yamashita and A. Nakai. Maintenance of olfactory neurogenesis requires
HSF1, a major heat shock transcription factor in mice. J. Biol. Chem. 281: 4931-4937, 2006

25

1) Biological Sciences1-1) Molecular Biology

Visualization of demyelinating neuropathic pain
by lysophosphatidic acid
MAKOTO INOUE
Nagasaki University
minoue@net.nagasaki-u.ac.jp

IntroductionMore recently, we have found the critical role for receptor-mediated LPA signaling in the initiationof neuropathic pain (Inoue et al., Nature Medicine, 10,712-718, 2004). However, we are still poorly
understood LPA synthesis and the relationship between demyelination and allodynia. In this research,I tried to be find following three topics. 1) The analysis of LPA synthesis. 2) The analysis ofmechanism of demyelination. 3) The visualization of allodynia and analysis of its mechanism.

Results
1) The analysis of LPA synthesis.
After intrathecal injection of antisense oligodeoxynucleotide (AS-ODN) for LPA synthetic enzyme,
nerve injury-induced nociceptive hyperalgesia and allodynia were abolished.
2) The analysis of mechanism of demyelination.
We carried out the in vitro explant culture of dorsal root.. In this model, LPA induced thedemyelinnation, and the decrease of myelinated protein mRNA and protein expression.
Furthermore, LPA-induced these events were completely abolished by Rho A inhibitor, but notother inhibitors.
3) The visualization of allodynia and analysis of its mechanism.
We developed the behavioral test, which we can detect specific primary afferent stimuli (each C,
Adelta, Abeta)-induced responses in nerve-injured mice (please see the reference).
In nerve-injured mice, both Adelta and Abeta-stimuli-induced responses were supersensitized,
whereas C-fiber stimuli-induced response was decreased.
In nerve injured mice, Adelta stimuli-induced signaling in spinal cord appeared in novel region,
which may be activate by Adelta-stimuli.

26

Discussion & Summary
From 1) experiments, we concluded that LPA is synthesized after nerve injury. Now we also carriedout the measure of LPA content in CSF following nerve injury.
From 2) experiments, we concluded that LPA induces the demyelination and the decrease of myelin
protein expression directly, but not through other molecules. And these signaling are mediatedthrough LPA1-G12/13 protein-RhoA pathway, but not Gi or Gq protein pathway, which are otherdownstream pathway of LPA1 receptor activation.
From 3) experiments, we concluded that both Abeta and Adelta fibers-induced signaling are mixed
after nerve injury. This may be related to the development of allodynia.

References
Matsumoto M, Inoue M, et al., Characterization of three different sensory fibers by use of neonatal
capsaicin treatment, spinal antagonism and a novel electrical stimulation-induced paw flexion test.
Molecular Pain, 2, 16, 2006

27

1) Biological Sciences1-1) Molecular Biology

Molecular mechanism of axonal guidance of the injured
central nervous system
Toshihide Yamashita
Chiba University
t-yamashita@faculty.chiba-u.jp

Introduction
We hypothesized that regenerating neurons can be navigated by guidance molecules that include
myelin-derived inhibitors of axon growth. They may change their responsiveness to the guidance
molecules, which allow regenerating neurons to make complex network. While neurons are normally
repulsive to myelin-associated glycoprotein, they are attracted by it if the neurons are priorly
exposed to neurotrophins. This phenomenon is designated neurotrophin priming and is a good
example for intrinsic mechanisms of neuronal navigations. Through exploring the underlying
molecular mechanisms of neurotrophins priming, we would be able to establish therapeuticstrategies to make correct axonal and dendritic connections.

Results
First, we cultured the cerebellar neurons from p7 rat, and treated them with brain derived
neurotrophic factor for 24 hours. We then screened the cDNAs using microarray techniques, andfound that more than several genes were upregulated after exposure to neurotrophins in the postnatal
cerebellar neurons in vitro. The enhanced expression of these genes was confirmed by real time
RT-PCR as well as Northern blot analysis. Time course experiments show that the expression ofsome of these genes was upregulated gradually at 2 to 6 hours after exposure to the neurotrophin. To
explore possible involvement of these genes in regeneration of the injured axons in the central
nervous system, we next determined the expression of these genes in vivo after the spinal cord injury.
Among the candidate genes, two genes were upregulated around the site of injury after spinal cord
injury. They turned out to be BMP-2 and Wnt4. BPM-2 was intensively expressed in reactive
astrocytes around the epicenter of the lesioned site after injury. Wnt4 was also expressed specificallyin the reactive astrocytes after the injury. These interesting observations suggest that the reactive
astrocytes as well as oligodendrocytes could be chemical barrier that inhibit regeneration of the
injured axons in the spinal cord. To address if these proteins work as inhibitors of axon growth invitro, we performed neurite outgrowth assay using the postnatal cerebellar neurons. Cultured
neurons were incubated in vitro for 24 hours in the presence or absence of BMP-2 or Wnt4. Both

28

proteins significantly inhibited neurite outgrowth compared to the control. Co-incubation of Y-27632,
a Rho-kinase inhibitor, completely abolished the effect of Wnt4, but not that of BMP-2. As expected,
Wnt4 induced activation of RhoA in the neurons. These results demonstrate that Wnt4 inhibits
neurite outgrowth by activating RhoA and Rho-kinase. Wnt family proteins comprise of several
members, and we determined the expression of these proteins in the spinal cord after injury. Wnt3 aswell as Wnt5a was expressed in the reactive astrocyes after injury. Thus, multiple members of Wnts
work together on the neurons. We further found that neutralizing antibody to Ryk, one of thereceptors for Wnts, totally abolished the effect of Wnt4, Wnt3 and Wnt5a. So, the inhibitory effecton the neurons is dependent on Wnt binding to Ryk. Interestingly, the effect of BMP-2 was not
mediated by activation of RhoA or Rho-kinase. Additional experiments demonstrate that the effect ofBMP-2 was dependent on the activity of LIM kinase, as the dominant negative form of LIM kinasesilenced the effect of BMP-2. Indeed, BMP-2 induced activation of LIM kinase in vitro. Although
BMP-2 also activated the Smad signal, transfection of inhibitory Smad did not modulate the effect ofBMP-2 on the neurite outgrowth. In addition, BMP-2 did not activate p38MAPK, though previous
reports showed that BMP-2 activated this signal in some cells. In addition, we found that theneutralizing antibody to RGM reduced the effect of BMP-2. As RGM and the LIM kinase receptors
form a receptor complex that transduces the signal of BMP-2. This antibody offers an effective
blocker of the BMP signal. Our findings, therefore, identified new signals involved in axon
regeneration inhibition (Figure).

29

Discussion & Summary
So far, several proteins were identified to inhibit axon outgrowth at least in vitro. Among them, threemolecules

expressedinoligodendrocytes,

myelin-associatedglycoprotein,

Nogo

and

oligodendrocyte myelin glycoprotein, have been extensively studied. All these proteins inhibit
neurite outgrowth from some neurons in vitro by activating RhoA and Rho-kinase. However, recent
studies show that inhibiting each molecule or the receptors for these molecules resulted in no or little
regeneration of the injured axons or no functional recovery. These findings suggest the presence ofother inhibitory molecules. We reported recently that RGM is another inhibitor of axon regeneration(Hata et al., 2006). Inhibition of RGM promoted massive axon regeneration and functional recovery.
RGM inhibits axon regeneration by activating RhoA and Rho-kinase, common signals of axon
regeneration. Our present study shows that two other proteins are implicated in inability of axon
regeneration in the central nervous system. Importantly, the effect of BMP-2 was not dependent on
RhoA or Rho-kinase, but was mediated by LIM kinase. These findings provide new moleculartargets to treat patients with injuries to the central nervous system.

References(Hata, K., Fujitani, M., Yasuda, Y., Doya, H., Saito, T., Yamagishi, S., Mueller, B.K. and Yamashita,
T. (2006) RGMa inhibition promotes axonal growth and recovery after spinal cord injury. J. Cell
Biol. 173, 47-58.

301) Biological Sciences1-1) Molecular Biology

Analysis of the new death receptor signal for the treatmentof cancers and immune diseases
Tadaaki Miyazaki
Department of Bioresources,Hokkaido University Research Center for Zoonosis Control
miyazaki@czc.hokudai.ac.jp

Introduction
DR6(death receptor 6) is the newest member of death receptor family(Pan et al., 1998). Disruptionof DR6 gene in mice caused a reduced apoptosis and hyper-proliferation of both T and B cells(Liu etal., 2001; Schmidt et al., 2003; Zhao et al., 2001). It is suggested that DR6 has important roles forregulating activation of peripheral T and B cells. On the other hands, DR6 gene disruption also
caused production of Th2 type cytokine production such as IL-4, but not Th1 type cytokine
production such as IFN-gamma, (Liu et al., 2001; Zhao et al., 2001). These findings suggested that
DR6 is one of critical factors for the regulation of Th balance. Interestingly, unlike other death
receptors, DR6 does not interact with FADD, RIP1 or RAIDD and over-expression of dominantnegative form of FADD failed to inhibit DR6-mediated apoptosis(Kasof et al., 2001; Pan et al.,1998). These data suggest that there is a unique and unknown mechanism in DR6-mediateddeath-signaling pathway.

Results
Identification of CLIPR-59 as a novel binding protein to the cytoplasmic region of DR6
To examine the signaling pathway mediated by DR6, we screened the molecules which bind to
DR6. By the yeast two-hybrid (Y2H) screening using the cytoplasmic domain of DR6 as a bait, we
identified 111 positive clones. It should be noted that among these genes, 28 independent clones
encoded the same region of CLIPR-59 (cytoplasmic linker protein-170, CLIP-170 related protein
59kDa), from amino acids 387 to 547. To clarify the specificity of the interaction of CLIPR-59 with
DR6 and CLIPR-59 binding region within DR6, the following Y2H and immunoprecipitation assay
were carried out using mutants of DR6. We showed that the region from amino acids 370 to 428 of
DR6 is necessary for its interaction with CLIPR-59.
CLIPR-59 induces apoptosis and inhibits NF-kB activation in DR6 signal
Since DR6 is known to induce apoptosis and the regulation of NF-kB activity, we examined the31functional roles of CLIPR-59 on DR6-mediated apoptosis and NF-kB activation. In NF-kB
dependent reporter gene assay, we found that the expression of CLIPR-59 could inhibit the
DR6-mediated NF-kB activation but CLIPR-59 delta C, lacking DR6-binding region (Figure 1D),
could not (Figure 2A). Contrary to the inhibition of NF-kB activation, CLIPR-59 significantly
augmented DR6-mediated apoptosis and this effect was not seen in CLIPR-59 delta C (Figure 2B).
These data suggested that CLIPR-59 could play negative roles for NF-kB activation and positiveroles for apoptosis in DR6 signaling. Next, we examined whether the interaction of DR6 withendogenous CLIPR-59 is involved in DR6-mediated cell death, the cell viability assay was carriedout using DR6WT or delta CLR. Expression of DR6WT significantly reduced the cell viability. On
the other hands, absence of CLR of DR6 partially but significantly abrogated this effect (Figure 2C).
In addition, in NF-kB dependent reporter gene assay, DR6WT induced NF-kB activation anddeletion of CLR of DR6 further augmented NF-kB activation (Figure 2D).

Discussion & Summary
CLIPR-59 was originally identified as a member of a family of proteins related to CLIP-170, which
is a cytoplasmic linker protein mediating interactions of organelles with microtubules(Perez et al.,2002).

CLIPR-59 consists of 547 amino acids, and several predicted protein interacting motifs,

including proline and glutamic acid rich region, ankyrin like repeats, two cytoskeleton-associatedprotein glycine-rich (CAP-Gly) domain and serine rich region (Perez et al., 2002). It was previously
shown that CAP-Gly domain of CLIPR-59 is responsible for its interaction with microtubule, andthis association was inhibited by its amino-terminal region (Lallemand-Breitenbach et al., 2004;
Perez et al., 2002), indicating that conformational change of CLIPR-59 is involved in the process of
association with microtubules. We showed that presence of amino-terminal region of CLIPR-59 did
not affect its binding to DR6. Our results suggested that the underlying mechanisms for theinteraction between DR6/CLIPR-59 and CLIPR-59 and microtubules are quite distinct. It should bepointed that DR6 mutant, lacking the region from amino acids 461 to 655 was unable to induce
apoptosis and mediate NF-kB activation (Pan et al., 1998).
domain (DD) and still possesses CLR.

Nevertheless, this mutant lacks deathThus, it is likely that primary region for mediating apoptosis

and NK-kB activation exists in the region from DD to the carboxy-terminal end of DR6.

CLR of

DR6 should play a positive role for apoptosis and negative role for NF-kB activation in DR6
signaling by interacting with CLIPR-59.
DR6 regulates the process of apoptosis and interacts with TRADD but not with FADD, RIP1 or
RAIDD, suggesting that there might be a unique DR6-mediated signaling pathway, which leads toapoptosis. Here, we showed a novel mode of action for DR6-mediated signaling pathway.
We identified CLIPR-59 as a novel DR6-binding molecule. CLIPR-59 is a novel signal transducer toinhibit DR6-mediated NF-kB activation and induce apoptosis.

32

Figure 1. CLIPR-59 interacts with DR6.

Amino-acid coordinates of the deletion mutants of

DR6(A) and CLIPR-59(B) are indicated. (C)DR6 wild type but not DCLR mutant could interact
with endogenous CLIPR-59. 293Tcells were transfected with indicated plasmid. At 48hr after
transfection, cell extracts were analysed by immunoblotting (IB) either directly (WCE) or after
immunoprecipitation with indicated antibodies (IP). (D)CLIPR-59 wild type but not DC mutant
could interact with DR6. 293T cells were transfected with the combinations of empty vector (-) andindicated plasmid, and analysed as shown in C.

33

Figure 2. CLIPR-59 could inhibit NF-kB activation and induce apoptosis in DR6-mediatedsignaling.

(A)Expression of CLIPR-59 could inhibit NF-kB activation in DR6 signaling. 293T

cells were co-transfected with either empty vector (-) or indicated expression plamid and NF-kB
reporter plasmids. NF-kB activation was analysed as shown in Methods. Relative expression of the
DR6 and CLIPR-59 or these mutants is shown in lower panel. (B)CLIPR-59 enhanced
DR6-mediated apoptosis. Cells were transfected with indicated plasmids. At 24hr after transfection,
apoptosis was determined as described in Method. Deletion of CLR on DR6 reduced cell death(C)and enhanced NF-kB activation(D). Hela cells (5x10^4 cells) were seeded in 24well culture plate.
Next day, cells were transfected with indicated plasmid, and at 48hr after transfection, cell viability
assay or luciferase assay were done as described in Methods.

34

References
1. Nitta T, Nasreen M, Seike T, Goji A, Ohigashi I, Miyazaki T, Ohta T, Kanno M, and Yousuke
Takahama Y, IAN family critically regulates survival and development of T lymphocytes “Role of
IAN family in T lymphocyte development”,
PLoS Biology 2006 7;4(4).
2. Masunaga T, Yamashita K, Sakihama H, Hashimoto T, Hua N, Imai A, Inobe M, Miyazaki T,
Todo S, Uede T. Dimeric but not monomeric soluble CD40 prolongs allograft survival and generates
regulatory T cells that inhibit CTL function. Transplantation. 2005 15;80(11):1614-22.
3. Miyazaki T, Shen M, Fujikura D, Tosa N, Kim HR, Kon S, Uede T, Reed JC., Functional role ofdeath associated protein 3 (DAP3) in anoikis. J. Biol. Chem. 2004

22;279(43):44667-72.

4. Watanabe K, Saito K, Kinjo M, Matsuda T, Tamura M, Kon S, Miyazaki T, Uede T. Molecular
dynamics of STAT3 on IL-6 signaling pathway in living cells.
Biochem. Biophys. Re.s Commun. 2004 26;324(4):1264-73.
5. Morimoto J, Inobe M, Kimura C, Kon S, Diao H, Aoki M, Miyazaki T, Denhardt DT, Rittling S,
Uede T., Osteopontin affects the persistence of beta-glucan-induced hepatic granuloma formationand tissue injury through two distinct mechanisms. Int. Immunol. 2004 Mar;16(3):477-88.
Kasof, G.M., Lu, J.J., Liu, D., Speer, B., Mongan, K.N., Gomes, B.C. and Lorenzi, M.V. (2001)
Tumor necrosis factor-alpha induces the expression of DR6, a member of the TNF receptor
family, through activation of NF-kappaB. Oncogene, 20, 7965-7975.
Liu, J., Na, S., Glasebrook, A., Fox, N., Solenberg, P.J., Zhang, Q., Song, H.Y. and Yang, D.D.
(2001) Enhanced CD4+ T cell proliferation and Th2 cytokine production in DR6-deficientmice. Immunity, 15, 23-34.
Pan, G., Bauer, J.H., Haridas, V., Wang, S., Liu, D., Yu, G., Vincenz, C., Aggarwal, B.B., Ni, J. andDixit, V.M. (1998) Identification and functional characterization of DR6, a novel death
domain-containing TNF receptor. FEBS Lett, 431, 351-356.
Perez, F., Pernet-Gallay, K., Nizak, C., Goodson, H.V., Kreis, T.E. and Goud, B. (2002) CLIPR-59, a
new trans-Golgi/TGN cytoplasmic linker protein belonging to the CLIP-170 family. J Cell
Biol, 156, 631-642.
Schmidt, C.S., Liu, J., Zhang, T., Song, H.Y., Sandusky, G., Mintze, K., Benschop, R.J., Glasebrook,A., Yang, D.D. and Na, S. (2003) Enhanced B cell expansion, survival, and humoral
responses by targeting death receptor 6. J Exp Med, 197, 51-62.
Zhao, H., Yan, M., Wang, H., Erickson, S., Grewal, I.S. and Dixit, V.M. (2001) Impaired c-Junamino terminal kinase activity and T cell differentiation in death receptor 6-deficient mice. J
Exp Med, 194, 1441-1448.

351) Biological Sciences1-1) Molecular Biology

Neuronal cell death and malfunction of intracellular-trafficking
Shinji Hadano
Department of Molecular Neuroscience, The Institute of Medical Sciences, Tokai University
shinji@is.icc.u-tokai.ac.jp

Introduction
ALS2, a causative gene product for a number of juvenile recessive motor neuron diseases (MNDs),
acts as a guanine nucleotide exchange factor (GEF) for small GTPase Rab5 and enhances earlyendosome fusion through the activation of Rab5. Thus, ALS2 may play an important role in thesurvival and maintenance of motor neurons via regulating membrane trafficking. However, detailed
functions of ALS2 are still unclear. The aim of this study was to delineate molecular function of
ALS2 and its interactors, thereby clarifying the molecular mechanisms for neuronal cell dysfunction
and death in the ALS2-linked MNDs.

Results
First, to examine the ALS2 localization in the cells with different developmental stages,
hippocampal neurons were isolated from hippocampi dissected from post neonatal day 1 mouse, andcultured for a different period of time. In stage 3 neurons, ALS2 was distributed to cytoplasm andpatchy membrane structures. ALS2 was also enriched in membrane ruffles at growthcone
colocalizing with F-actin. In stage 5 neurons, ALS2 was localized to endosomes both in dendrites
and in axon, which were partially labelled with Alexa-594 transferrin.
Recently, we have identified a novel ALS2 homolog, ALS2 C-terminal like (ALS2CL), which ishighly homologous to the C-terminal half of ALS2. To delineate the molecular and cellular functionsof ALS2CL, and the functional relationship between ALS2 and ALS2CL in the cells, we next
conducted yeast two-hybrid assays, immunoprecipitation, and gel-filtration analysis. The results
showed that a majority of ALS2CL was present as a homo-dimeric form, which could turn into
interaction with the ALS2-oligomer, resulting in the formation of the large ALS2/ALS2CL
heteromeric complex. Although formation of such heteromeric complex exhibited no significant
impact on their Rab5GEF activity in vitro, overexpression of ALS2CL significantly increased thedetergent-insoluble fraction of ALS2 with concomitant increase of the vesicular colocalization of
ALS2CL with ALS2 in the cells. Further, ALS2CL dominantly suppressed the endosome
enlargement induced by a constitutively active form of ALS2, and induced extensive perinuclear

36

tubulo-membranous phenotypes instead. Collectively, ALS2CL is implicated in the modulation of
ALS2-mediated endosome function in the cells.
Finally, to further investigate the effects of ALS2 absence on cellular function, particularly
receptor-mediated endocytosis in detail, we have prepared primary fibroblasts from new born Als2-/and Als2+/+ mice. Fibroblasts were exposed to Alexa Fluor-488 labeled epidermal growth factor(EGF) for 10 min, allowing the internalization of EGF via receptor-mediated endocytosis, and thenanalyzed at the 10 min, 30 min, and 60 min time points. Internalized EGF forms a punctate patternof vesicles, representing EGF-positive endosome compartments. No gross abnormality was observedat all time points analyzed both in Als2-null and wild-type cells. These results imply that endocytosisof the EGF receptor per se does not require ALS2. However, a quantitative analysis of thefluorescence intensities of the EGF-labeled endosomes/vesicles revealed that frequency of thevesicles with stronger fluorescent signals, thus larger in size, was significantly lower at a 10 min
point in the Als2-null mutants than in the wild-type cells. Notably, signal intensities in the wild-type
cells were gradually decreased thereafter, while those in Als2-null mutants rather increased withhighest at a 30 min point. The results imply that trafficking and fusion of EGF-positiveendosomes/vesicles in fibroblasts were significantly delayed by the lack of ALS2. Thus, ALS2
might control efficiency of vesicles/endosomes trafficking and fusion in the cells.

Discussion & Summary
We here demonstrated the evidences representing a unique endosomal localization of ALS2 inmatured neurons and its positive effect on endosome fusion in the cells. These findings imply thatALS2 might regulate membrane trafficking through the endosome fusion, thereby mediating thesurvival and maintenance of motor neurons. Further, our results suggest that ALS2CL is a novel
ALS2 binding protein and may
play an important role in the
ALS2-mediatedmembrane/endosome trafficking
in the cells (Fig.1).Thus,further
studies on the neuronal functionsof ALS2 and ALS2CL will leadto a better understanding of thepathogenesis for the ALS2-linked
as well as other MNDs.

37

References
1）Suzuki-Utsunomiya K, et al: Amyotroph Lateral Scler Other Motor Neuron Disord 6, 22, 2005.
2）Hadano S and Ikeda JE: Methods in Enzymology vol. 403, 310-321, 2005.
3）Hadano S, et al: Hum Mol Genet 15, 233-250, 2006.

381) Biological Sciences1-1) Molecular Biology

Proteomic Studies of Hepatitis C Virus
Tohru Ichimura
Tokyo Metropolitan University
ichimura@mail.comp.metro-u.ac.jp

Introduction
Hepatitis C virus (HCV) has recently been discovered to be the cause of majority of blood born
non-A and non-B hepatitis. This virus encodes a single polypeptide of 3011 amino acids, which isthen processed into 10 mature structural and regulatory proteins in infected cells. A number of hostfactors that could bind with these virus proteins have already been identified by use of yeast two
hybrid system and GST-pull down assay. However, the molecular mechanism by which the HCV
infection induces pathogenesis in host cells is still largely unknown.

Results
We recently developed a novel tandem affinity purification approach coupled with mass
spectrometry-based proteomics technology to identify novel protein-protein interactions (1). The
purification strategy employs an amino-terminal affinity tag (we call it MEF tag) composed of two
independent epitope tags, myc and FLAG, that are fused in tandem and separated by the spacer
sequence containing a TEV protease cleavage site. A series of immunoprecipitation, cleavage with
TEV protease, and elution with FLAG-peptide allowed us to identify very specific protein-protein
interactions (1-3). Here, we applied the MEF method to isolate novel host factors associated withvirus proteins in human hepatoblastoma HepG2 cells.
We used the adenovirus expression system to express recombinant proteins, because this system
permits suitable expression of exogenous proteins in most mammalian cells. The MEF cassette was
fused to the N terminus of HCV structural and regulatory proteins (core, NS3, NS5A and NS5B)(Fig.
1), and the MEF-core, -NS3, -NS5 or –NS5B cDNA was inserted into the multicloning sites in an
adenovirus expression vector. HepG2 cells were then transformed with recombinant adenovirus toexpress each MEF-fused protein. Immunoblotting using anti-myc monoclonal antibody
demonstrated that all the fused proteins were expressed in HepG2 cell with relatively high amounts(data not shown).

After the HepG2 cells expressing MEF-core were lysed, the MEF-core wasrecovered with its binding partners by the procedures of MEF method (1). Ultimately, the protein
complexes bound to FLAG beads were dissociated with a synthetic FLAG peptide.

SDS-PAGE

39

analysis revealed that several host factors including E6AP, an E3 ubiqutin ligase that had previously
been identified as a core binding partner in human kidney Hek293 cells, were specifically interactedwith the expressed MEF-core in HepG2 cells (Fig. 2, marked).

This procedure was also similarly useful to characterize the binding partners for NS3 and NS5B
in HepG2 and Hek293 cells (data not shown), suggesting that the expression system using
adenovirus and MEF cassette is suitable for the functional proteomic analysis of HCV virus proteins.
In a separate study, we further characterized the previously identified interaction between HCVcore protein and E6AP.

We confirmed that E6AP directly binds HCV core both in vivo and in vitro.

Immunofluorescence study revealed that HCV core and E6AP are predominantly colocalized incytoplasm. Exogenous expression of E6AP promoted proteasomal degradation of the core protein in
hepatic cells and in non-hepatic cells. Knockdown of endogenous E6AP by siRNA resulted inincreasing the levels of HCV core protein. Furthermore, in vivo and in vitro ubiquitylation assays
revealed that HCV core protein is polyubiquitylated by E6AP. Taken together, these results suggestthat E6AP functions as the E3 ubiquitin-protein ligase that mediates ubiquitylation and degradationof HCV core protein in vivo.

Discussion & Summary
In summary, our results identified E6AP as a novel HCV core binding partner in both Hek293 cells
and HepG2 cells. We also confirmed that HCV core protein directly interacts with E6AP in vitro.
HCV core and E6AP are found to colocalize in the cytoplasm. We further suggested that E6AP
mediates ubiquitylation and degradation of HCV core protein. From these results, we propose thatthe abundance of HCV core protein is regulated by E6AP-mediated ubiquitin proteasome pathway.
Further studies will help to understand new targets of HCV pathogenesis in hepatoblastoma cells.

40

References(1) Ichimura, T., Yamamura, T., Sasamoto, K., Tominaga, Y., Taoka, M., Kakiuchi, K., Shinkawa,T., Takahashi, N., Shimada, S. & Isobe, T. (2005) 14-3-3 proteins modulate the expression ofepithelial Na+ channels by phosphorylation-dependent interaction with Nedd4-2 ubiquitin
ligase. J. Biol. Chem. 280, 13187-13194.
(2) Tanaka, Y., Kanai, F., Ichimura, T., Tateishi, K., Asaoka, Y., Guleng, Y., Jazag, A., Ohta, M.,
Imamura, J., Ikenoue, T., Ijichi, H., Kawabe, T., Isobe, T., & Omata. M. (2006) The hepatitis B
virus X protein enhances AP-1 activation through interaction with Jab1. Oncogene 25,
633-642.
(3) Tsuda, Y., Mori, Y., Abe, T., Yamashita, T., Okamoto, T., Ichimura, T., Moriishi, K. & Matsuura,
Y. (2006) Nucleolar protein b23 interacts with Japanese encephalitis virus core protein andparticipates in viral replication. Microbiol. Immunol. 50, 225-234.

411) Biological Sciences1-1) Molecular Biology

Analysis of physiological roles of catecholamines and vasopressin on
fat and carbohydrate metabolism in insulin resistance, obesity anddiabetes.
Akito Tanoue
National Research Institute for Child Health and Development
atanoue@nch.go.jp

Introduction
Catecholamines and vasopressin play critical roles in cardiovascular regulation and endocrine
systems. In addition, they are involved in regulating carbohydrate and fat metabolism via insulin
secretion from pancreas or glycogenolysis in liver. In order to investigate the functional roles ofcatecholamines and vasopressin on carbohydrate metabolism as well as in developing obesity,mutant mice lacking or overexpressing alpha 1 adrenergic receptor or vasopressin receptor are
generated and assessed in vivo.

Results
We found that mutant mice lacking V1a vasopressin receptor have higher blood glucose level during
feeding compared to wild type mice. By analyzing with GTT and ITT, the mutant mice showeddecreased insulin sensitivity. The levels of blood glucose in the mutant mice were 165 and 103
mg/dL at feeding and fasting, respectively, while the levels in control mice were 124 and 83 mg/dL.
On the other hands, fat tissue in the mutant mice was significantly heavier than that in controls,suggesting that the mutant mice could be prone to become diabetic mellitus and obesity. In addition,
during feeding with high fat diet, the mutant mice showed increased weight gain and decreased
insulin sensitivity.

Discussion & Summary
Our study implicated that antagonism for V1a vasopressin receptor could lead to diabetic mellitus
and obesity and specific ligands for the receptor would be useful for treating these disorders.

421) Biological Sciences
1-3） Cell Biology

The molecular mechanism of activity-dependent vesicle transport
regulated by synaptotagmin IV
Mitsunori Fukuda
Fukuda Initiative Research Unit, RIKEN
mnfukuda@brain.riken.go.jp

Introduction
Synaptotagmin (Syt) is a putative membrane trafficking protein that contains an N-terminal single
transmembrane domain and C-terminal tandem C2 domains. Among the Syt family members, Syt
IV has been suggested to control synaptic plasticity, because its mRNA is rapidly increased bymembrane depolarization (or synaptic activity) and Syt IV protein is sorted from the Golgi to the cell
periphery (referred to as “activity-dependent vesicle transport”) (refs. 1-3).

However, the preciserole of Syt IV protein in synaptic plasticity and the cargos of Syt IV-containing vesicles still
completely remains unknown.

The aim of this research is to determine the specific cargo of Syt

IV-containing vesicles

Results
To isolate Syt IV-containing vesicles from membrane fractions of PC12 cells or mouse brain, we
produced rabbit polyclonal antibody against Syt IV, and the specificity of the antibody wasconfirmed by probing with COS-7 cell lysates expressing Syt I-XV as described previously (ref. 1).
Specific antibody against Syt I or Syt IX was obtained from commercial sources.

We coupledpurified antibody against Syt I, IV, or IX with magnetic beads (Dynabeads M-280), and the beads
coupled with either anti-Syt I, IV, or IX antibody was incubated for 1 hr with crude membrane
fractions of PC12 cells or developing mousebrain in 0.03M sucrose, 5 mM HEPES-KOH,
pH 7.2, and 5 mM EDTA (ref. 4).

Afterwashing the beads with PBS containing 2 mM
EDTA, the bound fractions were analyzed by
10% SDS-polyacrylamide gel electrophoresis(PAGE) followed by immunoblotting with
anti-synaptophysin (a marker for synaptic
vesicles), anti-Rab27A (a marker for dense-core43

vesicles), anti-Syt I, anti-Syt IV, and anti-Syt IX antibodies.

We found that Syt IV protein in PC12

cells was not co-purified with Syt I-containing vesicles, Syt IX-containing vesicles, or secretory
vesicle markers (Figure 1), whereas Syt I and Syt IX proteins were co-purified with synaptophysin(i.e., synaptic-like microvesicles) and Rab27A (i.e., dense-core vesicles).

We further showed bysubcellular fractionation study using a 0.15 - 0.9 M sucrose gradient that Syt IV protein was absent
in the Syt I-containing vesicles from mouse brain (i.e., synaptic vesicles) and instead present in themembrane fractions larger or denser than synaptic vesicles.

Our result strongly indicates that Syt

IV-containing vesicles derived from the Golgi are different from conventional synaptic vesicles inbrain or dense-core vesicles in PC12 cells.

Discussion & Summary
In the present study, we have succeeded in isolating Syt IV-containing vesicles from developing
mouse brain or PC12 cells and found that Syt IV protein is not present on conventional secretory
vesicles, where Syt I (or IX) is normally present.

We speculate that Syt IV is present on a novel

type of vesicles that may contain molecules involved in synaptic plasticity or activity-dependentchanges of synapse.

We are now trying to determine the specific cargo molecule(s) of Syt

IV-containing vesicles by two-dimensional electrophoresis combined with tandem Mass
spectroscopy.

Future study will clarify the mechanism of how Syt IV is involved in synaptic

plasticity at the molecular level.

References
1. Ibata, K., Fukuda, M., Hamada, T., Kabayama, H. and Mikoshiba, K. (2000) J. Neurochem. 74,
518-526.
2. Ibata, K., Hashikawa, T., Tsuboi, T., Terakawa, S., Liang, F., Mizutani, A., Fukuda, M. and
Mikoshiba, K. (2002) Neurosci. Res. 43, 401-406.
3. Fukuda, M., Kanno, E., Ogata, Y., Saegusa, C., Kim, T., Peng Loh, Y. and Yamamoto, A. (2003) J.
Biol. Chem. 278, 3220-3226.
4. Fukuda, M. (2006) NeuroReport 17, 179-182.

441) Biological Sciences
1-3） Cell Biology

Relationship between carcinogenesis and the controlof the ribosome and translation
Daita Nadano
Nagoya University
nadano@agr.nagoya-u.ac.jp

Introduction
The cytoplasmic ribosome, a huge RNA-protein complex essential for protein synthesis, has beentreated as a “black box”, especially in the research on higher organisms. The structures, functions,and regulations of ribosomal proteins are largely unknown. In the present study, proteomics
screening has been performed to clarify qualitative changes in the mammalian ribosome.

Results
For the purpose of this study, we selected the translational machinery in the mouse mammary glandas an analytical target, because this tissue repeats a dynamic change in protein synthesis during theperiods of proliferation, differentiation, and a regressive phase, involution. The mammary tissue
samples were collected from female mice on lactation day 10 (L10; time point of large production ofmilk proteins) and 48 hours after a forced weaning (involution day 2, I2; end point of milk protein
production). The polysome fractions wereisolated by biochemical methods such as
sucrose density gradient ultracentrifugation.

highProteins in these fractions were separated by
two-dimensional gel electrophoresis, and the↑electrophoretic patters were compared between
L10 and I2. Most of ribosomal proteins areMW

known to be very basic (isoelectric point > 10).
The radical-free and highly reducing (RFHR)↓

method, which we have used (instead of aconventional method based on the O’Farrell
method) and is specialized for such very basic
proteins, provided good separation of many
protein components in the ribosomes (Fig. 1).

low
low

←

pI

→

high

Fig.1.
Two-dimensional electrophoretogram of ribosomal
proteins from the polysome collected at L10.

45

Proteins were stained with a Coomassie brilliant blue method modified for low-molecular-weightproteins, collected from the gels, digested in gel with trypsin, and subjected to MALDI-TOF mass
spectrometry (including tandem mass spectrometry). Protein spots specific to each time point wereintensively analyzed. Proteins were identified using the Mascot program. Part of the results is shownin Fig. 2. By this comprehensive screening, specific ribosomal proteins have been found to be more
phosphorylated at L10 than at I2, suggesting that the post-translational modifications may be linked
to efficiency of protein synthesis. In addition, Rack1, receptor for activated C-kinase 1, wasidentified at L10 and I2. Immunoblotting using antibodies against phosphorylated ribosomal S6 andRack1 has been a method suitable for confirmation and further analysis of these data. The findings
implicate an intriguing relationship between protein phosphorylation and translational control. We
have also obtained some candidates for new functional components in the ribosomes, analysis ofwhich is in progress in our laboratory.

Fig. 2.
Comparison of protein bands between L10 and I2: qualitative changes in
the components of the ribosome. S6, ribosomal protein S6; RACK1,
receptor for activated C-kinase 1. These two proteins were identified bymass spectrometry.

Discussion & Summary
Proliferation of cancer cells accompanies the upregulation of protein synthesis. Therefore, it is easy
to speculate the enhancement of functions of the ribosome and increased synthesis of the ribosome
itself in these cells. Actually, fragmental data have been obtained to support this hypothesis. In otherwords, it is natural to consider that these mechanisms (for translation and ribosome biogenesis) aregood targets for cancer therapy. The comprehensive analysis of the ribosome will be fundamental
basic knowledge from this therapeutic viewpoint.

46

References
Aoyama, J., Nakayama, Y., Sugiyama, D., Nadano, D., Fukuda, M. N., and Yamaguchi, N.

Apicalcell adhesion molecule, trophinin, localizes to cell surface, cytoplasm and nuclear envelope. FEBS
Lett., 579 (28): 6326-6332, 2005.

47

1) Biological Sciences
1-3） Cell Biology

Studies of the regulatory mechanism in EMT
mediated by the Zn signaling
Susumu Yamashita
Laboratory of Developmental Immunology, Graduate School of Frontier Biosciences,and Graduate School of Medicine, Osaka University, Suita, Osaka, Japansyamasi@molonc.med.osaka-u.ac.jp

Introduction
During embryonic development, organ and tissue regeneration, and cancer progression, epithelial
cell subpopulations actively downregulate cell-cell adhesion systems, and leave their “local
neighborhood” to move into new microenvironments. This regulated phenotypic modulation
including cell-cell dissociation and cytoskeleton remodeling, is called epithelial-mesenchymaltransition (EMT). The Snail/Slug family zinc-finger transcription factors, master regulators of EMT,
are responsible for the transcriptional repression of epithelial genes, and for the transcriptional
activation of mesenchymal genes. The transcriptional activity of Snail is regulated by its intracellularlocation. We previously showed that LIV1, a breast cancer associated zinc transporter protein, isessential for the nuclear localization of Snail, however its precious mechanism is unknown. In thisproject, we try to reveal molecular mechanism of zinc transporter LIV1 mediated zinc signalling,which regulates subcellular location of zinc finger transcription factor Snail and induces EMT.

Results
Zinc is a structural constituent of a great number of proteins, including enzymes in cellular signaling
pathways and transcriptional factors, and it is essential for their biological activity; however, inexcess it is toxic to cells. Thus, the intracellular zinc concentration must be tightly controlled by thezinc importers ZIPs/SLA39s, exporters ZNTs/SLC30s, and binding proteins metallothioneins, tomaintain its homeostasis. In the course of the cell cycle and differentiation, the cellular zinc level isaltered, and zinc accumulates in the nucleus; however, zinc, unlike calcium, has not been thought ofas a cellular signaling molecule. We previously showed that the Stat3-Liv1/Zip6 cascade is criticallyinvolved in the epithelial-mesenchymal transition (EMT) during zebrafish gastrulation, and isrequired for the nuclear localization of Snail1, a master regulator of the EMT. Intriguing questions
are how the membrane protein Liv1/Zip6 moves Snail1 into the nucleus and whether zinc is
involved in this process. Here we show that zinc transduces the signal from the zinc transporters,

48ZIPs, to the zinc-finger transcriptional factor SNAIL1 during EGF-induced EMT in wound healing
and cancer invasion. EGF induces the expression of ZIP10, which leads to the uptake and nuclear
accumulation of zinc. This actively transported zinc is coordinated by the finger domain on SNAIL1
and localizes SNAIL1 to the nucleus to initiate the EMT in keratinocytes and cancer cells. Thus, zinc
is a regulator of the EMT during wound healing and cancer progression, and may act as a second
messenger to transduce extracellular stimuli to zinc metalloproteins via zinc transporters.

Discussion & Summary
Our results established that zinc is a regulator of the EMT during wound healing in vitro and cancer
progression in vitro and in vivo, and suggest that zinc may act as a second messenger transducing
extracellular stimuli to zinc metalloproteins via zinc transporters. Zinc was actively transported byZIPs and coordinated at the zinc–finger domain of SNAIL1, precluding its nuclear export, andresulting in increased levels of SNAIL1 in the nucleus, thereby enabling the induction of EMT.
These results provide a molecular explanation for the zinc transporter ZIP-dependent signaling and
its relevance in the nuclear accumulation of the zinc-finger protein SNAIL that is induced by EGF
stimulation, as well as a conceptual basis for understanding the intracellular zinc signaling used byother zinc metalloproteins.

References
Kagara, N., Yamashita, S., Miyagi, C., Tanaka, N., Fukada, T., Francí, C., Virtanen, I., García de
Herreros, A., Noguchi, S. & Hirano, T. EGF-induced zinc signaling regulates nuclear accumulationof zinc-finger protein SNAIL in epithelial-mesenchymal transition. Submitted

491) Biological Sciences
1-3） Cell Biology

The function of human BIG family proteins in the membranetrafficking between TGN and endosomes
Hye-Won Shin
Graduate school of pharmaceutical sciences, Kyoto University
shin@pharm.kyoto-u.ac.jp

Introduction
Membrane traffic between organelles is mediated by vesicular and tubular intermediates that carry
cargo proteins.

Small GTPases of the ADP-ribosylation factor (ARF) family trigger budding ofcoated vesicles by recruiting coat protein complexes onto organelle membranes (Fig.1). ARF
switches between a GDP-bound (inactive) state and a GTP-bound (active) state capable oftrasmitting signals to its effectors. The exchange of GDP for GTP is catalyzed by guanine nucleotide
exchange factors (GEFs) (1). Among the ARF-GEFs, we have focused on the high molecular weightand brefeldin A (BFA) sensitive BIG family, BIG1 and BIG2, and investigated their function in theintracellular membrane trafficking.

Results
In order to investigate the specific and redundant function of BIG family ARF-GEFs, we set out todeplete BIG1 or BIG2, or both ARF-GEFs by utilizing an RNAi approach. First, we confirmed thespecific knockdown of BIG1 or BIG2, or both by immunoblot analysis of lysate from HeLa cells
subjected to RNAi. In cells subjected to RNAi for BIG1, there was a significant decrease in the levelof BIG1 (70 %) but not in that of BIG2. Reciprocally, RNAi for BIG2 suppressed specifically theexpression of BIG2 (90 %) but not that of BIG1. Cells subjected to RNAi for both ARF-GEFs
showed depletion of both proteins (3).
Consistent with our previous study (2) showing that the expression of catalytically inactive BIG2
mutant induces the membrane tubulation of recycling endosomes, depletion of BIG2, but not that of
BIG1, induces the tubulation of the recycling endosomes but not that of the Golgi complex. On theother hand, depletion of both BIG1 and BIG2, but not each of them, resulted in the disappearance ofAP-1 from the Golgi complex. Consistent with this result, it has been demonstrated that AP-1 isrecruited to the Golgi membrane by active ARF and its recruitment is inhibited by BFA treatment.
Moreover, TGN46, which recycles between TGN and plasma membrane, partially disappeared fromthe TGN suggesting that depletion of BIG1 and BIG2 affects the TGN46 recycling pathway. The

50

depletion of BIG1 and BIG2 did not
significantly affect the localization of other
Golgi marker proteins, such as golgin-245
and GM130, excluding disintegration of theGolgi structure. These results indicate that
BIG2 alone play a role in the integrity ofrecycling endosomes and BIG1 and BIG2function redundantly in the AP-1 recruitment
to the Golgi membrane (Fig.1). Moreover,
BIG1 and BIG2 may function redundantly inthe transport pathway between Golgi and

Fig.1 Intracellular membrane trafficking

plasma membrane via endosomes.
We therefore, have examined whether BIG1 and BIG2 is involved in the anterograde transport
pathway from the Golgi to the plasma membrane and the retrograde transport from the endosomes to
the Golgi complex. To investigate the former pathway, we make use of the temperature sensitive
vesicular somatitis virus glycoprotein (VSVG). In a permissive temperature, VSVG is deliveredfrom the ER to the plasma membrane via the Golgi complex. The VSVG delivery from the Golgi
complex to the plasma membrane, however, was delayed in BIG1 and BIG2 depleting cells, butneither BIG1 nor BIG2 depleting cells (Fig.2). In contrast, the VSVG transport from the ER to theGolgi complex was not affected. Interestingly, we have frequently observed tubular VSVG carriersin BIG1 and BIG2 depleting cells suggesting that ARF dependent budding step seems to be inhibited(Fig. 2). Now we are trying to dissect this phenomena using time lapse video microscope.

Fig.2 Depletion of both BIG1 and BIG2 inhibits the VSVG transport from theGolgi to the plasma membrane. EGFP-VSVG tsO52 was transfected in HeLa
cells pretreated with con, BIG1, BIG2 or BIG1 and BIG2 siRNAs and the cells
were incubated in the non-permissive temperature (42℃) for overnight. Cells
were incubated in 20℃ for 3hrs (0hr, VSVG is localized to the Golgi) in thepresence of cycloheximide and then shifted to the permissive temperature (32℃)
for 1hr (1hr, VSVG is localized to the plasma membrane (PM) except the last
lower panel). Cells were fixed and performed the immunofluorescence analysis.

51

Next, to examine latter pathway, we made use of a chimera protein of CD4-furin which is known
to recycle the Golgi and the plasma membrane via endosomes like TGN46. It has been shown that
AP-1 adaptor complex is required for the retrograde transport of furin from endosomes to the TGN.
The transport of furin from endosomes to the Golgi was also delayed in both BIG1 and BIG2
depleting cells but neither BIG1 nor BIG2 depleting cells.

Discussion & Summary
Here, we have extended our previous studies (2) and made three important findings concerning
BIG1 and BIG2. First, BIG2 but not BIG1 is specifically required for the integrity of recycling
endosomes that is consistent with our previous observations (2). Second, both BIG1 and BIG2
redundantly act on the AP-1 recruitment to the Golgi complex that is an excellent agreement with thedissociation of AP-1 from the Golgi complex upon BFA treatment. Third, BIG1 and BIG2 function
redundantly in the anterograde transport of VSVG as well as the retrograde transport of furin (Fig.1).
Further dissection of the function of BIG1 and BIG2 in these pathways will bring an important key
to understanding how ARF regulates various transport pathways in an appropriate time and place.

References
1. Shin, H.-W. & Nakayama, K. (2004) Guanine Nucleotide-Exchange Factors for Arf GTPases:
Their Diverse Functions in Membrane Traffic. J.Biochem. 136, 761-767
2. Shin, H.-W., Morinaga, N., Noda, M., & Nakayama, K. (2004) BIG2, a Guanine Nucleotide
Exchange Factor for ADP-Ribosylation Factors: Its Localization to Recycling Endosomes and
Implication in the Endosome Integrity. Mol. Biol. Cell

15, 5283-5294

3. Ishizaki, R, Shin, H.-W., Iguchi-Ariga, Sanae M.M., Ariga, Hiroyoshi, and Nakayama, K.
(2006) AMY-1 (Associate of Myc-1) Localization to the trans-Golgi Network throughInteracting with BIG2, a Guanine-Nucleotide Exchange Factor for ADP-Ribosylation Factors.
Genes to Cells (accepted)

521) Biological Sciences1-4） Developmental Biology

Studies on human hereditary disease using medaka as a model
An analysis of pc medaka mutant showing a phenotype
of polycystic kidney disease.
Hisashi Hashimoto
Gene Engineering Division, BioResouce Center, RIKEN
yokoyama@brc.riken.jp

Introduction
Polycystic kidney disease (PKD) is a common hereditary disease, which leads to massive kidney
enlargement by the cystic dilation of renal tubules and chronic renal failure. Various murine modelsof PKD have been generated from naturally occurring or experimentally induced mutants and areused for the understanding of PKD. In medaka, pc (polycystic) mutant is a fish model for this kidney
disorder developing bilateral massive enlargement in adulthood in an autosomal recessive trait.

Results
1) Developmental stage when kidney tubules start to enlarge was examined in pc mutant. In
pronephros, which form during embryogenesis, enlargement of the tubule was observed justafter hatching, consequently to form cysts.
2) Mesonephros, generated in the surrounding tissue of the pronephros, developed a number ofcysts although we could not distinguish between mesonephric and pronephric.
3) By positional cloning, we identified the responsible gene of pc mutant. pc gene consists of atleast ten exons and encodes a novel transcription factor containing five repeats of C2H2-zincfinger. In pc mutant, a large fragment of transposon-like sequence was found to be inserted in
the intron 4 of this gene. This insertion was considered to alter splicing site of pc mRNA toproduce C-terminal truncated pc protein.
4) In wild type medaka, pc gene was specifically expressed in the epithelial cells of tubules andducts in both pronephric and mesonephric kidneys., which was shown by in situ hybridization.
5) Knock-down of pc gene with antisense-oligo led to cyst formation in pronephros

53

Discussion & Summary
Our histological analysis has shown that pc mutant medaka is the counterpart of human PKD. pc
mutant develops renal cysts at an early stage in pronephros as well as in mesonephros. In medaka
early initiation of PKD was not lethal, which might be due to the complementary function of thegill.
pc gene is a novel PKD gene, which is not known as a causal gene in mammalian models andhuman, provides a new insight into the mechanism underlying generation and maintenance of tube
structure in kidney.
Our findings here have demonstrated that medaka is a good model animal for human genetic disease.

Figures & Tables

Appearance of transparent medaka pc mutant
left-side: wild-typeright-side: pc mutant
kidney enlargement is obvious in the living transparent medaka.
ab, air bladder； b, brain； g, gill； h, heart； k, kidney； li, liver； s,
spleen； sc, spinal cord
Mochizuki et al. Kidney International, 68, 23-34 (2005)

541) Biological Sciences1-4） Developmental Biology

The role of Notch ligand for organogenesis and regenerationof various tissues.
Katsuto Hozumi
Department of Immunology, Tokai University School of Medicine
hozumi@is.icc.u-tokai.ac.jp

Introduction
Organ-specific stem cells are maintained in organ-specific environment, which is called as niche,
and produce various progenies during organogenesis and tissue repair after various injuries. These
stem cells will be useful to transplant into patient who bears serious problem of an organ. However,molecular basis of these phenomena, especially molecule(s) that composes the environment, has notbeen determined. Recently, Notch/Notch ligand was reported to contribute for these events. In thisstudy, I use the conditional targeting mice of Notch ligands, Dll11) or Jagged2), I’ve recently
established, to know their physiological significance for various organogenesis and tissue repair.

Results
Jagged1- or Dll1-floxed mice were bred with Mx- (IFN-inducible) or K5-Cre (epithelial
cell-specific) transgenic mice (Tgm), and administrated with IFN-inducer, poly I-C after birth for theformer system, resulting in the gene deletion of the floxed allele. In these mice, the gene deletion
was completely observed in bone marrow, liver (Mx-Cre) or skin (K5-Cre).
[A] The role of Notch ligand for the maintenance of the hematopoietic stem cells in vivo
At 4 weeks after the treatment of Poly I-C in Mx-Cre Tgm for the gene deletion, the cells resided inbone marrow were obtained and analyzed by flowcyometry. All hematopoietic lineages (myeloid(Gr1- or CD11b-positive), erythroid/megakaryocytic (Ter119-positive), lymphoid (CD19-positive)
and their stem cells (as linege markers-negative, Sca1-positive, c-kit-positive cells; HSC) weredetected in Jagged1- or Dll1-null mice as well as that in WT mice. To know the role of Notch ligands
for the maintenance of HSC precisely, GFP Tgm-derived BM cells were transferred into Jagged1- or
Dll1-null mice (poly I-C treated) and traced by the expression of GFP. The development of blood
cells derived from GFP Tgm was also investigated by flowcytometry. Moreover, 2nd BM chimeras
were prepared and analyzed for their chimerism of blood cells. However, all blood cells developed
normaly in 1st and 2nd BM chimeras, and there is no difference between Notch ligand-deficient andWT mice as environment for the maintenance of HSC.

55

[B] The role of Notch ligand for hepatocytes
After the induction of gene deletion by the treatment of Poly I-C, lots of white spots were observedat the liver in Jagged1-null mice. In the HE staining of the liver section of Jagged1-null mice, thedistinctive feature of cell death appeared at the middle area of hepatic lobules with no infiltration(Figure).This suggested that the cell death found in Jagged1-null mice was due to their intrinsic
property but not infection. The dead cells lost clear-defined nucleus and seemed to fuse each other,
but their boundary could be recognized. In contrast, Mx-Cre(-) or Mx-Cre(+), Dll1-floxed mice did
not show this phenotype.
[C] The role of Notch ligand for the generation of epidermis.
To investigate the role of Jagged1 in adult skin, we crossed Jagged1-floxed mice with K5-Cre Tgm.
The K5-Cre, Jagged1-floxed mice appeared normal at birth but went on to lose their hair andwhiskers. Subsequent hair growth was sparse and the hairs were short, resembling the Notch1
deletion phenotype3).

Discussion & Summary
We had established two floxed mice for Notch ligands, Dll1 and Jagged1, to know their
physiological significance as a environmental factor for organogenesis or regeneration of various
tissues. In this study, we investigated for the maintenance of HSC in bone marrow, of hepatocyte
viability or the generation of skin and hair, by breeding with Mx- or K5-Cre Tgm.
For the maintenance of HSC, both Notch ligands were not necessary. Recently, it was reported
that Jagged1 is expressed on the osteoblasts which is estimated to provide the stem cell niche, thesuitable place for the maintenance of HSC in bone marrow. We could see the gene deletion of
Jagged1 in CD45-negative, adherent cells including enriched osteoblast, but its efficiency was about
80% evaluated by quantitative PCR analysis. This means that definitive conclusion could not be
obtained from this experiment. To induce the gene deletion in osteoblasts efficiently, we’ve bred the
Jagged1-floxed mice with a1Col-Cre Tgm where Cre is expressed in osteoblasts. Moreover, it ispossible to establish Jaged1, Dll1, double-floxed mice, to make clear their significance for that.
For the hepatocyte viability and the generation of normal hair, it was demonstrated that Jagged1,
but not Dll1, is indispensable. However, its molecular machinery remains to be determined. The
relationship with the apoptosis in hepatocytes, and with regulation of the cell cycle in hair follicular
cells should be investigated.

56

Figures & Tables

References
1) Hozumi, K. et al. Delta-like 1 is necessary for the generation of marginal zone B cells but not T
vells in vivo. Nature Immunology 5:638-643, 2004.
2) Brooker, R., Hozumi, K. and Lewis, J. Notch ligands with contrasting functions: Jagged1 andDelta1 in the mouse inner ear. Development 133:1277-1286, 2006.
Vauclair, S. et al. Notch1 is essential for postnatal hair follicle development and homeostasis.
Developmental Biology 284:184, 2005.

57

1) Biological Sciences1-4） Developmental Biology

Regulation of mesenchymal stem cell differentiationby phosphoinositide metabolism
Kiyoko Fukami
Tokyo University of Pharmacy and Life Sciencekfukami@ls.toyaku.ac.jp

Introduction
Phosphoinositide metabolism is an important intracellular signaling system that is involved in avariety of cell functions, including secretion of hormones, transduction of neurotransmitter, growth
factor signaling, and regulation of the cytoskeleton. Phospholipase C (PLC) is a key enzyme in thissystem that acts by hydrolyzing phosphatidylinositol 4,5-bisphosphate (PIP2) to generate
two-second messengers, diacylglycerol and inositol 1,4,5-trisphosphate. Among the PLC family
isozymes, PLCδ is evolutionally conserved and is thought to be the primary form in mammals.
We have reported that PLCδ1 is required for skin stem cell lineage commitment and PLCδ4 isinvolved in acrosome reaction in sperm by generating PLCδ1- and PLCδ4 gene-deficient mice. We
also found that PLCδ4 gene-deficient mice got fat, suggesting that PLC delta-type may be involved
in differentiation of mesenchymal stem cells to adipocytes, osteoblasts and myocytes.

Results
PLCδ4 gene-deficient mice showed obesity
First of all, we compared the weight of PLCδ4 gene-deficient mice to that of wild-type mice (WT).
Both male and female PLCδ4 gene-deficient mice were about 20% heavier than WT at any age. We
also observed the accumulation of many lipid-drops in liver and skeletal muscle of PLCδ4gene-deficient mice by histochemical staining of Oil-red-O.
Enhanced adipogenesis of primary MEF derived from PLC delta-type gene-deficient micePrimary MEFs have multi-potencies to differentiate into adipocyte or cardiocyte. Then we
examined the difference of adipogenesis in primary MEFs derived from PLCδ1 gene-deficient,
PLCδ3 gene-deficient, and PLCδ4 gene-deficient mice. Insulin-induced adipogenesis of MEFs
from PLCδ4KO mice was quite enhanced in compared to MEFs from WT mice by oil-red
O-staining. On the other hand, basal level of adipogenesis was increased in MEFs from PLCδ3gene-deficient mice.

58

Abnormal differentiation of mesenchymal stem cells from PLCd3 gene-deficient mice into
adipocytes and osteoblasts
We observed the remarkable decrease in osteogenesis, instead, the marked increase in adipogenesisof mesenchymal stem cells from PLCδ3 gene-deficient mice. In addition, we noted a correlation
between the expression of PLCδ3 and the inductive osteogenesis by measuring alkaline phosphataseactivity of C310T1/2 cells, which is a mesenchymal stem cell-line and established from bonemarrow. Furthermore, when we carried out PLCδ3-RNAi in C310T1/2 cells, osteogenesis of
C310T1/2 cells was partially inhibited. These results suggest that PLCδ3 is involved in lineage
commitment of mesenchymal stem cells between adipocytes and osteoblasts.

Discussion & Summary
This time we have indicated that PLCδ4 gene-deficient mice got fat and adipogenesis of MEFs
derived from PLCδ4 gene-deficient mice was enhanced. These results suggest that PLCδ4 may haveimportant role in adipogenesis. We also showed that mesenchymal stem cells from PLCδ3gene-deficient mice differentiated abnormally into adipocytes and osteoblasts, indicating that PLCδ3
may be involved in lineage commitment of mesenchymal stem cells. It may be necessary tounderstand the mechanism of how these PLC delta-type proteins are involved in adipogenesis or
osteogenesis.

Figures & Tables

59

References* Qin, Q. et al; A novel GTPase, CRAG, mediates PML-associated nuclear body formation anddegradation of expanded polyglutamine protein. J. Cell Biol. 172, 497-504 (2006)* Nakamura, Y. et al;development.

Phospholipase Cd1 and d3 are essential in trophoblast for placental

Mol. Cell. Biol. 25, 10979-10988 (2005)* Nakahara, M. et al; A novel phospholipase C, PLCeta2, is a neuron-specific isozyme. J. Biol.
Chem. 280, 29128-34 (2005)*Kurokawa, M. et al; Functional, biochemical, and chromatographic characterization of thecomplete [Ca2+]i oscillation-inducing activity of porcine sperm.

Dev Biol. 285, 376-392 (2005)

601) Biological Sciences1-4） Developmental Biology

Molecular genetic analysis of the signaling pathway of the DIF, an
anti-tumor substance involved in the development of a modelorganism, Dictyostelium discoideum.
Masashi Fukuzawa
Faculty of Agriculture and Life Science, Hirosaki University
fukuzawa@cc.hirosaki-u.ac.jp

Introduction
DIF (Differentiation-Inducing Factor) made by Dictyostelium discoideum cells acts as an anti-tumor
and differentiation-inducing substance on mammalian cells. In order to understand the DIF action on
mammalian cells it is necessary to reveal the intrinsic DIF signaling pathway in Dictyostelium. I
have characterized two transcription factors, MybE (a SHAQKY family Myb) and DimB (a bZIP
family), as the endpoint of the pathway. In this report I have cloned gene regulatory regions of these
two factors and analyzed their expression pattern to define the place of action within multicellular
stage.

Results
Analysis of the expression patterns of the promoters
Promoter fragments to each transcription factor gene were generated by PCR and cloned into
pDdGal, a beta-galactosidase (lacZ) reporter with G418 selection. Figure 1 shows the structure of
the reporter constructs. To dissect the promoter region roughly, a half-length of the longer ones (ca-600 fragment of mybEp and dimBp, respectively) was also tested for its expression. The -621
promoter fragment of mybE was inactive, showing that the cis regulatory region locates between-1571 to -621. For dimBp, the -610
fragment is sufficient for its expression.
When multicellular structure (slug) was
stained for beta-galactosidase activity, both
mybEp and dimBp showed ubiquitous
expression, with a slight gradient ofexpression

fromanterior

tip

towards

posterior (Fig. 2, A and C). The promoter
activity of the transformants was so highand,together

with

the

stabilityof

61

beta-galactosidase, the strong uniform staining observed might be due to saturation of the staining.
To overcome this the transformant cells were diluted 10 times with unlabelled wild-type cells to
reduce the overall staining intensity. The resultant slug with mybEp construct showed a distinct
pattern of expression; high expression at the tip and the front 1/3 of the posterior prespore region,and no expression within pstO region (Fig. 2B). To my knowledge, this complex pattern has neverbeen shown by any other promoter construct previously described.

In Arabidosis,

CAPRICE(CPC), a Myb-like protein, is transported from atrichoblasts (where it is expressed) totrichoblasts (where it functions). It is very interesting if MybE would be transported from the frontof the prespore region (where it is expressed) to pstO region (where it functions).
Staining-positive cells for dimBp are distributing throughout the front half of a slug when they werediluted with unlabeled cells and there was not much difference to undiluted staining in terms ofpatterning (Fig. 2D). Taken together, theseresults suggest that the promoter structureand expression pattern is totally different
between mybE and dimB.
I also analyzed cudCp, whose expression is
confined to pstA cells. Although the -1128
and -845 fragments of cudCp exhibit strong
pstA expression,neither

of

them

are

DIF-inducible (not shown). Since recent
studies suggest that differentiation of pstA
population is not directly related to DIF
signaling, cudCp was not analyzed further.
DIF inducibility of the promoters
The mybEp and dimBp constructs were examined on microtiter assay for DIF inducibility and raw
data was presented with duplicate wells for each experimental point (Fig. 1, right). Both promoterswere constitutively activated in a DIF-independent manner, suggesting that DIF regulations of MybE
and DimB are at post-transcriptional level. This notion is likely to be directly applied for DimB,
where nuclear translocation is induced by DIF treatment within 3 minutes (ref. 2). However, it seems
that MybE does not respond to DIF in terms of nuclear translocation, because either anti-MybE
antibody or GFP-MybE are shown to be constitutively nuclear localized with or without DIF (ref.1).
This would lead to a prediction that DIF acts on MybE indirectly; there should be an interacting
protein that regulates MybE activity within pstO cells.

62Discussion & Summary
Evidences suggest that DIF induces pstO cell differentiation, hence ecmO-lacZ expression within
pstO region. MybE was identified as a binding factor for ecmO promoter fragment, and when
MybE is inactivated by gene knockout, the ecmO-lacZ is not expressed (ref. 1). The simple
explanation for this activation is that MybE localizes in pstO region. However, this study suggeststhat the promoter for mybE is not active in pstO region but mainly active in the tip and in theanterior part of prespore region. This implies that expression pattern of MybE and the place ofaction do not coincide, and suggests another mode of complexity for its regulation. The gradientspatial pattern of expression observed in dimBp is consistent with the proposed role of DimB, thatis to establish a gradient of ecmA gene expression in the slug tip (ref. 2). This suggests thattranscriptional regulation of dimB is partly involved in maintaining the identity of the prestalk cells
within a slug. Because of lack of good antibodies, the precise localizations of these factors are still
not clear. It is necessary to combine the promoter results and antibody localization to define theplace of action for these two factors. Also, identification of binding partners would greatly help toreveal the signaling pathway.
This study showed spatial expression patterns of mybE and dimB, transcription factors that
regulate DIF responses in Dictyostelium.

References
1)

Fukuzawa M., Zhukovskaya N.V., Yamada Y., Araki T. and Williams J.G.
Regulation of Dictyostelium prestalk-specific gene expression by a SHAQKY family MYB
transcription factor.
Development, Vol. 133:1715-1724 (2006).

2)

Zhukovskaya N.V., Fukuzawa M.(co-first authors), Yamada Y., Araki T. and Williams J.G.
The Dictyostelium bZIP transcription factor DimB regulates prestalk-specific gene expression.
Development, Vol. 133:439-448 (2006).

631)Biological Sciences
1-5） Physioiogy

Analysis of primary afferent-evoked synaptic plasticity in spinal
dorsal horn by simultaneous imaging of nitric oxide and presynaptic
neuronal excitation.
Hiroshi Ikeda
University of Fukui
ikeda@synapse.his.fukui-u.ac.jp

Introduction
Synaptic long-term potentiation (LTP) in the spinal dorsal horn is believed as a mechanism forinduction of hyperalgesia, an increased response to noxious stimuli, following peripheral
inflammation or nerve injury. Recently, we have shown that LTP at primary afferent synapses onlamina I neurons projecting to the periaqueductal gray matter (PAG) may be induced by the action of
nitric oxide (NO) released from glial cells on presynaptic terminals (Ikeda et al., J. Neurosci., inpress). The purpose of this study, therefore, is to construct optical imaging system to visualize the
distribution of NO and the pre- and postsynaptic neuronal excitation in spinal dorsal horn
simultaneously, and to reveal the action of NO on presynaptic terminals in detail so as to clarify theunderling mechanism of hyperalgesia.

Results
As in our previous reports (Ikeda & Murase, 2004), high-intensity single-pulse test stimulation to
the dorsal root, which activates A- and C-afferent fibers, evoked an increase in light absorption, an
indication of neuronal excitation, in the spinal dorsal horn. The neuronal excitation was strongest inlamina I-III of dorsal horn, which contains nociceptive neurons (Fig. 1).
After LFS, the neuronal excitation was gradually potentiated to as much as 141 ± 5% of control at
2 hours after LFS (Fig.1, n = 5). The LTP of neuronal excitation was strongest in the superficial
dorsal horn, which includes many projection neurons that convey nociceptive signals to the brain.
Although LFS-induced LTP of optically recorded neuronal excitation in the spinal dorsal horn isstrongly inhibited by NOS inhibitors As in our previous reports (Ikeda & Murase, 2004), it is notclear if NO is produced as a result of this stimulation. Thus, we measured the spatiotemporal
distribution of NO during LFS in the spinal dorsal horn with the fluorescent NO indicator, DAR-4M.
The NO signal gradually increased during LFS (Fig. 2A,B,C, 2.4 ± 0.3%, n = 5) especially in lamina
I and II. The same region as that LTP was induced.

64

Depending on the slice, the degree of LTP varied from 130 % to 160 %. In five slices showing
different degrees of LTP, there is close linear correlation between degree of LTP in the neuronal
excitation and the intensity of NO signal (Fig. 2D)
We also examined the effect of NOS inhibitors on both neuronal excitation and NO production.
100 µM L-NAME significantly inhibited both the LTP (105 ± 2%, p < 0.01 compared with control, n= 5) and NO signals induced by LFS (0.5 ± 0.3%, p < 0.01 compared with control, n = 5). L-NMMA
also strongly inhibited both LTP (10 µM, 141 ± 6%, n = 5; 50 µM, 117 ± 5%, n = 5; 100 µM, 99 ±3%, n = 4) and NO signals (10 µM, 2.6 ± 0.6%, n = 5; 50 µM, 1.1 ± 0.4%, n = 5; 100 µM, 0.5 ±
0.3%, n = 4) induced by LFS in a dose-related manner.
It is well known that NO synthase activity is controlled by NMDA receptors (Kawamata & Omote,1999; Takata et al., 2005). Therefore, we examined contribution of NMDA receptor to LTP and NO
signals induced by LFS. A NMDA receptor antagonist D-AP5 (50 µM) did not inhibited neither LTP(166 ± 8%, n = 5) nor NO signals (2.8 ± 0.5%, n = 5).
Since it is reported that carbon monoxide (CO) also acts as a retrograde messenger during LTP(Zhuo et al. 1999), we tested the inhibitor of heme oxygenase, the synthetic enzyme for CO. BothLTP and NO signal induced by LFS were significantly inhibited in the presence of a heme oxygenase
inhibitor zinc protoporphyrin IX 10 µM (LTP: 100± 1%, n = 5; NO signal: 0.9 ± 0.3%, n = 5).

Discussion & Summary
In behavioral studies, hyperalgesia induced by intrathecal injection of NMDA is blocked byadministration of the NOS inhibitor, L-NAME (Kitto et al., 1992). The enhancement offormalin-induced paw licking behavior in rats treated with L-glutamate or substance P is reversed by
pretreatment with L-NAME (Coderre & Yashpal, 1994). Topical application of L-NAME onto spinal
cord reduces both the first and second peaks of the response of dorsal horn neurons to formalin
injection (Haley et al., 1992). The sensitization of spinothalamic neurons induced by intradermal
injection of capsaicin is prevented by pretreatment of the dorsal horn with the NOS inhibitor
L-NAME or 7-NINA (Lin et al., 1999). The allodynic response, and increased response of dorsal
horn neurons to noxious and innoxious stimuli induced by intradermal capsaicin injection are
reversed by the administration of the NOS inhibitors 7-NINA or AMT (Wu, 2001). The
NO-dependent LTP in the spinal dorsal horn reported in this study indicates a possible cellularmechanism for the induction of these NO-dependent abnormal pains.

65

Fig.2

Fig.1

References
1.

Ikeda, H. & Murase, K. (2004) Glial nitric oxide-mediated long-term presynaptic facilitation
revealed by optical imaging in rat spinal dorsal horn. J. Neurosci., 24, 9888-9896.

2.

Kawamata, T. & Omote, K. (1999) Activation of spinal N-methyl-D-aspartate receptors
stimulates a nitric oxide/cyclic guanosine 3,5-monophosphate/glutamate release cascade in
nociceptive signaling. Anesthesiology, 91, 1415-1424.

3.

Takata, N., Harada, T., Rose, J.A. & Kawato, S. (2005) Spatiotemporal analysis of NO
production upon NMDA and tetanic stimulation of the hippocampus. Hippocampus, 15,427-440.

66

4.

Zhuo, M., Laitinen, J.T., Li, X.C. & Hawkins, R.D. (1999) On the respective roles of nitricoxide and carbon monoxide in long-term potentiation in the hippocampus. Learn. Mem., 6,
63-76.

5.

Kitto, K.F., Haley, J.E. & Wilcox, G.L. (1992) Involvement of nitric oxide in spinally mediatedhyperalgesia in the mouse. Neurosci. Lett., 148, 1-5.

6.

Coderre, T.J. & Yashpal, K. (1994) Intracellular messengers contributing to persistentnociception and hyperalgesia induced by L-glutamate and substance P in the rat formalin pain
model. Eur. J. Neurosci., 6, 1328-1334.

7.

Haley, J.E., Dickenson, A.H. & Schachter, M. (1992) Electrophysiological evidence for a roleof nitric oxide in prolonged chemical nociception in the rat. Neuropharmacology, 31, 251-258.

8. Lin, Q., Wu, J., Peng, Y.B., Cui, M. & Willis, W.D. (1999) Nitric oxide-mediated spinal
disinhibition contributes to the sensitization of primate spinothalamic tract neurons. J.
Neurophysiol., 81, 1086-1094.
9. Wu, J., Fang, L., Lin, Q. & Willis, W.D. (2001) Nitric oxide synthase in spinal cord central
sensitization following intradermal injection of capsaicin. Pain, 94, 47-58.

671)Biological Sciences
1-5） Physioiogy

Roles of presynaptic calcium store in synaptic plasticity
Haruyuki Kamiya
Hokkaido University School of Medicine
kamiya@med.hokudai.ac.jp

Introduction
Calcium ions play important roles in neurotransmitter release and its use-dependent modulation.
However, the roles of intracellular calcium stores in regulating calcium dynamics within presynaptic
terminals were not examined well. In this study, I aimed to elucidate the roles of presynaptic calcium
stores in synaptic plasticity at the hippocampal mossy fiber synapses by using simultaneousrecordings of presynaptic calcium dynamics and EPSPs in the slice preparations.

Results
To examine the presence of presynaptic calcium stores at the hippocamplal mossy fiber synapses, weused simultaneous recordings of fluorescent measurement of presynaptic calcium dynamics andelectrophysiological recording of EPSPs in mouse hippocampal slices. We first examined the effectof caffeine, which causes calcium release through ryanodine receptor channels. Application ofcaffeine caused robust enhancement of mossy fiber synaptic transmission. This synaptic
enhancement was accompanied by reduction of paired-pulse facilitation of field EPSPs (excitatory
postsynaptic potentials) or monosynaptic NMDA receptor-mediated EPSCs (excitatory postsynaptic
currents) measured in the presence of AMPA receptor blocker CNQX. Caffeine also caused anincrease in frequency, but not in amplitude, of miniature EPSCs recorded from CA3 neurons.
Frequency of kainate receptor-mediated miniature EPSCs recorded in the presence of AMPA
receptor-selective antagonist GYKI 53655, which reflect spontaneous transmitter release exclusively
from mossy fiber terminals, were also increased by application of caffeine. Optically monitored
presynaptic calcium level was also increased by application of caffeine. These results suggest
presynaptic origin of the caffeine-induced synaptic enhancement, i.e. caffeine may cause calcium
release from the presynaptic calcium stores via ryanodine receptors and thereby increasingtransmitter release from the presynaptic terminals. To elucidate the physiological roles of presynaptic
ryanodine receptors, the effect of ryandine receptor blocker TMB-8 was also examined. EPSCs
evoked at low frequency (e.g. 0.1 Hz) stimulation of mossy fibers were not significantly affected byapplication of TMB-8. However, presynaptic calcium transients evoked by train of high frequency

68

stimulation (100 Hz for 1 s) were significantly suppressed by TMB-8. Notably, initial phase of
calcium transients was hardly affected by TMB-8, but prominent inhibition was occurred at the latephase during high frequency stimulation. These results suggested the notion that presynaptic
ryanodine receptors were activated in a use-dependent manner, and caused amplification ofpresynaptic calcium transients by calcium-induced calcium release (CICR) mechanism. Sincelong-term potentiation (LTP) and long-tem depression (LTD) at the mossy fiber-CA3 synapse are
known to be a unique form of synaptic plasticity independent from postsynaptic NMDA-receptors
but dependent on presynaptic calcium accumulation within the mossy fiber terminals, it wassuggested that activity dependent amplification of presynaptic calcium transients facilitate inductionof the presynaptic LTP and LTD at this synapse. In support of this notion, prolonged application ofcaffeine reliably induced long-term depression lasting longer than at least 60 min after washout ofthe drug. This caffeine-induced LTD was accompanied by enhancement of paired-pulse facilitation,suggesting that presynaptic LTD could be induced by calcium release within the presynaptic
terminals. We also examined different conditions to activate presynaptic ryanodine receptors at the
mossy fiber-CA3 synapse. Synaptic strength at this synapse exhibits robust frequency facilitation(FF), another form of presynaptic short-term synaptic plasticity elicited by repeated stimulation ofafferent fibers at relatively low frequency (e.g. 1 - 5 Hz). Application of CPA, an inhibitor of calcium
uptake into intracellular stores, reversibly suppressed the magnitude of FF tested at 1Hz stimulation.
This suggested that amplification of presynaptic calcium raise by ryanodine receptors could be
activated at rather low frequency stimulation of mossy fibers.

Ca2+
RyR

mossy fiber

(+)
CICR

Ca2+ channnel

Glu

Figure 1
Ryanodine receptors reside within presynaptic terminals of
hippocampal mossy fiber synapses. They are activated by calcium
accumulation following repetitive stimuli, and cause amplification ofpresynaptic calcium raise by calcium-induced calcium release (CICR)
mechanism.

69

Discussion & Summary
It has been demonstrated that presynaptic calcium stores are specifically localized in certain
synapses in the central nervous system. In this study, I examined the roles of presynaptic calcium
stores at hippocampal mossy fiber synapses using fluorescence measurement of presynaptic calcium
and electrophysiological recordings. All the results are consistent with the notion that ryanodine
receptors within the presynaptic terminals are involved the presynaptic forms of short- and long-tem
synaptic plasticity at the hippocampal mossy fiber terminals. Since these forms of presynaptic
plasticity has been shown to depend on accumulation of calcium in the presynaptic terminals,
presynaptic ryanodine receptors may serve to amplify robust presynaptic plasticity at the mossy
fiber-CA3 synapse.

References
Presence and functional significance of presynaptic ryanodine receptors. R Bouchard, R Pattarini, J.
D. Geiger, Progress in Neurobiology, 69: 391-418, 2003.

701) Biological Sciences
1-6） Pharmacology

Photodynamic therapy for cancer using photoreactive nano-micelles
with the favorable property of accumulation in a tumor
Yuji Morimoto
Department of Medical Engineering, National Defense Medical College
moyan@ndmc.ac.jp

Introduction
Photodynamic therapy (PDT) is a minimally-invasive local therapy for solid cancers. In order toenhance efficacy of PDT, the property of photosensitizer requires achievement of high singlet
oxygen quantum efficiency, accomplishment of their tumor-selective delivery, and minimization ofside effects such as skin photosensitivity.
We have synthesized a novel potent photosensitizing agent composed of dendrimer-based
photosensitizers and polymeric micelle incorporating them. Dendrimer-based photosensitizers
achieve high quantum efficiency by preventing photosensitizers from aggregating. Polymeric
micelle can accomplish tumor-selective delivery by EPR (enhanced permeability and retention)effect and prolonged blood circulation.
In this study, we therefore evaluated the therapeutic effect of PDT using polymeric micelleincorporating dendrimer-based phthalocyanine.

Results
We designed the novel photosensitive nano-carrier using Zn(II)-phthalocyanine as a hotosensitizer
since it can be excited by longer wavelength light (~ 680 nm). In the region ranging from visible tonear-infrared, a light can penetrate deeper in tissue dependent on its wavelength. In addition,~680-nm light is less absorbed by hemoglobin than 630-nm light, which corresponds to thesecond-maximum absorption of Photofrin, the most popular photosensitizer in clinical practice. As aresult, the light at ~680 nm can penetrate tissues approximately 2 times deeper than that at 630 nm
can.
The synthesis of the ionic dendrimer phthalocyanine (Pc/d) has been reported previously (1). The
synthesized Pc/d is composed of the second-generation aryl ether dendrimer with aZn(II)-phthalocyanine center and 32 negatively charged (COO-) groups on its periphery (Fig. 1A).
Poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-b-PLL) (Fig. 1B) was synthesized by
the polymerization of the N-carboxy anhydride of Nε-Z-L-lysine initiated by CH3O-PEG-NH2 in71

dimethylformamide (DMF), followed by deprotection of the Z group according to a previously
reported method (2). The polymeric micelle encapsulating Pc/d (Pc/m) was prepared with astoichiometric ratio of negatively charged Pc/d and positively charged PEG-b-PLL (Fig. 1D). The
synthesized Pc/m showed a size of ~ 50 nm. Zn(II)-phthalocyanine tetrasulfonic acid (Pc) (Fig. 1C)
was used as a comparative photosensitizer.
The photo-cytotoxicities of Pc, Pc/d, and Pc/m were quantitatively evaluated by MTT assay 24 hafter photoirradiation. 50% inhibitory concentration (IC50) in human lung cancer cells (A549) by
PDT using Pc/m was 7 - 44 folds lower than PDT using Pc or Pc/d.
Morphological changes of A549 cells after PDT using Pc, Pc/d or Pc/m were also observed intime lapse manner by a differential interference contrast (DIC) microscope. As a result, PDT with
Pc/m induced a cellular expansion with blebs on cellular membrane, whereas PDT with Pc or Pc/dcaused cellular shrinkage. Remarkable morphological changes of cells in PDT using Pc/m wereconsidered to be associated with the immediate cell death.
Anti-tumor effect of PDT using Pc, Pc/d or Pc/m was evaluated in tumor xenograft mice model.
One of the tested photosensitizing agents was administered intravenously as the administration
amounts were proportional to molecular weights. 24 h after the administration, tumor sites were
irradiated. This animal study protocol was approved by the Ethics Committee for Laboratory
Animals of the National Defense Medical College. Mice with PDT using Pc/m showed significantly
slow proliferation of tumor compared with those using Pc or Pc/d, or control (untreated) mice. The
ratio of tumor volume in mice with PDT using polymeric micelle was about half as large as that in
PDT-untreated mice on 30 days after PDT. Note that single PDT after the single injection of Pc/m
was enough to significantly reduce the proliferation rate of solid tumors. The dosage of agent andeffective photoirradiation should be further studied to achieve more effective PDT in in vivo study.
In addition, tumor-selective delivery may be accomplished by polymeric micelle formation with
PEG shell, so that side effects such as skin photosensitivity can be minimized.

Discussion & Summary
We have demonstrated that PDT using polymeric micelle encapsulating dendrimer-based
phthalocyanines induced a unique and high cytotoxic effect in in vitro study. This treatment modality
also exerted anti-tumor effect in tumor-bearing animal model. By incorporating Pc/d into polymeric
micelle, more effective cytotoxicity, immediate cell death and characteristic morphological change
can be induced in PDT. We expect that PDT using polymeric micelle encapsulating dendrimer-based
photosensitizers can be more effective and safe treatment, and research in this direction is now goingin our laboratory.

72

Figures & Tables

Fig. 1 Chemical structure of ionic dendrimer Zn(II)-phthalocyanine (A), poly(ethylene
glycol)-poly(L-lysine) (PEG-b-PLL) block copolymer (B) and Zn(II)-phthalocyanine tetrasulfonicacid (C). (D) Formation of Pc/m via electrostatic assembly of Pc/d and PEG-b-PLL block
copolymers.

References
1. Ng, A.C.H., X. Li and D.K.P. Ng (1999) Synthesis and photophysical properties ofnonaggregated phthalocyanines bearing dendritic substituents. Macromolecules 32, 5292-5298.
2. Jang, W.D., N. Nishiyama, G.D. Zhang, A. Harada, D.L. Jiang, S. Kawauchi, Y. Morimoto, M.
Kikuchi, H. Koyama, T. Aida and K. Kataoka (2005) Supramolecular nanocarrier of anionic
dendrimer porphyrins with cationic block copolymers modified with polyethylene glycol toenhance intracellular photodynamic efficacy. Angew Chem Int Ed Engl 44, 419-23

731) Biological Sciences
1-6） Pharmacology“Studies on the molecular mechanisms for physiological regulation ofurinary bladder smooth muscle and etiology of overactive bladder
Analysis from the viewpoint of large-conductance,
Ca2+-sensitive K+ (MaxiK) channel role”
Yoshio Tanaka, Ph.D.
Toho University School of Pharmaceutical Sciences
yotanaka@phar.toho-u.ac.jp

Introduction
Overactive bladder (OAB) is a urinary bladder dysfunction usually accompanied with frequenturination and urinary incontinence. Although OAB itself is not life-threatening, its incidence isincreasing in developed countries decreasing patients’ QOL. Against OAB, new therapeutic drugs
other than anticholinergic drugs are expected to emerge. One of the key molecules targeted at theunderstanding of OAB and its drug therapy, is the large conductance, voltage-dependent andCa2+-sensitive K+ (MaxiK) channel. In the present study, roles of MaxiK channel in the regulation ofurinary bladder smooth muscle electrical and mechanical activities and the effects of its openers
were examined.

Results
2+

1) Effects of Ca channel modulators on the generation of action potential (AP)In the isolated guinea-pig urinary bladder smooth muscle (UBSM) preparation, spontaneous AP was
generated when membrane potential changes of UBSM cells were recorded with microelectrode. AP
was myogenic in nature since its generation was not inhibited by receptor antagonists for neuronaltransmitters in the UBSM wall. On the other hand, the amplitude and frequency of AP weresignificantly decreased by L-type Ca2+ channel inhibitors (nisoldipine and diltiazem), while BayK
8644, its channel activator, dramatically increased them, indicating that the upstroke of the APrecorded is provided by an activation of L-type Ca2+ channel.
2) Effects of K+ channel modulators on AP
UBSM cell AP was composed of depolarizing phase followed by repolarizing phase without aclearly defined plateau phase. Since the repolarizing phase of AP is assumed to be attributed to theactivation of K+ channels, effects of several K+ channel inhibitors on the AP were examined: they

74

were iberiotoxin (IbTx) vs. MaxiK channel; apamin vs. small conductance Ca2+-activated K+
channel; 4-aminopyridine (4-AP) vs. voltage-gated K+ channel; E-4031 vs. HERG channel. Amongthese K+ channel inhibitors, IbTx (50 nM) was found to affect most dramatically the generation of
AP: both the amplitude and frequency of AP were significantly increased by IbTx (Fig. 1A). IbTx
was also the most powerful enhancer of the spontaneous mechanical activity of the guinea-pig
UBSM (Fig. 1B). These electrical and mechanical studies with IbTx indicate that (1) IbTx enhances
spontaneous mechanical activity of UBSM in part by enhancing AP frequency and (2) MaxiK
channel functions as a primary negative feedback element to limit extracellular Ca2+ influx throughaffecting AP generation in the UBSM contraction.
To confirm the primary role for MaxiK channel in the UBSM electrical and mechanical activities,
effects of openers of this channel were examined: they are NS-1619 and niflumic acid (MaxiK
channel α-subunit activator), and estradiol and tamoxifen (MaxiK channel β-subunit activator). As
expected, both types of MaxiK channel activators abolished the AP generation and significantly
reduced the UBSM spontaneous contraction (Fig. 2B, C). NS-1619 also suppressed the spontaneouscontractions generated in the balloon-inserted urinary bladder of the anesthetized guinea-pig. Thesefindings strongly support the central role for MaxiK channel in the UBSM functional regulation andimply a usefulness of the opener of this K+ channel in suppressing excessive UBSM excitability andcontractility.
3) Effects of MaxiK channel openers on the channel activity of the isolated UBSM cells
In the enzymatically isolated guinea-pig UBSM cells, MaxiK channels with a slope conductance of≈200pS were recorded. The channel activity was enhanced by BayK 8644

and a Ca2+-pump

Ca2+-ATPase inhibitor, thapsigargin. In this UBSM tissue, both α- and β-subunits of MaxiK channels
were abundantly detected at the mRNA level. These findings indicate that MaxiK is expressed inguinea-pig UBSM cells and functions as a negative feedback element to suppress cell excitability
after the intracellular Ca2+ concentrations are increased. MaxiK channel openers, both α-subunit
activators (NS-1619, niflumic acid) and β-subunit activators (estradiol and tamoxifen), significantly
increased the channel open probability (Po) (Fig. 2A). These results substantiate a crucial role forthis channel as indicated by the electrical and mechanical studies mentioned above.
4) Possible roles for MaxiK channel in β3-adrenoceptor (β3-AR)-mediated inhibitory effect on thecontractile responses of UBSM
Isoprenaline (ISO) and noradrenaline (NA) suppressed acetylcholine-induced contractions of UBSM
in the presence of propranolol. These inhibitory effects were judged to be mediated through β3-AR
since they were suppressed by bupranolol that is able to block β3-AR. The β3-AR-mediatedrelaxations by ISO and NA were significantly reduced in the presence of IbTx, which suggests a

75possible functional coupling between MaxiK channel and β3-AR.

76

Discussion & Summary
In the present study, we clearly indicate a key role for MaxiK channel which functions as anegative-feedback element to restrain over excitability and contractility of UBSM. This inhibitory
effect of MaxiK channel is attained in part through suppressing the generation of AP. Therefore,
functional disturbance of this type of K+ channel would lead to causing OAB. On the other hand, theactivation of MaxiK channel limits the generation of AP and contraction of UBSM. Accordingly,
MaxiK channel openers would be useful drugs for the treatment of OAB. Another important findingof this study is that MaxiK channel might be functionally coupled with β3-AR. This is of interest
since β3-AR is also expressed in UBSM cells and its agonist is a potent candidate for OAB treatment.
For the better understanding of the UBSM functions and OAB, establishment of molecularmechanisms between MaxiK channel and β3-AR coupling will be a critical issue that should besettled in future.

References
Imai, T., Okamoto, T., Yamamoto, Y., Tanaka, H., Koike, K., Shigenobu, K. and Tanaka, Y.: Effects
of different types of K+ channel modulators on the spontaneous myogenic contraction of guinea-pig
urinary bladder smooth muscle. Acta Physiol. Scand. 173(3): 323–333 (2001).
Imai, T., Tanaka, Y., Okamoto, T., Yamamoto, Y., Horinouchi, T., Tanaka, H., Koike, K. andShigenobu, K.: Evidence that action potential generation is not the exclusive determinant to trigger
spontaneous myogenic contraction of guinea-pig urinary bladder smooth muscle. Acta Physiol.
Scand. 176(1): 57–63 (2002).
Tanaka, Y. and Koike, K.: Spontaneous rhythmic contraction of urinary bladder smooth muscle:
pharmacological characteristics and its correlation with action potential generation. Current Topics
in Pharmacology 7(1): 117–124 (2003).
Tanaka, Y., Okamoto, T., Imai, T., Yamamoto, Y., Horinouchi, T., Tanaka, H., Koike, K. andShigenobu, K.: BKCa channel activity enhances with muscle stretch in guinea-pig urinary bladdersmooth muscle. Res. Commun. Mol. Pathol. Pharmacol. 113–114: 247–252 (2003).
Nakahara, T., Mitani, A., Kubota, Y., Maruko, T., Sakamoto, K., Tanaka, Y., Koike, K., Shigenobu, K.
and Ishii, K.: MaxiK channel-triggered negative feedback system is preserved in the urinary bladdersmooth muscle from streptozotocin-induced diabetic rats. J. Smooth Muscle Res. 40(3): 97–109(2004).

77

Tanaka, Y., Koike, K., Alioua, A., Shigenobu, K., Stefani, E. and Toro, L.: β1-Subunit of MaxiK
channel in smooth muscle: a key molecule which tunes muscle mechanical activity. J. Pharmacol.
Sci. 94(4): 339–347 (2004).
Tanaka, Y., Horinouchi, T. and Koike, K.: New insights into β-adrenoceptor in smooth muscle:
distribution of receptor subtypes and molecular mechanisms triggering muscle relaxation. Clin. Exp.
Pharmacol. Physiol. 32(7): 503–514 (2005).

781) Biological Sciences
1-6） Pharmacology

Molecular dissection of the role of CLICK-III/CaMKIγ in fear- andeating-related behavior
Haruhiko Bito
University of Tokyo Graduate School of Medicine
hbito@m.u-tokyo.ac.jp

Introduction
CLICK-III/CaMKIγ is a novel CaMKI isoform isolated and cloned by the Bito laboratory. This
CaM kinase distinguishes itself from all other CaM kinases through three features: 1) the expressionof this kinase is CNS- and neuron-specific; 2) it is physically inserted into lipid bilayers through its
lipidifiable C-terminal domain; 3) this kinase is abundant in the CeA of the amygdala and the VMHof the hypothalamus, indicating a possible function in regulation of fear- and eating-related behavior.

Results
In our 3-year proposal plan (to be ended on March 2008), we sought to identify the biologicalsignificance of CLICK-III/CaMKIγ through histological, biochemical and behavioral analyses of
CLICK-III/CaMKIγ-KO mice. As a first step towards this goal, heterogyzous mice for a
CLICK-III/CaMKIγ-KO line were successfully generated in a BL/6 ES cell background.
Analysis of expression pattern of CLICK-III/CaMKIγ in embryonic and adult CNS revealed that
this kinase is relatively ubiquitously expressed in the central nervous system, and in particular in thecortical plate. This was unexpected as its expression pattern was largely confined to the CeA and the
VMH in the adult tissues. In a preliminary analysis of the substrates of this kinase, we haveidentified a membrane-bound signaling molecule whose role is known in neuritogenesis.
In the next two years, the availability of the knockout mice will be capitalized to further help us
investigate the role of CLICK-III/ CaMKIγ in CeA- and VMH-based regulation of fear andeating-related behavior.

Discussion & Summary
CLICK-III/CaMKIγ is a novel CaMKI isoform that has no similarity with other CaM kinases interms of cellular and subcellular distribution. Thus, a knockout analysis and identification of its
physiological substrates will be critical to dissect its role. Availability of the knockout mice will now
facilitate intensive screening of membrane-bound substrates while also allowing behavioral analyses.

79

References
Takemoto-Kimura S, Ishihara N, Nonaka M, Adachi-Morishima A, Mano T, Fujii H, Hoshino M,
Okuno

H,

Bito

H.

Regulationofdendritogenesis

via

a

lipid

raft-associated2+

Ca /calmodulin-dependent protein kinase CLICK-III/CaMKIγ., submitted.
Takemoto-Kimura S, Terai H, Takamoto M, Ohmae S, Kikumura S, Segi E, Furuyashiki T, Arakawa
Y, Narumiya S, Bito H. Molecular cloning and characterization of CLICK-III /CaMKIγ, a novel
membrane-anchored neuronal Ca2+/calmodulin-dependent protein kinase (CaMK). J. Biol. Chem.
278: 18597-18605, 2003.

801) Biological Sciences

1-7） Plant Biology

Molecular mechanism of stem cell maintenancein the plant root meristem
Keiji Nakajima
Nara Institute of Science and Technology
k-nakaji@bs.naist.jp

Introduction
In contrast to animal development where basic body plans are completed in embryogenesis, higher
plants continue to form new organs after germination. This post-embryonic organ formation relies on
the maintenance of undifferentiated cell populations (stem cell pools) in both shoot and root
meristems (Ref. 1). How plants control stem cell numbers has not been clearly elucidated, though a
few genes have been implicated in the shoot stem cells maintenance. In this study the role of
WUSCHEL-LIKE HOMEOBOX 5 (WOX5) in the root stem cell maintenance was investigated byusing mutants and transgenic plants. A new approach using the novel activation tagging method was
also applied.

Results
1. Functional analysis of WOX5 geneIn the Arabidopsis root meristem, stem cells surround a small number of cells termed quiescentcenter (QC). It has been proposed that QC signals the surrounding stem cells and maintains theirundifferentiated status (Fig.1A). A homeobox transcription factor, WOX5, is expressed specificallyin the QC. Here the role of WOX5 in the root stem cell maintenance was studied by two approaches.
First, loss-of-function wox5 phenotype was analyzed by using T-DNA insertion mutants. wox5
mutants displayed abnormal cell patterns in the root cap. Marker analysis indicated differentiationof the stem cells that would give rise to the root cap cell lineage (Fig. 1B). In contrast, stem cells
proximal to the QC were not affected. Because the latter stem cells account for the root growth,wox5 mutants showed comparable root growth rate to that of wild-type. Second, transgenic plantsthat express WOX5 throughout the root in an inducible manner were generated. Upon induction,these plants accumulated supernumerary layers of undifferentiated cells in the root cap region (Fig.
1B). When WOX5 expression was turned off, these cells rapidly differentiated into root the cap
cells (data not shown). These observations indicate that WOX5 acts to maintain undifferentiatedstatus of the root cap stem cells non-cell-autonomously. This strongly suggests that WOX5 is a part

81of the postulated QC-derived intercellular signal that maintains stem cell activity in the Arabidopsis
root meristem.

2. Identification of novel genes involved in root stem cell maintenance by the GAL4/UAS
activation-tagging system.
The number of Arabidopsis root meristem mutants is considerably fewer than the expected
number of genes involved. This is most likely due to the gene redundancy. I am establishing a newactivation-tagging system in which random genes in the genome are forced to express ectopicallyby the use of artificial transcription activator GAL4:VP16 (GV) and its target sequence UAS. Thismethod allows isolation of gain-of-function mutants of redundant genes for which loss-of-function
would not cause a visible phenotype (Fig. 2). Here this method was applied to screen for the
mutants defective in the cell pattern formation in the root meristem (Ref. 2).
After screening about 17,000 independent lines, eight UAS-tagged root patterning (urp) mutants,
urp1D through urp8D were isolated. Four of the eight mutants were defective in the root meristem
maintenance, with their root growth terminating early after germination, whereas other four mutants
showed severe patterning defects likely arose from disorganized cell divisions in the root meristem.
Genes causal to the mutant phenotype were identified by determining the UAS insertion sites,
followed by co-segregation and recapitulation experiments. In all cases UAS was found within the 5’

82

upstream regions of annotated genes, except for one, in which novel ORF was found to cause themutant phenotype. All seven annotated genes encoded putative transcription factors belonging to theAP2, NAC, Zinc finger, MYB, DOF and RWP-RK families. Expression studies indicated that allthese genes are expressed specifically in certain cell types in the root meristem, and two of them
have been already implicated in root patterning by other research groups through an elaborated
marker-based screening or homology-based functional studies.

Discussion & Summary
Based on classical laser-ablation studies, it has been postulated that QC cells in the root meristem
function as a “stem cell niche” that emits unknown cellular signals to the surrounding stem cells andmaintains their undifferentiated status. Results obtained in this study strongly suggest that ahomeobox transcription factor WOX5 plays a pivotal role in the QC-derived niche signal for the83root cap stem cells. The current data, however, does not provide evidence that WOX5 acts forproximal stem cells. Because root growth depends on the activity of proximal stem cells, in the
future it is essential to identify genes responsible for the proximal stem cell maintenance.
The second part of this study proved that the GAL4/UAS activation tagging system is effective in
identifying novel genes that have escaped from conventional mutant screenings. In the future thissystem will be applied to identify genes that can rescue known root meristem mutants.

References
1. Nakajima, K., and Benfey, P.N. (2002). Signaling in and out: control of cell division anddifferentiation in the shoot and root. Plant Cell 14, S265-S276.
2. Nakajima, K. et al. (2006) Identification of genes implicated in Arabidopsis root patterning by a
GAL4/UAS activation tagging system. Plant Cell Physiol. Supplement to vol.47, in press.

841) Biological Sciences

1-7） Plant Biology

Molecular and genetic studies on the function of polyaminesin plant development
Taku Takahashi
Graduate School of Bioscience and Technology, Okayama University
perfect@cc.okayama-u.ac.jp

Introduction
Stem elongation is a trait of particular significance in determining gross morphology of higher
plants. Loss-of-function mutants of the Arabidopsis thaliana ACAULIS5 (ACL5) gene, which
encodes spermine synthase, result in severe defects in the stem elongation (1). To elucidate how
ACL5 regulates the stem elongation, we isolated four dominant or semi-dominant suppressor of
acaulis (sac) mutants that suppress the acl5 dwarf phenotype. The sac51-d mutation disrupts a short
upstream open reading frame (uORF) of the SAC51 gene which encodes a basic helix-loop-helix(bHLH) transcription factor. We suggest that ACL5 plays a role in the translational control ofuORF-containing genes.

Results
Mutations that suppressed the dwarf phenotype of the acl5 mutant were identified by screening fortall individuals among M2 plants descended from EMS-mutagenized homozygous acl5-1 seeds. A
total of four putative suppressors were isolated and designated as sac (suppressor of acaulis) 51
through 54 (Fig. 1). Microscopic observation of stem longitudinal sections revealed that the recoveryof the plant height is attributable to that of the cell length in all sac acl5 mutants. The Arabidopsis
genome has two genes encoding spermine synthase, ACL5 and SPMS. acl5-1 spms-1 double mutant
plants have no detectable levels of endogenous spermine but are morphologically indistinguishable
from acl5-1 plants (2). To examine the effect of sac alleles on the stem elongation under thecomplete depletion of spermine, we generated sac acl5 spms triple mutants. All sac mutants
suppressed the acl5 phenotype in the absence of SPMS in a similar degree to those in the presence of
SPMS, indicating that the recovery of the stem growth involves no spermine.
We chose sac51-d acl5-1, which showed the recovery of the shoot growth comparable to
the wild type, for further study. Fine mapping experiments placed the SAC51 locus on a 60-kb
region of chromosome V. We found a single C-to-T mutation in the At5g64340 gene. To confirm that
At5g64340 is the SAC51 gene, a genomic fragment including 990 bp upstream of the transcription

85

start site and 723 bp downstream of the stop codon of At5g64340 was cloned from sac51-d plants
and introduced into acl5-1 plants. The resulting transgenic plants reproduced sac51-d acl5-1 that iswild-type morphology in four independent lines, thus confirming that At5g64340 is SAC51. SAC51
encodes a bHLH protein and includes a long 5’ leader region with five overlapping upstream open
reading frames (uORFs). The sac51-d allele has a C to T nucleotide exchange that creates apremature stop codon in the fourth uORF.
To examine the effect of the sac51-d mutation on the translation of the main ORF, weprepared chimeric gene constructs that consist of a 990-bp SAC51 promoter fragment, the entire 5’
leader region of the wild-type SAC51 or sac51-d transcript containing five uORFs and three introns,
and the GUS reporter gene. The GUS activity was determined in seedlings for each individualtransformant and related to the normalized GUS reporter transcript levels to provide an indication of
the translational efficiency of each construct. The sac51-d-GUS yielded from 27.0- to 87.6-fold more
GUS activity than did the SAC51-GUS wild-type construct. In parallel with the SAC51 transcriptlevels in sac51-d acl5-1, however, steady-state levels of the GUS transcript was increased insac51-d-GUS plants compared to SAC51-GUS plants. Consequently, our results indicated that thesac51-d mutation caused from 3.9- to 7.3-fold increase in the GUS translational efficiency comparedto the wild-type.
To address possible regulatory interactions between ACL5 and SAC51, we examined theeffect of the acl5-1 allele on the translation of SAC51 by using the wild-type SAC51-GUS construct.
The construct was introduced into acl5-1 and sac51-d acl5-1 mutants by crossing. The GUS activity
in acl5-1 and sac51-d acl5-1 seedlings was about half of that in the wild-type background, while thesteady-state levels of the GUS transcript were not affected in these mutant seedlings. Thus, the GUS
translational efficiency in acl5-1 and sac51-d acl5-1 was estimated respectively to be 47.4% and64.3% of that in the wild-type background, suggesting a relevant function of ACL5 in the translationof SAC51.
Figure 1. Morphological phenotypes of sac acl5 mutants.

86

Discussion & Summary
The sac51-d dominant allele was found to have a point mutation in one of these uORFs, which
introduces a premature stop codon. Our results suggest that the sac51-d allele results in the
deregulation of translational repression of the SAC51 bHLH protein and its overproduction is
responsible for suppression of the acl5 phenotype in sac51-d acl5-1. This is consistent with thedominant trait of sac51-d.
We propose a model whereby ACL5 directly or indirectly acts as a negative regulator of
the translational repression of SAC51 and probably its homologues with their uORFs (Fig. 2). In thismodel, we hypothesize that the premature termination of a 53-amino-acid polypeptide encoded by
the fourth uORF of SAC51 facilitates the release of the ribosome from the uORF and the translation
reinitiation at the main ORF in sac51-d. Restoration in sac51-d acl5-1 of the acl5-1 mutant transcriptlevel, which is increased in acl5-1, suggests that negative feedback control of the ACL5 expression
does not require spermine itself. In conclusion, our findings shed first light on the uORF-mediatedtranslational control in plant development.
Figure 2. A model of uORF-mediated translational control of SAC51 expression via ACL5 function

87

References
1) Hanzawa, Y., Takahashi, T., Michael, A.J., Burtin, D., Long, D., Pineiro, M., Coupland, C. andKomeda, Y. (2000) ACAULIS5, an Arabidopsis gene required for stem elongation, encodes aspermine synthase. EMBO J. 19: 4248-4256
2) Imai, A., Akiyama, T., Kato, T., Sato, S., Tabata, S., Yamamoto, K.T. and Takahashi, T. (2004)
Spermine is not essential for survival of Arabidopsis. FEBS Lett. 556: 148-152

88

Biological Sciences

1-7） Plant Biology

Molecular mechanism of phototropin kinase regulation by blue light
Satoru Tokutomi
Osaka Prefecture University
toxan@b.s.osakafu-u.ac.jp

Introduction
Phototropin [1] is a blue light receptor in plants for phototropism, chloroplast relocation,light-induced stomata opening and so on.

Phototropin molecule has two photoreceptive domains

and a kinase domain. The kinase activity is regulated by blue light received by the photoreceptive
domains, however, the details of the regulation mechanism has been obscure.

We tried to elucidate

the role of the photoreceptive domains in this photoregulation using a newly developed
phosphorylation assay system.

Results
Phototropin molecule has two photoreceptive domains named LOV in their N-terminal region and a
Ser-Thr kinase domain at the C-terminus (Fig. 1a). We prepared 3 constructs of Arabidopsis
phototrorpin 2 (phot2), 1) S/T kinase domain (KD), 2) KD + LOV2, 3) KD + LOV2 + LOV1 by an
E-coli expression system.

Until this study, only autophosphorylation has been reported and noother substrate than phot itself has been detected for phot kinase [2].

We surveyed systematicallycommon substrates for Ser/Thr kinase if it can be a substrate for phot kinase, and found casein is thebest substrate.

Using casein as a substrate, we found the followings [3].

1) KD constitutively phosphorylates casein independently on light conditions.
2) In KD + LOV2 polypeptide, LOV2 prohibit the kinase activity of KD in the dark, that is canceledupon light illumination indicating light activation of kinas.

This light effect was not observed on

introduction of an amino acid mutation that prohibited the photoreaction of LOV2.
3) In KD + LOV2 + LOV1 polypeptide, LOV1 attenuate the light activation of the kinase by LOV2.
4) When isolated LOV2 is mixed with KD, the LOV still bind to KD and shows the inhibition of thekinase activity.

Light illumination induced desorption of LOV2 and the kinase activation that were

not observed with isolated LOV1.
These are summarized schematically in Fig. 2.
To go into the details of the molecular mechanism underlying the light regulation of pot kinase,photoreaction and conformational changes leading to the regulation were studied by low-temperature

89

FTIR of hydrated films of Adiantum phytochrome 3 that comprises a N-terminal chromophoric
domain of plant phytochrome and a C-terminal full-length phototropin (Fig. 1b).

We found that

LOV2 showed a secondary structural changes, however, it was partially frozen in LOV1 suggestinga different conformational changes between them [3].

Furthermore, we found a mutation in aβ-strand E that forms so-call β-scaffold abolished the structural changes in the LOV2.

The

mutation is suggested to cancel the interaction between the β-scaffold and one α-helix resides
downstream of LOV2 domain to KD.

Importance of α-helical unfolding upon light perception issuggested including our present results.

These are also illustrated schematically in Fig. 2.

Fig.1 a) N -

Lov1

Lov2

S/T kinase

-C

b)
GAF

Target Proteins?

Fig. 2

LOV1

LOV2

Jα

Kinase Domain
Blue light
LOV1

P

P

attenuator

LOV2

switchJα

autophosphorylation

Target Proteins?

P substrate-phosphorylation

Kinase Domain

Discussion & Summary
We have proved for the first time that phot kinase can phosphorylate the other substrate that phot
itself.

This suggests presence of a possible substrate(s) in vivo that is involved signal transductionof phot.

The present LOV2 + KD polypeptide gives a useful in vitro assay system to detects the“real substrate(s)” involved in the light-signal transduction.
progress.

Investigation along this line is under90

References[1] Briggs, W. R. and J. M. Christie (2002) Phototropin 1 and 2: versatile plant blue-light receptors.
Trends Plant Sci. 7, 204-210.
[2] Matsuoka, D. and S. Tokutomi (2005) Blue light-regulated molecular switch of Ser/Thr kinase inphototropin. Proc. Natl Acad. Sci. USA 102, 13337-13342[3] Iwata, T., D. Nozaki, S. Tokutomi, and H. Kandori (2005) Comparative Investigation of the
LOV1 and LOV2 Domains in Adiantum Phytochrome3, Biochemistry 44, 7427-7434.

911) Biological Sciences

1-7） Plant Biology

Plant-plant communication mediated by volatile compounds.
〜Elucidation of its molecular mechanisms〜
Kenji MATSUI
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University
matsui@yamaguchi-u.ac.jp

Introduction
It has been reported that plants emit a special blend of volatile compounds when herbivorous
insects attack them. The surrounding plants can perceive the volatiles. Upon perception of thevolatiles, the plants evoke the defense systems in order to manage the future disaster. The instancesof this ‘plant-plant communication’ are accumulating, however, it has not been known what
compounds mediate the communication and how plants can perceive them. In this study, I would
like to identify the compounds and also to dissect the molecular machinery to perceive the volatiles
with a model plant, Arabidopsis.

Results
When intact and healthy Arabidopsis plants were exposed to volatile C6-aldehydes,(E)-2-hexenal and (Z)-3-hexenal, a subset of defense genes was induced, and plants’ resistanceagainst a necrotrophic pathogen (Botrytis cinerea) was induced. We attempted to dissect thecomponents involved in the induced resistance. The penetration of B. cinerea hyphae into
Arabidopsis epidermis and the growth of hyphae after penetration were suppressed on theC6-aldehyde-treated leaves when compared with those on the control leaves. We found that after thevolatile-treatment, the vascular bundles and cell walls of the leaves were intensely lignified. Higher
lignification could be observed with (E)-2-hexenal treatment compared to (Z)-3-hexenal treatment,
which coincided with the degree of overall resistance. Increase of high antifungal activities could befound with the treated leaves. C6-aldehydes also induced accumulation of PDF1.2 (plant
defensin1.2) and PR-3 (chitinaseB) transcripts, and camalexin. Taken together, these data suggestthat C6-aldehydes made Arabidopsis resistant to the pathogen due to induced lignification, which
formed a physical barrier to the penetration of the hyphae, and to the accumulation of antifungal
substances including PDF1.2, PR-3 and camalexin, which inhibited the growth of the hyphae in theplant cells.

92

In order to elucidate the signaling pathway mediating the defense responses induced by
C6-aldehydes in Arabidopsis, we compared the responses of Arabidopsis mutants deficient in thesignaling pathways; i.e., etr1-1 (ethylene resistant), jar1-1 (jasmonate resistant), npr1-1 (salicylic
acid insensitive), or pad2-1 (phytoalexin-deficient) with those of wild type (WT) plants. Induction ofsome, but not all of the defense genes in response to C6-aldehydes was significantly repressed in
jar1-1, etr1-1, and pad2-1, but not at all in npr1-1. C6-aldehyde-treatment enhanced accumulation of
camalexin with WT and npr1-1, but only partially with etr1-1 and jar1-1. pad2-1 showed little
accumulation of camalexin. npr1-1 accumulated the antifungal substances as WT did, however,
etr1-1, jar1-1 and pad2-1 exhibited only partial accumulation. The treatment enhanced resistance of
etr1-1, jar1-1 and npr1-1 against B. cinerea, but failed to enhance the resistance of pad2-1. Taken
together, it was suggested that ETR1-, JAR1-, and PAD2-dependent signaling pathways were
simultaneously activated by C6-aldehyde-treatment. Among these, PAD2-dependent signaling
appeared to be most important. In contrast, involvement of NPR1-dependent signaling was minimal.
Other than C6-aldehydes, monoterpenes are also emitted from plants after biotic attack.
Previously we found that volatile allo-ocimene enhanced resistance of Arabidopsis thaliana against
Botrytis cinerea. Because the structure of allo-ocimene is totally different from C6-aldehydes, it has
been assumed that responses caused by allo-ocimene are different from those caused by
C6-aldehydes. The penetration of B. cinerea hyphae into Arabidopsis epidermis and the growth ofhyphae after penetration were suppressed on allo-ocimene-treated leaves. allo-Ocimene also induced
lignification on cell walls and veins of the leaves. The treatment induced accumulation of antifungal
substances including Arabidopsis phytoalexin, camalexin. allo-Ocimene enhanced resistance against
B. cinerea in etr1-1, jar1-1 or npr1-1. Thus, it was suggested that a signaling pathway independent
on ETR1, JAR1 and NPR1 was operative to induce the resistance. The series of responses observed
after allo-ocimene-treatment was mostly similar to that observed after C6-aldehyde-treatment. The
effect of C6-aldehyde-treatment has been largely accounted to the chemical reactivities of thecompounds, however, from this result it was suggested that resistance responses of Arabidopsis
could be induced by the volatiles mostly independent on their reactivities and that a common
signaling pathway unaffected by the reactivities of compound was activated by the volatiles.

Discussion & Summary
Volatile compounds formed and emitted by plants while they were attacked by invaders can beperceived by neighboring plants, and they could start defense responses even without real invaders.
In this study, we used Arabidopsis-Botrytis system to dissect molecular mechanism of volatile
perception. It has been shown that C6-aldehydes could cause pleiotropic effects on the defense
system including lignification of plant tissues and accumulation of antifungal substances. Jasmonate
and ethylene signalings were at least partly involved in this response, and a signaling mediated by

93

glutathione must be also important. Interestingly, there was no apparent structure-dependency for thevolatiles to affect defense systems in plants. Thus, there might be a volatile perceiving system
independent of specific receptors. It is still controversial whether this kind of plant-plantcommunication, or eavesdropping, has ecological and evolutional relevance; thus, further study toreveal whole the system must be carried out. The novel knowledge waiting for us must be useful todevelop a novel tactics for the sustainable agriculture.

94

References[1] Components of C6-aldehyde-induced resistance in Arabidopsis thaliana against a necrotrophic
fungal pathogen, Botrytis cinerea. Kishimoto K, Matsui K, Ozawa R, & Takabayashi J (2006) Plant
Sci. 170, 715-723[2] Biosynthesis of fatty acid derived aldehydes is induced upon mechanical wounding and its
products show fungicidal activities in cucumber. Matsui K, Minami A, Hornung E, Shibata H,
Kishimoto K, Ahnert V, Kindl H, Kajiwara T, & Feussner I (2006) Phytochemistry 67, 649-657.
[3] Green leaf volatiles: hydroperoxide lyase pathway of oxylipin metabolism. Matsui K (2006) Curr
Opin Plant Biol (2006) 9, 274-280.
[4] ETR1-, JAR1- and PAD2-dependent signaling pathways are involved in C6-aldehyde-induced
defense responses of Arabidopsis. Kishimoto K, Matsui K, Ozawa R, & Takabayashi J (2006) Plant
Sci, in press.
[5] Analysis of defensive responses activated by volatile allo-ocimene treatment in Arabidopsis
thaliana. Kishimoto K, Matsui K, Ozawa R, & Takabayashi J (2006) Phytochemistry, in press.

951) Biological Sciences
1-7） Plant Biology

Non-Cell-autonomous flowering signal of FT is conferredby its protein trafficking.
Koji Goto
Research Institute for Biological Sciences, Okayama
kgoto@v004.vaio.ne.jp

Introduction
FLOWERING LOCUS T (FT) is one of the flowering pathway integrators of Arabidopsis.
FT integrates long-day responsive pathway of flowering and promotes flowering strongly.

FT isexpressed in the vascular tissue of leaf apical region and its expression is not detectable in the shootapical meristem (SAM).

FT function is, however, required in the SAM because flowering is

promoted when FT is expressed in the SAM specific manner.

These results suggest that FT

functions non-cell-autonomously, that is FT is expressed in the leaves and functions in the SAM.

Results
In order to visualize FT protein, we made FT-GFP fusion gene and introduced into ft mutants.
By using constitutive promoter, 35S, FT-GFP fusion gene rescued ft mutant phenotype dependent on
the FT-GFP conjunction sequences.

This suggests protein function of FT is impaired by theaddition of some peptide sequences.

Then, we transformed ft mutants by FT and functional

FT-GFP gene by using FT promoter.

We found that FT gene complemented ft, but FT-GFP did not.

This suggesting that FT-GFP lost its protein mobility. We examined protein localization of FT and
FT-GFP, but we failed to detect due to the low level of expression driven from FT promoter.
We moved our assay system to N. tabacum (tobacco), because tobacco is one of the best plants
for grafting experiments.

First, we tested whether Arabidopsis FT can promote flowering of

tobacco or not, because FT functions as a "florigen" which works in the wide range of flowering
plants. As expected, FT as well as functional FT-GFP promoted flowering in the tobacco driven by
35S promoter.

These transgenic tobacco, 35S::FT and 35S::FT-GFP were used as rootstock andgrafted wild type tobacco var. Maryland Mammoth (MM) as scions.

MM is a cultivar that flowers

in the short day conditions and never flowers when it is grown in the long day conditions, thoughnative tobacco is a day-neutral plant. While scions grafted on 35S::FT flowered after producing
eight to twelve leaves, scions on 35S::FT-GFP never flowered. These results suggest that FT
produced graft transmissible flowering signal and FT-GFP did not.

This is consistent with the

96Arabidopsis experiments. Together, these results suggest that FT or flowering signal produced by
FT can move beyond the grafting surface but FT-GFP cannot.
Recently, Huang et al., (2005) showed that FT RNA moves from Arabidopsis leaves to the
SAM and which is the molecular basis of systemic flowering signal transduction.

Our previous

data, however, show that FT as well as its homolog TFL1 proteins moves cell to cell in the SAM.
This protein trafficking is not diffusion but targeted movement (manuscript in preparation).

These

results suggest protein trafficking of FT contributes flowering signal transduction. Or at least, FT
trafficking helps to deliver FT protein, which is translated from RNA after unloaded from vascularsystem, to the SAM because vascular bundle is not developed in the SAM.
We next tried to detect FT RNA and protein in the grafted scion.

In the scions, grafted on the

35S::FT, we could detect FT protein about 10 ng per 1 g-fresh weight, but protein is under detectableamount in the scions grafted on 35S::FT-GFP.

FT RNA was not detected in both grafted scions,even by using Taqman real-time quantitative RT-PCR, which is the most sensitive detection method
at present.

It is pretty hard to prove that RNA is not transferred from rootstock to scion because it

remains a possibility that underdetectable small amount of RNA still exists.

However, our results

strongly suggest that protein trafficking through the grafting surface plays primary role for flowering
signal transmission produced by FT.

(Stock)(11+)
35S::FT:GFP
(32+)
+MM
(32+)
Scion
(31+)
1/10
35S::FT:GFP

Marker
1/10
35S::FT
1/50(Stock)
1/100
(6)
(7)
35S::FT+MM
(10)
Scion
(18+)
MM (SD)

FT protein was detected in the scion but FT-GFP was not.

anti FT
antibody

FT-GFP
FT

Discussion & Summary
While graft transmissible FT protein is detected in the scion, not transmissible FT-GFP protein
is not detected.

These results suggest the possibility that FT protein trafficking is the molecularbasis for the systemic flowering signal transduction produce by FT.

Our results do not contradict

the previous report showing that FT RNA movement transfer the flowering signal.
however, no molecular mechanism of FT RNA movement is shown.
FT RNA transmission or how FT RNA selectively transferred?

There is,What sequence is essential for

Farther analyses are required todetermine whether protein trafficking or RNA movement is the molecular basis or essential for theflowering signal transduction produced by FT.

97References
Abe, M., Kobayashi, Y., Yamamoto, S., Daimon, Y., Yamaguchi, A., Ikeda, Y., Ichinoki, H.,
Notaguchi, M., Goto, K., and Araki, T. (2005). FD, a bZIP protein mediating signals fromthe floral pathway integrator FT at the shoot apex. Science 309, 1052-1056.
Blazquez, M.A. (2005). Plant science. The right time and place for making flowers. Science 309,
1024-1025.
Huang, T., Bohlenius, H., Eriksson, S., Parcy, F., and Nilsson, O. (2005). The mRNA of the
Arabidopsis gene FT moves from leaf to shoot apex and induces flowering. Science 309,
1694-1696.
Wigge, P.A., Kim, M.C., Jaeger, K.E., Busch, W., Schmid, M., Lohmann, J.U., and Weigel, D.
(2005). Integration of spatial and temporal information during floral induction in Arabidopsis.
Science 309, 1056-1059.

982) Medical Sciences2-1) Immune System

Role of negative regulators of cytokine signaling in atopic disorders
Hiromasa Inoue, MD
Research Institute for Diseases of the Chest, Graduate School of Medical Sciences,
Kyushu University
inoue@kokyu.med.kyushu-u.ac.jp

Introduction
Atopic diseases, such as asthma, allergic rhinitis, and atopic eczema, have increased dramaticallyin prevalence, and patients with severe/refractory asthma are at great risk of death. Allergic airway
inflammation is an important feature of asthma, and its inflammatory process is mediated via various
cytokines.

T helper 2 (Th2)-type cytokines, interleukin (IL)-4, IL-5, and IL-13, play a critical rolein the development of asthma. These cytokines transmit signals through the JAK/STAT and theRas/ERK signaling pathways. The suppressor of cytokine signaling (SOCS) family proteins andSprouty-related EVH1-domain-containing protein (SPRED) family proteins have been shown toregulate the STAT pathway and the ERK pathway, respectively. However, the precise roles of IL-13,
SOCS, and SPRED in the pathophysiology of allergic asthma have not been clarified.

Results
Local administration of recombinant IL-13 to non-immunized mice induced the asthma
phenotype, and glucocorticoids were not sufficient to suppress IL-13-induced airway
hyperresponsiveness or goblet cell hyperplasia. Chloride channels have been implicated in theregulation of mucus production in epithelial cells, and we studied the effects of chloride channel
inhibitor, niflumic acid, in IL-13-induced asthma. Niflumic acid inhibited not only IL-13-induced
goblet cell hyperplasia but also airway hyperresponsiveness and eosinophilic infiltration. Niflumic
acid suppressed JAK2/STAT6 activation and eotaxin expression in epithelial cells.
High levels of SOCS3 expression in T cells resulted in Th2 skewing, and relate to allergic
diseases, and this was because SOCS3 binds to IL-12Rβ2 and inhibits IL-12 mediated STAT4
activation, thereby blocking Th1 development. T cell-specific SOCS3 conditional knockout mice
showed reduced responses to OVA-induced asthma, while little difference in the Th1 responses.
STAT3 and SOCS3 reciprocally modulate Th3 differentiation characterized by the production of
TGF-β1 and IL-10.
Using SPRED1-deficient mice, we demonstrated that SPRED1 negatively regulates

99

allergen-induced asthma without affecting helper T cell differentiation. SPRED1 suppresses
IL-5-dependent cell proliferation and ERK activation.

Discussion & Summary
These findings suggest that a) chloride channel inhibitor can control IL-13-mediated airway
features at least by suppressing JAK/STAT6 activation, b) the high expression of SOCS3 in T cells
led to skewing to Th2 differentiation.

SOCS3 also regulates Th2/Th3 balance, and the low levels of

SOCS3 in T cells enhanced Th3 differentiation. c) SPRED1 negatively controls eosinophil numbers
and functions by modulating IL-5 signaling in allergic asthma.
We consider the possibility that SOCS and SPRED proteins might be novel therapeutic targets forthe treatment of allergic diseases.

100

References
1) Nakano T, et al. Niflumic acid suppresses IL-13-induced asthma phenotypes. Am J Respir Crit
Care Med. 2006, 173: 1216.
2) Kinjyo I, et al. Loss of SOCS3 in T helper cells resulted in reduced immune responses andhyperproduction of IL-10 and TGF-β1. J Exp Med. 2006, 203:1021
3) Inoue H, et al. Spred-1 negatively regulates allergen-induced airway eosinophilia andhyperresponsiveness. J Exp Med. 2005, 201:73

1012) Medical Sciences2-1) Immune System

Identification of novel immune adjuvants from parasites or fungi
Tsuneyasu Kaisho
RIKEN Research Center for Allergy and Immunology
tkaisho@rcai.riken.jp

Introduction
Toll-like receptors (TLR) 7 and 9 recognize ssRNA and CpG DNA, respectively. Their signaling can
induce production of not only inflammatory cytokines but also type I interferons (IFNs). It remains
unclear whether TLR7 and TLR9 are involved in recognizing helminthes or how TLR7/9 induces
type I IFNs.

Results(1) IL-12 inducing activity from murine DCs was detected in nematode extracts. This activity wasabolished in mutant DCs lacking a cytoplasmic TLR adapter, MyD88, indicating possible
involvement of certain TLRs. Among several TLR-deficient DCs, TLR7 or TLR9 deficient DCs
were found to deteriorate the responses to nematode extracts.
(2) A DC subset, plasmacytoid DC (PDC), express TLR7 and TLR9 exclusively among TLRs. TLR7
and TLR9 are expressed also on non-PDC (conventional DC, cDC) and both PDC and cDC could
produce inflammatory cytokines in response to TLR7/9 agonists. However, only PDC can produce
IFN-α in response to TLR7/9 agonists. All of TLR7/9 functions require a cytoplasmic adapter,
MyD88, but at the downstream of MyD88, signaling cascades are bifurcated into NF-κB and IRF-7
activation pathways, which can lead to induction of inflammatory cytokines and type I IFNs,
respectively. IRF-7 should be phosphorylated to be activated, but the responsible kinases were notclear. We focused on IκB kinase-α, because it is a member of IKK family and the other three IKK
family members were known for their critical roles in TLR signaling. We have found that DCs fromthe mutant mice lacking IKKα, failed to produce IFN-α in response to TLR7/9 agonists. TLR7/9
signaling could activate the mutant DCs to produce inflammatory cytokines and the mutant DCs
could normally respond to other TLR agonists. Meanwhile, virus-induced type I IFN production inembryonic fibroblasts, which depends on cytosolic virus recognition system, was normal in theabsence of IKKα. These results indicate TLR7/9-induced type I IFN production specifically requires
IKKα (Fig.). IKKα could enhance the ability of IRF-7 to activate the IFN-α promoter and dominantnegative form of IKKα could inhibit MyD88-mediated activation of IRF-7. IKKα could associate

102with IRF-7 in DCs and in vitro kinase assays revealed that IKKα can phosphorylate IRF-7. Notably,
electrophoretic mobility shift assay showed TLR-induced IRF-7 activation was impaired in
IKKα-deficient PDC. Taken together, these results demonstrate that IKKα is critically involved in
TLR7/9-induced IRF-7 activation. and IFN-α production (Fig and ref. Hoshino et al.).

Discussion & Summary
Our results indicate that TLR7 and TLR9 may be involved in immune responses against helminth. It
should be investigated in the future how TLR7 or TLR9-deficient mice respond to helminth infection.
We could also identify IKKα as a critical molecule for TLR7/9-induced type I IFN production.
TLR7/9-induced type I IFN induction is supposed to be critical not only for antiviral immunity but
also for pathogenesis of certain autoimmune disorders such as SLE. Therefore, IKKα should be apotential target for manipulating host defense and autoimmunity.

References
K. Hoshino, T. Sugiyama, M. Matsumoto, T. Tanaka, M. Saito, H. Hemmi, O. Ohara, S. Akira,T. Kaisho. 2006. IκB kinase-α is critical for interferon-α production induced by Toll-like receptors
7 and 9. Nature. 440: 949-953.

1032) Medical Sciences2-1) Immune System

Molecular mechanism of the immune regulation maintained by
CD8+CD122+ immuno-regulatory T cells.
Haruhiko Suzuki
Nagoya University, Graduate School of Medicine
K46200a@nucc.cc.nagoya-u.ac.jp

Introduction++

We identified CD8 CD122 regulatory T cells that are important in regulating other T cells. In theabsence of these regulatory cells, other T cells become dangerously activated T cells that are harmful
for self and kill the individual host. Understanding the molecular action of the regulatory cells is anecessary step for the application of these cells to diseases. The aim of this study is to determine themolecular mechanism responsible for the suppressive activity of CD8+CD122+ regulatory T cells.

Results
We have already established the in vitro culture system to evaluate the activity of CD8+CD122+
regulatory T cells. We proceeded to identify the molecules involved in the regulation by adding
specific neutralizing antibodies into the culture system.
1. Molecular mechanism in the recognition of target cells to be regulated.
First, CD8+CD122+ regulatory T cells need to recognize the target cells that should be regulated.
We proved that CD8+CD122+ regulatory T cells recognize already-activated T cells stimulated by
specific antigen or non-specific reagent such as anti-CD3 antibody. In the recognition phase,interaction between MHC class I on the regulated cells and T cell receptor (TCR) on the regulatory
cells is essential. Furthermore, interaction between CD28 and CD80/CD86 molecules is criticallyinvolved for the regulatory cells to become active regulatory cells. We also performed experiments
using T cells from MHC class I-deficient mice and those from CD28 knockout mice, and confirmedthat these molecules are involved in the process of recognition or development into active regulatory
T cells.
2. Molecular mechanism in the effect-transmitting phase.
Culture supernatant of CD8+CD122+ cells under the stimulation by plate-bound anti-CD3
antibody effectively suppressed the proliferation and IFN-γ production of CD8+CD122- cells. This
result means that CD8+CD122+ cells produce medium-soluble factor(s) that transmit the suppressive

104effect. Experiments using specific antibodies that neutralize the function of various cytokines
revealed that IL-10 was the most important and essential factor that transmitted the effect of
CD8+CD122+ cells. Experimental results using the CD8+CD122+ cells derived from IL-10 knockout
mice further confirmed that IL-10 was the responsible factor in vitro. However, CD8+CD122+ cells
from IL-10 knockout mice showed reduced but significant effect to regulate CD8+CD122- cells,
indicating that the function of IL-10 is partially compensated by other factor(s) in vivo.

Discussion & Summary
In this study, we investigated on the molecular mechanism of CD8+CD122+ regulatory T cells. These
regulatory cells first recognize the target cells depending on the cell-cell interaction with a MHC
class I-restricted manner. It was highly suggested that TCR on the regulatory cells recognized theantigen on MHC class I molecule on the target cells. Once the CD8+CD122+ regulatory cells
recognize target cells, they become active regulatory cells. In the step of recognition and activationof the regulatory cells, interaction between CD28 on the regulatory cells and CD80/CD86 on thetarget cells is also required. Then, the active regulatory T cells produce soluble factors to suppress
proliferation and IFN-γ production of the target cells. The most important and essential soluble
factor produced by CD8+CD122+ regulatory cells is IL-10. However, the function of IL-10 may becompensated by other factors in an in vivo condition.

References
Rifa’i M, Kawamoto Y, Nakashima I, and Suzuki H. Essential roles of CD8+CD122+ regulatory T
cells in the maintenance of T cell homeostasis. J. Exp. Med. 200:1123-1134, 2004.
CD8+CD122+ regulatory T cells produce IL-10 to suppress IFN-γ production and proliferation of
CD8+ T cells. J. Immunol. 175:7093-7097, 2005.

105

106

107

2) Medical Sciences2-1) Immune System

Studies on Immune Regulation by Interleukin (IL)-27,
A New Member of The IL-12 Cytokine Family
Takayuki Yoshimoto
Intractable Immune System Disease Research Center, Tokyo Medical University
yoshimot@tokyo-med.ac.jp

Introduction
IL-27 is a novel IL-6/IL-12 family cytokine that is considered to play a role in Th1 differentiation,
whereas the exact role of IL-27 in Th1 differentiation and its molecular mechanism remain unclear.
Here, we demonstrate a role for IL-27 in the early regulation of Th1 differentiation and its possible
molecular mechanism.

Results
To first elucidate the role of IL-12 in IL-27-induced Th1 differentiation in vivo, we examined
whether IL-27 induces an antitumor activity even in IL-12 p40-deficient mice, and found that IL-27
can induce an antitumor activity by augmenting type 1 cell-mediated immunity in vivo in
IL-12-independent manner. We then examined the effect of IL-27 on in vitro Th1 differentiation withvarious concentrations of IL-12. Naive CD4+ T cells were purified from spleen cells and stimulated
in vitro by plate-coated anti-CD3 and anti-CD28 in Th1 polarizing conditions, neutral conditions, or
Th2 polarizing conditions in the presence or absence of IL-27. These primed cells were expanded in
IL-2-containing medium on day 3 and restimulated on day 6 by plated-coated anti-CD3 for 24 h.
Resultant supernatants were analyzed for cytokine production by ELISA. Consistent with the in vivo
results, IL-27 induced in vitro Th1 differentiation most efficiently in the absence of IL-12, and thatthe presence of higher concentration of IL-12 overruled the ability of IL-27 to induce Th1
differentiation. We then examined the requirement for STAT4, which is an essential
signal-transducing molecule to IL-12/IL-12R signaling, in IL-27-induced Th1 differentiation using
STAT4-deficient naive CD4+ T cells, whereas STAT4 was not required for IL-27-induced Th1
differentiation. Since IL-27 synergizes with IL-12 in primary IFN-γ production and IFN-γ is wellknown to play a critical role in Th1 differentiation through induction of T-bet and subsequent
IL-12Rβ2 expression, we next examined the requirement for IFN-γ in IL-27-induced Th1
differentiation using IFN-γ-deficient naive CD4+ T cells and neutralizing mAb against IFN-γ.
However, IFN-γ was not required for IL-27-induced Th1 differentiation. Several previous studies

108

revealed that ICAM-1/LFA-1 interaction is important for Th1 polarization and that ICAM-1
expressed on T cells also can provide a costimulatory signal for T cell activation and Th1 cytokine
production. More recently, it has been demonstrated that ICAM-1/LFA-1 interaction favors humanTh1 development and this Th1 differentiation is overruled by IL-12 in a dose-dependent manner.
Therefore, we next examined the cell surface expression of ICAM-1 and its counterpart LFA-1 onnaive CD4+ T cells. IL-27 induced rapid and marked up-regulation of ICAM-1 expression on naive
CD4+ T cells stimulated with plate-coated anti-CD3 and anti-CD28. Blocking experiments using
anti-ICAM and/or anti-LFA-1 revealed that the ICAM-1/LFA-1 interaction plays an important rolein the IL-27-induced Th1 differentiation. Since IL-27 induces expression of T-bet, we next examined
the requirement for T-bet in IL-27-induced Th1 differentiation using T-bet-deficient mice. However,T-bet was revealed not to be essential to IL-27-induced rapid up-regulation of ICAM-1 expression
and Th1 differentiation. We then investigated the role of STAT1 in IL-27-induced Th1 differentiation
using STAT1-/- and STAT1+/- mice. In STAT1-/- naive CD4+ T cells stimulated with plate-coatedanti-CD3 and anti-CD28, induction of T-bet expression was almost abolished even in the presence of
IL-27, IL-12 or both, although efficient induction of T-bet expression by IL-27 was observed in
STAT1+/- naive CD4+ T cells. Moreover, the absence of STAT1 markedly inhibited the Th1
differentiation induced by IL-27 as well as IL-12. These results suggest that STAT1 is required for
IL-27-induced rapid up-regulation of ICAM-1 expression and Th1 differentiation. It was previously
demonstrated that STAT1 activated by IFN-γ binds to the GAS located in the 5’-flanking region of
ICMA-1 gene and confers IFN-γ responsiveness. To definethemechanism

whereby IL-27 induces up-regulation of ICAM-1 expression, we finally performed a reporter assay
and EMSA, and reveled that STAT1 directly mediates IL-27-induced transcriptional up-regulation of
ICAM-1 gene expression.

Discussion & Summary
The present results suggest that the ability of IL-27 to induce Th1 differentiation is most prominent
under Th1 polarizing conditions but without IL-12 and overruled by IL-12 dose-dependently. The
IL-27-induced Th1 differentiation was mainly mediated by rapid and marked up-regulation of
ICAM-1 expression on naive CD4+ T cells through ICAM-1/LFA-1 interaction in STAT1-dependent
but T-bet-, IFN-γ- and STAT4-independent mechanism. Considering that IL-27 is produced frommacrophages and DCs earlier than IL-12, the present results suggest that IL-27 may play a pivotal
role in early efficient induction of Th1 differentiation until enough amounts of IL-12 are produced(Figure 1).

109

References
T. Owaki, N. Morishima, M. Asakawa, Y. Iwakura, F. Fukai, M. Matsui, J. Mizuguchi, and T.
Yoshimoto. A role for IL-27 in early regulation of Th1 differentiation. J. Immunol., 175, 2191-2200,
2005.

110

2) Medical Sciences2-2) Central Nervous System

The analysis of molecular mechanism s of schizophrenia
and bipolar disorder caused by 22q11.2 deletion
Kenji Tanigaki
Research Institute, Shiga Medical Center
tanigaki@shigamed.jp

Introduction
Children with del 22q11.2 syndrome have learning difficulties, deficits of motor development, andattention deficit disorders1. They have high risk for developing schizophrenia and bipolar disorder.
Df1 is a mouse homologous region of human chromosome 22q11.2. Mice that are heterozygously
deleted for Df1 region have deficits in pre-pulse inhibition, learning and memory2.

Results
Df1 region contains about 30 genes including COMT and PRODH. These genes are shown to be
associated with schizophrenia and bipolar disease by genetical studies. Knockout mice of thesegenes show behavioral abnormalities such as hyper-locomotion and pre-pulse inhibition deficit,
which are characteristic physiological disorders of patients with schizophrenia. Our aim is toelucidate which gene and which neural circuit is responsible for the psychiatric disorders of
Df1-deleted mice. We are planning to use lenti virus and adeno virus mediated gene delivery to
rescue these phenotypes of Df1 deleted mice. The neural circuit of pre-pulse inhibition includes
hippocampus (HP), prefrontal cortex (PFC), ventral tegmental area (VTA), nucleus accumbens(NAc), ventral pallidum, basolateral amygdala (BLA), mediodorsal thalamus (MD) andpedunculopontine nucleus (PPTg), which modulates responses ofthe primary startle circuitcomposed of the auditory nerve, the cochlear root neurons, the nucleus reticularis pontis caudalis(PnC), and the spinal motor neuron3.
It has been reported that adenovirus injection to embryonic ventricles enables introduction ofexogenous genes to specific layers of cerebral cortex4. However, It has not been known which neuralcircuit is affected by this adenovirus mediated gene delivery method, because adenovirus mediatedgene introduction is only transient and introduced genes are lost by cell divisions after gene
introduction. To identify all neural substrates effected bythis adenovirus-mediated gene delivery atembryonic stage, we injected Cre-expressing adenovirus to embryonic ventricles of ROSA26 Cre
reporter (R26R) mice5 at various developmental stages, E13.5, E14.5, and E15.5. The R26R consists

111of a floxed PGK neo cassette with four polyadenylation sites and a ß-galactosidase gene (ß-Gal)
downstream of the distal LoxP. In the absence of Cre the expression of ß-Gal is blocked by the PGK
neo polyadenylation sites. In the presence of Cre the LoxP sequences flanking the PGK neo cassette
are deleted and ß-Gal is expressed. With their ß-Gal activity we can trace all progeny of
adenovirus-infected cells. In brains infected at E13.5 or E14.5, ß-Gal-positive cells were observed incortex (layer IV and II/III), BLA, NAc, and CA1, CA2, CA3 and dentate gyrus of HP, and MD.

Inbrains infected at E 15.5, lacZ-expressing cells were found only in cortex (layer II/III), NAc, andhippocampus CA1, CA2 region (Figure1,2).
We are also planning to search anatomical and developmental abnormalities of Df1-deleted mice.
The migration pattern of differentiated neurons of early postnatal mice and the histology ofhippocampus will be examined. This is because Disc1, which is known to be genetically associatedwith schizophrenia, is important for migration of differentiated neurons and damage to ventral
hippocampus cause similar behavioral deficits.

Discussion & Summary
Our Results showed that adenovirus injection to embryonic ventricle can introduce exogenous genes
not only to cerebral cortex but to BLA, NAc, and CA1, CA2, CA3 and dentate gyrus of HP, and MD,
which play pivotal roles in PPI, though their expression is lost by cell divisions after birth. This
results suggest potentials of this adenovirus-mediated method to modulate PPI neural circuit directly.
Gene delivery to mature neurons is very difficult. However, our method of virus-mediated genedelivery allowed us to elucidate which gene in which neural circuits are responsible for behavioral
abnormalities of schizophrenia and bipolar disorder. This research can contribute to the exploitationof new remedy for schizophrenia and bipolar disease.

112

References
1.
2.
3.
4.
5.

Karayiorgou, M. & Gogos, J. A. The molecular genetics of the 22q11-associated sch
izophrenia. Brain Res Mol Brain Res. 132, 95-104. (2004).
Paylor, R. et al. Mice deleted for the DiGeorge/velocardiofacial syndrome region sho
w abnormal sensorimotor gating and learning and memory impairments. Hum Mol G
enet. 10, 2645-50. (2001).
Swerdlow, N. R., Geyer, M. A. & Braff, D. L. Neural circuit regulation of prepulse
inhibition of startle in the rat: current knowledge and future challenges. Psychophar
macology (Berl). 156, 194-215. (2001).
Hashimoto, M. & Mikoshiba, K. Neuronal birthdate-specific gene transfer with adeno
viral vectors. J Neurosci. 24, 286-96. (2004).
Soriano, P. Generalized lacZ expression with the ROSA26 Cre
reporter strain.
NAT Genet. 21, 70-1. (1999).

1132) Medical Sciences2-2) Central Nervous System

Role of forkhead transcription factor Foxo1 in feeding behavior
Jun Nakae
Department of Clinical Molecular Medicine, Division of Diabetes, Digestive and Kidney
Disease, Kobe University Graduate School of Medicine
nakaej@med.kobe-u.ac.jp

Introduction
Obesity should be a cause of type 2 diabetes, cardiovascular disease, hypertension andhyperlipidemia. Therefore, it is very important to elucidate molecular mechanism of obesity and todevelop drugs for preventing obesity. Obesity is defined by both increased food intake and decreased
energy expenditure. Now two important hormones are known well for regulating food intake. The
first one is leptin, which is secreted from white adipose tissue, and the second one is insulin, which
is secreted from pancreatic β-cell. Both hormones act through arcuate nucleus in hypothalamus.
However, the molecular mechanism by which insulin regulates food intake, especially downstreamof Irs-2, has not been known yet. Forkhead transcription factor FoxO1 is phosphorylated byinsulin/IGF-1 through PI3-kinase-dependent manner and known to be a negative regulator of insulin
signaling. Recently, we found out that FoxO1 is also expressed in the arcuate nucleus in
hypothalamus. The purpose of this project is to clarify role of Foxo1 in feeding behavior. For thispurpose, we construct knock-in mice of two kinds of mutant FoxO1 (3A; constitutively active typeand ∆256; dominant negative type) in ROSA26 genomic locus. Using Cre-loxP system, we willcross ROSA26/ mutant Foxo1 knock-in mice with Pomc Cre or Agrp Cre in order to overexpress
mutant Foxo1 in arcuate nucleus-specific manner and analyze effects of overexpression of mutant
Foxo1 on feeding behavior and glucose metabolism.

Results
1. Construction of Rosa26-mutant FoxO1 knock-in mice
In order to investigate roles of FoxO1 in arcuate nucleus, we tried to overexpress two kinds ofmutant FoxO1s which include 3A (a constitutively active mutant FoxO1) and ∆256 (a dominantnegative FoxO1) in a neuron-specific manner using Cre recombinase transgenic mice(Figure 1a).
During this research period, we established both Rosa26-3A and Rosa26-∆256 knock-in mice
(Figure 1b).
2. Overexpression of mutant FoxO1s in Agrp/Npy neuron or Pomc neuron

114

For overexpression of mutant FoxO1s in a neuron-specific manner, we crossed Rosa26-FoxO1
knock-in mice with Agrp Cre or Pomc Cre transgenic mice.
In this report, we described data from Rosa26-3A FoxO1-Agrp Cre mice. About other knock-in
mice of FoxO1, projects are going on and not finished.
3. Growth curve of Rosa26-3A FoxO1-Agrp Cre mice
In both male and female Rosa26-3A-Agrp Cre mice, we could not see any significant differencescompared with other genotyped mice.
4. Food intake of Rosa26-3A-Agrp mice
There were any significant differences of food intake in Rosa26-3A-Agrp Cre mice compared withother genotyped mice.
5. Glucose metabolism of Rosa26-3A-Agrp Cre mice1) IPGTT(intraperitoneal glucose tolerance test)
In both male and female Rosa26-3A-Agrp mice, glucose tolerance was better than in othergenotyped mice (Figure 2a).
2) ITT (insulin tolerance test)
In male Rosa26-3A-Agrp Cre mice, we could observe better insulin sensitivity (Figure 2b).
6. Oxygen consumption (VO2)
In male Rosa26-3A-Agrp Cre mice, increased oxygen consumption was observed.

115

Discussion & Summary
We established two kinds of Rosa26-mutant FoxO1 (3A and ∆256) knock-in mice. Using one(Rosa26-3A FoxO1) of them, we analyzed effects of overexpression of 3A FoxO1 in Agrp/Npy
neurons on growth, food intake and glucose metabolism. Although there are no significant
differences in body weight and food intake, mutant knock-in mice showed better glucose tolerance
and insulin sensitivity. Furthermore, they showed increased oxygen consumption. The 3A FoxO1 is aconstitutively nuclear mutant and reflects defect of insulin signaling. Therefore, these data suggestthat insulin signaling in Agrp/Npy neuron worsens glucose metabolism and also transcriptional
activation of FoxO1 may improve insulin sensitivity.

References
1. Barsh GS and Schwarts MW. 2002. Genetic approaches to studying energy balance: perception
and integration. Nature Genet Rev.3: 589-600.
3. Accili, D., and Arden, K.C. 2004. FoxOs at the crossroads of cellular metabolism, differentiation,and transformation. Cell 117:421-426.

1162) Medical Sciences2-4) Arthritis/Bone Metabolism

Cell cycle regulation therapy to treat rhueumatoid arthritis
Hitoshi Kohsaka,M.D.
Tokyo medical and Dental University
Kohsaka.rheu@tmd.ac.jp

Introduction
Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by synovial hyperplasia
with massive infiltration of inflammatory cells in the affected joins. This leads to functional loss ofthe joints. Current treatments try to suppress inflammatory mediators including tumor necrosis alpha,but do not effective to all patients. We have been developing cell cyecle regulation treatment since
we showed that intraarticlar gone transfer of cyclin-dependent kinase inhibitors (SDKIs) to suppress
synovial hyperplasia has been effective in treating animal models of rheumatoid arthitis (RA) (1-3).
CDKIs inhibt cell cycling by inhibition of cyclin-dependent kinases (CDKs) and also modulate
immune function in a CDKindependent pathway. Therefore, we whold like to see whether systemic
administration of small-molecule (sm) CDK inhibiting compounds can ameliorate arthritis.

Results
The effects of smCDKIs, clinically tolerated flavopiridol and a newly synthesized CDK4/6-selective
inhibitor, compound A , on synovial cell growth were determined using proliferation and cell cycle
analyses. Mice were subjected to immunization with typeII collagen (CII) or serum transfer of
K/BxN mice to develop arthritis. They were then treated with systemic administration of smCDKIs
and the severity of arthritis was assessed clinically and histologically.
In vitro, smCDKIs inhibited proliferation of synovial fibroblasts without inducing apoptosis.
Treatment of collagen-induced arthritis (CIA) with flavopiridol suppressed synovial hyperplasia andjoint destruction. However, serum levels of anti- CII antibodies were preserved. The treated micedeveloped arthritis after termination of treatment. There facts argue that immune responses to CII
were not impaired. The same treatment ameliorated K/BxN serum-induced arthritis elicited inlymphocytedeficient mice. Similarly, a CDK4/6-selective inhibitor suppressed CIA, and did so evenwhen administered after the onset of clinical disease.
Figure 1 depicsts Arthritis scores of CIA mice treated with flavopiridol. Mice with CIA were treatedwith intraperitoneal injection of 1mg/kg, 2.5 mg/kg, flavopiridol in 0.01 % DMSO, or DMSO saline
for 10 consecutive days (a) or twice a week for 5 weeks (b). Treatment was started 25 (a) or 24 (b)117days after the initial immunization. Severity of arthirtis was assessed with artritis acore. Shown are
the mean score ± SD of 7 mice per group. Another set of 2.5 mg/kg flavopiridol treatment wasstarted concurrently, but was terminated after 10 days (b, open aquare). *: p<0.05, **: p<0.01,***: p<0.001 Figure 2 depicts Treatment of CIA mice with compound A. CIA mice were treatedwith compound A orally (200 mg/kg) every 12 hours for 7 days. Treatment was started at the day
when arthritis became evident (24 days after the initial immunization). CIA mice were also
therapeutically treated with compound A intraperitoneally (30 mg/kg) for 5 days and repeated again
after a day interval. Control mice were treated with buffer including DMSO. Severity of arthritis wasassessed by the arthritis score, ankle width, or paw thickness. Shown are the mean ±SD of 8 mice
per group. *: p<0.05, **: p<0.01, ***: p<0.001

Discussion & Summary
We have demonstrated that systemic administration of smCDK inhibitors is anti-arthritic, with noapparent impairment of immune responses. In addition, the smCDK inhibitors were effective intreating lymphocyte-independent arthritis. Although we cannnot formally exclede their effects on
immune cells, these results argue that the effects of smCDK inhibitors on synovial fibroblasts
primarily account for the therapeutic effects. Previous studies have shown that endogenous CDKIs
have an immunomodulatory effect, which done not depend on the kinase activity of CDKI, and thus
could be responsible at least partly for the therapeutic effects of CDKI gene therpy. 16,17 However,the present study has detemined that inhibition of CDK activity alone is sufficient to ameliorate
animal models of RA. We propose that smCDKIs be designated a new class of anti-rheumatic drugs,
which inhibit a distinct phase in rheumatoid pathology.

Figere1

118

Figure2

References
1.Taniguchi K, kohsaka H, et al. Induction of the p16INK4asenescence gone as a new therapeuticstrategy for the treatment of rheumatoidarthritis. Nat Med 1999;5(7):760-7.
2.Nasu K,kohsaka H, Nonomura Y, et al. adenoviral transfer of cyclindependent kinase inhibitor
genes suppresses collagen-induced arthritis in mice. J Immunol 2000;165(12):7246-52.
3.Nonomura Y, Kouhsaka H, et al. Suppression of arthritis by forced expression of cyclin-dependent
kinase inhibitor p21(Cip1) gene into the joints. Int Immunol 2001;13(6):723-31.

1192) Medical Sciences2-5) Cardiovascular /Metabolic/Endocrine

Creation of knock-down transgenic rabbits using RNAi technology
for the study of atherosclerosis
Jianglin Fan
Department of Molecular Pathology, Interdisciplinary Graduate School of Medicine
and Engineering, University of Yamanashi
Jianglin@yamanashi.ac.jp

Introduction
Rabbits are considered an excellent experimental model for the study of atherosclerosis. We
laboratory has been using transgenic rabbits to investigate many gene functions and theirrelationship with atherosclerosis. However, compared to mice, rabbit ES cells are still unavailable, it
is not possible to generate knock-out (KO) rabbits using gene target technology. Nuclear transfer
may be used as another method to create KO rabbits, however, there are many technical problems
which remain unsolved. In this study, we performed a pioneer study aiming at generating novel
knock-down (KD) rabbit models using RNAi technology. Although the technology is still in theimmature stage, it will provide a new avenue to create new rabbit models for biomedical research.

Results
In this study, we performed the following studies. First, we cloned rabbit lipoprotein lipase cDNA
sequences, which will be used as a potential target for gene-knock-down. Second, we created atRNA-promoter-directed construct for the gene transfer. This construct contains GFP sequencewhich will allow the detection of the target gene in transfected cells or tissues in vivo. The constructalso has two copies of insulator, which keep the tissue-specific expression at the stable levels
independent upon the copy number and location of the transgene. The efficacy of this construct was
confirmed in vitro using cultured cells. The major difficulties in developing KD rabbits are infinding effective sequences used for gene silencing of the target gene and methods for the evaluationof gene-silencing ability. To explore a new method in this regard, we selected rabbit cholesteryl estertransfer protein (CETP), an important protein for the lipoprotein metabolism, which is not present inmice. Using the internet sequence information, we identified several sequences for rabbits CETP
silencing sequences. After that, we attempted to find a hepatocyte cell line of rabbits. For thispurpose, we received 16 hepatocyte cells lines of rabbit produced by Dr. Nakanishi’s lab. This work
is still under way and hopefully we can have such cell lines for the evaluation of any genes

120expressed in the liver before we can start microinjection into rabbits.

Discussion & Summary
In this study, we have performed a pioneer study aiming at the creation of KD transgenic rabbits.
Apparently, there are still several obstacles in technology, including the establishment of the easy
method for the evaluation of gene-silencing efficacy in vitro, construct that can be used for
microinjection, and germline transmission from founders to the offspring. To target thehepatic-specific gene, the hepatocyte lines are urgently needed. We also work out these proceduresand hopefully we can find a new way to generate KD rabbits in future.

References
1. Fan J, Chen YE: Source(s) of CRP in atherosclerotic lesions, Am J Pathol 2006, 168:1054-1056
2. Liang J, Liu E, Koike T, Yu Y, Kitajima S, Yingji J, Morimoto M, Watanabe T, Sasaguri Y,
Watanabe S, Fan J: Macrophage elastase (MMP-12) accelerates the progression of atherosclerosis intransgenic rabbits, Circulation 2006, 113:1993-2001
3. Sun H, Koike T, Ichikawa T, Hatakeyama K, Shiomi M, Zhang B, Kitajima S, Morimoto M,
Watanabe T, Asada Y, Chen YE, Fan J: C-reactive protein in atherosclerotic lesions: its origin andpathophysiological significance, Am J Pathol 2005, 167:1139-1148
4. Kitajima S, Liu E, Morimoto M, Koike T, Yu Y, Watanabe T, Imagawa S, Fan J: Transgenic
rabbits with increased VEGF expression develop hemangiomas in the liver: a new model for
Kasabach-Merritt syndrome, Lab Invest 2005, 85:1517-1527

1212) Medical Sciences2-6) Gastro-intestinal

Regulation of macrophage responses to commensal flora
by the colonic mucosal microenvironment
Taeko Dohi
Research Institute, International Medical Center of Japan
dohi@ri.imcj.go.jp

Introduction
In spite of enormous numbers of Gram-negative bacteria with outer membrane lipopolysaccharide(LPS) in the colon, inflammatory responses normally do not occur.

Our previous study showed thatmacrophages in the large intestinal mucosal site are hyporesponsive to lipopolysaccharide (LPS) in
terms of release of inflammatory cytokines, such as TNF-α.

Since we found that various

inflammatory cytokines and chemokines are constitutively produced in the colonic mucosa, wehypothesized that particular microenvironment in the colon comprised of these molecules renderedmacrophages to be hyporesponsive to LPS in steady state.

Results
1. Murine bone-marrow (BM) derived macrophages or a macrophage cells lineRAW264.7 werepretreated with various cytokines and chemokines known to be produced in colonic mucosa,
including IL-4, IL-13, activin A, GM-CSF, MIF, lymphotactin, TGF-β, IL-1β, and IL-6 for 12 hours
and then stimulated with LPS to measure release of TNF-α. Among these cytokine and chemokine
molecules, fractalkine (CX3CL1, FKN) was a potent inhibitor of TNF-α release induced by LPS
stimulation, which was aound 50 %.

Dependence of this effect on IL-10 was limited because FKN

inhibited TNF-α release by cells derived from IL-10 knockout mice, although less extent than as
seen in cells from wild type mice.

The levels of IFN-γ, IL-12, MCP-1, IL-10 or IL-6 release after

LPS stimulation was not alterf by pretreatment with FKN.

FKN itself showed no effect on the

TNF-α, IL-10 or other cytokine production tested in both BM-derived macrophages or RAW267.4
cells.
2. In immunohistological examination, we found that FKN was abundantly found in both small andlarge intestine of mouse, in epithelial cells and mesenchymal cells.

Colonic tissue showedstronger staining of FKN than small intestine. We isolated myofibroblasts from the small andlarge intestine and established cell lines. Myofibroblasts derived from colonic tissue showed muchhigher expression of FKN than those from the small intestine, as determined by RT-PCR and122

Western-blotting.
3. When BM-derived macrophages or RAW267.4 cells were pretreated with low concentration of
FKN (1 ng/ml), expression of cell-surface Toll-like receptor 4 and MD-2 were decreased,
determined as mean fluorescence intensity in flow cytometry.
CD14 did not change.

On the other hand, expression of

The levels of mRNA of both molecules determined by RT-PCR also

decreased after treatment with FKN when compared with cells without pretreatment.
4. MAP kinase activation was analyzed by western blotting using antibodies against
phophorylated ERK1 and ERK2.
activated in RAW264.7 cells.

Six hours after LPS stimulation, MAP kinases were dramatically

In contrast, when cells were pretreated with FKN for 12 hours, suchstrong MAP kinase activation after LPS stimulation was not seen.
5. One hour after stimulation of RAW267.4 cells with LPS, nuclear translocation of NF-κB subunits,
p65/p50, was observed by EMSA; however, pretreatment with FKN prevented nuclear translocationof p50. We found that translocation of p65 was not inhibited but rather enhanced in FKN-treatedcells using transfactor assay and immunostaining of cells analyzed with fluorescent microscopy.
When cells were treated with high concentration of FKN (100 ng/ml), mild activation of NF-κB
was seen; however this activation was blocked in the presence of anti-FKN antibody in the culture.
This indicated that effect of addition of FKN solution to the cells was not due to the contamination
with endotoxin but FKN itself.
6. We stimulated RAW264.7 cells with LPS derived from different Gram negative bacteria strains(Salmonella minnesota, Echerichia coli, and Bacteroides vulgatus) as well as CpG-ODN,
peptidoglycan and Pam3CSK4, after pretreatment with FKN.

Of interest, inhibitory effect of FKN

on TNF-α release was seen only when cells were stimulated with LPS, ligand for Toll-like receptor
4, irrespective of their origin and potential to induce TNF-α release.

Peptidoglycan, Pam3CSK4,

and CpG-ODN, which are ligands for Toll-like receptor 2 and 9, efficiently induced TNF-α
secretion, which was comparable to the levels of LPS; however, it was not affected by pretreatment
with FKN at all.

These results indicated that FKN modulated the signaling pathway transduced by

Toll-like receptor 4, but not Toll-like receptors 2 or 9.

Discussion & Summary
The treatment of macrophages with FKN attenuated the TNF-α production induced by LPS
stimulation. FKN modified the signaling pathway and activation of nuclear transcriptional factor
after LPS stimulation.

Our findings, decrease of the expression of TLR4-MD-2 molecules,

modulated NF-κB activation and suppression of MAPKs phophorylation, caused by pretreatment
with fractalkine, all supported the suppression of TNF-α secretion after LPS stimulation.

Since wefound the constitutive production of fractalkine by myofibroblasts and epithelial cells in the colon,

123

FKN may contribute to hyporesponsiveness of colonic macrophages to LPS.
Possible mechanism of specific modification of signaling via Toll-like receptor 4 in the macrophage
responses to LPS is under further investigation.

1242) Medical Sciences2-10）Dermatology

A study of metastatic trait of malignant melanoma with expressionof MCM3-binding β-primase GANP correlated withhighly metastatic clinical cases
Kazuhiko Kuwahara
Department pf Immunology, Graduate School of Medical Sciences, Kumamoto University
kazukuwa@kaiju.medic.kumamoto-u.ac.jp

Introduction
Radical treatment of malignant melanoma is hampered by the highly metastatic trait of cells.

It isimportant to detect the nevus cells or melanoma cells that are potent to be transformed into highly
metastatic property at the primary resection.

Here, we found that MCM3-binding β-primase GANP

is up-regulated in the severe stage malignant melanoma cells. Moreover, we hypothesized that theabnormal expression of the GANP is involved in transformation of malignant metastatic trait intomelanoma cells.

Results
The department of dermatology has a largest collection of Japanese patients of malignant melanoma
patients in the western area and has long records of clinical courses.

We have studied the pastclinical samples to evaluate our system of detecting a highly metastatic cell at a single cell level2).
First, we examined if melanoma cell lines also up-regulated the GANP expression compared tomelanocytes and next observed the subcellular localization of GANP and P-GANP in the nucleus
and cytoplasm.

GANP and P-GANP were weakly expressed in normal melanocytes but they were

up-regulated in melanoma cell lines (G361, HM3KO and MeWo).

RT-PCR products of MCM3 andBCL-6 mRNAs were detected in human melanocytes and all three human melanoma cell lines.
There was no significant difference in the level of MCM3 message.

Second, we analyzed theexpression of GANP and P-GANP in various melanocytic lesions.

GANP and P-GANP were

widely expressed at various levels both in benign and malignant melanocytic lesions.

Most of themelanocytic cells co-expressed GANP and P-GANP, which was compatible to human melanoma celllines. Intensity of GANP expression was not associated with disease progression. On the other
hand, P-GANP in primary and metastatic melanoma lesions was significantly stronger than that ofmelanocytic nevus lesions (p<0.001 and p=0.015, respectively) (Table 1).

Intracellular localization

125

of GANP and P-GANP in melanocytic nevus cells and melanoma cells was not significantly
different.

Third, we analyzed the levels of MCM3, BCL-6, and Ki67 expression in 10 melanocytic

nevi, 11 primary melanoma, and 5 metastatic melanoma lesions.

MCM3 and Ki67 were

exclusively detected in primary and metastatic melanoma cells but not in melanocytic nevi cells.
BCL-6 was only detected in 1 primary lesion with 20% positive cells, although BCL-6 waspreviously shown as a highly indicative marker for poor prognosis of melanoma patients.

We

compared the levels of MCM3 and/or Ki67 expression with GANP and P-GANP in 17 primarymelanoma lesions.

Higher expression of MCM3 and/or Ki67 (more than 20% of positive cells) was

not associated with intensity of GANP and P-GANP expression in melanoma cells. In addition,cytoplasmic GANP and P-GANP expression showed a trend to higher MCM3 and Ki67 positive
cells than those of nuclear localization, but this may not be significant, since the number of theselesions studied was too small for statistical analysis (Figure 1).

These results suggested that MCM3

and GANP co-localizations are functionally related with cell cycle progression or cell proliferationin the melanoma cells.

Finally, we established two kinds of mutant mice carrying ganp-gene in themenocytes using tyrosinase promoter, because we recently reported lymphomagenesis in
ganp-transgenic mice1).

Although we observed melanocyte-specific ganp-transgenic of

ganp-deficient mice for about one year, the mice did not show any abnormalities in skin cells.

As it

is very important to ask if the onset of malignant transformation is spontaneous or if it is affected by
the various environmental stresses on the mice, we are now doing contaneous observation of thephenotypes in these mice after exposure by ultraviolet and other chemical agents.

126

Table 1. Intensities of GANP and P-GANP expression in surgically removed melanocytic lesions
GANP

P-GANP# Strong(%) Moderate(%) Weak(%) Strong(%) Moderate(%) Weak(%)

Melanocytic nevus

140

10 (71)

4 (29)

Primary melanoma

36

3(8)

28(78)

Metastatic melanoma 9

2(22)

6(67)

0

8(57)

6(43) P = 0.001

5(14) 11(31)22(61)

3(8)

1(3)

5(56)

1(11)

3(33)

P = 0.023#: total number of lesions, %:Perceutage of lesions

Discussion & Summary
The present study has demonstrated for the first time that GANP and P-GANP were up-regulated inhuman melanoma cell lines compared to human melanocytes.

GANP and P-GANP were expressedin the nucleus of melanocytes but co-expressed both in the nucleus and slightly in the cytoplasm of
melanoma cells.

Intensity of P-GANP staining in melanoma cells showed stronger than that ofbenign melanocytic nevus cells. However, intensity of GANP staining in melanoma cells did notreach significant difference.

These findings suggest that certain high level of GANP as well as

P-GANP expression may be associated with malignant transformation through direct or indirectinteractions with other factors that must play an important role in melanoma disease progression.
Most of the melanocytic cells showed cytoplasmic and nuclear localizations of GANP and P-GANP,
but nevus cells at lower portions of the dermis showed nuclear localization exclusively.

On theother hand, cytoplasmic localizations of GANP and P-GANP in melanoma cells were associatedwith higher MCM3 and Ki67 expressions than those of nuclear localization suggesting that GANP
expression is shuttled between the cytoplasm and the nucleus during cell cycle progression. These
data were agreeable to those of cultured melanocytes and melanoma cells, since melanocytes showed
only nuclear expression of GANP and P-GANP.

References
1) Fujimura, S., Xing, Y., Takeya, M., Yamashita, Y., Ohshima, K., Kuwahara, K. & Sakaguchi, N.
Increased expression of germinal center-associated nuclear protein RNA-primase is associated withlymphomagenesis. Cancer Res. 65(13):5925-5934 (2005)
2) Kageshita, T., Kuwahara, K., Oka, M., Ma, D., Ono, T. & Sakaguchi, N.
Increased expression of germinal center-associated nuclear protein (GANP) is associated withmalignant transformation of melanocytes. J. Dermatol. Sci. 42(1):55-63 (2006)

1272) Medical Sciences2-12) Hamatology

Role of Polycomb-group complexes in Geminin degradation which is
crucial for hematopoietic stem cell regulation
Yoshihiro Takihara
Research Institute for Radiation Biology and Medicine
takihara@hiroshima-u.ac.jp

Introduction
Hematopoietic stem cell (HSC) transplantation is now routinely performed in clinical practice for thetreatment of patients with hematological malignancies and bone marrow failure.

And new

strategies with HSCs are developed for the treatment of patients with the other malignancies andvascular failure including myocardial infarction, attracting further attention to HSCs.
molecular mechanisms regulating HSCs still remain unclear.

However,In this study, focusing on

Polycomb-group genes we attempted to uncover the role underlying stemness.

Results
Polycomb-group genes (PcG) were uncovered by Drosophila genetics as genes supporting thecellular memory system during development.

We for the first time reported that PcG are required

for sustaining HSC activity. Although PcG are recently reported to support stemness through therepression activity to ink4A locus encoding p16CDKI and p19ARF, so far the molecular bases
underlying PcG functions supporting stemness are not fully understood.
DNA replication was impaired in mice lacking PcG.

We found that licensing of

And PcG complexes interact with Geminin, an

inhibitor of a DNA replication licensing factor Cdt1 through Scmh1, a member of PcG, which weoriginally identified based on the conserved protein structure in mammals. We also found Hoxb4, awell known factor regulating HSCs, also interacts with Geminin.

Furthermore, by using

retrovirus-mediated gene transfer method, we examined in detail the effect of affected Geminin
regulation on HSCs.

And we showed that PcG complexes regulate Geminin at the level of protein

through ubiquitination and that Geminin regulation is pivotal in hematopoiesis and HSC regulation.

Discussion & Summary
These results indicate that DNA replication licensing is pivotal in sustaining HSC activity. And
PcG complexes play a crucial role in regulation of Geminin.

Now by using in vitro reconstituting

system further detailed analysis of PcG complexes are in progress. These findings may help future
progress in regenerative medicine with stem cells.

128

PcG complex
Cdt1(active)

Geminin

rae28
Scmh1

Ub Ub

Cdt1

rae28
scmh1

DNA replication
licensing

Activity of hematopoietic
stem cells (self renewal)

transport
proteasome

Fig.１．Molecular basis regulatinghematopoietic stem cells

degradation

Ub: Ubiquitination

References
1. Takihara, Y., Tomotsune, D., Shirai, M., et al. Targeted disruption of the mouse homologue of the
Drosophila polyhomeotic gene leads to altered anteroposterior patterning and neural crest defects.
Development 124:3673-3682, 1997.
2. Tomotsune, D., Takihara, Y. , et al. A novel member of murine Polycomb-group proteins, Sex
comb on midleg homolog protein, is highly conserved, and interacts with RAE28/mph1 in vitro.
Differentiation 65:229-239, 1999.
3. Ohta, H., Sawada, A., Kim, J. Y., Tokimasa, S., Nishiguchi, S., Humphries, R. K., Hara, J., andTakihara, Y. The Polycomb-group gene rae28 is required forsustaining activity of hematopoietic

stem cells. J. Exp. Med. 195:759-770, 2002.
4. Luo, L., Yang, X., Takihara, Y., Knoetgen, H., and Kessel, M. The cell cycle regulator Geminin
inhibits Hox function via direct and Polycomb-mediated interactions. Nature 427:749-752, 2004.
5. Kim, J. Y., Sawada, A., Tokimasa, S., Ohta, H., Endou, H., Hara., J., and Takihara, Y. Defective
long-term repopulating ability of hematopoietc stem cells lacking Polycomb-group gene rae28. Eur.
J. Haematol. 73:75-84, 2004.
6. Mihara, K., Chowdhury, M.,

Nakaju, N., Hidani, S., Ihara, A., Hyodo H., Yasunaga, S., Takihara,Y., Kimura, A. Bmi-1 is useful as a novel molecular marker for predicting progression of
myelodysplastic syndrome and prognosis of the patients. Blood 107: 305-308, 2006.

1293) Chemical Sciences3-1) Organic Chemistry

Design and Synthesis of Optically Active Supramolecular Capsules in
Aqueous Solution and Their Application to Asymmetric Recognition.
Shin Aoki
Faculty of Pharmaceutical Sciences, Tokyo University of Scienceshinaoki@rs.noda.tus.ac.jp

Introduction
Zinc(II) complex of 12-membered cyclic tetraamine 1 (ZnL1) gives a stable complex with anion(X–) in aqueous solution (Figure 1).

We have reported that a 1:1 mixture of a tris(Zn2+-cyclen) 2(Zn3L2) and a trianion of trithiocyanuric acid (TCA3–) yielded a 4:4 self-assembly complex, whose
exterior is represented by a cuboctahedral framework, through Zn2+-S– coordination bond inaqueous solution at neutral pH. It was also discovered that the supramolecular complex has an
inner cavity, in which hydrophobic guest molecules are encapsulated.
More interestingly, the supramolecular complex is a chiral molecule, since the three C-S-Zn2+
bonds around all of the four TCA3– units in 3 are bent by 100˚ in a clockwise or counterclockwise
fashion, as shown in 4 (Figure 1).
In this project, therefore, we have decided to synthesize optically active supramolecular cagefor selective recognition of chiral guest molecules and asymmetric synthesis in aqueous solution.

ResultsWe

havestartedtosynthesizeachiralcyclen,(S,S,S,S)-2,5,8,11-tetramethyl-1,4,7,10-tetraazacyclododecane) 5 (L3) as an important unit for an optically active tris(Zn2+-cyclen) 6(Zn3L4) (Figure 2).

First, L-alanine was reduced to L -alaninol, which was N-alkylated by

reductive amination with benzaldehyde to give N-benzyl- L –alaninol.

N-benzyl- L –alaninol was

reacted under the conditions for Mitsunobu reaction to yield an N-benzyl-1-(S)-methylaziridine.
N-Benzyl-1-(S)-methylaziridine was reacted with acid to yield ) 5 (L3), whose three nitrogens were
protected with (Boc)2O to give 3Boc- L3.

Discussion & Summary
As described above, we have succeeded to synthesize 3Boc-L3.

So, we are now reacting

3

3Boc-L with 1,3,5-tris(bromomethyl)benzene followed by deprotection to obtain an optically activetris(cyclen) (L4), which will be converted to trimeric Zn2+ complex 6 (Zn3L4). The attempts to130construct optically active supramolecular capsule 7 will be made.
Fig. 1 Supramolecular capsule formed from Zn3L2 and TCA3–
Zn2+

Guest

R

Zn2+
S- N
SN
N
100Þ
S-

S-

S–

X–
HN

NH

HN

NH

+

R

N
–S

N

R

R=

Anion (X–) complexof Zn2+–cyclen(ZnL1)
1

N
S–

TCA3-

Clockwise

in waterat neutral pH

4

H

N

N

HN

N

S-

H

Zn3L2
2

Cuboctahedral supramolecular capsule
formed by 4:4 self-assembly of 2 and TCA3(racemic when a guest is achiral)

3
Fig. 2 Design and synthesis of optically active supramolecular capsule
Me
Me
H2N

CO2H

Me

R

H
N

N

HN

N

H Me

in water

H

R

Me

L-Alanine

TCA3-

R

Chiral cyclen
(L3)

R=

7

Me
NH Me

N

5

Optically active
cuboctahecral
supramolecular
capsuleChiral recognition
Optical resolution
Asymmetric synthesis

NH

Me H NMe

Chiral tris(Zn2+-cyclen)( Zn3L4)

6

References
1. Tamaki Fujiwara, Shin Aoki, Hitoshi Komatsuzawa, Tetsuya Nishida, Masaru Ohara, Hidekazu
Suginaka, and Motoyuki Sugai, “Mutation Analysis of the Histidine Residues in the Glycylglycine
Endopeptidase ALE-1.” J. Bacteriol. 2005, 187(2), 480–487.
2. Shiin Aoki, Mohd Zulkefeli, Motoo Shiro, Masanori Kohsako, Kei Takeda, and Eiichi Kimura, “A
Luminescence

Sensorof

Inositol

1,4,5-Triphosphate

and

Its

Model

Compound2+

byRuthenium-Templated Assembly of a Bis(Zn -cyclen) Complex Having a 2,2-Bipyridyl Linker(Cyclen = 1,4,7,10-Tetraazacyclododecane).” J. Am. Chem. Soc., 2005, 127(25), 9129–9139.

1313) Chemical Sciences3-1) Organic Chemistry

Development of New Oxidation Reactions and Synthesis
of Bio-active Compounds
Jun-ichi Matsuo
Kanazawa University
jimatsuo@p.kanazawa-u.ac.jp

Introduction
1. Oxidation of N-carboalkoxy amines
N-carboalkoxy ketimines should be attractive electrophilic targets for creating chiral quaternarycarbon centers; however, only a few N-carboalkoxy ketimines have been prepared, and most did nothave acidic α-protons.

We expected that such N-carboalkoxy ketimines would be synthesizedunder mild conditions by N-t-butylbenzenesulfinimidoyl chloride(1)-mediated oxidation of
N-carboalkoxy amines.
2. Stereoselective glycosylation
For an efficient O-glycosyl bond formation, it is desirable that glycosyl donors should be prepared
conveniently, and thus-prepared glycosyl donors should be activated under mild conditions to realize
stereoselective glycosylation.

Results
1. Oxidation of N-carboalkoxy amines
Oxidation of N-carbobenxyloxy(Cbz)-amines 2 to the corresponding N-Cbz ketimine 3 was
performed by the following procedure: 2 was treated with n-BuLi in THF at –78 oC and then 11 wasadded at the same temperature.

As expected, the 1-mediated oxidation of 2 proceeded rapidly ato–78 C, and the desired product 3 was obtained in high yields.

It was noted that ene carbamate 3’

was not obtained by this procedure (Scheme 1).
It was also found that yields of N-Cbz aryl ketimines 3 depended strongly on substituents of thephenyl ring.

In the presence of an electron-attracting group, such as chloride, ketimine wasisolated in 96% yield as a sole product, while a mixture of ketimine and ene carbamate wasobtained in the presence of electron-donating substituents such as methyl and methoxy groups.

It

was thought that protonation to the nitrogen atom of the ketimine took place more easily whenelectron-donating groups were attached on the phenyl ring.

132

It should be noted that only one isomer of ketimine was obtained by the present procedure. We
speculated that anti-N-Cbz ketimines 3 were selectively formed, judging from 13C NMR chemical
shift differences between the α-carbons of N-Cbz ketimines and the corresponding ketones.
Cyclic and acyclic dialkyl ketimines were generated by the above-mentioned method and
MeOH-addition products were obtained in good to high yields by trapping ketimines with MeOH.
Kugelrohor distillation of the MeOH-addition product gave ene carbamate as a sole product.
Therefore, oxidation of N-Cbz amine with 1, followed by addition of MeOH and successive
distillation would give a useful method for preparation of ene carbamates.
2. Stereoselective glycosylation
N-Trichloroacetylcarbamate donor 5 was prepared from 2,3,4,6-tetra-O-benzyl-D-glucopyranose
4 (α/β = 86/14) by the reaction of 1.1 equivalents of trichloroacetyl isocyanate in dry CH2Cl2 atroom temperature (Scheme 2). The reaction completed within 30 min, and evaporation of CH2Cl2
in vacuo gave 5 quantitatively as a mixture of anomers (α/β = 89/11, determined by 1H NMR).
It was found that 1.2 equivalents of Me3SiOTf smoothly catalyzed the glycosylation with 5 at 0o

C to afford a dissacharide 6 in 95% yield as a mixture of anomers (α/β = 61/39).

The samereaction proceeded slowly by using other Lewis acids such as Mg(ClO4)2 and Cu(OTf)2 even atroom temperature, and Zn(OTf)2 did not activate the donor 5.
As in glycosylation with other glycosyl donors, such as glycosyl fluorides, good α-selectivity(α/β = 87/13) was observed in the Me3SiOTf-catalyzed glycosylation of 5 in Et2O.

The

α-selectivity was further improved to a ratio of α/β = 93/7 by changing the activator Me3SiOTf to
Me3SiClO4.2 Interestingly, the present catalytic glycosylation had to be carried out in the presenceof MS5A because the Me3SiClO4-catalyzed glycosylation did not proceed in the presence of MS4A
or MS3A.
β-Selective glycosylation of 5 was performed by using MeCN3 as a solvent and a stoichiometricor catalytic amount of Me3SiOTf.
Glycosyl acceptors having primary, secondary, and even tertiary hydroxy groups readily reactedwith the glycosyl donor 5 in Et2O to give α-glycosides in high yields and with high α-selectivity byusing 20 mol% of Me3SiClO4. On the other hand, though high β-selectivity was observed in
Me3SiOTf-catalyzed glycosylation in EtCN with glycosyl acceptors having a primary hydroxygroup, moderate β-selectivities were observed in the cases of acceptors having secondary andtertiary hydroxy groups.
Stoichiometric one-pot dehydrative glycosylation of 4 proceeded smoothly to afford α- or
β-glycoside, respectively, in high yields by preparing glycosyl donor 5 in situ with trichloroacetyl
isocyanate, followed by activating 5 with 1.5 equivalents of Me3SiClO4 in Et2O or Me3SiOTf in
EtCN.

Also, catalytic one-pot dehydrative glycosylation proceeded stereoselectively in the133presence of MS5A.

Discussion & Summary
1. Oxidation of N-carboalkoxy amines
Since a variety of N-Cbz ketimines are now available and anti-isomers of ketimines wereselectively formed by the present oxidation, it is expected that the N-Cbz ketimines would be useful
synthetic intermediates in organic synthesis.
2. Stereoselective glycosylation
α- or β-Selective glycosylation of using glycosyl N-trichloroacetylcarbamate 5 as a glycosyl
donor proceeded efficiently by activating the donor with a catalytic amount of Me3SiClO4 in Et2O or
Me3SiOTf in EtCN, respectively, in the presence of MS5A. Preparation of 5 from a 1-hydroxycarbohydrate 4 and successive stereoselective and catalytic glycosylation were also realized in a
one-pot manner, and the desired α- or β-glycoside was obtained directly from 4.

The easy

operation of the present glycosylation, especially one-pot dehydrative glycosylation, would be usefulin the synthesis of oligosaccharides or bioactive compounds having carbohydrate parts.

134

References
1

Matsuo, J.-i.J. Synth. Org. Chem. Jpn. 2004, 62, 574-583

2

Nakarahra, Y.; Ogawa, T. Carbohydr. Res. 1990, 200, 363-375.

3

Kreuzer, M.; Thiem, J. Carbohydro. Res. 1986, 149, 347-361.

1353) Chemical Sciences3-1) Organic Chemistry

Development of enantioselective reactions based on organocatalysis
directing at the total synthesis of biologically active compounds
Makoto Nakajima
Kumamoto University
nakajima@gpo.kumamoto-u.ac.jp

Introduction
The development of asymmetric organocatalysis (catalytic asymmetric reaction utilizing a
metal-free organic small molecule a catalyst) is one of the most important and challenging topics inrecent organic chemistry because of their environmentally benign nature.

Considering that highly

nucleophilic Lewis-bases form hypervalent silicates with trichlorosilyl compounds to afford thealdol and allylation adducts, the development of novel organocatalyses based on chiral Lewis bases
should be a significant addition of a new type of catalyst to the field of asymmetric synthesis.

Results
Phosphine oxides are highly polar to form complexes with various compounds, however, examples
of applications of chiral phosphine oxides to asymmetric organocatalyses are still limited.
BINAPO is a precursor of BINAP which is the most common chiral phosphine ligand. We havedeveloped the enantioselective allylation, aldol reactions and ring opening reaction withtrichlorosilyl compounds promoted by a chiral phosphine oxide as an organocatalyst.
The asymmetric allylation of carbonyl compounds to generate two successive stereogenic centers
has been the subject of investigation in recent years. We found that enantioselective allylations ofaldehydes with allyltrichlorosilanes in the presence of 10 mol % of BINAPO, gave the allylated
adducts in good enantioselectivities, where the use of the combination of diisopropylethylamine andtetrabutylammonium iodide as additives was crucial for the acceleration of the catalytic cycle.

The

best enantioselectivity was obtained in the reaction of 3,5-dimethylbenzaldehyde andmethallyltrichlorosilane (equation 1).1

γ-Allylated syn-homoallylic alcohol was obtained from(Z)-crotyltrichlorosilane,

while

corresponding(E)-crotyltrichlorosilane.

These results suggest that these allylations mediated by BINAPO proceed

theanti-alcohol

wasproducedfromvia cyclic chair-like transition structures that involve hypervalent silicates.
The asymmetric aldol reaction is one of the most powerful methods for constructing two successive
chiral carbon centers.

BINAPO was found to promote the enantioselective aldol reactions of

136

aldehydes with trichlorosilyl enol ethers in the presence of diisopropylethylamine as an additive,
affording the corresponding aldol adducts in high diastereo- and enantioselectivities.

It isnoteworthy that anti-adducts were predominantly produced from the E-enol ethers and the
syn-adduct was predominantly produced from the Z- enol ether, suggesting that the reaction
proceeded via a chair-like six-membered transition state.

The best result was obtained in thereaction of 4-nitrobenzaldehyde and the trichlorosilyl enol ether derived from cyclohexanone(equation 2).2
BINAPO also promoted the enantioselective ring opening of meso-epoxides with tetrachlorosilane in
the presence of diisopropylethylamine, affording the corresponding chlorohydrins in highenantioselectivities.

For example, the reaction of cis-stilbene oxide and tetrachlorosilane in thepresence of diisopropylethylamine and BINAPO as a catalyst afforded the corresponding
chlorohydrin in 90 % ee (equation 3).3

This result provides a new methodology ofdesymmetrization of meso-epoxide by the use of a organocatalyst.
To explore novel silyl enol ethers, which are more stable than trichlorosilyl enol ethers, we examined
aldol reactions of trimethoxysilyl enol ethers with aldehydes.

Trimethoxysilyl enol ethers, which

are easily prepared from the corresponding lithium enolate with chlorotrimethoxysilane or enone
with trimethoxysilane, are so stable that they survive the aqueous work-up or silica gel column
chromatography.

However, examples of base-catalyzed aldol reactions of trimethoxysilyl enol

ether have yet to be reported and the reactivity of hypervalent silicate derived from thetrimethoxysilyl enol ether remains unknown.

We found that addition of trimethoxysilyl enol ether

derived from cyclohexanone to benzaldehyde with 10 mol % of lithium binaphthoxide as a catalyst
in THF afforded the corresponding anti-adduct predominantly in high chemical yield. To oursurprise, the above aldol reaction in the presence of H2O (1.5 equiv) afforded syn-adductpredominantly.

The best enantioselectivity was obtained in the reaction of trimethoxysilyl enol

ether derived from indanone to hydrocinnamaldehyde (equation 4).4

Discussion & Summary
The enantioselective reactions catalyzed by BINAPO is the first examples of the use of a chiral
phosphine oxide as an organocatalyst for the asymmetric reactions, which demonstrated the novel
importance of chiral phosphine oxides in synthetic organic chemistry.

The aldol reaction oftrimethoxysilyl enol ether is the first example of base-catalyzed aldol reactions of trimethoxysilyl
enol ethers with carbonyl compounds, which exhibit the high potentiality of trimethoxysilyl enol
ethers as enol equivalents, one of the most valuable reactive intermediate.

These results will

exploit a new chemistry in asymmetric synthesis, which will receive much attention of theresearchers in the field of organic chemistry.

137

References
1. Chiral Phosphine Oxide as a Catalyst for Enantioselective Allylation of Aldehydes with
Allyltrichlorosilane, Nakajima, M.; Kotani, S.; Ishizuka, T.: Hashimoto, S. Tetrahedron Lett. 2005,
46, 157-159.
2. Enantioselective aldol reactions of trichlorosilyl enol ethers catalyzed by the chiral phosphineoxide BINAPO, Kotani, S.; Hashimoto, S.; Nakajima, M. Synlett 2006, 1116-1118.
3. Asymmetric Ring Opening of meso-Epoxides Catalyzed by the Chiral Phosphine Oxide BINAPO,
Tokuoka, E.; Kotani, S.; Matsunaga, H.; Ishizuka, T.; Hashimoto, S.; Nakajima, M. Tetrahedron:
Asymmetry 2005, 16, 2391-2392.
4. Chiral Base-Catalyzed Aldol Reaction of Trimethoxysilyl Enol Ethers: Effect of Water as an
Additive on Stereoselectivities, Orito, Y.; Hashimoto, S.; Ishizuka, T.; Nakajima, M. Tetrahedron
2006, 62, 390-400.

1383) Chemical Sciences3-3) Protein Chemistry

Development of real-time sensors to simultaneously assess in vivo
productions of two different second messengers
TAKASHI MORII
Institute of Advanced Energy, Kyoto University
t-morii@iae.kyoto-u.ac.jp

Introduction
Intracellular second messengers regulate many critical cellular functions. The fluorescent sensor
would be an ideal tool for the convenient measurements of these second messengers. An attractive
framework for the design of highly efficient biosensors is the natural protein domain with highaffinity and selectivity to the second messenger. An intracellular second messenger
inositol-1,4,5-trisphosphate (IP3) is a key biological signaling molecule that controls the cellular
calcium concentration. We have designed a protein-based sensor for IP3 by exploring the selective
IP3 binding properties of pleckstrin homology (PH) domain. Fluorescent signal transduction is
imparted to the protein by mutating proximal residues to cysteine and then alkylation of the active
site by various fluorophore derivatives. This creates functionalized proteins that show micromolar
affinity for IP3, reasonably strong fluorescence emission and wavelength changes in the fluorophoreand selectivity higher than the original PH domain among different inositol phosphate derivatives invitro (J. Am. Chem. Soc. 2002, 124, 1139) and in vivo (EMBO J. 2003, 22, 4677).

Results
In order to understand further signaling mechanisms related to calcium ion concentration changes
and productions of inositol phosphates, it is necessary to assess simultaneously productions of otherinositol phosphates, such as inositol-1,3,4,5-tetrakisphosphate (IP4). We have focused on
development of fluorescent sensors that enable simultaneous detection and quantitation of two
different inositol phosphates derivatives.
Tailoring IP4 sensors.
We have developed fluorescent inositol-1,3,4,5-tetrakisphosphate (IP4) sensors that emit at various
wavelengths upon binding to IP4. Receptors for IP4 have been made from a GRP1 PH domain
according to our previously published method for fluorescent IP3-sensors (J. Am. Chem. Soc. 2002,
124, 1139). An amino acid sequence of five arginine residues (R5) was added at the C-terminus of

139

the GRP1 PH domain to help the sensor permeabilize the cell membrane. Two amino acid residues
that located near the binding pocket and did not interact with IP4 directly, Val15 and Glu82, waschosen to mutate into a Cys residue （ V15C, E82C ） so that a selective modification withfluorophores could be possible (Figure 1). Four different IP4 sensors were constructed with different
fluorophores and mutated GRP1 PH domains. The mutant GRP1 PH domains were modified at theCys

residue

with

thiol

reactivefluorophores,

6-iodoacetamidofluorescein

(6F)

and

6-bromoacetyl-2-dimethyl-aminonaphthalene (DAN), at each of these positions, and successive
purification with a gel filtration followed by a cation exchange chromatography gave four different
fluorophore-labeled PH domains. CD spectra of these fluorophore-labeled GRP1 PH domains,
V15F, E82F, V15DAN, and E82DAN, were indistinguishable from that of the wild type, indicatingthat the secondary structure was not perturbed by the introduction of fluorophore.
Each fluorophore-labeled PH domain was tested for IP4 binding by monitoring the fluorescentemission spectra of a 100 nM solution. Distinct changes of emission spectra were observed for all
the sensors, V15F, E82F, V15DAN, and E82DAN. Though the affinity of sensors to IP4 varied bothwith the labeled position and with fluorophore, the equilibrium dissociation constants obtained by
the fluorescent titrations were comparable to that reported for the parent GRP1 PH domain. These
results suggest that a combinatorial approach with various fluorophore and with attachment
positions is necessary to optimize the sensitivity of the biosensor. The selectivity of biosensor tovarious inositol phosphate derivatives was next analyzed. All the IP4 sensors eficiently
discriminated IP4 from other inositol phosphate derivatives.
Proper placement of an environmentally sensitive fluorophore within the PH domain successfully
afforded a variety of IP4 biosensors that enables to monitor the IP4 concentration at various
wavelength. The sensor targets IP4 with a submicromolar dissociation constant and with aselectivity to IP4 over other inositol derivatives.

140

Sensing of IP3 and IP4 in the cell.
Cellular functions are regulated by intracelluar second messengers, such as Ca2+ and inositol
phosphates. However, metabolizing pathways of these second messengers are not completely
understood. We have succeeded quantitatively asess in vivo regulation of inositol 1,4,5-trisphosphateproduction in intact cells. The IP4 sensor obtained in this study was incorporated into HeLa cells byelectroporation. We next analyzed the intracellular production of inositol-1,3,4,5-tetrakisphosphateby using the IP4 sensor. The fluorecent intensity of the IP4 sensor incorporated HeLa cells increased
when stimulated by histamine, suggesting that the IP4 sensor detected the concentration changes of
IP4.

Discussion & Summary
We have succeeded in constructing optical IP4 sensors from a naturaly existing PH domain which
already has high affinity and selectivity to IP4. It is possible to incorporate these sensors into
mammalian cells and detect the concentration change of IP4. These IP4 sensors are expected to be
utilized to clearify the behavior of IP4 during signal transduction.
The fluorescent IP4-sensor obtained in this study do not interfere the emission of IP3 sensor. We will
covalently connect the IP3-sensor and the fluorescent peptide portion of IP4-sensor by a disulfide
bond or a chemical ligation reaction. This allows us to obtain a dual sensor that contains both IP3and IP4-sensors within the same body. Introduction of the dual sensor into a cell would be tested in
the conditions used for our in vivo IP3 sensor to acess IP3 and IP4 concentrations at the same locationin the cell.

References
In preparation

1413) Chemical Sciences3-4) Analytical Chemistry

Role of chymase in the development of aortic aneurysm
Shinji Takai
Osaka Medical College
Pha010@art.osaka-med.ac.jp

Introduction
Chymase contributes not only angiotensin II (AII) formation but also pro-matrix
metalloproteinase activation and its function may play an important role in the development of aortic
aneurysm.

In this study, we clarified the role of chymase in the development of A II-induced

abdominal aortic aneurysm in mice.

Results
Twenty male apolipoprotein E (apo E)-deficient mice on C57BL/6 background were obtained
from Jackson Laboratories. 10-week-old Apo E-deficient mice (n = 10) were infused with A II (1000ng/kg per min) for 4 weeks.

The remaining mice were used as control mice. At 10 weeks (before A

II infusion), 14 weeks (just after A II infusion), 20 weeks, 30 weeks and 40 weeks of age, wemeasured the blood pressure and the luminal diameter of abdominal aorta using ultrasound imaging
system. At 14 weeks of age, the blood pressure in the AII-infused apo E-deficient mice wassignificantly higher than that in the control mice, but it was not observed at the other point. At over
30 weeks of age, the luminal diameter of abdominal aorta was significantly larger in the AII-infused
apo E-deficient mice than in the age-matched control mice (Figure 1). At 40 weeks of age, mice
were sacrificed and directly measured the diameters of abdominal aorta.

The diameters of supra-

and infra-renal abdominal aortas were significantly larger in the in the AII-infused apo E-deficient
mice than in the control mice (Figure 2). Aortic chymase and matrixmetalloproteinase-9 levels weresignificantly higher in the A II-infused apo E-deficient mice than in the control mice.

Discussion & Summary
We found the upregulation of chymase in the A II-induced abdominal aortic aneurysm in mice,and this finding suggest that chymase may play an important role in the development of the aortic
aneurysm.

Furthermore, in this study, we established the method which could evaluate themeasurement of aortic aneurysm time-dependently. This method may be useful for evaluating theregressive effects of drugs.

142

143

Part Ⅱ
Reports from the Recipients of Garants
for International Meetings(Fiscal Year 2004)

144

4th European-Japanese Bioorganic Conference (EJBC-4)
1. Representative
Tetsuo Toraya, the chairman of the organizing committee

2. Date and location
March 15-19, 2005
Hotel Limani (Conference room: Olympus), Ushimado, Setouchi,
Okayama-Pref., Japan

3. Number of participants
Approximately 75 persons (including 20 from abroad)4. Total cost:
4,168,498 yen

5. Main use of subsidy
As a part of the rent for the Conference rooms

6. Results and impression
The recent development of chemical biology is amazing. The chemical biology in European
countries is at a top level of the world. On the other hand, the bioorganic chemistry in Japan is
also at a high level. Based on such backgrounds, this European-Japanese Bioorganic Conference(EJBC-4) was held with bioorganic chemistry and chemical biology as central themes.
Twenty European speakers from 5 countries (one of them was absent because of illness) and 17
Japanese speakers from 9 universities gave the invited lectures. The invited speakers are listed
below. In the two poster sessions, 31 posters were presented by European and Japaneseparticipants. In addition, there were a get-together party, excursion, and banquet as well in themeeting. The total number of the participants were approximately 75.
In the meeting, European and Japanese invited speakers presented newest results and insightsof their works, followed by very active discussion. The aim of the meeting to promote theprogress of both fields of increasing importance was achieved by exchanging new results and new
insights. From close interactions between European and Japanese participants, it can be expected145

to stimulate fruitful collaborations between Europe and Japan in the future. Relatively young
Japanese and European researchers presented their results in the poster sessions. They were
stimulated by the discussion with famous scientists from oversea counties and Japan, which was
also an important fruit of the meeting from the viewpoint of growing young scientists of Japan.
After the meeting, many European participants sent me “thank you” e-mails. They indicated that
European people were also satisfied with the high quality of the science in the meeting. One ofthem wrote to me that this meeting was the most successful EJBC ever held. I proposed in themeeting that the title of the meeting (EJBC) is changed to EJCB (European-Japanese Chemical
Biology) in the future. This proposal was accepted by the co-organizers. It was decided that thenext “EJCB” meeting will be held in Munich in 2007.

7. Additional description
The invited speakers were: Prof. B. T. Golding (Newcastle upon Tyne, UK); Prof. R. J. Griffin(Newcastle upon Tyne, UK); Prof. C. J. Schofield (Oxford, UK); Prof. N. J. Turner (Edingburgh,UK); Prof. T. D. H. Bugg (Warwick, UK); Prof. C. Abell (Cambridge, UK); Dr. W. P. Watson(Cheshire, UK); Dr. P. L. Roach (Southampton, UK); Prof. W. Buckel (Marburg, Germany); Prof.
M. Marahiel (Marburg, Germany); Prof. T. Carell (Muenchen, Germany); Prof. H. Dobbek(Bayreuth, Germany); Dr. A. J. Pierik (Marburg, Germany); Dr. B. M. Martins (Bayreuth,
Germany); Dr. T. Selmer (Marburg, Germany); Dr. M. Boll (Freiburg, Germany); Prof. A. M.
Papini (Firenze, Italy); Dr. M. Toernqvist (Stockholm, Sweden); Dr. M. Sahlin (Stockholm,
Sweden); Prof. M. Shionoya (Univ. of Tokyo); Dr. H. Shimada (Keio Univ.); Prof. N. Esaki(Kyoto Univ.); Prof. H. Kato (Kyoto Univ.); Prof. K. Tanizawa (Osaka Univ.); Prof. H. Hayashi(Osaka Med. Coll.); Prof. S. Itoh (Osaka City Univ.); Prof. J. Kikuchi (NAIST); Prof. Y. Hisaeda(Kyushu Univ.); Prof. M. Sisido (Okayama Univ.); Prof. K. Uneyama (Okayama Univ.); Prof. K.
Takai (Okayama Univ.); Prof. H. Tanaka (Okayama Univ.); Prof. Y. Wataya (Okayama Univ.);
Prof. T. Sakai (Okayama Univ.); Prof. T. Toraya (Okayama Univ.).

146

6th Asian-Pacific Conference on Medical and Biological Engineering(APCMBE 2005)
1.Representative (Chair of the meeting)Name: Katsuhiko Tsujioka
Institution: Kawasaki Medical School
Position: Professor
2. Venue of the Meeting: Tsukuba International Congress Center
Date: April 24-27, 2005
3. Number of Participants
376(including 128 from abroad)4. Total cost
¥13,599,000
5. Use of the Grant:
1. personnel expenses: a part-time job, PC operator
2. travel expenses: staff travel expenses
3. conference hall: Epochal Tsukuba
4. borrowing expenses: PC projector
6. Result and impression of the meeting:
Asian-Pacific Conference on Medical and Biological Engineering (APCMBE) has been playing
a leading role as an international meeting in Asian-Pacific region on medical and biologicalengineering research, and is held every 3 years as a regional meeting of International Federation
for Medical and Biological Engineering (IFMBE) since 1990. IFMBE is an international scientificorganization for research of human function and clinical application. The scope of IFMBE resides
in development of diagnostic and therapeutic equipment.
The present meeting (6th APCMBE) was successfully held in Tsukuba International Congress
Center in Tsukuba city, Ibaraki, from April 24 to 27, 2005. Nearly 400 researchers (including
about 130 from abroad) from 19 countries got together to present and discuss about diagnostic andtherapeutic equipment including Cardiovascular Bioengineering，Molecular - Cell - Tissue
engineering, Biorheology, Biomechanics (Bone, Musculo-skeletal, Vasculature, etc.), Dental
Bioengineering, Electromagnetic Radiation and Ultrasound, Bio-micro-nanotechnology, Drug

147

Delivery, Artificial Organs, Biomedical Engineering Education, Bioinformatics, Healthcare,
Rehabilitation Engineering, Elderly Persons Assist Technology, Virtual Reality, Biomedical
Instrumentation, Neural Engineering, Computational Bioengineering, In Silico Medicine, Medical
Imaging, Computer-Aided Surgery, etc. This meeting greatly contributed to a progress of medical
and biological engineering research in this area.
On each day, nearly 100 posters were presented, and a total of about 300 posters in three days
were discussed. As a highlight of the meeting, 3 special lectures, chairperson’s lecture, 8 invitedlectures, 1 evening session, 6 symposia (32 presentations) by foreign and domestic leading
scientists were presented as follows, and discussed actively with the audience.
Special Lecture
1. Robert M. Nerem (USA): Bioengineered Tissues and Organs: The Emergence of Cell-Based
Therapies
2. P. Åke Öberg (Sweden): Bio-Optical Sensors: Diagnostic and Monitoring Possibilities in HealthCare
3. Joachim H. Nagel (Germany): Perspectives for Advanced Therapeutic Applications of
UltrasoundChairperson’s Lecture
Katsuhiko Tsujioka (Japan): Micromechanics of Endothelial Cells
Invited Lecture
1. Takeyoshi Dohi (Japan): Computer Aided Surgery
2. Jun Miyake (Japan): Tissue and Cell Technologies Directing to Regenerative Medicine
3. Kenji Sunagawa (Japan): Bionic Cardiology: New Therapeutic Modality in the 21st Century
4. Myoungho Lee (Korea): Current Status and Future Perspectives on e-Health in Korea
5. Xiaochuan Pan (USA): Imaging Sciences and Their Medical Applications
6. Kunio Awazu (Japan): Free Electron Laser as New Diagnosis and Therapy for Bio-Molecules
7. Si-Shen Feng (Singapore): Nanoparticle Technology for New-Concept Chemotherapy
8. Koji Ikuta (Japan): Optical-Driven Nanorobotics for Minimally Invasive Microsurgery of
Intra-Cell
Evening Session
Alfred Dolan (Canada): Risk Management for Medical Devices -Ensuring Safety and Efficacy

148

Symposium
Symposium I: IAMBE (International Academy for Medical and Biological Engineering)
symposium: Nanotechnology and Physiome (Coordinator: Fumihiko Kajiya (Japan),
Chairpersons: Fumihiko Kajiya (Japan), Niilo Saranummi (Finland))
Symposium II: Neural and Rehabilitation Engineering (Coodrinator: Toshiyo Tamura (Japan),
Metin Akay (USA), Chairpersons: Toshiyo Tamura (Japan), Metin Akay (USA))
Symposium III: Promotion of Biomedical Engineering in Asian-Pacific Region -Academic,
Clinical, and Industry's Perspective (Coordinator: Makoto Kikuchi (Japan), Chairpersons:
Makoto Kikuchi (Japan), Joachim H. Nagel (Germany))
Symposium IV: Mechanobiology of the Vascular System (Coordinator: Joji Ando (Japan),
Chairpersons: Joji Ando (Japan), Xiaoqiang Yao (Hong Kong))
Symposium V: Cellular Biomechanics (Coordinator: Masaaki Sato (Japan), Chairperson: Masaaki
Sato (Japan))Symposium VI: Past, Present and Future of MBE in Asian-Pacific Region (Coordinator:
Katsuhiko Tsujioka (Japan), Chairpersons: Masatsugu Hori (Japan), Walter H. Chang(China-Taipei))
7. Topics of the meeting
In the meeting, Young Investigator’s Award (YIA) Competition was held for encouraging young
scientists in the field in Asian-Pacific countries. Over 50 papers were applied to YIA, and 8 papers
were selected in advance by the program committee to present orally at the conference. Finally thetwo best papers were selected and awarded as the YIA best presentation award.

149

The 58th Meeting of the Japan Society for Cell Biology
1. Representative
Keiji Tanaka, the president of the annual meeting
2. Opening period and place
June 15-17, 2005
3. Number of participants
703 persons (42 persons from foreign countries)
4. The total cost
Twenty-seven million yen
5. Main use of the subsidy
Support for travel expenses of invited speakers
6. Result and Impression
The 58th Annual Meeting of the Japan Society for Cell Biology was held to encourage theinternational and inter-generational interaction of the scientists working in various fields of thecell biology.
For further development of the Society, the annual meeting last year has adopted a variety of
new attempts. For example, English is now used as the official language and many distinguishedscientists have been invited from overseas and Japan to provide an overview of topics from all
fields of cell biology. Furthermore, many young scientists, who were recommended by
outstanding researchers from overseas, have been invited to present posters and to discuss the
topics with Japanese scientists. All invited presenters are requested to participate in all to thesessions so as to provide for a more stimulating meeting.
In addition to these innovations, Computer Based Presentation has newly adopted at the poster
session this year to allow presenters to use visual data and movies. To concentrate on the sessions
in a relaxed atmosphere, we announced the participants to dress casual to attend the meeting, justlike the other meetings in the U.S. and Europe. Any formal or business wear with a tie was notrecommended.
I think that all participants discussed their ideas extensively with many scientists from overseas
and Japan, and created an international relationship during the meeting. Symposia had beenorganized to cover all fields of cell biology and no more than two symposia will run
simultaneously. So, the participants could attend all symposia and obtained current information of
all fields of cell biology.

150

The scientific program consisted of 7 symposia and poster presentations. 341 contributed papers
including invited ones were accepted for poster presentation and 35 distinguished speakers for
symposia have been invited from Japan and overseas. These played a prominent role in theexchange of ideas and provided us with new research results on various topics in cell biology.
Particularly, the main focus of the Meeting in this year was to discuss the basic mechanisms,
pathophysiological roles, and technological developments of the modern biology in the
post-genome era. In addition, the Meeting aimed that the young Japanese scientists take theopportunity to make friends with leading scientists from overseas and exchange their opinions on
their work. On the second day of the meeting, a mixer reception was held at a low price and mostinvited presenters participated. I believe this meeting was the most informative one in cell biology
and related scientific fields.
7. Additional description
Number of participating countries : 9

151International Symposium on Ran and the Cell Cycle
1. Representative
Yoshihiro Yoneda, the chairperson of organizing committee
2. Opening Period and Place
October 2 (Sunday) ~ October 4 (Tuesday), 2005
Awaji Yumebutai International Conference Center (Hyogo, Japan)
3. Number of participants
111 persons (7 persons from foreign countries)
4. The total cost
4,612,350 yen
5. Main use of the subsidy
Support for travel expenses and hotel accommodations of invited speakers
6. Results and Impression
A remarkable range of insights into nuclear structure and function has resulted from theestablishment by Nishimoto et al. (1978) of the temperature-sensitive mutant tsBN2 cell line that
rapidly undergoes mitosis during S phase without completion of DNA replication.

Because of

the rapid and inappropriate nature of chromosome condensation under non-permissive
temperature, the end result was a striking chromosomal fragmentation, resulting in pulverized
mitotic chromosomes and the formation of micronuclei upon mitotic exit.

These intriguing

observations have developed the Ran GTPase field, which has become a key interface between
the control of mitotic progression, nuclear-cytoplasmic trafficking, the formation of the interface
nuclear envelope and the mitotic spindle.

More than one hundred former lab members,

collaborators and other scientific colleagues from around the world gathered October 2-4, 2005
to celebrate the career and scientific achievements of Professor Takeharu Nishimoto.

The

International Symposium on Ran and the Cell Cycle, held in his honor at the Awaji Island, Japan,
was organized by Yoshihiro Yoneda (Osaka University) and Mary Dasso (National Institutes of
Health) into sessions reflecting major areas under investigation: cell cycle and chromosomes;
nuclear import and export of proteins and RNA; nuclear envelope and the nuclear pore complex;
and RCC1 and chromatin.

In addition to the many excellent scientific presentations (toomany to discuss individually) and reminiscences about and tributes to Dr. Nishimoto from the152participants, highlights of the meeting included a special lecture by Dr. Nishimoto on his career
and accomplishments (see below) and a guitar performance by his son Takeshi Nishimoto, who
is a professional musician.

We feel that Dr. Nishimoto's retirement from Kyushu University isa perfect time to look back at the milestones of his career, to assess the current state of the field,
and to discuss his scientific legacy and the challenges that remain in order to unravel thecomplexities of the Ran GTPase system.

Thus, at this symposium, the participants discussed

the progress of the intervening 27 years and revealed the broad significance of Nishimoto’s
original finding.

In addition, many of the talks at this meeting and the formal and informal

discussions among participants implicitly or explicitly raised many interesting questions andissues that remain to be addressed in the future.
7. Additional description:
In a special lecture, Professor Takeharu Nishmoto provided both a historical perspective on
the development of the Ran field and a current outlook of the unresolved questions that havearisen from genetic studies conducted within his laboratory.

In the 1980s, Nishmoto andcolleagues accomplished the impressive feat of cloning the gene that was mutated in tsBN2 cellsby complementation.

They found that tsBN2 phenotype was rescued by a protein named

RCC1 (Regulator of Chromosome Condensation 1).

This chromatin-associated protein was

rapidly proteolyzed upon temperature shift, suggesting that tsBN2 cells were functionally nullfor RCC1 activity at their restrictive temperature. After the discovery that RCC1 acts as
exchange factor for the Ran GTPase, Nishimoto and colleagues identified many now
well-known Ran pathway components, including Yrb2p in yeast and RanBP2 in vertebrate cells.
They also identified a large number of components whose roles have not been fully established.
In their recent studies on one of these interactions, Nishimoto and colleagues have examined thegenetic relationships between the S. pombe homologue of RanGAP1, Rna1, and the histone H3
K9 methyltransferase Clr4. On the basis of in vitro studies and chromatin immunoprecipitation,
Nishimoto proposed that a small amount of Rna1 within nuclei works to activate Clr4 andpromote the formation of silenced chromatin near centromeres.

This model is particularly

intriguing with respect to the previous finding that RanGAP1 mutants show altered responses tocentromeric heterochromatin, disrupting the accurate packaging of sperm chromosomes in
Drosophila. Indeed, this model would provide another novel function for the Ran pathway
within eukaryotic nuclei.

153

International Interdisciplinary Conference on Vitamins, Coenzymes,
and Biofactors 2005
1. Representative

Hiroyuki Kagamiyama, the president of the conference and the chairman of the organizing
committee.
2. Opening period and place

6–11 November, 2005, Awaji Yumebutai International Conference Center, Hyogo, Japan
3. Number of participants

217 persons (46 persons from foreign countries)
4. The total cost

Eighteen million yen
5. Main use of the subsidy

Support for the local expenses of the excellent young researchers (7 persons)
6. Result and Impression

This conference is the first joint meeting of the 4th International Symposium on Vitamin B6,
PQQ, Carbonyl Catalysis and Quinoproteins, and the 4th International Congress on Vitamins andRelated Biofactors, both of which have been held every 3 to 4 years.
The recent advances in genomic science, structural biology, and biomedical applications haveenhanced mutual communications between the individual studies on vitamins, coenzymes, andbiofactors. It is, therefore, one of the best times to hold an interdisciplinary meeting on the
biologically important small molecules. These molecules include vitamin A, D, E, K, ubiquinone,
essential fatty acids, carotenoids, vitamin B1, B2, B6, B12, niacin, biotin, folic acid, pantothenic
acid, vitamin C, PQQ, quinoproteins, and biofactors.
To take the full advantage of the joint meeting, some closely related areas were combined, andmany sessions included contributions from both sides. Accordingly, sessions were divided bytechnical aspects. These include enzyme science, chemical aspects, metabolic aspects, molecularand cellular biology, nutritional aspects, pharmaceutical aspects, medical aspects, andbiotechnology. Sixty-three invited speakers and 14 persons selected from poster presentationlectured on these topics. One-hundred-one posters were presented.
The largest group was enzyme science, which occupied nearly half of the both oral and poster

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presentations. This certainly reflects the rapid technical advancement in the field of structural
biology. The enzyme actions are now discussed in detail based on the structural data of thewild-type and mutant enzymes, and reaction intermediates. Even the transient species could be
structurally analyzed by kinetic crystallography. Combined with the further advancement in the“classical” analytical method, such as the global analysis of the spectral change using the singularvalue deconvolution and mass spectrometry, the detailed reaction mechanism has been elucidated
for many enzymes presented at the conference. These enzymes are mainly pyridoxal 5'-phosphate(PLP)-dependent enzymes and PQQ/quinoprotein enzymes. Actually, the reaction mechanisms ofthese enzymes have been the main topic of the former 3 meetings of the “International
Symposium of Vitamin B6, PQQ, Carbonyl Catalysis and Quinoproteins”, and the antecedent 8
meetings “the Vitamin B6 Meeting” which has been held since 1962. However, this is the firstmeeting in which the research papers on PLP (B6) enzyme were presented and discussed together
with those on flavins (B2) and NAD (niacin). There were many active discussions between the
research groups, both inside and outside the meeting room.
Because of the number of the oral presentations (62) and the fact that half of the titles belonged
to the area of enzyme science, the speeches were presented at two meeting rooms, Main Hall wasfor enzyme science and Reception Hall was for other fields. In the area other than enzyme science,biomedical aspects of small molecules have been discussed. It should be pointed out that, also inthese fields, the research is growing to be based on structural biology. That is, biological andmedical processes of small molecules such as vitamins and biofactors are now understood in termsof the interaction with macromolecules, especially proteins.
This trend has much relevance to the debate on the issue that whether or not PQQ is a vitamin,
which emerged since the publication of the paper in Nature (2003 Apr 24;422(6934):832) thatclaims PQQ is a vitamin involved in the lysine degradation pathway. These authors used“bioinformatics” as a tool in addition to nutritional studies and proposed that α-aminoadipate
δ-semialdehyde dehydrogenase is a PQQ enzyme. However, the proposal lacked structuralevidence. In this conference, two groups, one is a structural biology group and the other anutritional group, gave oral presentations on this problem. Both groups addressed remarks that
there is no evidence for the proposal that PQQ is a vitamin and pointed out the possible flaws in
the Nature's report. This is a good example to show that such biomedical studies must be based on
exact molecules, and detailed analyses on the biomolecules are important not only in enzyme
science but also in all the fields of bioscience. This also indicates that too much reliance on today's“bioinformatics” sometimes leads to the erroneous conclusions.
The two meeting rooms are adjacent to each other on the same floor, and coffee breaks are heldat the foyer between the two rooms. The participants at each meetings rooms could communicate
at the coffee break. Breakfast was also served at the same place.

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The poster session was held at the corridor connecting the conference center and the hotel. Thiswas highly appreciated by the participants. “In most meetings, posters are being displayed at someplaces that are not easy to access. In this meeting, we could see many interesting contributions
from young researches on the way to and back from the meeting room.” a participant from UK
said.
Have done travel expense help to 6 overseas (5 countries) and 1 domestic young participants.
This was especially important for those scientists who succeed in continuously doing good science
under quite difficult circumstances.
It was decided that the next conference is held at Athens, Georgia, USA in 2008, by the
initiative of Prof. Robert S. Phillips, University of Georgia, Athens.
7. Additional description
8. Participating countries (in alphabetical order)

Bangladesh, China, Finland, France, Germany, Greece, Israel, Italy, Japan, Korea, Pakistan,
Russia, Slovenia, Switzerland, UK, USA (16 countries)
９. Approvals
Approved by the International Union of Biochemistry and Molecular Biology (IUBMB) as the
IUBMB Symposium No. 344
10. Satellite symposium
After the conference, a satellite symposium on redox bioscience (organized by Prof. Etsuo
Niki, AIST, Japan) was held at the same conference center.