Researcher Database

Toshiaki Koda
Faculty of Advanced Life Science Functional Life Sciences Embryonic and Genetic Engineering
Specially Appointed Professor

Researcher Profile and Settings


  • Faculty of Advanced Life Science Functional Life Sciences Embryonic and Genetic Engineering

Job Title

  • Specially Appointed Professor


  • 1988.6 Doctor of Medical Science(Hokkaido University)


J-Global ID

Research Interests

  • 疾患モデル動物   ハンチントン病   包括脳ネットワーク   実験病理学   実験動物学   分子生物学   

Research Areas

  • Life sciences / Experimental pathology
  • Life sciences / Laboratory animal science
  • Life sciences / Molecular biology


  •        - 1982  Hokkaido University  School of Medicine
  •        - 1982  Hokkaido University  Faculty of Medicine

Association Memberships

  • 北海道医学会   日本免疫学会   日本癌学会   日本分子生物学会   日本実験動物学会   

Research Activities

Published Papers

  • Nobutaka Hanagata, Taro Takemura, Keiko Kamimura, Toshiaki Koda
    Scientific reports 10 (1) 21197 - 21197 2020/12/03 
    Osteogenesis imperfecta (OI) type V is an autosomal dominant disorder caused by the c.-14C > T mutation in the interferon-induced transmembrane protein 5 gene (IFITM5), however, its onset mechanism remains unclear. In this study, heterozygous c.-14C > T mutant mice were developed to investigate the effect of immunosuppressants (FK506 and rapamycin) on OI type V. Among the mosaic mice generated by Crispr/Cas9-based technology, mice with less than 40% mosaic ratio of c.-14C > T mutation survived, whereas those with more than 48% mosaic ratio exhibited lethal skeletal abnormalities with one exception. All heterozygous mutants obtained by mating mosaic mice with wild-type mice exhibited a perinatal lethal phenotype due to severe skeletal abnormalities. Administration of FK506, a calcineurin inhibitor, in the heterozygous fetuses improved bone mineral content (BMC) of the neonates, although it did not save the neonates from the lethal effects of the mutation, whereas rapamycin, an mTOR inhibitor, reduced BMC, suggesting that mTOR signaling is involved in the bone mineralization of heterozygous mutants. These findings could clarify certain aspects of the onset mechanism of OI type V and enable development of therapeutics for this condition.
  • Neu-medullocytes, sialidase-positive B cells in the thymus, express autoimmune regulator (AIRE).
    Kijimoto-Ochiai S, Kamimura K, Koda T
    Scientific Reports 9 (1) 858  2019/01 [Refereed][Not invited]
  • Kan Yaguchi, Takahiro Yamamoto, Ryo Matsui, Yuki Tsukada, Atsuko Shibanuma, Keiko Kamimura, Toshiaki Koda, Ryota Uehara
    The Journal of cell biology 217 (7) 2463 - 2483 0021-9525 2018/07/02 [Refereed][Not invited]
    In animals, somatic cells are usually diploid and are unstable when haploid for unknown reasons. In this study, by comparing isogenic human cell lines with different ploidies, we found frequent centrosome loss specifically in the haploid state, which profoundly contributed to haploid instability through subsequent mitotic defects. We also found that the efficiency of centriole licensing and duplication changes proportionally to ploidy level, whereas that of DNA replication stays constant. This caused gradual loss or frequent overduplication of centrioles in haploid and tetraploid cells, respectively. Centriole licensing efficiency seemed to be modulated by astral microtubules, whose development scaled with ploidy level, and artificial enhancement of aster formation in haploid cells restored centriole licensing efficiency to diploid levels. The ploidy-centrosome link was observed in different mammalian cell types. We propose that incompatibility between the centrosome duplication and DNA replication cycles arising from different scaling properties of these bioprocesses upon ploidy changes underlies the instability of non-diploid somatic cells in mammals.
  • Shigeko Kijimoto-Ochiai, Tokuko Matsumoto-Mizuno, Daisuke Kamimura, Masaaki Murakami, Miwako Kobayashi, Ichiro Matsuoka, Hiroshi Ochiai, Hideharu Ishida, Makoto Kiso, Keiko Kamimura, Toshiaki Koda
    Glycobiology 28 (5) 306 - 317 1460-2423 2018/05/01 [Refereed][Not invited]
    Membrane-bound sialidases in themouse thymus are unique and mysterious because their activity at pH 6.5 is equal to or higher than that in the acidic region. The pH curve like this has never been reported in membrane-bound form. To clarify this enzyme, we studied the sialidase activities of crude membrane fractions from immature-T, mature-T and non-T cells from C57BL/6 mice and from SM/J mice, a strain with a defect in NEU1 activity. Non-T cells fromC57BL/6 mice had high activity at pH 6.5, but those from SM/J mice did not. Neu1 and Neu3 mRNA was shown by real-time PCR to be expressed in T cells and also in non-T cells, whereas Neu2 was expressed mainly in non-T cells and Neu4 was scarcely expressed. However, the in situ hybridization study on the localization of four sialidases in the thymus showed that Neu4 was clearly expressed. We then focused on a sialidase on the thymocyte surface because the possibility of the existence of a sialidase on thymocytes was suggested by peanut agglutinin (PNA) staining after incubation of the cells alone in PBS. This activity was inhibited by NEU1-selective sialidase inhibitor C9-butyl-amide-2-deoxy-2,3-dehydro-N-acetylneuraminic acid. The natural substrate for the cell surface sialidase was identified as clustered differentiation 5 (CD5) by PNA-blot analysis of anti-CD5 immunoprecipitate. We conclude that NEU1 exists on the cell surface of mouse thymocytes and CD5 is a natural substrate for it. Although this is not themain reaction of themembrane-bound thymus-sialidases, itmust be important for the thymus.
  • Kan Yaguchi, Takahiro Yamamoto, Ryo Matsui, Masaya Shimada, Atsuko Shibanuma, Keiko Kamimura, Toshiaki Koda, Ryota Uehara
    Communicative & integrative biology 11 (4) e1526605  2018 [Refereed][Not invited]
    Recently, we observed that tetraploidization of certain types of human cancer cells resulted in upregulation of centrosome duplication cycles and chronic generation of the extra centrosome. Here, we investigated whether tetraploidy-linked upregulation of centrosome duplication also occurs in non-cancer cells using tetraploidized parthenogenetic mouse embryos. Cytokinesis blockage at early embryonic stage before de novo centriole biogenesis provided the unique opportunity in which tetraploidization can be induced without transient doubling of centrosome number. The extra numbers of the centrioles and the centrosomes were observed more frequently in tetraploidized embryos during the blastocyst stage than in their diploid counterparts, demonstrating the generality of the newly found tetraploidy-driven centrosome overduplication in mammalian non-cancer systems.
  • Gizaw ST, Koda T, Amano M, Kamimura K, Ohashi T, Hinou H, Nishimura S
    Biochimica et biophysica acta 1850 (9) 1704 - 1718 0006-3002 2015/09 [Refereed][Not invited]
  • Solomon T. Gizaw, Toshiaki Koda, Maho Amano, Keiko Kamimura, Tetsu Ohashi, Hiroshi Hinou, Shin-Ichiro Nishimura
    BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS 1850 (9) 1704 - 1718 0304-4165 2015/09 [Refereed][Not invited]
    Background: Huntington's disease (HD) is an autosomal, dominantly inherited and progressive neurodegenerative disease, nosologically classified as the presence of intranuclear inclusion bodies and the loss of GABA-containing neurons in the neostriatum and subsequently in the cerebellar cortex. Abnormal processing of neuronal proteins can result in the misfolding of proteins and altered post-translational modification of newly synthesized proteins. Total glycomics, namely, N-glycomics, O-glycomics, and glycosphingolipidomics (GSL-omics) of HD transgenic mice would be a hallmark for central nervous system disorders in order to discover disease specific biomarkers. Methods: Glycoblotting method, a high throughput glycomic protocol, and matrix-assisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) were used to study the total glycome expression levels in the brain tissue (3 mice of each sex) and sera (5 mice of each sex) of HD transgenic and control mice. All experiments were performed twice and differences in the expression levels of major glycoforms were compared between HD transgenic and control mice. Results: We estimated the structure and expression levels of 87 and 58 N-glycans in brain tissue and sera, respectively, of HD transgenic and control mice. The present results clearly indicated that the brain glycome and their expression levels are significantly gender specific when compared with those of other tissues and serum. Core-fucosylated and bisecting-GlcNAc types of N-glycans were found in increased levels in the brain tissue HD transgenic mice. Accordingly, core-fucosylated and sialic acid (particularly N-glycolylneuraminic acid, NeuGc) for biantennary type glycans were found in increased amounts in the sera of HD transgenic mice compared to that of control mice. Core 3 type O-glycans were found in increased levels in male and in decreased levels in both the striatum and cortexes of female HD transgenic mice. Furthermore, serum levels of core 1 type O-glycans decreased and were undetected for core 2 type O-glycans for HD transgenic mice. In glycosphingolipids, GD1a in brain tissue and GM2-NeuGc serum levels were found to have increased and decreased, respectively, in HD transgenic mice compared to those of the control group mice. Conclusion: Total glycome expression levels are significantly different between HD transgenic and control group mice. General significance: Glycoblotting combined with MALDI-TOF/MS total glycomics warrants a comprehensive, effective, novel and versatile technique for qualitative and quantitative analysis of total glycome expression levels. Furthermore, glycome-focused studies of both environmentally and genetically rooted neurodegenerative diseases are promising candidates for the discovery of potential disease glyco-biomarkers in the post-genome era. (C) 2015 Elsevier B.V. All rights reserved.
  • Miwako Kobayashi, Toshiyuki Nakatani, Toshiaki Koda, Ken-ichi Matsumoto, Ryosuke Ozaki, Natsuki Mochida, Keizo Takao, Tsuyoshi Miyakawa, Ichiro Matsuoka
    MOLECULAR BRAIN 7 12  1756-6606 2014/02 [Refereed][Not invited]
    Background: We have previously identified BRINP (BMP/RA-inducible neural-specific protein-1, 2, 3) family genes that possess the ability to suppress cell cycle progression in neural stem cells. Of the three family members, BRINP1 is the most highly expressed in various brain regions, including the hippocampus, in adult mice and its expression in dentate gyrus (DG) is markedly induced by neural activity. In the present study, we generated BRINP1-deficient (KO) mice to clarify the physiological functions of BRINP1 in the nervous system. Results: Neurogenesis in the subgranular zone of dentate gyrus was increased in BRINP1-KO mice creating a more immature neuronal population in granule cell layer. The number of parvalbumin expressing interneuron in hippocampal CA1 subregion was also increased in BRINP1-KO mice. Furthermore, BRINP1-KO mice showed abnormal behaviors with increase in locomotor activity, reduced anxiety-like behavior, poor social interaction, and slight impairment of working memory, all of which resemble symptoms of human psychiatric disorders such as schizophrenia and attention-deficit/hyperactivity disorder (ADHD). Conclusions: Absence of BRINP1 causes deregulation of neurogenesis and impairments of neuronal differentiation in adult hippocampal circuitry. Abnormal behaviors comparable to those of human psychiatric disorders such as hyperactivity and poor social behavior were observed in BRINP1-KO mice. These abnormal behaviors could be caused by alteration of hippocampal circuitry as a consequence of the lack of BRINP1.
  • Shigeko Kijimoto-Ochiai, Naoko Doi, Miwako Fujiiy, Shinji Go, Kazuya Kabayamaz, Setsuko Moriya, Taeko Miyagi, Toshiaki Koda
    MICROBIOLOGY AND IMMUNOLOGY 57 (8) 569 - 582 0385-5600 2013/08 [Refereed][Not invited]
    Compared to other organs, the mouse thymus exhibits a high level of sialidase activity in both the soluble and crude membrane fractions, as measured at neutral pH using 4MU-Neu5Ac as a substrate. The main purpose of the present study was to identify the sialidase with a high level of the activity at neutral pH in the crude membrane. Several parameters were analyzed using the soluble (S) fraction, N and D fractions that were obtained by NP-40 or DOC/NP-40 solubilization from the thymus crude membrane. The main sialidase activity in the N fraction exhibited almost the same pI as that of soluble Neu2 and 60% of the activity was removed from the membrane by three washes with 10mM Tris-buffer, at pH 7.0. The N fraction preferentially hydrolyzed the sialic acid bond of glycoprotein and exhibited sialidase activity with fetuin at pH 7.0 but not at pH 4.5. The same activity was observed in a plasma membrane-rich fraction. To date, the removal of sialic acid from fetuin at pH 7.0 was reported only with soluble Neu2 and the membrane fraction from Neu2-transfected COS cells. We analyzed the gene that controls the sialidase activity in the crude membrane fraction at pH 7.0 using SMXA recombinant mice and found that compared with other three genes, Neu2 presented the best correlation with the activity level. We suggest that Neu2 is most likely responsible for the main activity in the N fraction, due to its association with the membrane by an unknown mechanism.
  • Kobayashi Miwako, Miyake Makiko, Motomiya Makoto, Takao Keizo, Koda Toshiaki, Miyakawa Tsuyoshi, Matsuoka Ichiro
    NEUROSCIENCE RESEARCH 71 E138 - E139 0168-0102 2011 [Refereed][Not invited]
  • Iori Sakurai, Toshiaki Kouda, Takahiro Fukumoto, Ikuji Hatamura, Yuki-Hisa Hirao
    Journal of Mammalian Ova Research 27 (4) 220 - 224 1341-7738 2010/10 [Not refereed][Not invited]
    Polycystic ovary syndrome (PCOS) is the most common etiology of menstrual disorders and hyperandrogenism, and The understanding of PCOS has advanced significantly. However, a fully convincing animal model for study of polycystic ovaries, or of PCOS, has not been established in the mouse. In the present study, polycystic ovary (PCO) was induced by a single intraperitoneal injection of testosterone propionate (TP 0.1 mg, dissolved in sesame oil) in female mice at 45 days of age. The mice exhibited either constant estrus or diestrus for 5 weeks after TP administration, and this resulted in anovulatory polycystic ovaries. These ovaries contained multiple large follicles and no corpus luteum. The morphology and ovulatory failure were similar to those of the PCOs of humans and other animals. In these ovaries, we found that only the oocytes in the secondary follicles had the following cytological changes: first meiotic division metaphase, second meiotic division metaphase, parthenogenesis and fragmentation. This mouse model has potential for application in studies of folliculogenesis and the mechanism of androgen-induced PCO.
  • Toshiaki Koda, Shigeko Kijimoto-Ochiai, Satoshi Uemura, Jin-ichi Inokuchi
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 387 (4) 729 - 735 0006-291X 2009/10 [Refereed][Not invited]
    Neu2 mRNA from the mouse thymus, as we have reported [K. Kotani, A. Kuroiwa, T. Saito, Y. Matsuda, T. Koda, S. Kijimoto-Ochiai, Cloning, chromosomal mapping, and characteristic 5'-UTR sequence of murine cytosolic sialidase, Biochem. Biophys. Res. Commun. 286 (2001) 250-258], has a novel sequence at the 50 terminus that shows the ability to encode 6 extra amino acids in the N-terminus than that of the muscle. In this paper, we analyzed the cDNA and EST database and found the five types of alternative splicing of Neu2 mRNA: A, B, C, D and N. We studied the expression of these types in the immune tissues and found that the thymus expressed only type B. We constructed 2 types of plasmid that encode long (B) or short (C) form of Neu2 protein, and transfected them into COS7 cells to study them under the same conditions. We found that 30-40% of the both forms of Neu2 activity was located in the crude membrane-fraction, and hydrolyzed ganglioside effectively, while both soluble fraction showed particular behavior with substrate specificity. Microscopic study by active staining with X-NANA showed that they located not only in the cytoplasm but also in areas surrounding the nucleus and in the peripheral ruffled spot. (C) 2009 Elsevier Inc. All rights reserved.
  • S. Kijimoto-Ochiai, T. Koda, T. Suwama, H. Matsukawa, M. Fujii, K. Tomobe, M. Nishimura
    GLYCOCONJUGATE JOURNAL 25 (8) 787 - 796 0282-0080 2008/11 [Refereed][Not invited]
    We have already reported that the homogenate of the A/J mouse thymus shows a high sialidase activity at the neutral pH region and that in both soluble and membrane fractions optimal pH was 6.5-7 (Kijimoto-Ochiai et al., Glycoconj. J., 20:375-384, 2004). In the present study, we investigated the level of sialidase activities in the thymus of the SM/J mouse, a mouse strain that we know to have a Neu1(a) allele that reveals a low level of sialidase activity in the liver. We found that while in the A/J thymus the soluble sialidase activity at pH 6.5 was high, the SM/J thymus lacked all such activity. A QTL analysis of SMXA recombinant inbred strains showed that soluble sialidase activity correlated well with the D1Mit8/9 marker on chromosome 1. The murine whole DNA-sequence data and the results of our FISH analysis (Kotani et al., Biochem. Biophys. Res. Comm., 286:250-258, 2001) showed that this location is consistent with the position of Neu2 gene. We confirmed that it is hard to detect the Neu2 enzyme of the SM/J mouse thymus by an anti-Neu2 antibody using a Western blot analysis. We also found that while the mRNA expression of Neu2 was quite normal in the SM/J mouse liver, it was very low in the SM/J mouse thymus. We therefore conclude that the lack of soluble sialidase activity in the SM/J mouse thymus is due to the thymus-specific low expression level of the Neu2 gene. We have previously shown that the sialidase positive cell which contains the Mac-1 and immunoglobulin, and which is located sparsely in the corticomedullar region or medullary region of the A/J mouse thymus (Kijimoto-Ochiai et al., Glycoconj. J., 20:375-384, 2004). We showed now in this paper that the detection of this cell in the SM/J mouse thymus at pH 7.0 was difficult. We propose, therefore, to name the cell "Neu-medullocyte".
  • Reiko Sadamoto, Takeshi Matsubayashi, Masataka Shimizu, Taichi Ueda, Shuhei Koshida, Toshiaki Koda, Shin-Ichiro Nishimura
    CHEMISTRY-A EUROPEAN JOURNAL 14 (33) 10192 - 10195 0947-6539 2008 [Refereed][Not invited]
  • A Yamaguchi, T Koda, H Abe, M Sato, J Li, T Sakai, Y Togashi, Y Shinohara, H Ikeda, T Nishimura
    IMMUNOLOGY LETTERS 101 (1) 95 - 103 0165-2478 2005/10 [Refereed][Not invited]
    The immune balance controlled by CD4(+) helper T cell subsets (T helper I (Th1) and T helper 2 (Th2)) is crucial for immunoregulation and its imbalance causes various immune diseases including infections, allergic disorders and autoimmune diseases. Therefore, it is of great importance to develop a system of diagnosing Th1/Th2 imbalances for curing immune diseases. Here we developed a functional cDNA array filter useful for assessing the Th1/Th2 balance in mice. To overcome the disadvantages of conventional microarrays carrying thousands of genes, we prepared an array filter containing 40 Th1-specific and 32 Th2-specific genes, which were selected from over 8700 genes based on (i) the specificity of expression in Th1 or Th2 cells and (ii) an expression level which is high enough for detection using a DNA array. This array filter provided a prompt and precise evaluation for the skewing of the Th1/Th2 balance combined with our calculation algorithm. The bias toward Th1 or Th2 was evaluated visually at a glance by aligning the genes on the filter. Moreover, we succeeded in evaluating the skewing of the Th1/Th2 balance in vivo during acute graft versus host disease (GVHD). Thus, this array filter will provide a novel tool for evaluation of the Th1/Th2 balance in a variety of immune diseases. (c) 2005 Elsevier B.V. All rights reserved.
  • Aki Yamaguchi, Yuji Togashi, Toshiaki Koda, Takashi Nishimura
    Japanese Journal of Clinical Immunology 日本臨床免疫学会 28 (2) 86 - 91 1349-7413 2005 [Refereed][Not invited]
    DNA arrays are useful for determining the expression levels of a number of genes at once. We utilized this technique to evaluate the Th1/Th2 balance in vivo. Immune responses are controlled by two types of helper T cells, Th1 and Th2. Once the balance of Th1/Th2 immunity is disrupted, various immune diseases can develop. Thus, it is important to evaluate the Th1/Th2 balance in each patient for diagnosis, treatment and/or prophylaxis of immune diseases. We have identified a number of genes specifically expressed in Th1 or Th2 cells, and developed a DNA array filter spotted with these genes. We confirmed that this filter is useful for the evaluation of changes in the immune balance in vivo. Clinical application of this technology may lead to the tailor-made therapy of immune diseases through the evaluation of the immune balance in each patient. © 2005, The Japan Society for Clinical Immunology. All rights reserved.
  • T Fujimura, K Chamoto, T Tsuji, T Sato, H Yokouchi, S Aiba, H Tagami, J Tanaka, M Imamura, Y Togashi, T Koda, T Nishimura
    IMMUNOLOGY LETTERS 93 (1) 17 - 25 0165-2478 2004/04 [Refereed][Not invited]
    Leukemic dendritic cells (DC) were induced from the peripheral blood (PB) or bone marrow (BM) of leukemia patients by culture with (i) GM-CSF + IL-3 (neutral condition); (ii) GM-CSF + IL-3 + IL-12 + IFN-gamma (type 1-condition); or (iii) GM-CSF + IL-3 + IL-4 (type 2-condition). Although leukemic cells rapidly differentiated into adhesive leukemic DC in all culture conditions, type 1-conditions were the most suitable for inducing leukemic DC expressing high levels of HLA and costimulatory molecules. Addition of IL-2 after 2 days of culture induced a preferential growth of minor T cell populations interacting with leukemic DC. In particular, IFN-gamma-producing CD4(+) Th1 cells were efficiently expanded in type I culture conditions but nor in neutral or type 2-conditions. However, CD4+ T cells expanded in neutral conditions showed Th1-like functions if they were pulsed with IFN-gamma for 2 days before harvest. Such Th1 cells produced IFN-gamma and exhibited cytotoxicity in response to autologous leukemia cells. We further dernonstrated that IFN-gamma production of leukemia-specific Th1 cells was blocked by anti-HLA-DR mAb. Thus, we established a novel culture system for inducing leukemia-specific Thl cells. (C) 2004 Elsevier B.V. All rights reserved.
  • T Sato, R Saito, T Jinushi, T Tsuji, J Matsuzaki, T Koda, S Nishimura, H Takeshima, T Nishimura
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 314 (2) 468 - 475 0006-291X 2004/02 [Refereed][Not invited]
    The production of eotaxin, which is a critical mediator for airway inflammation, is inhibited by IFN-gamma. Here, we investigated the precise mechanisms underlying IFN-gamma-dependent inhibition of eotaxin production using mouse embryonic fibroblasts (MEF). MEF produced high levels of eotaxin in STAT6-dependent manner when they were cultured with both IL-4 and TNF-alpha. However, the eotaxin production by MEF was strongly inhibited by addition of IFN-gamma. Western-blotting analysis demonstrated that IFN-gamma downmodulated STAT6 phosphorylation induced by IL-4 and TNF-alpha. Moreover, IFN-gamma did not exhibit its inhibitory effect on both STAT6-phosphorylation and eotaxin production in MEF obtained from deficient mice in STAT1, a key molecule of IFN-gamma signaling. We also demonstrated that SOCS-1, a potent inhibitory molecule of IL-4 signaling, was induced by IFN-gamma in STAT1-dependent manner. This indicated that SOCS-1 might be involved in IFN-gamma-mediated STAT1-dependent inhibition of cotaxin production. In SOCS-1(-/-) MEF, IFN-gamma inhibited neither STAT6 phosphorylation nor eotaxin production induced by IL-4 and TNF-a. Conversely, retroviral transduction of SOCS-1 into MEF inhibited STAT6 phosphorylation and eotaxin production induced by IL-4 and TNF-alpha, in the absence of IFN-gamma. Thus, we demonstrated that IFN-gamma-induced inhibition of STAT6 phosphorylation and eotaxin production were mediated by SOCS-1 induced in STAT1-dependent manner. (C) 2003 Elsevier Inc. All rights reserved.
  • Functional expression of the TrkC gene, encoding a high affinity receptor for NT-3, in antigen-specific T helper type 2 (Th2) cells.
    Sekimoto M, Tsuji T, Matsuzaki J, Chamoto K, Koda T, Nemoto K, Degawa M, Nishimura S, Nishimura T
    Immuno Lett. 95 5 - 11 2004 [Refereed][Not invited]
  • J Li, T Koda, A Yamaguchi, S Yamamoto, T Sato, Y Togashi, T Nishimura
    BIOMEDICAL RESEARCH-TOKYO 24 (6) 299 - 307 0388-6107 2003/12 [Not refereed][Not invited]
    CD4(+) helper T (Th) cells are subdivided into Th1 and Th2 cells in terms of their differential cytokine production pattern. These cells are key immunoregulatory cells in host defense mechanisms. Therefore, identification of differential gene expression in Th1 and Th2 cells is crucial for immunomodulation. Here we describe identification of Th1- or Th2-specific genes by DNA microarray analysis. We prepared mRNA from anti-CD3 mAb stimulated or unstimulated Th1 and Th2 cells, then gene expression profiles were analyzed by using a microarray spotted with 8,700 EST clones. We detected preferential expression of 57 genes in Th1 cells and 37 genes in Th2 cells. By semi-quantitative RT-PCR analysis, we identified 11 genes specifically expressed in Th1 cells, and 7 genes in Th2 cells. These genes may contribute to the differentiation and function of helper T cells and thereby regulation of immune response. Further analysis would help to develop a method to modulate immune response.
  • K Chamoto, A Kosaka, T Tsuji, J Matsuzaki, T Sato, T Takeshima, K Iwakabe, Y Togashi, T Koda, T Nishimura
    CANCER SCIENCE 94 (10) 924 - 928 1347-9032 2003/10 [Refereed][Not invited]
    Th1 and Th2 cells obtained from OVA-specific T cell receptor transgenic mice completely eradicated the tumor mass when transferred into mice bearing A20-OVA tumor cells expressing OVA as a model tumor antigen. To elucidate the role of Tc1 or Tc2 cells during tumor eradication by Th1- or Th2-cell therapy, spleen cells obtained from mice cured of tumor by the therapy were restimulated with the model tumor antigen (OVA) for 4 days. Spleen cells obtained from mice cured by Th1-cell therapy produced high levels of IFN-gamma, while spleen cells from mice cured by Th2-cell therapy produced high levels of IL-4. Intracellular staining analysis demonstrated that a high frequency of IFN-gamma-producing Tc1 cells was induced in mice given Th1-cell therapy. In contrast, IL-4-producing Tc2 cells were mainly induced in mice after Th2-cell therapy. Moreover, Tc1, but not Tc2, exhibited a tumor-specific cytotoxicity against A20-OVA but not against CMS-7 fibrosarcoma. Thus, immunological memory essential for CTL generation was induced by the Th1/Tc1 circuit, but not by the Th2/Tc2 circuit. We also demonstrated that Th1-cell therapy is greatly augmented by combination therapy with cyclophosphamide treatment. This finding indicated that adoptive chemoimmunotherapy using Th1 cells should be applicable as a novel tool to enhance the Th1/Tc1 circuit, which is beneficial for inducing tumor eradication in vivo.
  • M Sekimoto, T Tsuji, J Matsuzaki, K Chamoto, T Koda, K Nemoto, M Degawa, SI Nishimura, T Nishimura
    IMMUNOLOGY LETTERS 88 (3) 221 - 226 0165-2478 2003/09 [Refereed][Not invited]
    Neurotrophins, including nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 (NT-3) are essential factors for the development of the nervous system. In this report, we demonstrate gene expression of neurotrophins and their receptors in T helper 1 (Th1) and T helper 2 (Th2) cells induced from naive CD4(+) CD45RB(+) T cells of ovalbumin-specific DO11.10 T cell receptor transgenic mice. Interestingly, the TrkC gene, which encodes a high affinity receptor for NT-3, was expressed in Th2 cells, but not in Th1 and naive CD4(+) T cells. Expression of the TrkC gene was markedly augmented by addition of anti-IFN-gamma monoclonal antibody (mAb) into the culture, whereas it was blocked by anti-IL-4 mAb. Moreover, NT-3 synergistically enhanced anti-CD3 mAb-induced IL-4 production by Th2 cells, but did not affect IFN-gamma production by Th1 cells. These data suggest that NT-3, through its receptor TrkC, plays a critical role in regulating the Th1/Th2 balance. (C) 2003 Elsevier B.V. All rights reserved.
  • K Sasaki, T Tsuji, T Jinushi, J Matsuzaki, T Sato, K Chamoto, Y Togashi, T Koda, T Nishimura
    INTERNATIONAL IMMUNOLOGY 15 (7) 893 - 893 0953-8178 2003/07 [Refereed][Not invited]
  • K Sasaki, T Tsuji, T Jinushi, J Matsuzaki, T Sato, K Chamoto, Y Togashi, T Koda, T Nishimura
    INTERNATIONAL IMMUNOLOGY 15 (6) 701 - 710 0953-8178 2003/06 [Refereed][Not invited]
    We found that T(h)1 cells derived from ovalbumin (OVA)-specific TCR transgenic (DO11.10) mice showed significantly higher levels of VLA-2 (CD49b/CD29) expression than T(h)2 cells. In the early days (until 6 days) during induction of T(h)1 or T(h)2 cells, the expression of VLA-2 was gradually increased on both T-h subsets. Thereafter, VLA-2 expression was further up-regulated on TO cells until 13 days, while a significant decrease of VLA-2 was observed in T(h)2 cells, resulting in a marked difference of expression at day 13. Up-regulation of VLA-2 on T(h)1 cells was not impaired in IFN-gamma(-/-) T-h cells nor blocked by anti-IL-12 mAb treatment on wild-type T-h cells, suggesting that up-regulation of VLA-2 on T(h)1 cells occurs in an IFN-gamma- and IL-12-independent manner. In contrast, T-h cells cultured under IL-4-depleted T(h)2 conditions abrogated the down-regulation of VLA-2 expression, suggesting that down-regulation of VLA-2 expression on T(h)2 cells was dependent on IL-4. The finding that STAT6(-/-) T(h)2 cells did not show any down-regulation of VLA-2 expression and expressed the same levels of VLA-2 as T(h)1 cells indicated a critical role for the IL-4 receptor/STAT6 signaling pathway in IL-4-depedent down-regulation of VLA-2 on T(h)2 cells. Stimulation of T(h)1 cells by VLA-2 ligands such as collagen type I or agonistic mAb provided co-stimulation for anti-CD3 mAb-induced IFN-gamma production. However, these ligations had little effect on the IL-4 production of T(h)2 cells. Together, these results indicate that VLA-2 is a novel functional marker that dissociates T(h)1 from Th2 cells, and thus might be useful for therapeutic monitoring of T(h)1-dependent immune diseases such as rheumatoid arthritis or Crohn's disease.
  • T Tsuji, K Chamoto, H Funamoto, A Kosaka, J Matsuzaki, H Abe, K Fujio, K Yamamoto, T Kitamura, Y Togashi, T Koda, T Nishimura
    CANCER SCIENCE 94 (4) 389 - 393 1347-9032 2003/04 [Refereed][Not invited]
    Genes encoding 2C T cell receptor (TCR) alpha, beta chains from H-2(b)-restricted L-d-specific CD8(+) cells were successfully transduced into polyclonally activated CD8(+) cells by retroviral modification to generate antigen-specific cytotoxic T lymphocytes (CTL). Antigen-nonspecific CD8+ T cells polyclonally expanded in the presence of interleukin (IL)-2, Th1 cytokines (interferon (IFN)-gamma and IL-12) and anti-IL-4 monoclonal antibody showed neither cytokine production nor cytotoxicity in response to L-d-expressing P815 tumor cells. However, 2C-TCR gene-modified CD8(+) T cells exhibited both IFN-gamma production and cytotoxicity in response to P815 tumor cells. The antitumor activity of TCR gene-modified Tc1 cells was also demonstrated in vivo by Winn's assay. Thus, we have developed an efficient method to induce TCR gene-modified antigen-specific TO cells that exhibit antitumor activity both in vitro and in vivo.
  • Kouda Toshiaki, Togashi Yuuji, Nishimura Takashi
    The Hokkaido journal of medical science 北海道大学 77 (2) 151 - 156 0367-6102 2002/03 [Refereed][Not invited]
  • R Valsdottir, H Hashimoto, K Ashman, T Koda, B Storrie, T Nilsson
    FEBS LETTERS 508 (2) 201 - 209 0014-5793 2001/11 [Refereed][Not invited]
    The role of rab33b, a Golgi-specific rab protein, was investigated. Microinjection of rab33b mutants stabilised in the GTP-specific state resulted in a marked inhibition of anterograde transport within the Golgi and in the recycling of glycosyltransferases; from the Golgi to the ER, respectively. A GST- rab33b fusion protein stabilised in its GTP form was found to interact by Western blotting or mass spectroscopy with Golgi protein GM130 and rabaptin-5 and rabex-5, two rab effector molecules thought to function exclusively in the endocytic pathway. A similar binding was seen to rab1 but not to rab6, both Golgi rabs. In contrast, rab5 was as expected, shown to bind rabaptin-5 and rabex-5 as well as the endosomal effector protein EEA1 but not GM130. No binding of EEA1 was seen to any of the Golgi rabs. (C) 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.
  • M Sato, K Chamoto, T Tsuji, Y Iwakura, Y Togashi, T Koda, T Nishimura
    JOURNAL OF IMMUNOLOGY 167 (7) 3687 - 3691 0022-1767 2001/10 [Refereed][Not invited]
    Bone marrow-derived dendritic cell (BMDC) subsets have distinct immunoregulatory functions. Th1 cytokine-induced BMDC (BMDC1), compared with Th2 cytokine-induced BMDC2, have superior activities for the differentiation and expansion of CTL. To evaluate the cellular interactions between dendritic cells and CD8(+) T cells for the induction of CTL, BALB/c-derived BMDC subsets were cocultured with purified CD8(+) T cells from C57BL/6 mice. Our results demonstrate that BMDC1 support the generation of allogeneic CD8(+) CTL in the absence of CD4(+) Th cells. In contrast, BMDC0 (GM-CSF- plus IL-3-induced BMDC) and BMDC2 failed to promote the differentiation of CD8(+) CTL. Using Ab-blocking experiments and studies with gene knockout mice, IL-2 and LFA-1 are demonstrated to be critical for BMDC1-induced CTL differentiation. Unexpectedly, BMDC1 were able to induce CTL from CD8(+) T cells isolated from IFN-gamma (-/-) and IFN-gamma receptor(-/-) mice. However, BMDC1 produced higher levels of IFN-beta than other BMDC subsets, and anti-IFN-beta mAb blocked BMDC1-dependent CTL generation. These results indicated an indispensable role of IFN-beta, but not IFN-gamma, during BMDC1-induced CTL differentiation. We conclude that Th1-cytokine-conditioned BMDC1 can bypass Th cell function for the differentiation of naive CD8(+) T cells into CTL.
  • A Takaoka, Y Tanaka, T Tsuji, T Jinushi, A Hoshino, Y Asakura, Y Mita, K Watanabe, S Nakaike, Y Togashi, T Koda, K Matsushima, T Nishimura
    JOURNAL OF IMMUNOLOGY 167 (4) 2349 - 2353 0022-1767 2001/08 [Refereed][Not invited]
    Ag-specific Th1 and Th2 cells have been demonstrated to play a critical role in the induction of allergic diseases. Here we have investigated the precise mechanisms of Th1-induced airway inflammation. Airway inflammation was induced in BALB/c mice by transfer of freshly induced OVA-specific Th1 or Th2 cells followed by OVA inhalation. In this model, both Th1 and Th2 cells induced airway inflammation. The former induced neutrophilia in airways, whereas the latter induced eosinophilia. Moreover, we found that Th1 cells induced more severe airway hyperresponsiveness (AHR) than Th2 cells. The eosinophilia induced by Th2 cell infusion was almost completely blocked by administration of anti-IL-5 mAb, but not anti-IL-4 mAb. In contrast, Th1-induced AHR and pulmonary neutrophilia were inhibited by the administration of anti-human IL-8R Ab, which blocks the function of mouse CXC chemokine(s). These findings reveal a critical role of mouse CXC chemokine(s) in Th1-dependent pulmonary neutrophilia and AHR.
  • K Kotani, A Kuroiwa, T Saito, Y Matsuda, T Koda, S Kijimoto-Ochiai
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 286 (2) 250 - 258 0006-291X 2001/08 [Refereed][Not invited]
    We have totally sequenced a cytosolic sialidase [EC] by RT-PCR from the murine thymus (murine thymic sialidase, NITS) which has a 1844-base length (encoding 385 amino acids including two sialidase motifs) and is the longest cytosolic sialidase ever reported. MTS has high and relatively low homologies with those of mammalian cytosolic sialidases from the mouse brain (99%), rat (91%), and human skeletal muscle (75%), and those of the mouse lysosomal (47%) and membrane-bound (51%) sialidases, respectively. Chromosomal mapping, being the first report of mouse cytosolic sialidase gene, showed that the MTS gene is localized to the distal part of mouse chromosome 1D and to rat chromosome 9q36. RT-PCR with the site-specific primers revealed that the coding region was expressed in all organs tested, but expressions including the 5'-UTR were barely detectable except for in the upper-thymic fraction. Also, soluble sialidase activity in the thymus was the highest of these organs. There were mRNA instability signals and AT-rich regions in 143 bp of NITS 5'-end. (C) 2001 Academic Press.
  • Differential regulation of VLA-2 expression on Th1 and Th2 cells: a novel marker for the classification of Th subsets.
    Sasaki K, Tsuji T, Jinushi T, Matsuzaki J, Sato T, Chamoto K, Togashi Y, Koda T, Nishimura T
    Int Immunol. 167 3687 - 3691 2001 [Refereed][Not invited]
  • A Ohta, M Sekimoto, M Sato, T Koda, S Nishimura, Y Iwakura, K Sekikawa, T Nishimura
    JOURNAL OF IMMUNOLOGY 165 (2) 956 - 961 0022-1767 2000/07 [Refereed][Not invited]
    We report the development and characterization of a novel model of severe hepatitis induced against hepatitis B virus surface Ag (HBsAg), HBsAg was successfully targeted into the liver in soluble form. Using this unique property of HBsAg, we established a liver injury model induced by HBsAg-specific Th1 cells. Severe liver injury was induced in C57BL/6 mice by injection of HBsAg together with HBsAg-specific Th1 cells. Histochemical examination demonstrated extensive necroinflammatory hepatic lesions in these animals. Application of this liver injury model to mutant or gene knockout mice enabled us to define the effector mechanisms of Th1 cells in fulminant hepatitis, When Fas-deficient lpr mice were used as recipients, a similar degree of liver injury was induced as in wild-type mice. Moreover, HBsAg-specific Th1 cells obtained from perforin(-/-) mice could induce severe liver injury in both wild-type and lpr mice. These results indicated that neither Fas ligand nor perforin are essential for Th1-mediated liver injury in this model. Pretreatment with anti-TNF-alpha mAb prevented liver injury, whereas severe liver injury was induced in TNF-alpha(-/-) mice. Moreover, IFN-gamma receptor-deficient mice were resistant to Th1-mediated liver injury. Therefore, TNF-alpha and IFN-gamma, which were produced by HBsAg-specific Th1 cells during the effector phase, appeared to be indispensable in the pathogenesis of fulminant hepatitis.
  • T Nishimura, H Kitamura, K Iwakabe, T Yahata, A Ohta, M Sato, K Takeda, K Okumura, L Van Kaer, T Kawano, M Taniguchi, M Nakui, M Sekimoto, T Koda
    INTERNATIONAL IMMUNOLOGY 12 (7) 987 - 994 0953-8178 2000/07 [Refereed][Not invited]
    In vivo administration of NKT cell ligand, alpha-galactosylceramide (alpha-GalCer), caused the activation of NKT cells to induce a strong NK activity and cytokine production by CD1d-restricted mechanisms. Surprisingly, we also found that alpha-GalCer induced the activation of immunoregulatory cells involved in acquired immunity. Specifically, in vivo administration of alpha-GalCer resulted in the induction of the early activation marker CD69 on CD4(+) T cells, CD8(+) T cells and B cells in addition to macrophages and NKT cells, However, no significant induction of CD69 was observed on cells from CD1d- or V(alpha)14 NKT-deficient mice, indicating an essential role for the interaction between NKT cells and CD1d-expressing dendritic cells (DC) in the activation of acquired immunity in response to alpha-GalCer, Indeed, in vivo injection of alpha-GalCer resulted not only in the activation of NKT cells but also in the generation of CD69(+)CD8(+) T cells possessing both cytotoxic T lymphocyte (CTL) activity and IFN-gamma-producing ability, Tumor-specific CTL generation was also accelerated by alpha-GalCer, The critical role of CD40-CD40 ligand (CD40L)-mediated NKT-DC interaction during the development of CD69(+)CD8(+) CTL by alpha-GalCer was demonstrated by blocking experiments using anti-CD40L mAb, These findings provide direct evidence for a critical role of CD1d-restricted NKT cells and DC in bridging innate and acquired immunity.
  • T Yahata, C Yahata, A Ohta, M Sekimoto, H Kitamura, K Iwakabe, S Habu, S Azuma, M Nakui, M Sato, T Koda, T Nishimura
    JOURNAL OF NEUROIMMUNOLOGY 105 (2) 103 - 108 0165-5728 2000/06 [Refereed][Not invited]
    Naive Th cells obtained from OVA(323-339)-specific DO11.10TCR-Tg mice did not express preproenkephalin (PPE) mRNA. However, culture of naive Th cells with OVA(323-339) peptide (OVA-pep) plus IL-2 under Th2-inducing conditions for 7 days resulted in an induction of PPE mRNA. The PPE mRNA was also induced by culturing with OVA-pep plus IL-2 (neutral condition). However, PPE mRNA induction under neutral conditions was totally abrogated by addition of anti-IL-4 mAb. The existence of methionine-enkephalin was also demonstrated in peptidase-digested peptides derived from Th2 cell lysate. These results demonstrate that IL-4 is a critical factor for the induction of PPE mRNA in freshly expanded antigen-specific Th2 cells. (C) 2000 Elsevier Science B.V. All rights reserved.
  • T Nishimura, M Nakui, M Sato, K Iwakabe, H Kitamura, M Sekimoto, A Ohta, T Koda, S Nishimura
    CANCER CHEMOTHERAPY AND PHARMACOLOGY 46 (Supplement) S52 - S61 0344-5704 2000/06 [Refereed][Not invited]
    To investigate the precise role of antigen-specific Th1 and Th2. cells in tumor immunity, we developed a novel adoptive tumor-immunotherapy model using OVA-specific Th1 and Th2 cells and an OVA gene-transfected tumor. This therapeutic model demonstrated that both antigen-specific Th1 and Th2 cells had strong antitumor activity in vivo with distinct mechanisms. However, immunological memory suitable for the generation of tumor-specific cytotoxic T lymphocytes was induced only when tumor-bearing mice received Th1 cell therapy, but not Th2 cell therapy. Thus it was strongly suggested that Th1-dominant immunity is critically important for the induction of antitumor cellular immunity in vivo. We also proposed that several immunomodulating protocols using interleukin (IL)-12, IL-12 gene, the natural killer T cell ligand alpha-galactosylceramide, or Th1 cytokine-conditioned dendritic cells might be useful strategies for the induction of Th1-dominant immunity essential for the development of tumor-specific immunotherapy.
  • M Sato, K Iwakabe, A Ohta, M Sekimoto, M Nakui, T Koda, S Kimura, T Nishimura
    INTERNATIONAL IMMUNOLOGY 12 (3) 335 - 342 0953-8178 2000/03 [Refereed][Not invited]
    Three distinct bone marrow (BM)-derived dendritic cells (BMDC) were expanded from BALB/c BM cells by culture with (i) granulocyte macrophage colony stimulating factor (GM-CSF) plus IL-3, (ii) GM-CSF, IL-3 plus T(h)1-biasing cytokines (IL-12 and IFN-gamma) or (iii) GM-CSF, IL-3 plus T(h)2-biasing cytokines (IL-4), All of these cells expressed the DC-specific marker CD11c, and were designated as BMDC0, BMDC1 and BMDC2 cells respectively. BMDC1 cells exhibited superior T cell-stimulating activity in allogeneic mixed lymphocyte culture (MLC), while BMDC2 showed inferior stimulating activity. Specifically, BMDC1, as compared with BMDC2, induced a higher frequency of IFN-gamma-producing CD8(+) T cells in MLC, Moreover, BMDC1, but not BMDC2, were strong inducers of H-2(d)-specific cytotoxic T lymphocytes (CTL) in MLC, BMDC0 always showed intermediate stimulatory activity; however, when BMDC0 were cultured with IFN-gamma, they differentiated into BMDC1-like stimulator cells concomitant with the up-regulation of both MHC antigens and costimulatory molecules. In contrast, BMDC2 were refractory to differentiation into superior stimulator cells by treatment with IFN-gamma, although this treatment enhanced MHC expression. These findings indicate that T(h)1- and T(h)2-biasing cytokines, in addition to their effect on Th cell differentiation, may play a critical role in the functional skewing of DC. These findings have important implications for the development of DC-based immunotherapies.
  • H Kitamura, A Ohta, M Sekimoto, M Sato, K Iwakabe, M Nakui, T Yahata, HX Meng, T Koda, S Nishimura, T Kawano, M Taniguchi, T Nishimura
    CELLULAR IMMUNOLOGY 199 (1) 37 - 42 0008-8749 2000/01 [Refereed][Not invited]
    alpha-Galactosylceramide (alpha-GalCer), a glycolipid antigen, specifically activates natural killer T (NKT) cells by a CD1d-restricted mechanism. In this work, we found that in vivo administration of alpha-GalCer resulted in the activation of B cells in addition to NKT cells, namely, alpha-GalCer administration caused upregulation of the early activation marker, CD69, on both NKT and B cells. In addition, expression of B7.2 and I-A(b) on B cells was greatly upregulated by alpha-GalCer. However, serum levels of IgE, IgG1, and IgG2a were not significantly changed within 48 h. In the present experiments, it was also demonstrated that the upregulation of CD69 expression by alpha-GalCer was strongly blocked by anti-IL-4 monoclonal antibody. Moreover, B-cell activation by alpha-GalCer was not observed in NKT-deficient mice. These results suggested that antigen-stimulated NKT cells might play a critical role not only in early defense mechanisms but also in early B-cell activation through IL-4 production. (C) 2000 Academic Press.
  • M Nakui, A Ohta, M Sekimoto, M Sato, K Iwakabe, T Yahata, H Kitamura, T Koda, T Kawano, H Makuuchi, M Taniguchi, T Nishimura
    CLINICAL & EXPERIMENTAL METASTASIS 18 (2) 147 - 153 0262-0898 2000 [Refereed][Not invited]
    The combined therapeutic effect of natural killer T (NKT) cell ligand alpha -galactosylceramide (alpha -GalCer) and IL-12 against highly metastatic B16-BL6-HM melanoma cells was investigated. In comparison with a single administration of alpha -GalCer or IL-12, the combined treatment of tumor-bearing mice with alpha -GalCer plus IL-12 caused a super-induction of serum IFN-gamma levels, though alpha -GalCer-induced IL-4 production was rather inhibited. In parallel with the augmented IFN-gamma production, the natural killing activity against YAC-1 cells and syngeneic B16- BL6-HM melanoma was greatly augmented by the combined therapy. The major effector cells responsible for natural killing activity induced by alpha -GalCer plus IL- 12 were enriched in both NK1.1(+)TCR alpha beta (+) NKT cells and NK1.1(+)TCR alpha beta (-) NK cells. The preventing effect of alpha -GalCer or IL-12 alone against lung metastasis of B16-BL6-HM was also enhanced by the combination therapy. The antitumor activity of alpha -GalCer was totally abolished in NKT-deficient mice. However, IL- 12-induced antitumor activity was not eliminated in NKT-deficient mice though it was inhibited by anti-asialo GM1 Ab treatment. These findings suggested that alpha -GalCer synergistically act with IL-12 to activate both NKT cells and NK cells, which may play a critical role in the strong prevention of distant tumor metastasis at early stages of tumor-bearing. These data will provide a novel tool for the prevention of tumor metastasis using NKT-specific ligands alpha -GalCer and IL-12.
  • T Koda, JY Zheng, M Ishibe
    MOLECULAR BIOLOGY OF THE CELL 10 214A - 214A 1059-1524 1999/11 [Refereed][Not invited]
  • Y Shoya, T Kobayashi, T Koda, K Ikuta, M Kakinuma, M Kishi
    JOURNAL OF VIROLOGY 72 (12) 9755 - 9762 0022-538X 1998/12 [Refereed][Not invited]
    Borna disease virus (BDV) uses a unique strategy of replication and transcription which takes place in the nucleus, unlike other known, nonsegmented, negative-stranded RNA viruses of animal origin. In this process, viral constituents necessary for replication must be transported to the nucleus from the cytoplasm. We report here the evidence that BDV P protein, which may play an important role in viral replication and transcription, is transported into the nucleus in the absence of other viral constituents. This transportation is accomplished by its own nuclear localization signals (NLSs), which are present in both N-terminal (29PRPRKIPR36) and C-terminal ((181)PPRIYPQLPSAPT(193)) regions of the protein. These two NLSs can function independently and both have several Pro residues as key amino acids.
  • Kuriyama T, Fujinaga M, Koda T, Nishihira J
    Biochimica et biophysica acta 2 1388 506 - 512 0006-3002 1998/11 [Refereed][Not invited]
  • M Ishibe, T Ishibashi, K Kaneda, T Koda, RN Rosier, JE Puzas
    CALCIFIED TISSUE INTERNATIONAL 63 (1) 36 - 38 0171-967X 1998/07 [Refereed][Not invited]
    Insulin-like growth factor-II (IGF-II) plays an important role in skeletal remodeling, however, little is known about its effect on bone formation in vivo. In our study of the stimulation of bone formation in vivo by IGF-II we injected recombinant human IGF-II into the parietal bones of neonatal rats once a day for 12 days. The bone mineral density measured by dual energy X-ray absorptiometry and the thickness of IGF-II-injected parietal bones increased in a dose-dependent manner. The layers of osteoblasts were observed along the IGF-II-injected side.
  • WR Tang, T Koda, M Kishi, M Kakinuma
    IMMUNOGENETICS 48 (1) 67 - 72 0093-7711 1998/06 [Refereed][Not invited]
  • JY Zheng, T Koda, T Fujiwara, M Kishi, Y Ikehara, M Kakinuma
    JOURNAL OF CELL SCIENCE 111 1061 - 1069 0021-9533 1998/04 [Refereed][Not invited]
    Small GTP-binding proteins of the Rab family play important roles at defined steps of vesicular transport in protein secretion and the endocytosis pathway. In mammals, more than 30 proteins belonging to the Rab family have been reported to date. We report here the molecular cloning and characterization of a novel Rab protein, Rab33B. The amino acid sequence of Rab33B shows 55.3% identity to the Rab33A protein (previously called S10), and these two proteins share unique amino acid sequences at the effector domain. The genomic organization of rab33B was the same as rab33A: it consists of two exons. Thus, these two proteins make a subclass within the Rab family Northern blot analysis showed that rab33B is expressed ubiquitously in mouse tissues, in contrast to rab33A whose expression is restricted to the brain and the immune system. A 26 kDa protein was detected by western blotting using a Rab33B-specific monoclonal antibody. Using immunofluorescence studies, Rab33B was shown to co-localize with alpha-mannosidase II, a Golgi-specific marker. Immunoelectron microscopy analysis further defined the localization of Rab33B to the medial Golgi cisternae. These results suggest Rab33B plays a role in intra-Golgi transport.
  • T Kobayashi, Y Shoya, T Koda, Takashima, I, PK Lai, K Ikuta, M Kakinuma, M Kishi
    VIROLOGY 243 (1) 188 - 197 0042-6822 1998/03 [Refereed][Not invited]
    The Borna disease virus (BDV) replicates in the nucleus. The viral p40 protein (N), which is found abundantly in the nucleus in BDV-infected cells, may play an important role in virus replication. To analyze the amino acid residues involved in the nuclear targeting of BDV N, a series of eukaryotic expression plasmids encoding deletion mutants of N was constructed and transfected into COS-7 cells. In indirect immunofluorescence assays with a rabbit anti-BDV N antiserum, wild-type N was located in the nucleus of transfected cells in the absence of other viral constituents. In contrast, mutants tacking the 13 NH2-terminal amino acid residues (1)MPPKRRLVDDADA(13) in common gave a cytoplasmic localization pattern. Similarly, a mutant with substitution of 4KRR6 by (4)NSG(6) was retained in the cytoplasm. Furthermore, a nonapeptide, (3)PKRRLVDDA(11), derived from the NH2-terminal region of N conferred nuclear targeting activity to beta-galactosidase, which normally resides in the cytoplasm. Thus, we have identified the nuclear targeting signal of the BDV N and narrowed it to the NH2-terminal region where 4KRR6 basic amino acid residues are located. (C) 1998 Academic Press.
  • M Kakinuma, WR Tang, Y Arimura, T Koda, M Kishi
    TRANSPLANTATION PROCEEDINGS 29 (3) 1670 - 1670 0041-1345 1997/05 [Refereed][Not invited]
  • Zheng JY, Koda T, Arimura Y, Kishi M, Kakinuma M
    Biochimica et biophysica acta 1-2 1351 47 - 50 0006-3002 1997/03 [Refereed][Not invited]
  • JY Zheng, T Koda, Y Arimura, M Kishi, M Kakinuma
    BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION 1351 (1-2) 47 - 50 0167-4781 1997/03 [Refereed][Not invited]
    We previously reported the cloning of a human S10 cDNA which encodes a small GTP-binding protein belonging to the Rab subfamily. Here we describe a mouse S10 cDNA and its genomic structure. Mouse S10 is 92.3% homologous at the nucleotide level and 98.3% identical at the amino acid level compared to human SIG. The mouse S10 gene is comprised of two exons and a single intron. Northern blotting of tissue RNAs indicates that the S10 gene is predominantly expressed in brain. (C) 1997 Elsevier Science B.V.
  • Y Shoya, T Kobayashi, T Koda, PK Lai, H Tanaka, T Koyama, K Ikuta, M Kakinuma, M Kishi
    MICROBIOLOGY AND IMMUNOLOGY 41 (6) 481 - 486 0385-5600 1997 [Refereed][Not invited]
    We have developed a novel reverse transcriptase-polymerase chain reaction (RT-PCR) to amplify the full-length 8.9 kilobase (kbp) cDNA of the Borna disease virus (BDV) RNA genome from the total cellular RP;A of MDCK cells persistently infected with BDV (MDCK/BDV). Antigenomic BDV cDNA was reverse transcribed using a 53-mer oligonucleotide primer, corresponding to the 5'-terminus of a putative 3'-leader sequence of the BDV RNA genome, for 2 hr at 42 C followed by 30 min at 55 C. PCR was performed in the presence of this 53-mer antigenomic primer and a 25-mer primer, corresponding to the 3'-terminus of the BDV antigenomic cDNA, by use of an rTth DNA polymerase with proof-reading activity. The amplified full-length BDV cDNA was detected in as little as 20 ng of total cellular RNA of MDCK/BDV. This RT-PCR method should be a useful technique to study the molecular quasispecies of BDV.
  • Y Arimura, T Koda, M Kishi, M Kakinuma
    IMMUNOGENETICS 43 (3) 152 - 155 0093-7711 1996 [Refereed][Not invited]
    CALCIFIED TISSUE INTERNATIONAL 57 (6) 430 - 435 0171-967X 1995/12 [Refereed][Not invited]
    It is well known that 17 beta-estradiol (E(2)) and 1,25-dihydroxyvitamin D-3 (1,25(OH)(2)D-3) have important roles in bone metabolism. This study was undertaken to examine E(2) regulation of 1,25(OH)(2)D-3 receptor (VDR) expression and the biological action of 1,25(OH)(2)D-3 in human osteoblastlike cells. When human osteosarcoma-derived osteoblastlike cells were treated with varying concentrations of E(2), the VDR levels increased by up to 100% in a dose-dependent manner. VDR levels significantly increased at 10 nM E(2), and this increase plateaued at 100 nM E(2). E(2)-dependent increase of VDR was time dependent, plateauing at 24 hours and was maintained for at least 48 hours in human osteoblast-like cells. Scatchard analysis showed that E(2) increased the number of VDR (12.3 +/- 0.4 versus 26.5 +/- 0.3 fmol/mg protein; mean +/- SE of three independent experiments) rather than the Kd (0.15 +/- 0.02 versus 0.16 +/- 0.01 nM; mean +/- SE of three independent experiments). Tamoxifen (50 nM), a specific competitor with E(2), completely abolished the E(2)-induced increase of VDR. The levels of VDR mRNA (4.5 kb) from the cells increased in a dose-dependent manner after E(2) treatment. With regard to the biological effects, E(2) increased by 10-25% the inhibitory effect of 1,25(OH)(2)D-3 on cell growth. However, E(2) did not increase the stimulation of alkaline phosphatase activity by 1,25(OH)(2)D3. The present study suggests that E, modulates the biological action of 1,25(OH)(2)D-3 through VDR levels in bone cells.
    JOURNAL OF ORTHOPAEDIC RESEARCH 13 (5) 643 - 648 0736-0266 1995/09 [Refereed][Not invited]
    Insulin-like growth factor-II is known to stimulate the proliferation and differentiation of osteoblasts in part through activation of the type-2 insulin-like growth factor receptor. The present study examined the type-2 insulin-like growth factor receptors of three normal osteoblast-like cells and three osteosarcoma derived osteoblast-like cells (OGA, SU, and IMAI) from humans. [I-125]insulin-like growth factor-II was used for the binding studies. All of the cell types had high affinity binding sites for insulin-like growth factor-II (dissociation constants [Kd] less than or equal to 1 nM). The concentration of these sites was 10 to 24-fold higher in normal osteoblasts than in the osteosarcoma cells studied. Unlabeled insulin-like growth factor-II inhibited the binding of [I-125]insulin-like growth factor-II to the cells in a dose-dependent manner; however, unlabeled insulinlike growth factor-I and insulin were less effective. Covalent crosslinking of insulin-like growth factor-II binding sites gave molecular mass estimates of M(r) 250,000 in human osteoblast cells, 250,000 and 130,000 in OGA cells, 240,000 in SU cells, and 250,000 and 130,000 in IMAI cells. Unlabeled insulin-like growth factor-II inhibited all affinity labeling. In Northern blot analysis, the type-2 insulin-like growth factor receptor mRNA of normal osteoblasts was seen in greater abundance than it was in osteosarcoma cells. These results indicate that the numbers of type-2 insulin-like growth factor receptors differ between normal and transformed osteoblasts and that the differential expression of the receptor may be due to the differentiation of osteoblasts.
    IMMUNOGENETICS 42 (2) 156 - 158 0093-7711 1995/06 [Refereed][Not invited]
    TRANSPLANTATION PROCEEDINGS 27 (2) 1505 - 1506 0041-1345 1995/04 [Refereed][Not invited]
    IMMUNOGENETICS 41 (5) 320 - 325 0093-7711 1995/03 [Refereed][Not invited]
    JOURNAL OF BIOLOGICAL CHEMISTRY 269 (40) 25042 - 25048 0021-9258 1994/10 [Refereed][Not invited]
    Expression of hst (k-FGF, FGF-4), a member of the fibroblast growth factor gene family, is restricted to early stages of developing embryos and to embryonal carcinoma cells. In F9, which is a prototype of embryonal carcinoma cells expressing hst, the expression of hst gene is positively regulated by a downstream octamer motif that functions as an enhancer. We have investigated, by chloramphenicol acetyltransferase (CAT) reporter fusion gene analysis in F9, the cis-acting regulatory element within the hst promoter region that interacts with this enhancer. Electrophoretic mobility shift assay and methylation interference analysis showed that the hst promoter contains, in a segment termed Y, the sequence 5'-CTGATTGGCA-3', which closely resembles the consensus binding motif for the CCAAT-binding factor NF-Y. Deletions or mutations in this element substantially reduced expression of hst-CAT constructs. The nuclear factor binding to the Y segment of the hst promoter was indistinguishable from NF-Y, as inferred from interactions with specific anti-NF-Y monoclonal and polyclonal antibodies. We conclude that the expression of the hst gene in F9 is positively regulated by the coordinated interaction between an NF-Y-binding site and an octamer motif.
    FEBS LETTERS 342 (1) 71 - 75 0014-5793 1994/03 [Refereed][Not invited]
    The hst-1 gene, which is implicated in mammalian embryonic development and morphological transformation of NIH3T3 cells, is expressed in undifferentiated F9 cells, but not in differentiated F9 and other well-differentiated cells, such as PYS-2, NIH3T3 and HeLa cells. An octamer element present in the 3' untranslated region acts as an enhancer. Although Oct3 is down-regulated when F9 cells are differentiated, transient expression of Oct3 did not enhance the hst-1 promoter activity in HeLa, NIH3T3 or PYS-2 cells. Thus, the role of Oct3 on hst-1 expression remains elusive, and an additional transcription factor which interacts may regulate hst-1 transcription in association with Oct1, Oct3 or both.
    FEBS LETTERS 328 (1-2) 21 - 24 0014-5793 1993/08 [Refereed][Not invited]
    A cDNA encoding a small GTP-binding protein, S10, was cloned from Jurkat cells. The deduced amino acid sequence of S10 had the structural features characteristic to this family of proteins with highest homology to rab subfamily. Northern blot analysis revealed that this gene is expressed only in lymphoid cell lines and a histiocytic leukemia, U937. Hence, it should have a specialized function in cells derived from the hematopoietic stem cell.
    MICROBIOLOGY AND IMMUNOLOGY 37 (8) 633 - 640 0385-5600 1993 [Refereed][Not invited]
    A genomic HLA-G clone named 7.OE was isolated from a Japanese placenta. The deduced amino acid sequence of the 7.0E was identical to two HLA-G genomic clones and two cDNA clones previously described. The DNA sequences of alpha1 and alpha2 domains of the HLA-G gene from 5 cell lines also encoded the same amino acids. However, a 14 bp insertion, ATTTGTTCATGCCT, was present in the 3' untranslated region of 7.OE compared with the originally described HLA-G clone (HLA 6.0). Polymerase chain reaction (PCR)/single strand conformational polymorphism (SSCP) analysis of exon 8 allowed the HLA-G gene to be classified into two alternative types, G6.0 and 7.0 E, those correlated to the absence or the presence of the 14 bp stretch. Each group had minor sequence variant(s), and the alleles of the 7.OE-type were more heterogeneous than those of the G6.0- type. The 14 bp deletion is present only in the G6.0-type of HLA-G alleles among HLA class I genes.Thus it was suggested that G6.0 alleles were generated after diversification of the HLA-G.
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 187 (1) 507 - 514 0006-291X 1992/08 [Refereed][Not invited]
    EMBO JOURNAL 11 (2) 391 - 404 0261-4189 1992/02 [Refereed][Not invited]
    We report the identification and molecular characterization of Dtrk, a Drosophila gene encoding a receptor tyrosine kinase highly related to the trk family of mammalian neurotrophin receptors. The product of the Dtrk gene, gp160Dtrk, is dynamically expressed during Drosophila embryogenesis in several areas of the developing nervous system, including neurons and fasciculating axons. gp160Dtrk has structural homology with neural cell adhesion molecules of the immunoglobulin superfamily and promotes cell adhesion in a homophilic, Ca2+ independent manner. More importantly, this adhesion process specifically activates its tyrosine protein kinase activity. These findings suggest that gp160Dtrk represents a new class of neural cell adhesion molecules that may regulate neuronal recognition and axonal guidance during the development of the Drosophila nervous system.
    CELL GROWTH & DIFFERENTIATION 2 (2) 95 - 105 1044-9523 1991/02 [Refereed][Not invited]
    In this study, we report the isolation of complementary DNA clones containing the entire coding sequence of the mouse vav protooncogene. Antisera raised against a peptide corresponding to a predicted hydrophilic domain have allowed us to identify the product of the vav gene as a 95,000 Da protein. Analysis of the deduced amino acid sequence of p95vav revealed an amino-terminal leucine-rich region not present in the activated vav oncogene. This region consists of an amphipathic helix-loop-helix followed by a leucine zipper, a structure reminiscent of the carboxy-terminal region of myc proteins and the steroid binding domain of nuclear receptors. In vitro mutagenicity studies have indicated that removal of the amphipathic helix-loop-helix is sufficient to activate the transforming potential of human and mouse vav protooncogenes. vav proteins also possess a cysteine-rich domain whose sequence predicts the formation of two putative metal binding-like domains, Cys-X2-Cys-X13-Cys-X2-Cys and His-X2-Cys-X6-Cys-X2-His. Replacement of some of these cysteine and histidine residues completely abolished the transforming activity of vav genes. Further examination of the alignment of cysteine residues in this region revealed an alternative structure, Cys-X2-Cys-X13-Cys-X2-Cys-X7-Cys-X6-Cys, which is reminiscent of the phorbol ester binding domain of protein kinase C. A similar domain has been recently identified in a second enzyme, diacylglycerol kinase. These structural similarities, along with its expression pattern, suggest that the vav protooncogene codes for a new type of signal-transducing molecule that may play an important role in controlling hematopoiesis.
    JAPANESE JOURNAL OF CANCER RESEARCH 80 (3) 200 - 203 0910-5050 1989/03 [Refereed][Not invited]
    北海道医学雑誌 63 (6) 913 - 924 0367-6102 1988/11 [Not refereed][Not invited]
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 85 (13) 4861 - 4864 0027-8424 1988/07 [Refereed][Not invited]
    綜合臨床 37 (1) 11 - 16 0371-1900 1988/01 [Not refereed][Not invited]
    JAPANESE JOURNAL OF CANCER RESEARCH 78 (4) 325 - 328 0910-5050 1987/04 [Refereed][Not invited]
    JAPANESE JOURNAL OF CANCER RESEARCH 76 (7) 551 - 554 0910-5050 1985 [Refereed][Not invited]


Research Grants & Projects

  • 疾患モデルマウスを利用した治療法開発
    Date (from‐to) : 1997
  • Devolopment of new treatment through studies on model mice
    Date (from‐to) : 1997

Educational Activities

Teaching Experience

  • Bioethics
    開講年度 : 2021
    課程区分 : 修士課程
    開講学部 : 生命科学院
    キーワード : 生命倫理、被験者保護、インフォームド・コンセント、個人情報、薬害、動物実験、胚研究、遺伝子改変、遺伝子特許、データ捏造、デュアルユース
  • Inter-Graduate School Classes(General Subject):Inter-Disciplinary Sciences
    開講年度 : 2021
    課程区分 : 修士課程
    開講学部 : 大学院共通科目
    キーワード : 生命倫理、被験者保護、インフォームド・コンセント、個人情報、薬害、動物実験、胚研究、遺伝子改変、遺伝子特許、データ捏造、デュアルユース

Campus Position History

  • 2013年4月1日 

Position History

  • 2013年4月1日 

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