2021/10/27 更新

写真a

カナヤマ ナオキ
金山 直樹
KANAYAMA Naoki
所属
ヘルスシステム統合科学学域 准教授
職名
准教授
外部リンク

学位

  • 博士(工学) ( 京都大学 )

研究キーワード

  • Antibody

  • Lymphocyte

  • 抗体

  • リンパ球

  • B細胞

  • 細胞工学

  • 免疫

研究分野

  • ライフサイエンス / 免疫学

  • ものづくり技術(機械・電気電子・化学工学) / バイオ機能応用、バイオプロセス工学

  • ナノテク・材料 / 生体化学

  • ライフサイエンス / 機能生物化学

学歴

  • 京都大学 大学院   工学研究科   合成・生物化学専攻

    - 1998年

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    国名: 日本国

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  • 京都大学   Graduate School, Division of Engineering  

    - 1998年

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  • 京都大学   Faculty of Engineering  

    - 1992年

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  • 京都大学   工学部   工業化学

    - 1992年

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    国名: 日本国

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経歴

  • 岡山大学   ヘルスシステム統合科学研究科   准教授

    2018年4月 - 現在

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  • 岡山大学自然科学研究科機能分子化学専攻

    2007年4月 - 2018年3月

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  • - Associate Professor,Chemistry and Biochemistry,Graduate School of Natural Science and Technology,Okayama University

    2007年

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  • Associate Professor,Chemistry and Biochemistry,Graduate School of Natural Science and Technology,Okayama University

    2005年 - 2007年

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  • 岡山大学自然科学研究科機能分子化学専攻 助教授

    2005年 - 2007年

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  • Senior Assistant Professor,Dept. of Bioscience and Biotechnology,Faculty of Engineering,Okayama University

    2002年 - 2005年

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  • 岡山大学工学部生物機能工学科 講師   Faculty of Engineering, Department of Bioscience and Biotechnology

    2002年 - 2005年

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  • Research Associate,Dept. of Bioscience and Biotechnology,Faculty of Engineering,Okayama University

    1998年 - 2002年

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  • 岡山大学工学部生物機能工学科 助手   Faculty of Engineering, Department of Bioscience and Biotechnology

    1998年 - 2002年

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  • Postdoctoral Fellowships of Japan Society for the Promotion of Science

    1996年 - 1998年

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  • 京都大学大学院工学研究科合成・生物化学専攻 日本学術振興会特別研究員   Graduate School of Engineering, Department of Synthetic Chemistry and Biological Chemistry

    1996年 - 1998年

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▼全件表示

所属学協会

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論文

  • Regulation of the tubulin polymerization-promoting protein by Ca2+/S100 proteins. 国際誌

    Seita Doi, Naoki Fujioka, Satomi Ohtsuka, Rina Kondo, Maho Yamamoto, Miwako Denda, Masaki Magari, Naoki Kanayama, Naoya Hatano, Ryo Morishita, Takafumi Hasegawa, Hiroshi Tokumitsu

    Cell calcium   96   102404 - 102404   2021年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    To elucidate S100 protein-mediated signaling pathways, we attempted to identify novel binding partners for S100A2 by screening protein arrays carrying 19,676 recombinant glutathione S-transferase (GST)-fused human proteins with biotinylated S100A2. Among newly discovered putative S100A2 interactants, including TMLHE, TRH, RPL36, MRPS34, CDR2L, OIP5, and MED29, we identified and characterized the tubulin polymerization-promoting protein (TPPP) as a novel S100A2-binding protein. We confirmed the interaction of TPPP with Ca2+/S100A2 by multiple independent methods, including the protein array method, S100A2 overlay, and pulldown assay in vitro and in transfected COS-7 cells. Based on the results from the S100A2 overlay assay using various GST-TPPP mutants, the S100A2-binding region was identified in the C-terminal (residues 111-160) of the central core domain of a monomeric form of TPPP that is involved in TPPP dimerization. Chemical cross-linking experiments indicated that S100A2 suppresses dimer formation of His-tagged TPPP in a dose-dependent and a Ca2+-dependent manner. In addition to S100A2, TPPP dimerization is disrupted by other multiple S100 proteins, including S100A6 and S100B, in a Ca2+-dependent manner but not by S100A4. This is consistent with the fact that S100A6 and S100B, but not S100A4, are capable of interacting with GST-TPPP in the presence of Ca2+. Considering these results together, TPPP was identified as a novel target for S100A2, and it is a potential binding target for other multiple S100 proteins, including S100A6 and S100B. Direct binding of the S100 proteins with TPPP may cause disassembly of TPPP dimer formation in response to the increasing concentration of intracellular Ca2+, thus resulting in the regulation of the physiological function of TPPP, such as microtubule organization.

    DOI: 10.1016/j.ceca.2021.102404

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  • Identification and Biochemical Characterization of High Mobility Group Protein 20A as a Novel Ca2+/S100A6 Target. 国際誌

    Maho Yamamoto, Rina Kondo, Haruka Hozumi, Seita Doi, Miwako Denda, Masaki Magari, Naoki Kanayama, Naoya Hatano, Ryo Morishita, Hiroshi Tokumitsu

    Biomolecules   11 ( 4 )   2021年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    During screening of protein-protein interactions, using human protein arrays carrying 19,676 recombinant glutathione s-transferase (GST)-fused human proteins, we identified the high-mobility protein group 20A (HMG20A) as a novel S100A6 binding partner. We confirmed the Ca2+-dependent interaction of HMG20A with S100A6 by the protein array method, biotinylated S100A6 overlay, and GST-pulldown assay in vitro and in transfected COS-7 cells. Co-immunoprecipitation of S100A6 with HMG20A from HeLa cells in a Ca2+-dependent manner revealed the physiological relevance of the S100A6/HMG20A interaction. In addition, HMG20A has the ability to interact with S100A1, S100A2, and S100B in a Ca2+-dependent manner, but not with S100A4, A11, A12, and calmodulin. S100A6 binding experiments using various HMG20A mutants revealed that Ca2+/S100A6 interacts with the C-terminal region (residues 311-342) of HMG20A with stoichiometric binding (HMG20A:S100A6 dimer = 1:1). This was confirmed by the fact that a GST-HMG20A mutant lacking the S100A6 binding region (residues 311-347, HMG20A-ΔC) failed to interact with endogenous S100A6 in transfected COS-7 cells, unlike wild-type HMG20A. Taken together, these results identify, for the first time, HMG20A as a target of Ca2+/S100 proteins, and may suggest a novel linkage between Ca2+/S100 protein signaling and HMG20A function, including in the regulation of neural differentiation.

    DOI: 10.3390/biom11040510

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  • Development and Characterization of Novel Molecular Probes for Ca2+/Calmodulin-Dependent Protein Kinase Kinase, Derived from STO-609. 査読 国際誌

    Satomi Ohtsuka, Yui Ozeki, Moeno Fujiwara, Tomoyuki Miyagawa, Naoki Kanayama, Masaki Magari, Naoya Hatano, Futoshi Suizu, Teruhiko Ishikawa, Hiroshi Tokumitsu

    Biochemistry   59 ( 17 )   1701 - 1710   2020年5月

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    記述言語:英語  

    Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) activates particular multifunctional kinases, including CaMKI, CaMKIV, and 5'AMP-activated protein kinase (AMPK), resulting in the regulation of various Ca2+-dependent cellular processes, including neuronal, metabolic, and pathophysiological pathways. We developed and characterized a novel pan-CaMKK inhibitor, TIM-063 (2-hydroxy-3-nitro-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one) derived from STO-609 (7H-benzimidazo[2,1-a]benz[de]isoquinoline-7-one-3-carboxylic acid), and an inactive analogue (TIM-062) as molecular probes for the analysis of CaMKK-mediated cellular responses. Unlike STO-609, TIM-063 had an inhibitory activity against CaMKK isoforms (CaMKKα and CaMKKβ) with a similar potency (Ki = 0.35 μM for CaMKKα, and Ki = 0.2 μM for CaMKKβ) in vitro. Two TIM-063 analogues lacking a nitro group (TIM-062) or a hydroxy group (TIM-064) completely impaired CaMKK inhibitory activities, indicating that both substituents are necessary for the CaMKK inhibitory activity of TIM-063. Enzymatic analysis revealed that TIM-063 is an ATP-competitive inhibitor that directly targets the catalytic domain of CaMKK, similar to STO-609. TIM-063 suppressed the ionomycin-induced phosphorylation of exogenously expressed CaMKI, CaMKIV, and endogenous AMPKα in HeLa cells with an IC50 of ∼0.3 μM, and it suppressed CaMKK isoform-mediated CaMKIV phosphorylation in transfected COS-7 cells. Thus, TIM-063, but not the inactive analogue (TIM-062), displayed cell permeability and the ability to inhibit CaMKK activity in cells. Taken together, these results indicate that TIM-063 could be a useful tool for the precise analysis of CaMKK-mediated signaling pathways and may be a promising lead compound for the development of therapeutic agents for the treatment of CaMKK-related diseases.

    DOI: 10.1021/acs.biochem.0c00149

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  • Phosphorylation and dephosphorylation of Ca2+/calmodulin-dependent protein kinase kinase β at Thr144 in HeLa cells. 査読 国際誌

    Shota Takabatake, Yusei Fukumoto, Satomi Ohtsuka, Naoki Kanayama, Masaki Magari, Hiroyuki Sakagami, Naoya Hatano, Hiroshi Tokumitsu

    Biochemical and biophysical research communications   2020年2月

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    記述言語:英語  

    Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) acts as a regulatory kinase that phosphorylates and activates multiple downstream kinases including CaMKI, CaMKIV, 5'AMP-activated protein kinase (AMPK) and protein kinase B (PKB), resulting in regulation of wide variety of Ca2+-dependent physiological responses under normal and pathological conditions. CaMKKβ is regulated by Ca2+/calmodulin-binding, autophosphorylation, and transphosphorylation by multiple protein kinases including cAMP-dependent protein kinase (PKA). In this report, we found that phosphorylation of CaMKKβ is dynamically regulated by protein phosphatase/kinase system in HeLa cells. Global phosphoproteomic analysis revealed the constitutive phosphorylation at 8 Ser residues including Ser128, 132, and 136 in the N-terminal regulatory domain of rat CaMKKβ in unstimulated HeLa cells as well as inducible phosphorylation of Thr144 in the cells treated with a phosphatase inhibitor, okadaic acid (OA). Thr144 phosphorylation in CaMKKβ has shown to be rapidly induced by OA treatment in a time- and dose-dependent manner in transfected HeLa cells, indicating that Thr144 in CaMKKβ is maintained unphosphorylated state by protein phosphatase(s). We confirmed that in vitro dephosphorylation of pThr144 in CaMKKβ by protein phosphatase 2A and 1. We also found that the pharmacological inhibition of protein phosphatase(s) significantly induces CaMKKβ-phosphorylating activity (at Thr144) in HeLa cell lysates as well as in intact cells; however, it was unlikely that this activity was catalyzed by previously identified Thr144-kinases, such as AMPK and PKA. Taken together, these results suggest that the phosphorylation and dephosphorylation of Thr144 in CaMKKβ is dynamically regulated by multiple kinases/phosphatases signaling resulting in fine-tuning of the enzymatic property.

    DOI: 10.1016/j.bbrc.2020.02.056

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  • SRSF1-3, a splicing and somatic hypermutation regulator, controls transcription of IgV genes via chromatin regulators SATB2, UBN1 and histone variant H3.3. 査読 国際誌

    Amit Kumar Singh, Anubhav Tamrakar, Ankit Jaiswal, Naoki Kanayama, Prashant Kodgire

    Molecular immunology   119   69 - 82   2020年1月

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    記述言語:英語  

    SRSF1, a member of the SR protein family, is an important splicing factor and regulator of splicing. Multiple splicing isoforms have been reported for this gene. SRSF1-3, a splicing isoform of SRSF1, is necessary for AID-dependent SHM of IgV genes. However, its precise role in SHM remains enigmatic. Transcriptomic analysis of SRSF1-3 reconstituted cells shows upregulation of transcription factor SATB2 and chromatin regulator UBN1. The increased SATB2 and UBN1 are strikingly enriched in the MAR and promoter regions of the IgL gene, respectively. Furthermore, UBN1 enrichment at the promoter region was coupled with a hundred-fold enhanced occupancy of the histone variant H3.3 at the IgL promoter, that is a hallmark of efficient SHM. The enhanced occupancy of SATB2 at the MAR, UBN1 and histone variant H3.3 at the IgL promoter leads to an increase in IgL transcription, revealing a role of SRSF1-3 in SHM. Thus, SRSF1-3 is likely involved in the regulation of SHM, via upregulation of a crucial transcription factor SATB2, as well as, by overexpression of a chromatin modulator of Ig genes, UBN1, which further assists in the recruitment of the histone variant H3.3. Furthermore, the splicing isoform SRSF1-3 regulates alternate splicing pattern of splicing isoforms for various crucial genes. The present study provides the first evidence that a splicing isoform of an SR protein can regulate the post-transcriptional processing of RNA in vivo.

    DOI: 10.1016/j.molimm.2020.01.005

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  • Splicing regulator SRSF1-3 that controls somatic hypermutation of IgV genes interacts with topoisomerase 1 and AID. 査読

    Kumar Singh A, Tamrakar A, Jaiswal A, Kanayama N, Agarwal A, Tripathi P, Kodgire P

    Molecular immunology   116   63 - 72   2019年10月

  • 新規S100A6標的分子(HMG20A)の同定と相互作用解析

    山本 真穂, 近藤 里奈, 傳田 美和子, 土居 青太, 金山 直樹, 曲 正樹, 森下 了, 徳光 浩

    日本生化学会大会プログラム・講演要旨集   92回   [1T07m - 06]   2019年9月

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    記述言語:日本語   出版者・発行元:(公社)日本生化学会  

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  • ニワトリB細胞株DT40におけるscFv型抗体の発現効率の改善

    野田 凌太郎, 岡 知里, 曲 正樹, 徳光 浩, 金山 直樹

    日本生物工学会大会講演要旨集   2019年   217 - 217   2019年8月

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    記述言語:日本語   出版者・発行元:(公社)日本生物工学会  

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  • scFv型抗体を発現するニワトリB細胞株DT40の樹立と利用

    岡 知里, 野田 凌太郎, 曲 正樹, 徳光 浩, 金山 直樹

    日本生物工学会大会講演要旨集   2019年   216 - 216   2019年8月

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    記述言語:日本語   出版者・発行元:(公社)日本生物工学会  

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  • Regulation of Ca<sup>2+</sup>/calmodulin-dependent protein kinase kinase β by cAMP signaling. 査読

    Takabatake S, Ohtsuka S, Sugawara T, Hatano N, Kanayama N, Magari M, Sakagami H, Tokumitsu H

    Biochimica et biophysica acta. General subjects   1863 ( 4 )   672 - 680   2019年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1016/j.bbagen.2018.12.012

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  • Interaction of S100A6 with Target Proteins In Vitro and in Living Cells. 査読

    Sakane K, Yamaguchi F, Tsuchiya M, Kondo R, Kanayama N, Magari M, Hatano N, Kobayashi R, Tokumitsu H

    Methods Mol. Biol.   1929   367 - 377   2019年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1007/978-1-4939-9030-6_23

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  • IL-34 cell surface localization regulated by the 78 kDa glucose-regulated protein facilitates the differentiation of monocytic cells. 査読

    Ogawa S, Matsuoka Y, Takada M, Matsui K, Yamane F, Kubota E, Yasuhara S, Hieda K, Kanayama N, Hatano N, Tokumitsu H, Magari M

    The Journal of biological chemistry   294 ( 7 )   2386 - 2396   2018年12月

  • CaMKKβシグナル伝達のAMPKおよびPKAによるリン酸化制御機構の解明

    大塚 里美, 高畠 翔太, 金山 直樹, 曲 正樹, 徳光 浩

    日本生化学会大会プログラム・講演要旨集   91回   [3T12a - 208)]   2018年9月

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    記述言語:日本語   出版者・発行元:(公社)日本生化学会  

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  • ヘルスシステムの理解とその応用 濾胞樹状細胞による抗体の親和性成熟の制御機構の解明(Interdisciplinary Science and Engineering in Health Systems Immunological functions of follicular dendritic cells on affinity maturation of antibody)

    Magari Masaki, Ogawa Sayaka, Matsuoka Yukiko, Takada Miho, Kanayama Naoki, Tokumitsu Hiroshi

    生物物理   58 ( Suppl.1-2 )   S197 - S197   2018年8月

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    記述言語:英語   出版者・発行元:(一社)日本生物物理学会  

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  • スプライシング因子の過剰発現及び標的遺伝子配列の操作によるニワトリB細胞株における遺伝子変異の増強

    金山 直樹, 太田 愛美, 西山 由美子, 岡 知里, 曲 正樹, 徳光 浩

    日本生物工学会大会講演要旨集   平成30年度   85 - 85   2018年8月

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    記述言語:日本語   出版者・発行元:(公社)日本生物工学会  

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  • AMP-activated protein kinase-mediated feedback phosphorylation controls the Ca2+/calmodulin (CaM) dependence of Ca2+/CaM-dependent protein kinase kinase β 査読

    Akihiro Nakanishi, Naoya Hatano, Yuya Fujiwara, Arian Sha'ri, Shota Takabatake, Hiroki Akano, Naoki Kanayama, Masaki Magari, Naohito Nozaki, Hiroshi Tokumitsu

    Journal of Biological Chemistry   292 ( 48 )   19804 - 19813   2017年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:American Society for Biochemistry {\&} Molecular Biology ({ASBMB})  

    DOI: 10.1074/jbc.M117.805085

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  • AMP-activated protein kinase?mediated feedback phosphorylation controls the Ca2+/calmodulin (CaM) dependence of Ca2+/CaM-dependent protein kinase kinase β 査読

    Akihiro Nakanishi, Naoya Hatano, Yuya Fujiwara, Arian Sha’ri, Shota Takabatake, Hiroki Akano, Naoki Kanayama, Masaki Magari, Naohito Nozaki, Hiroshi Tokumitsu

    Journal of Biological Chemistry   292 ( 48 )   19804 - 19813   2017年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1074/jbc.M117.805085

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  • スプライシング因子の過剰発現及び標的遺伝子配列の操作によるニワトリB細胞株における遺伝子変異の増強

    太田 愛美, 西山 由美子, 曲 正樹, 徳光 浩, 金山 直樹

    生命科学系学会合同年次大会   2017年度   [1P - 1380]   2017年12月

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    記述言語:日本語   出版者・発行元:生命科学系学会合同年次大会運営事務局  

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  • 濾胞樹状細胞の発現するCSF-1の単球系細胞分化への関与の解析

    安原 詩織, 松岡 由希子, 小川 紗也香, 金山 直樹, 徳光 浩, 曲 正樹

    生命科学系学会合同年次大会   2017年度   [3P - 1002]   2017年12月

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    記述言語:日本語   出版者・発行元:生命科学系学会合同年次大会運営事務局  

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  • 抗体遺伝子変異の転写関連過程におけるSRSF1-3の役割の解析

    金山 直樹, 川口 祐加, 横山 和輝, 石橋 朋之, 曲 正樹, 徳光 浩

    生命科学系学会合同年次大会   2017年度   [3P - 0586]   2017年12月

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    記述言語:日本語   出版者・発行元:生命科学系学会合同年次大会運営事務局  

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  • AID発現とSHM誘導におけるBCRシグナルの役割

    梶浦 雄也, 金廣 優一, 曲 正樹, 徳光 浩, 金山 直樹

    生命科学系学会合同年次大会   2017年度   [3P - 0587]   2017年12月

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    記述言語:日本語   出版者・発行元:生命科学系学会合同年次大会運営事務局  

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  • Identification and characterization of a centrosomal protein, FOR20 as a novel S100A6 target 査読

    Kyohei Sakane, Miyu Nishiguchi, Miwako Denda, Fuminori Yamagchi, Masaki Magari, Naoki Kanayama, Ryo Morishita, Hiroshi Tokumitsu

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   491 ( 4 )   980 - 985   2017年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS INC ELSEVIER SCIENCE  

    S100A6 is a Ca2+-signal transducer that interacts with numerous proteins and regulates their biochemical functions. Here we identified a centrosomal protein, FOR20 (FOP-related protein of 20 kDa) as a novel S100A6 target by screening protein microarrays carrying 19,676 recombinant GST-fused human proteins. Binding experiments revealed that S100A6 interacts with the N-terminal region (residues 1-30) of FOR20 in a Ca2+-dependent manner in vitro and in living cells. Several S100 proteins including S100A1, A2, A4, All, B also exhibited Ca2+-dependent interactions with FOR20 as well as S100A6. We found that two distantly related centrosomal proteins, FOP and OFD1, also possess N-terminal regions with a significant sequence similarity to the putative S100A6-binding site (residues 1-30) in FOR20 and are capable of binding to S100A6 in a Ca2+-dependent manner. Taken together, these results may indicate that S100A6 interacts with FOR20 and related centrosomal proteins through a conserved N-terminal domain, suggesting a novel Ca2+-dependent regulation of centrosomal function. (C) 2017 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.bbrc.2017.07.161

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  • 抗体遺伝子変異能力を操作できるニワトリB細胞を用いた動物細胞ディスプレイ

    金山 直樹, 太田 愛美, 中谷 元紀, 中谷 耕治, 植月 英智, 西山 由美子, 仲尾 祐輝, 曲 正樹, 徳光 浩

    日本生物工学会大会講演要旨集   平成29年度   280 - 280   2017年8月

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    記述言語:日本語   出版者・発行元:(公社)日本生物工学会  

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  • SRSF1-3 contributes to diversification of the immunoglobulin variable region gene by promoting accumulation of AID in the nucleus 査読

    Yuka Kawaguchi, Hiroaki Nariki, Naoko Kawamoto, Yuichi Kanehiro, Satoshi Miyazaki, Mari Suzuki, Masaki Magari, Hiroshi Tokumitsu, Naoki Kanayama

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   485 ( 2 )   261 - 266   2017年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS INC ELSEVIER SCIENCE  

    Activation-induced cytidine deaminase (AID) is essential for diversification of the Ig variable region (IgV). AID is excluded from the nucleus, where it normally functions. However, the molecular mechanisms responsible for regulating AID localization remain to be elucidated. The SR-protein splicing factor SRSF1 is a nucleocytoplasmic shuttling protein, a splicing isoform of which called SRSF1-3, has previously been shown to contribute to IgV diversification in chicken DT40 cells. In this study, we examined whether SRSFI-3 functions in IgV diversification by promoting nuclear localization of AID. AID expressed alone was localized predominantly in the cytoplasm. In contrast, co-expression of AID with SRSFI-3 led to the nuclear accumulation of both AID and SRSF1-3 and the formation of a protein complex that contained them both, although SRSF1-3 was dispensable for nuclear import of AID. Expression of either SRSF1-3 or a C-terminally-truncated AID mutant increased IgV diversification in DT40 cells. However, over expression of exogenous SRSF1-3 was unable to further enhance IgV diversification in DT40 cells expressing the truncated AID mutant, although SRSFI-3 was able to form a protein complex with the AID mutant. These results suggest that SRSF1-3 promotes nuclear localization of AID probably by forming a nuclear protein complex, which might stabilize nuclear AID and induce IgV diversification in an AID C terminus-dependent manner. (C) 2017 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.bbrc.2017.02.097

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  • Identification of striated muscle activator of Rho signaling (STARS) as a novel calmodulin target by a newly developed genome-wide screen 査読

    Yusui Furuya, Miwako Denda, Kyohei Sakane, Tomoko Ogusu, Sumio Takahashi, Masaki Magari, Naoki Kanayama, Ryo Morishita, Hiroshi Tokumitsu

    CELL CALCIUM   60 ( 1 )   32 - 40   2016年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:CHURCHILL LIVINGSTONE  

    To search for novel target(s) of the Ca2+-signaling transducer, calmodulin (CaM), we performed a newly developed genome-wide CaM interaction screening of 19,676 GST-fused proteins expressed in human. We identified striated muscle activator of Rho signaling (STARS) as a novel CaM target and characterized its CaM binding ability and found that the Ca2+/CaM complex interacted stoichiometrically with the N-terminal region (Ala13-Gln35) of STARS in vitro as well as in living cells. Mutagenesis studies identified Ile20 and Trp33 as the essential hydrophobic residues in CaM anchoring. Furthermore, the CaM binding deficient mutant (Ile20Ala, Trp33Ala) of STARS further enhanced its stimulatory effect on SRF-dependent transcriptional activation. These results suggest a connection between Ca2+-signaling via excitation-contraction coupling and the regulation of STARS-mediated gene expression in muscles. (C) 2016 Elsevier Ltd. All rights reserved.

    DOI: 10.1016/j.ceca.2016.04.004

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  • Differential AMP-activated Protein Kinase (AMPK) Recognition Mechanism of Ca2+/Calmodulin-dependent Protein Kinase Kinase Isoforms 査読

    Yuya Fujiwara, Yoshinori Kawaguchi, Tomohito Fujimoto, Naoki Kanayama, Masaki Magari, Hiroshi Tokumitsu

    JOURNAL OF BIOLOGICAL CHEMISTRY   291 ( 26 )   13802 - 13808   2016年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) is a known activating kinase for AMP-activated protein kinase (AMPK). In vitro, CaMKK phosphorylates Thr(172) in the AMPK subunit more efficiently than CaMKK, with a lower K-m (approximate to 2 m) for AMPK, whereas the CaMKI phosphorylation efficiencies by both CaMKKs are indistinguishable. Here we found that subdomain VIII of CaMKK is involved in the discrimination of AMPK as a native substrate by measuring the activities of various CaMKK/CaMKK chimera mutants. Site-directed mutagenesis analysis revealed that Leu(358) in CaMKK/Ile(322) in CaMKK confer, at least in part, a distinct recognition of AMPK but not of CaMKI.

    DOI: 10.1074/jbc.M116.727867

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  • Analysis of Distinct Roles of CaMKK Isoforms Using STO-609-Resistant Mutants in Living Cells 査読

    Yuya Fujiwara, Yuri Hiraoka, Tomohito Fujimoto, Naoki Kanayama, Masaki Magari, Hiroshi Tokumitsu

    BIOCHEMISTRY   54 ( 25 )   3969 - 3977   2015年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER CHEMICAL SOC  

    To assess the isoform specificity of the Ca2+/calmodulin-dependent protein kinase kinase (CaMKK)-mediated signaling pathway using a CaMKK inhibitor (STO-609) in living cells, we have established A549 cell lines expressing STO-609-resistant mutants of CaMKK isoforms. Following serial mutagenesis studies, we have succeeded in obtaining an STO-609-resistant CaMKK alpha mutant (Ala292Thr/Leu233Phe) and a CaMKK beta mutant (Ala328Thr/Val269Phe), which showed sensitivity to STO-609 that was 2-3 orders of magnitude lower without an appreciable effect on kinase activity or CaM requirement. These results are consistent with the results obtained for CaMKK activities in the extracts of A549 cells stably expressing the mutants of CaMKK isoforms. Ionomycin-induced 5'-AMP-activated protein kinase (AMPK) phosphorylation at Thr172 in A549 cells expressing either the wildtype or the STO-609-resistant mutant of CaMKK alpha was completely suppressed by STO-609 treatment but resistant to the inhibitor in the presence of the CaMKK beta mutant (Ala328Thr/Val269Phe). This result strongly suggested that CaMKK beta is responsible for ionomycin-induced AMPK activation, which supported previous reports. In contrast, ionomycin-induced CaMKIV phosphorylation at Thr196 was resistant to STO-609 treatment in A549 cells expressing STO-609-resistant mutants of both CaMKK isoforms, indicating that both CaMKK. isoforms are capable of phosphorylating and activating CaMKIV in living cells. Considering these results together, STO-609-resistant CaMKK mutants developed in this study may be useful for distinguishing CaMKK isoform-mediated signaling pathways in combination with the use of an inhibitor compound.

    DOI: 10.1021/acs.biochem.5b00149

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  • CSF-1 receptor-mediated differentiation of a new type of monocytic cell with B cell-stimulating activity: its selective dependence on IL-34 査読

    Fumihiro Yamane, Yumiko Nishikawa, Kazue Matsui, Miki Asakura, Eriko Iwasaki, Koji Watanabe, Hikaru Tanimoto, Hiroki Sano, Yuki Fujiwara, E. Richard Stanley, Naoki Kanayama, Neil A. Mabbott, Masaki Magari, Hitoshi Ohmori

    JOURNAL OF LEUKOCYTE BIOLOGY   95 ( 1 )   19 - 31   2014年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:FEDERATION AMER SOC EXP BIOL  

    Occurrence of a CSF-1 receptor-dependent monocyte differentiation process driven by IL-34, but not CSF-1. With the use of a mouse FDC line, FL-Y, we have been analyzing roles for FDCs in controlling B cell fate in GCs. Beside these regulatory functions, we fortuitously found that FL-Y cells induced a new type of CD11b(+) monocytic cells (F4/80(+), Gr-1(-), Ly6C(-), I-A/E-/lo, CD11c(-), CD115(+), CXCR4(+), CCR2(+), CX(3)CR1(-)) when cultured with a Lin(-)c-kit(+) population from mouse spleen cells. The developed CD11b(+) cells shared a similar gene-expression profile to mononuclear phagocytes and were designated as FDMCs. Here, we describe characteristic immunological functions and the induction mechanism of FDMCs. Proliferation of anti-CD40 antibody-stimulated B cells was markedly accelerated in the presence of FDMCs. In addition, the FDMC-activated B cells efficiently acquired GC B cell-associated markers (Fas and GL-7). We observed an increase of FDMC-like cells in mice after immunization. On the other hand, FL-Y cells were found to produce CSF-1 as well as IL-34, both of which are known to induce development of macrophages and monocytes by binding to the common receptor, CSF-1R, expressed on the progenitors. However, we show that FL-Y-derived IL-34, but not CSF-1, was selectively responsible for FDMC generation using neutralizing antibodies and RNAi. We also confirmed that FDMC generation was strictly dependent on CSF-1R. To our knowledge, a CSF-1R-mediated differentiation process that is intrinsically specific for IL-34 has not been reported. Our results provide new insights into understanding the diversity of IL-34 and CSF-1 signaling pathways through CSF-1R.

    DOI: 10.1189/jlb.0613311

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  • In vitro substrate phosphorylation by Ca2+/calmodulin-dependent protein kinase kinase using guanosine-5 '-triphosphate as a phosphate donor 査読

    Saki Yurimoto, Tomohito Fujimoto, Masaki Magari, Naoki Kanayama, Ryoji Kobayashi, Hiroshi Tokumitsu

    BMC BIOCHEMISTRY   13   27   2012年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:BIOMED CENTRAL LTD  

    Background: Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) phosphorylates and activates particular downstream protein kinases -including CaMKI, CaMKIV, and AMPK- to stimulate multiple Ca2+-signal transduction pathways. To identify previously unidentified CaMKK substrates, we used various nucleotides as phosphate donors to develop and characterize an in vitro phosphorylation assay for CaMKK.
    Results: Here, we found that the recombinant CaMKK isoforms were capable of utilizing Mg-GTP as a phosphate donor to phosphorylate the Thr residue in the activation-loop of CaMKI alpha (Thr(177)) and of AMPK (Thr(172)) in vitro. Kinetic analysis indicated that the K-m values of CaMKK isoforms for GTP (400-500 mu M) were significantly higher than those for ATP (similar to 15 mu M), and a 2- to 4-fold decrease in V-max was observed with GTP. We also confirmed that an ATP competitive CaMKK inhibitor, STO-609, also competes with GTP to inhibit the activities of CaMKK isoforms. In addition, to detect enhanced CaMKI phosphorylation in brain extracts with Mg-GTP and recombinant CaMKKs, we found potential CaMKK substrates of similar to 45 kDa and similar to 35 kDa whose Ca2+/CaM-induced phosphorylation was inhibited by STO-609.
    Conclusions: These results indicated that screens that use STO-609 as a CaMKK inhibitor and Mg-GTP as a CaMKK-dependent phosphate donor might be useful to identify previously unidentified downstream target substrates of CaMKK.

    DOI: 10.1186/1471-2091-13-27

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  • Activation-induced cytidine deaminase (AID)-dependent somatic hypermutation requires a splice isoform of the serine/arginine-rich (SR) protein SRSF1 査読

    Yuichi Kanehiro, Kagefumi Todo, Misaki Negishi, Junji Fukuoka, Wenjian Gan, Takuya Hikasa, Yoshiaki Kaga, Masayuki Takemoto, Masaki Magari, Xialu Li, James L. Manley, Hitoshi Ohmori, Naoki Kanayama

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   109 ( 4 )   1216 - 1221   2012年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATL ACAD SCIENCES  

    Somatic hypermutation (SHM) of Ig variable region (IgV) genes requires both IgV transcription and the enzyme activation-induced cytidine deaminase (AID). Identification of a cofactor responsible for the fact that IgV genes are much more sensitive to AID-induced mutagenesis than other genes is a key question in immunology. Here, we describe an essential role for a splice isoform of the prototypical serine/arginine-rich (SR) protein SRSF1, termed SRSF1-3, in AID-induced SHM in a DT40 chicken B-cell line. Unexpectedly, we found that SHM does not occur in a DT40 line lacking SRSF1-3 (DT40-ASF), although it is readily detectable in parental DT40 cells. Strikingly, overexpression of AID in DT40-ASF cells led to a large increase in nonspecific (off-target) mutations. In contrast, introduction of SRSF1-3, but not SRSF1, into these cells specifically restored SHM without increasing off-target mutations. Furthermore, we found that SRSF1-3 binds preferentially to the IgV gene and inhibits processing of the Ig transcript, providing a mechanism by which SRSF1-3 makes the IgV gene available for AID-dependent SHM. SRSF1 not only acts as an essential splicing factor but also regulates diverse aspects of mRNA metabolism and maintains genome stability. Our findings, thus, define an unexpected and important role for SRSF1, particularly for its splice variant, in enabling AID to function specifically on its natural substrate during SHM.

    DOI: 10.1073/pnas.1120368109

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  • IL-21-Dependent B Cell Death Driven by Prostaglandin E-2, a Product Secreted from Follicular Dendritic Cells 査読

    Masaki Magari, Yumiko Nishikawa, Yasumasa Fujii, Yumi Nishio, Koji Watanabe, Michiya Fujiwara, Naoki Kanayama, Hitoshi Ohmori

    JOURNAL OF IMMUNOLOGY   187 ( 8 )   4210 - 4218   2011年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER ASSOC IMMUNOLOGISTS  

    In germinal centers (GCs), B cells are selected through interaction with follicular dendritic cells bearing immune complexes and follicular helper T (Tfh) cells secreting Tfh cytokines, including IL-21. To analyze these cellular interactions, we have explored culture conditions that can simulate GC B cell selection in vitro using a mouse follicular dendritic cell line, FL-YB. FL-YB cells efficiently enhanced viability of cocultured mouse B cells in a BAFF-dependent fashion. Interestingly, we found that addition of IL-21, a major Tfh cytokine, readily induced death of B cells that were cocultured with FL-YB cells, whereas IL-21 alone sustained viability of B cells in the absence of FL-YB cells. The IL-21-induced death was dependent on a low m.w. soluble factor that was released from FL-YB cells, which was finally identified as PGE(2). Treatment of B cells with IL-21 plus PGE(2), but not either alone, resulted in enhanced expression of a proapoptotic protein Bim and the upstream transcription factor Foxo1. A PGE(2) receptor isoform, EP4, was responsible for IL-21/PGE(2)-induced B cell death. Thus, PGE(2) is an endogenous chemical mediator that can switch pleiotropic actions of IL-21 on B cells. IL-21/PGE(2)-induced B cell death was rescued if B cells were costimulated via CD40. In immunized mice, deficiency of IL-21R in B cells led to a significant decrease in the frequency of activated caspase-3-positive GC B cells concomitant with impaired affinity maturation of Abs. Taken together, results implicate a physiological role of IL-21/PGE(2)-induced B cell death in GC B cell selection. The Journal of Immunology, 2011, 187: 4210-4218.

    DOI: 10.4049/jimmunol.1100934

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  • Efficient affinity maturation of antibodies in an engineered chicken B cell line DT40-SW by increasing point mutation 査読

    Masamichi Kajita, Takahiro Okazawa, Mika Ikeda, Kagefumi Todo, Masaki Magari, Naoki Kanayama, Hitoshi Ohmori

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   110 ( 3 )   351 - 358   2010年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SOC BIOSCIENCE BIOENGINEERING JAPAN  

    The chicken B cell line DT40 undergoes hypermutation of immunoglobulin variable region (IgV) genes during culture, thereby constituting an antibody (Ab) library. We previously established an in vitro Ab generation system using an engineered line DT40-SW whose hypermutation machinery can be switched on and off. Abs for various antigens (Ags) can be obtained from the DT40-SW library and the specificity of the Ag-specific clones can be stabilized by stopping hypermutation. Furthermore, the affinity of obtained monoclonal Abs (mAbs) can be improved through further mutation followed by selection, a process analogous to "affinity maturation" that occurs in vivo. Although gene conversion dominantly diversifies the IgV genes in DT40 cells, point mutation is considered to be more favorable for fine-tuning Ab properties during affinity maturation. Here, we examined whether affinity maturation occurs more efficiently when the hypermutation pattern was transformed from gene conversion into point mutation in DT40-SW cells. To this end, we disrupted the XRCC3 gene that is essential for gene conversion. It was found that hemizygous disruption of the XRCC3 gene was sufficient to increase the point mutation frequency. Since hemizygous disruption is conducted more easily, we tested whether the XRCC3 (+/-) mutant generates high-affinity Abs through affinity maturation more efficiently than the wild type. Using this affinity maturation technique, we generated an improved 4-hydroxy-3-nitrophenylacetyl-specific mAb with similar to 600-fold lower K(D)) than that of the original mAb. Taken together, hemizygous disruption of the XRCC3 gene is considered to be useful for obtaining high-affinity mAbs from DT40-SW cells though affinity maturation. (C) 2010, The Society for Biotechnology, Japan. All rights reserved.

    DOI: 10.1016/j.jbiosc.2010.03.006

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  • Enhancement of hypermutation frequency in the chicken B cell line DT40 for efficient diversification of the antibody repertoire 査読

    Masaki Magari, Yuichi Kanehiro, Kagefumi Todo, Mika Ikeda, Naoki Kanayama, Hitoshi Ohmori

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   396 ( 2 )   353 - 358   2010年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS INC ELSEVIER SCIENCE  

    Chicken B cell line DT40 continuously accumulates mutations in the immunoglobulin variable region (IgV) gene by gene conversion and point mutation, both of which are mediated by activation-induced cytidine deaminase (AID), thereby producing an antibody (Ab) library that is useful for screening monoclonal Abs (mAbs) in vitro. We previously generated an engineered DT40 line named DT40-SW, whose AID expression can be reversibly switched on or off, and developed an in vitro Ab generation system using DT40-SW cells. To efficiently create an Ab library with sufficient diversity, higher hypermutation frequency is advantageous. To this end, we generated a novel cell line DT40-SW Delta C, which conditionally expresses a C-terminus-truncated AID mutant lacking the nuclear export signal. The transcription level of the mutant AID gene in DT40-SW Delta C cells was similar to that of the wild-type gene in DT40-SW cells. However, the protein level of the truncated AID mutant was less than that of the wild type. The mutant protein was enriched in the nuclei of DT40-SW Delta C cells, although the protein might be highly susceptible to degradation. In DT40-SW Delta C cells, both gene conversion and point mutation occurred in the IgV gene with over threefold higher frequency than in DT40-SW cells, suggesting that a lower level of the mutant AID protein was sufficient to increase mutation frequency. Thus, DT40-SW Delta C cells may be useful for constructing AID libraries for efficient screening of mAbs in vitro. (C) 2010 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.bbrc.2010.04.096

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  • Conditional transformation of immunoglobulin mutation pattern from gene conversion into point mutation by controlling XRCC3 expression in the DT40 B cell line 査読

    Masamichi Kajita, Masaki Magari, Kagefumi Todo, Naoki Kanayama, Hitoshi Ohmori

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   109 ( 4 )   407 - 410   2010年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SOC BIOSCIENCE BIOENGINEERING JAPAN  

    A hypermutating B cell line DT40 is useful for screening antibodies and improving affinity of the selected antibodies in vitro. To perform affinity maturation efficiently, we generated an engineered DT40 line whose immunoglobulin mutation pattern can be transformed from gene conversion into point mutation by conditional suppression of XRCC3 expression. (C) 2009, The Society for Biotechnology, japan. All rights reserved.

    DOI: 10.1016/j.jbiosc.2009.09.050

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  • Enhancement of antibody production from a chicken B cell line DT40 by reducing Pax5 expression 査読

    Masaki Magari, Takahiro Aya, Mika Ikeda, Kagefumi Todo, Naoki Kanayama, Hitoshi Ohmori

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   107 ( 2 )   206 - 209   2009年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SOC BIOSCIENCE BIOENGINEERING JAPAN  

    We developed a novel in vitro antibody (Ab) generation system using a hypermutating chicken B cell line (DT40-SW). We suppressed the expression of the Pax5 transcription factor by targeted disruption of the gene to increase Ab production in isolated clones and produce the desired Abs. This single genetic manipulation resulted in a significant enhancement of Ab production without significantly affecting maximum cell density. (C) 2008, The Society for Biotechnology, Japan. All rights reserved.

    DOI: 10.1016/j.jbiosc.2008.09.014

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  • Creation of Valuable Antibodies by an In Vitro Antibody Generation System Using a Hypermutating B Cell Line 査読

    Naoki Kanayama, Kagefumi Todo, Masaki Magari, Hitoshi Ohmori

    YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN   129 ( 1 )   11 - 17   2009年1月

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    記述言語:日本語   出版者・発行元:PHARMACEUTICAL SOC JAPAN  

    Monoclonal antibodies (mAb) have recently proven to be excellent biopharmaceutical agents. The generation of hybridomas from antigen-stimulated B cells has been a key technology for obtaining mAbs; however, it is a laborious and time-consuming process, and sometimes mAbs for molecules conserved between species are difficult to obtain because of immunological tolerance. Thus, it is of great importance to develop in vitro technologies for generating useful Abs as drug candidates. We have been attempting to develop a novel in vitro antibody generation system using a chicken B cell line DT40, which displays Abs and mutates Ig genes during Culture, thereby generating a useful Ab library for screening mAbs. First, we generated an engineered cell line DT40-SW whose mutation machinery can be reversibly switched on and off. The Ab generation system using DT40-SW is useful in the following ways: (1) mAbs for various model antigens including antoantigens can be obtained from the DT40-SW Ab library that is free from immunological tolerance; (2) the switching device of the mutation machinery enables fixing desirable Ig mutants by stopping mutation; (3) by repeated culture and sorting of clones bearing higher affinity for target antigens, affinity maturation can be mimicked in vitro. We have also genetically improved DT40-SW cells for mutation efficiency and Ab production. The Ab generation system will be applicable for obtaining valuable Abs such as antitumor Abs.

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  • 高親和性抗体の産生機構に関する研究とその工学的応用:平成20年度生物工学奨励賞(斎藤賞) 招待 査読

    金山直樹

    生物工学会誌   87 ( 3 )   116 - 122   2009年

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    記述言語:日本語   掲載種別:研究論文(学術雑誌)  

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  • Analysis of B cell selection in the germinal center reaction during a T-dependent antibody response at a single cell level 査読

    Takahiro Okazawa, Masaki Magari, Takafumi Kimoto, Emi Kouyama, Hitoshi Ohmori, Naoki Kanayama

    IMMUNOLOGY LETTERS   117 ( 1 )   96 - 105   2008年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE BV  

    The quasimonoclonal mouse is useful to examine B cell selection during T-dependent antibody (Ab) responses because of its limited B cell populations mainly expressing the knockin 17.2.25 V-H-encoded H chain (VHT) paired with the lambda 1 or lambda 2 L chain. It has been reported that both two VHT/lambda 1 and VHT/lambda 2 B cell populations responded to a T-dependent antigen conjugated with a hapten p-nitrophenylacetyl (pNP), but only VHT/lambda 2 B cells differentiated to secrete high affinity anti-pNP IgG Abs by acquiring a critical mutation (T313A) in the VHT. The VHT/lambda 2 B cells may be more potent in migrating to the germinal centers (GCs) due to about 50-fold higher affinity for pNP than VHT/lambda 1 B cells. Here, to uncover how VHT/lambda 2 B cells were preferentially recruited for affinity maturation during the anti-pNP Ab response, we examined the L chain usage and mutation frequency of VHT+ GC B cells at a single cell level. VHT/lambda 2 B cells bearing the unmutated VHT gene were found in the GCs more frequently than VHT/lambda 1 and mutated VHT/lambda 2 counterparts in an early phase of the Ab response. In the course of the GC reaction, the number of VHT/lambda 2 B cells that mutated their VHT genes preferentially expanded, and finally VHT/lambda 2 B cells bearing the T313A mutation occupied VHT+ GC B cell population. Thus, it is suggested that B cells with a higher affinity were selected not only for entry to the GCs but also in the affinity maturation process during a T-dependent Ab response. (C) 2008 Elsevier B.V. All rights reserved.

    DOI: 10.1016/j.imlet.2008.01.002

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  • Analysis of antigen-stimulated B cell migration into germinal centers during the early stage of a T-dependent immune response 査読

    Emi Kouyama, Yumiko Nishikawa, Takahiro Okazawa, Masaki Magari, Hitoshi Ohmori, Naoki Kanayama

    IMMUNOLOGY LETTERS   109 ( 1 )   28 - 35   2007年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE BV  

    The quasimonoclonal (QM) mouse provides a model to analyze B cell selection because major B cell antigen receptors (BCR) are composed of the knockin V(H)DJ(H) 17.2.25 (VHT) encoded H chain and the lambda 1 or lambda 2 L chain, thereby being specific for (4-hydoxy-3-nitrophenyl)acetyl (NP). We have reported that during a T-dependent antibody (Ab) response for a low-affinity NP analog p-nitrophenylacetyl (pNP), although VHT/lambda 1 and VHT/lambda 2 IgM were equally produced, VHT/lambda 2 IgG almost exclusively underwent affinity maturation toward pNP. The initial affinity of VHT/lambda 2 B cells for pNP was approximately 50-100-fold higher than that of VHT/lambda 1 B cells, suggesting a role of BCR affinity in recruiting B cells to affinity maturation processes. Here, we investigated whether the intensity of BCR signals could contribute to the selection of VHT/lambda 2 B cells for affinity maturation. VHT/lambda 2 B cells were more responsive to pNP than VHT/lambda 1 B cells in vitro. When CFSE-labeled QM B cells were transferred into the wild type mice where T cells had been primed with chicken gamma-globulin (CGG), QM B cells challenged by pNP-conjugated CGG could be observed to get activated and migrate to GCs in the early phase of the T-dependent response to pNP-CGG. Adoptive transfer of sorted populations revealed that the VHT/lambda 2 B cell population was more potent in migration into GCs than the VHT/lambda 1 counterpart. Thus, it is suggested that the higher BCR affinity of VHT/lambda 2 B cells may be an initial cue for their recruitment to GCs during a T-dependent Ab response. (c) 2007 Elsevier B.V. All rights reserved.

    DOI: 10.1016/j.imlet.2006.12.011

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  • Novel in vitro screening system for monoclonal antibodies using hypermutating chicken B cell library 査読

    Kagefumi Todo, Kenji Miyake, Masaki Magari, Naoki Kanayama, Hitoshi Ohmori

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   102 ( 5 )   478 - 481   2006年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SOC BIOSCIENCE BIOENGINEERING JAPAN  

    Here, we report an in vitro screening system for monoclonal antibodies using a hypermutating chicken B cell line, DT40-SW. When switching on hypermutation, cultured DT40-SW cells constituted an antibody library, from which clones secreting antibodies to a test antigen were successfully isolated, and genetically stabilized by switching off the mutation machinery.

    DOI: 10.1263/jbb.102.478

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  • B細胞免疫反応と免疫記憶 超変異ニワトリB細胞株を用いたin vitro抗原生産システム(In vitro antibody generation system using a hypermutating chicken B cell line)

    Kanayama Naoki, Todo Kagefumi, Okazawa Takahiro, Ikeda Mika, Fujita Risako, Magari Masaki, Ohmori Hitoshi

    日本免疫学会総会・学術集会記録   36   55 - 55   2006年11月

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    記述言語:英語   出版者・発行元:(NPO)日本免疫学会  

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  • Establishment of lymphotoxin beta receptor signaling-dependent cell lines with follicular dendritic cell phenotypes from mouse lymph nodes 査読

    Yumiko Nishikawa, Masaki Hikida, Masaki Magari, Naoki Kanayama, Masaharu Mori, Hiroshi Kitamura, Tomohiro Kurosaki, Hitoshi Ohmori

    JOURNAL OF IMMUNOLOGY   177 ( 8 )   5204 - 5214   2006年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER ASSOC IMMUNOLOGISTS  

    Follicular dendritic cells (FDCs) have been shown to play a crucial role in the positive selection of high-affinity B cells that are generated by somatic hypermutation in germinal center (GC). Because of technical difficulties in preparing and maintaining pure FDCs, a role for FDCs in this complicated process has not been fully elucidated. In this study, we established a cell line designated as pFL that retained major FDC phenotypes from a three-dimensional culture of mouse lymph node cells. pFL cells proliferated slowly in response to an agonistic anti-lymphotoxin beta receptor mAb and TNF-alpha. A more rapidly growing clone, named FL-Y, with similar requirements for growth was isolated from a long-term culture of pFL. Analysis of surface markers in these two cell lines by immunostaining, flow cytometry, and DNA microarray revealed the expression of genes, including those of CD21, Fc gamma RIIB, lymphotoxin beta receptor, ICAM-1, VCAM-1, IL-6, and C4, which have been shown to be characteristic of FDCs. In addition, B cell-activating factor was expressed in these two cell lines. At the pFL or FL-Y:B cell ratio of 1:100, the cell lines markedly sustained B cell survival and Ab production during 2 wk of culture, while most B cells collapsed within 1 wk in the absence of the FDC-like cells. Interestingly, expression of typical GC markers, Fas and GL-7, was notably augmented in B cells that were cocultured with Th cells on these two cell lines. Thus, pFL and FL-Y cells may be useful for providing insight into the functional role for FDCs in GC.

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  • Suppressive effects of mometasone furoate on an antigen-specific IgE antibody response and production of IL-4 in mice 査読

    Masaki Magari, Mika Ikeda, Miki Asakura, Naoki Kanayama, Masami Ogawa, Hitoshi Ohmori

    IMMUNOPHARMACOLOGY AND IMMUNOTOXICOLOGY   28 ( 3 )   491 - 500   2006年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:TAYLOR & FRANCIS INC  

    A nasal formulation of mometasone furoate (MF) is advantageous in avoiding systemic activity characteristic of glucocorticoids when it is applied topically. To confirm anti-allergic effects of this glucocorticoid formulation elaborately, we investigated whether the drug can suppress the production of IgE antibodies and related cytokines. It we showed that IgE production induced in mice immunized via intranasal route was significantly reduced when the mice were administered MF intranasally. Further, MF was effective in inhibiting production of type-2 helper T cell cytokines in vivo and in vitro. These results provide a immunopharmacological basis for clinical efficacy of this drug.

    DOI: 10.1080/08923970600928155

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  • Genetic manipulation of an exogenous non-immunoglobulin protein by gene conversion machinery in a chicken B cell line 査読

    N Kanayama, K Todo, S Takahashi, M Magari, H Ohmori

    NUCLEIC ACIDS RESEARCH   34 ( 2 )   2006年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:OXFORD UNIV PRESS  

    During culture, a chicken B cell line DT40 spontaneously mutates immunoglobulin (Ig) genes by gene conversion, which involves activation-induced cytidine deaminase (AID)-dependent homologous recombination of the variable (V) region gene with upstream pseudo-V genes. To explore whether this mutation mechanism can target exogenous non-Ig genes, we generated DT40 lines that bears a gene conversion substrate comprising the green fluorescent protein (GFP) gene as a donor and the blue fluorescent protein (BFP) gene as an acceptor. A few percent of the initially BFP-expressing cells converted their fluorescence from blue to green after culture for 2-3 weeks when the substrate construct was integrated in the Ig light chain locus, but not in the ovalbumin locus. This was the result of AID-dependent and the GFP gene-templated gene conversion of the BFP gene, thereby leading to the introduction of various sizes of GFP-derived gene segment into the BFP gene. Thus, G/B construct may be used to visualize gene conversion events. After switching off AID expression in DT40 cells, the mutant clones were isolated stably and maintained with their mutations being fixed. Thus, the gene conversion machinery in DT40 cells will be a useful means to engineer non-Ig proteins by a type of DNA shuffling.

    DOI: 10.1093/nar/gnj013

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  • Immunogenicity of an inflammation-associated product, tyrosine nitrated self-proteins 査読

    H Ohmori, N Kanayama

    AUTOIMMUNITY REVIEWS   4 ( 4 )   224 - 229   2005年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE BV  

    To understand the mechanism leading to autoantibody production; it is of importance to reveal how self-components that are otherwise inactive as antigens acquire immunogenicity. One possible mechanism is the generation of structurally modified self-proteins in apoptotic or inflamed tissues. The post-translational modification of proteins might give rise to the generation. of new epitopes to which T and B lymphocytes are not rendered tolerant. Among the protein modifications; this review is focussed on the generation and the immunogenicity of self-proteins carrying 3-nitrotyrosine (NT), an inflammation-associated marker. NT proteins are generated in vivo by nitration with peroxynitrite, which is formed from nitric oxide and superoxide that are released from activated inflammatory cells. Interestingly, many anti-DNA Abs from autoimmune mice have been shown cross-reactive with NT. Analysis of the immunogenicity of NT-carrying self-proteins has revealed that they elicit both Immoral and cellular immune responses in mice. Thus, NT containing epitopes created on self-proteins may serve as a trigger to impair or bypass immunological tolerance. (c) 2004 Elsevier B.V. All rights reserved.

    DOI: 10.1016/j.autrev.2004.11.011

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  • Reversible switching of immunoglobulin hypermutation machinery in a chicken B cell line 査読

    N Kanayama, K Todo, M Reth, H Ohmori

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   327 ( 1 )   70 - 75   2005年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS INC ELSEVIER SCIENCE  

    A chicken B lymphoma line, DT40, hypermutates immunoglobulin (Ig) genes spontaneously during culture. Thus, cultured DT 40 cells constitute a useful Ig library for screening antibodies(Abs) in vitro. To fix desirable Ig mutants by stopping hypermutation or to resume mutation for further improvement of Ab affinity, activation-induced cytidine deaminase (AID), a key enzyme responsible for the Ig mutation machinery, must be switched on or off. To this end, we generated a DT40 line whose one AID allele was disrupted, and the other allele was replaced by the loxP-flanked AID construct. In this engineered cell line designated as DT40-SW, AID expression could be switched reversibly by tamoxifen-regulated Cre recombinase. Devices were also introduced to discriminate between the "AID-ON" and the "AID-OFF" cells by GFP expression and puromycin resistance, respectively. Starting from a single DT40-SW cell, Ig gene repertoire was efficiently diversified during culture only when AID expression was on. (C) 2004 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.bbrc.2004.11.143

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  • Analysis of marginal zone B cell development in the mouse with limited B cell diversity: Role of the antigen receptor signals in the recruitment of B cells to the marginal zone 査読

    N Kanayama, M Cascalho, H Ohmori

    JOURNAL OF IMMUNOLOGY   174 ( 3 )   1438 - 1445   2005年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER ASSOC IMMUNOLOGISTS  

    The quasimonoclonal (QM) mouse provides an intelligible model to analyze the B cell selection as the competition between two major 4-hydroxy-3-nitrophenylacetyl-specific B cell populations whose BCR are comprised of the knockin V(H)17.2.25 (VHT)encoded H chain and the lambda1 or lambda2 L chain. In this study, we show the QM system is useful to examine how BCR signals guide a subset of B cells to the marginal zone (MZ). Compared with the control C57BL/6 mice, the QM mice had similar to2.7-fold increased number of B cells exhibiting the MZ B cell phenotype and a larger MZ area in the spleen. Interestingly, VHT/lambda2 B cells significantly predominated over VHT/lambda1 B cells in MZ-(VHT/lambda1:VHT/lambda2 approximate to 3:7) and transitional 2-B cell subsets, while these two populations were comparable in immature, transitional 1, and mature counterparts. Thus, the biased use of lambda2 in the MZ B cells may be the result of selection in the periphery. The enlargement of MZ B cell compartment and the preferred recruitment of the VHT/(lambda)2 B cells were further augmented by doubling the VHT gene, but dampened by the dysfunction of Bruton's tyrosine kinase, suggesting a positive role of BCR signaling in this selection. Comparison of Ag specificity between VHT/lambda1 and VHT/lambda2 IgM mAbs revealed a polyreactive nature of the VHT/lambda2 BCR, including the reactivity with ssDNA. Taken together, it is suggested that polyreactivity (including self-reactivity) of BCR is crucial in driving B cells to differentiate into the MZ phenotype.

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  • Immunogenicity of autologous IgG bearing the inflammation-associated marker 3-nitrotyrosine 査読

    H Ohmori, M Oka, Y Nishikawa, H Shigemitsu, M Takeuchi, M Magari, N Kanayama

    IMMUNOLOGY LETTERS   96 ( 1 )   47 - 54   2005年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE BV  

    To explore the link between inflammation and autoimmunity, we analyzed the immunogenicity of 3-nitrotyrosine(NT)-bearing self-proteins, an inflammation-associated marker that is formed by nitration of protein tyrosine residues with peroxynitrite generated during inflammation. An interesting feature of NT is its structural similarity to a synthetic hapten, 4-hydroxy-3-nitrophenylacetyl (NP), with which some anti-DNA antibodies (Abs) have been reported to show cross-reactivity. We confirmed that some of anti-DNA monoclonal Abs (mAbs) obtained from MRL/lpr mice also bound NT as well as NP. Based on these findings, we examined whether NIT-bearing autologous IgG (NT-1gG) as a model of NT-self proteins is immunogenic to induce a DNA-cross-reactive anti-NT Ab response in autologous normal mice. Anti-NT IgM and IgG Ab responses were elicited after the third immunization with NT-IgG. concomitant with an increase in anti-single stranded (ss)DNA titer. Interestingly, a part of anti-NT mAbs thus induced showed cross-reactivity with ssDNA. some of which used VH sequences that were highly homologous to those reported in anti-DNA Abs from NZB/WF1 mice. Splenic T cells primed with NT-IgG. but not with unmodified IgG. showed a proliferative response to the inducing antigen. Collectively, NT-IgG is immunogenic in autologous hosts. and can induce anti-NT Abs that are cross-reactive with ssDNA. (C) 2004 Elsevier B.V. All rights reserved.

    DOI: 10.1016/j.imlet.2004.07.004

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  • Generation of IgM and IgG1 monoclonal antibodies with identical variable regions: comparison of avidity

    Naoki Kanayama, Kimi Yamakoshi, Masaaki Kiyomi, Masaki Magari, Hitoshi Ohmori

    Memoirs of the Faculty of Engineering Okayama University   38   91 - 96   2004年

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  • Up-regulation of the peroxisomal beta-oxidation system occurs in butyrate-grown Candida tropicalis following disruption of the gene encoding peroxisomal 3-ketoacyl-CoA thiolase 査読

    M Ueda, N Kanayama, N Kamasawa, M Osumi, A Tanaka

    BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR AND CELL BIOLOGY OF LIPIDS   1631 ( 2 )   160 - 168   2003年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE BV  

    In the yeast Candida tropicalis, two thiolase isozymes, peroxisomal acetoacetyl-CoA thiolase and peroxisomal 3-ketoacyl-CoA thiolase, participate in the peroxisomal fatty acid p-oxidation system. Their individual contributions have been demonstrated in cells grown on butyrate, with C. tropicalis able to grow in the absence of either one. In the present study, a lack of peroxisomal 3-ketoacyl-CoA thiolase protein resulted in increased expression (up-regulation) of acetoacetyl-CoA thiolase and other peroxisomal proteins, whereas a lack of peroxisomal acetoacetyl-CoA thiolase produced no corresponding effect. Overexpression of the acetoacetyl-CoA thiolase gene did not suppress the up-regulation or the growth retardation on butyrate in cells without peroxisomial 3-ketoacyl-CoA thiolase, even though large amounts of the overexpressed acetoacetyl-CoA thiolase were detected in most of the peroxisomes of butyrate-grown cells. These results provide important evidence of the greater contribution of 3-ketoacyl-CoA thiolase to the peroxisomal beta-oxidation system than acetoacetyl-CoA thiolase in C tropicalis and a novel insight into the regulation of the peroxisomal beta-oxidation system. (C) 2003 Elsevier Science B.V. All rights reserved.

    DOI: 10.1016/S1388-1981(03)00002-7

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  • Mechanisms leading to autoantibody production: Link between inflamation and antoimmunity

    Hitoshi Ohmori, Naoki Kanayama

    Current Drug Targets-Inflamation & Allergy   2   232 - 241   2003年

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  • B cell selection and affinity maturation during an antibody response in the mouse with limited B cell diversity 査読

    N Kanayama, T Kimoto, K Todo, Y Nishikawa, M Hikida, M Magari, M Cascalho, H Ohmori

    JOURNAL OF IMMUNOLOGY   169 ( 12 )   6865 - 6874   2002年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER ASSOC IMMUNOLOGISTS  

    The quasi-monoclonal mouse has limited B cell diversity, whose major (similar to80%) B cell Ag receptors are comprised of the knockin V-H 17.2.25 (VHT)-encoded H chain and the lambda1 or lambda2 L chain, thereby being specific for 4-hydroxy-3-nitrophenylacetyl. The p-nitrophenylacetyl (pNP) was found to be a low affinity analog of nitrophenylacetyl. We examined affinity maturation of anti-pNP IgG by analyzing mAbs obtained from quasi-monoclonal mice that were immunized with this low affinity Ag. The results are: 1) Although VHT/lambda1 and VHT/lambda2 IgM were equally produced, VHT/lambda2 IgG almost exclusively underwent affinity maturation toward pNP. 2) A common mutation in complementarity-determining region 3 Of VHT (T313A) mainly contributed to generating the specificity for pNP. 3) Because mutated VHT-encoded gamma-chains could form lambda1-bearing IgG in Chinese hamster ovary cells, apparent absence of VHT/lambda1 anti-pNP IgG may not be due to the incompatibility between the gamma-chains and the lambda1-chain, but may be explained by the fact that VHT/lambda1 B cells showed 50- to 100-fold lower affinity for pNP than VHT/lambda2 B cells. 4) Interestingly, a pNP-specific IgM mAb that shared common mutations including T313A with high affinity anti-pNP IgG was isolated, suggesting that a part of hypermutation coupled with positive selection can occur before isotype switching. Thus, even weak B cell receptor engagement can elicit an IgM response, whereas only B cells that received signals stronger than a threshold may be committed to an,affinity maturation process.

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  • Role for complement receptors (CD21/CD35) in the regulation of recombination activating gene expression in murine peripheral B cells 査読

    H Ohmori, M Magari, Y Nakayama, N Kanayama, M Hikida

    IMMUNOLOGY LETTERS   83 ( 2 )   95 - 99   2002年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE BV  

    A population of peripheral B cells have been shown to express recombination activating gene products, RAG-1 and RAG-2, which are considered to be involved in revising the B cell antigen receptor (BCR) in the periphery. BCR engagement has been reported to turn off RAG expression in peripheral B cells, whereas the same treatment has an opposite effect on immature B cells in the bone marrow. In contrast to receptor editing that is involved in the removal of autoreactivity in immature B cells, it has been shown that secondary V(D)J rearrangement in peripheral B cells, termed receptor revision, contributes to affinity maturation of antibodies. Here, we show that RAG-2 expression in murine splenic B cells was abrogated by the coligation of BCR with complement receptors (CD21/CD35) much more efficiently than by the engagement of BCR alone. On the other hand, the same coligation augmented proliferation of anti-CD40-stimulated B cells. These findings suggest a crucial role for CD21/CD35 in directing the conservation or the revision of BCRs in peripheral B cells. (C) 2002 Elsevier Science B.V. All rights reserved.

    DOI: 10.1016/S0165-2478(02)00083-4

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  • Contribution of light chain rearrangement in peripheral B cells to the generation of high-affinity antibodies 査読

    M Magari, T Sawatari, Y Kawano, M Cascalho, M Wabl, N Kanayama, M Hikida, H Ohmori

    EUROPEAN JOURNAL OF IMMUNOLOGY   32 ( 4 )   957 - 966   2002年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-V C H VERLAG GMBH  

    Recently, peripheral B cells have been shown to undergo secondary V(D)J rearrangement of immunoglobulin genes, but the physiological role of this event has not been fully elucidated. To investigate whether rearrangement of L chain genes in the periphery is involved in the generation of high-affinity antibodies (Ab), we used the 17.2.25 rearranged VHDJH gene (VHT)-knockin mouse whose B cell diversity is limited due to the expression of the site-directed transgene. Immunization of the mouse with p-nitrophenylacetyl (pNP)-conjugated chicken gamma-globulin preferentially led to the production of anti-pNP IgG Ab comprised of non-VHT-encoded H chains and lambda chains. lambda(+) IgG constituted a majority of high-affinity Ab to this hapten. RAG-2 mRNA and the recombination signal sequence break of the lambda1 gene increased in the draining lymph node of immunized mice, but not of nonimmunized animals. There was a close correlation between the levels of these parameters implicating X, gene rearrangement and the production of lambda(+) high-affinity anti-pNP IgG. These observations were reproduced in RAG-1-deficient mice that were reconstituted with the spleen cells of the knockin mouse. Thus, our findings suggest that L chain rearrangement that occurs in the periphery can contribute to affinity maturation of Ab.

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  • Regulatory role for complement receptors (CD21/CD35) in the recombination activating gene expression in mouse peripheral B cells

    Masaki Hikida, Masaki Magari, Yasunori Nakayama, Naoki Kanayama, Hitoshi Ohmori

    Memoirs of the Faculty of Engineering Okayama Univiersity   36   51 - 60   2002年3月

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  • Use of secondarily revised VH genes in IgE antibodies produced in mice infected with the nematode Nippostrongylus brasiliensis 査読

    N Kanayama, C Hukue, M Magari, K Ohtani, M Hikida, M Yamada, S Matsuda, H Ohmori

    IMMUNOLOGY LETTERS   77 ( 3 )   181 - 186   2001年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE BV  

    Although a high level of IgE is produced after primary infection with Nippostrongylus brasiliensis (Nb), most of the IgE antibodies (Abs) are not specific to the worm. Analyses with Western blotting and enzyme-linked immunosorbent assay (ELISA) revealed that the IgE Abs from Nb-infected BALB/c mice did not show reactivity with Nb-derived excretory-secretory proteins (NES) and antigens present in the cell-free extracts of the worm. Monoclonal IEE Abs obtained from the Nb-infected mice were not reactive with these Nb antigen either. To characterize Nb-induced IgE response, we used (QM x C57BL/6)F1 (QBF1) mice that bear the knock-in 17.2.25 VHDJH segment (VHT) encoding a VH region specific to 4-hydroxy-3-nitrophenylacetyl hapten, and express VHT-encoded antigen receptors on 80-85% of their B cells. Consistent with the frequency of VHT-positive B cells, more than 80% of IgE Abs induced in QBF1 B cells that were cultured with LPS plus IL-4 were found to bear VHT-encoded H chains. In contrast, when QBF1 mice were infected with Nb, less than 10% of Nb-induced IgE Abs were found to use VHT. The QBF1-derived IgE did not react with Nb antigens either. Taken together, data suggest that Nb-induced IgE response in mice is not merely the result of polyclonal activation of B cells, but may involve a mechanism that revises Ig genes secondarily. (C) 2001 Elsevier Science B.V. All rights reserved.

    DOI: 10.1016/S0165-2478(01)00216-4

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  • Restoration of immunocyte functions by thymosin alpha 1 in cyclophosphamide induced immunodeficient mice 査読

    H Ohmori, M Kamo, K Yamakoshi, MHM Nitta, M Hikida, N Kanayama

    IMMUNOPHARMACOLOGY AND IMMUNOTOXICOLOGY   23 ( 1 )   75 - 82   2001年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:MARCEL DEKKER INC  

    Thymosin alpha1 (T alpha1) is an oligopeptide hormone originally isolated from the thymus gland, and has been reported to have stimulating effects on the differentiation of T cells and NK cells. These immunostimulating properties have been considered to be useful for improving immune disorders associated with various diseases including cancer, AIDS and hepatitis. Here, we characterized immunostimulating properties of Tal in experimental immunodeficiency of mice that was induced by the administration of cyclophosphamide (CY). Repeated injection of 30-300 mug/kg/day of Tal after CY-treatment significantly accelerated the restoration of the reduced number of CD4(+)CD8(+) T cells in the thymus. T alpha1 administration was effective in restoring the suppressed activities of helper T cells and cytotoxic T cells in CY-treated mice. Tal also had stimulating effects on reduced activity of lymphokine-activated killer cells in CY-treated mice. These results indicate that T alpha1 is stimulatory for both humoral and cellular immune responses, thus providing the immunological basis for the clinical benefit of this compound.

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  • Prevention of collagen-induced arthritis in DBA/1 mice by oral administration of AZ-9, a bacterial polysaccharide from Klebsiella oxytoca 査読

    R Sugihara, M Yoshimura, M Mori, N Kanayama, M Hikida, H Ohmori

    IMMUNOPHARMACOLOGY   49 ( 3 )   325 - 333   2000年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE BV  

    Collagen-induced arthritis (CIA) is an excellent model of rheumatoid arthritis (RA) in humans that is induced in DBA/1 mice immunized with bovine type II collagen (CII). Here, we report that the induction of CIA was effectively suppressed by oral administration of AZ-9, a purified polysaccharide with the average molecular weight of approximately 200 kDa that was produced by a soil bacterium, Klebsiella oxytoca. When AZ-9 was administered at 125-250 mg/kg/day orally for 9 consecutive days after immunization with CII followed by its administration every 3 days, resulted in a marked reduction of the incidence and the severity of CIA. The serum level of anti-CII IgG2a and the production of IFN-gamma and IL-12 in the draining lymph node (LN) cells were significantly lower in AZ-9-administered mice than the untreated control. These findings suggest that orally administered AZ-9 suppressed CIA through attenuating a Th1-type response to CII. AZ-9 could be fragmented into smaller molecules (3-4 kDa) without losing its suppressive activity. (C) 2000 Elsevier Science B.V. All rights reserved.

    DOI: 10.1016/S0162-3109(00)00247-2

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  • An n-alkane-responsive promoter element found in the gene encoding the peroxisomal protein of Candida tropicalis does not contain a C-6 zinc cluster DNA-binding motif 査読

    T Kanai, A Hara, N Kanayama, M Ueda, A Tanaka

    JOURNAL OF BACTERIOLOGY   182 ( 9 )   2492 - 2497   2000年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC MICROBIOLOGY  

    When an asporogenic diploid yeast, Candida tropicalis, is cultivated on n-alkane, the expression of the genes encoding enzymes of the peroxisomal beta-oxidation pathway is highly induced. An upstream activation sequence (UAS) which can induce transcription in response to n-alkane (UAS(ALK)) was identified on the promoter region of the peroxisomal 3-ketoacyl coenzyme A (CoA) thiolase gene of C. tropicalis (CT-T3A). The 29-bp region (from -289 to -261) present upstream of the TATA sequence was sufficient to induce n-alkane-dependent expression of a reporter gene. Besides n-alkane, UAS(ALK)-dependent gene expression also occurred in the cells grown on oleic acid. Several kinds of mutant UAS(ALK) were constructed and tested for their UAS activity, It was clarified that the important nucleotides for UAS(ALK) activity were located within 10-bp region from -273 to -261 (5'-TCCTGCACAC-3'). This region did not contain a CGG triplet and therefore differed from the sequence of the oleate-response element (ORE), which is a UAS found on the promoter region of 3-ketoacyl-CoA thiolase gene of Saccharomyces cerevisiae. Similar sequences to UAS(ALK) were also found on several peroxisomal enzyme encoding genes of C. tropicalis.

    DOI: 10.1128/JB.182.9.2492-2497.2000

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  • Selective augmenting effects of nitric oxide on antigen-specific IgE response in mice 査読

    H Ohmori, H Egusa, N Ueura, Y Matsumoto, N Kanayama, M Hikida

    IMMUNOPHARMACOLOGY   46 ( 1 )   55 - 63   2000年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ELSEVIER SCIENCE BV  

    Here, we report the enhancing effects of nitric oxide (NO) on an IgE antibody response in mice. Anti-trinitrophenyl (TNP) IgE production induced in vitro in TNP keyhole Limpet hemocyanin (KLH)-primed spleen cells was inhibited by approximately 70% when an NO synthase (NOS) inhibitor, L-N-G-monomethyl-L-arginine, was added at 10(-7)-10(-6) M to the lymphocyte culture. On the other hand, addition of NO-generating agents to the culture resulted in a marked enhancement of the IgE production. In contrast, anti-TNP IgM and IgG1 responses were affected only marginally when the IEE production was either suppressed or augmented by these agents. NO did not directly augment IgE class switching in normal B cells stimulated with lipopolysaccharide and interleukin (IL)-4. NO-mediated augmentation of the IgE response is considered to be of a physiological significance because administration of aminoguanidine (AG), an inhibitor of inducible NOS, to immunized mice resulted in a preferential suppression of anti-TNP IgE production in vivo. This may be explained by the observation that AG-administration increased interferon-gamma expression without changing that of IL-4 in the immunized mice. Taken together, these observations suggest a pathophysiological role of NO in the development of IEE-mediated allergic diseases. (C) 2000 Elsevier Science B.V. All rights reserved.

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  • Genetic Evaluation of Peroxisomal and Cytosolic Acetoacetyl-CoA Thiolase Isozymes in n-Alkane-Assimilating Diploid Yeast, Candida tropicalis 査読

    Mitsuyoshi Ueda, Naoki Kanayama, Atsuo Tanaka

    Cell Biochemistry and Biophysics   32   285 - 290   2000年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Humana Press  

    The n-alkane-assimilating diploid yeast, Candida tropicalis, possesses two acetoacetyl-CoA thiolase (Thiolase I) isozymes encoded by one allele: peroxisomal and cytosolic Thiolase Is encoded by both CT-T1A and CT-T1B. To clarify the function of peroxisomal and cytosolic Thiolase Is, the site-directed mutation leading Thiolase I ΔC6 without a putative C-terminal peroxisomal targeting signal was introduced on CT-T1A locus in the ct-t1bΔ-null mutant. The C-terminus-truncated Thiolase I was active and solely present in the cytosol. Although the ct-t1aΔ/t1bΔ-null mutants showed mevalonate auxotrophy, the mutants having the C-terminus-truncated Thiolase I did not require mevalonate for growth, as did the strains having cytosolic Thiolase I. These results demonstrate that the presence of Thiolase I in the cytoplasm is indispensable for the sterol synthesis in this yeast. It is of greater interest that peroxisomal and cytosolic Thiolase I isozymes, products of the same genes, play different roles in the respective compartments, although further investigations will be necessary to analyze how to be sorted into peroxisomes and the cytosol.

    DOI: 10.1385/CBB:32:1-3:285

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  • Construction of an autonomously replicating plasmid in n-alkane-assimilating yeast, Candida tropicalis 査読

    A Hara, M Ueda, T Matsui, K Furuhashi, N Kanayama, A Tanaka

    JOURNAL OF BIOSCIENCE AND BIOENGINEERING   87 ( 6 )   717 - 720   1999年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SOC BIOSCIENCE BIOENGINEERING JAPAN  

    A transformation system using the autonomously replicating plasmid in the n-alkane-assimilating and asporogenic diploid yeast, Candida tropicalis, was developed. For the cloning of a DNA fragment containing a potential autonomously replicating sequence (ARS) from the genomic DNA of C. tropicalis, the ura3 mutant obtained using ethylmethane sulfonate as the host and the URA3 gene amplified by PCR using the C. tropicalis genomic DNA as a selectable marker were prepared. Comparison of ARSs among yeasts revealed that the consensus sequence found in S. cerevisiae was also present in C. tropicalis. The autonomously replicating plasmid containing the putative ARS as the shuttle vector, capable of replicating in both E. coli and C. tropicalis, was first constructed. The transformation system using this plasmid, in addition to the integrative transformation system, will be applicable to genetic studies of C. tropicalis.

    DOI: 10.1016/S1389-1723(99)80143-1

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  • Genetic evaluation of physiological functions of thiolase isozymes in the n-alkane-assimilating yeast Candida tropicalis 査読

    N Kanayama, M Ueda, H Atomi, A Tanaka

    JOURNAL OF BACTERIOLOGY   180 ( 3 )   690 - 698   1998年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC MICROBIOLOGY  

    The n-alkane-assimilating diploid yeast Candida tropicalis possesses three thiolase isozymes encoded by trio pairs of alleles: cytosolic and peroxisomal acetoacetyl-coenzyme A (CoA) thiolases, encoded by CT-T1A and CT-TIE, and peroxisomal 3-ketoacyl-CoA thiolase, encoded by CT-T3A and CT-T3B. The physiological functions of these thiolases have been examined by gene disruption, The homozygous ct-t1a Delta/t1b Delta null mutation abolished the activity of acetoacctyl-CoA thiolase and resulted in mevalonate auxotrophy. The homozygous ct-t3a Delta/t3b Delta null mutation abolished the activity of 3-ketoacyl-CoA thiolase and resulted in growth deficiency on fz-alkanes (C-10 to C-13), All thiolase activities in this yeast disappeared with the ct-t1a Delta/t1b Delta and ct-t3a Delta/t3b Delta null mutations. To further clarify the function of peroxisomal acetoacetyl-CoA thiolases, the site-directed mutation leading acetoacetyl-CoA thiolase without a putative C-terminal peroxisomal targeting signal was introduced on the CT-TIA locus in the ct-t1b Delta null mutant. The truncated acetoacetyl-CoA thiolase vies solely present in cytoplasm, and the absence of acetoacetyl-CoA thiolase in peroxisomes had no effect on growth on all carbon sources employed. Growth on butyrate was not affected by a lack of peroxisomal acetoacetyl-CoA thiolase, while a retardation of growth hy a lack of peroxisomal 3-ketoacyl-CoA thiolase was observed. A defect of both peroxisomal isozymes completely inhibited growth on butyrate. These results demonstrated that cytosolic acetoacetyl-CoA thiolase was indispensable for the mevalonate pathway and that both peroxisomal acetoacetyl-CoA thiolase and 3-ketoacyl-CoA thiolase could participate in peroxisomal beta-oxidation. In addition to its essential contribution to the beta-oxidation of longer-chain fatty acids, 3-ketoacyl-CoA thiolase contributed greatly el en to the beta-oxidation of a C-4 substrate butyrate.

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  • Expression of acetoacetyl-CoA thiolase isozyme genes of n-alkane-assimilating yeast, Candida tropicalis: Isozymes in two intracellular compartments are derived from the same genes 査読

    N Kanayama, Y Himeda, H Atomi, M Ueda, A Tanaka

    JOURNAL OF BIOCHEMISTRY   122 ( 3 )   616 - 621   1997年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:OXFORD UNIV PRESS  

    In the n-alkane-assimilating yeast Candida tropicalis, there are two isozymes of acetoacetyl-CoA thiolase, peroxisomal acetoacetyl-CoA thiolase (peroxisomal Thiolase I), and cytosolic acetoacetyl-CoA thiolase (cytosolic Thiolase I). We have previously isolated two genes (CT-T1A and CT-T1B) which encode Thiolase I. In order to compare the expressed products of Thiolase I isozyme-encoding genes in C. tropicalis, cytosolic Thiolase I was first purified from glucose-grown C. tropicalis in which the proliferation of peroxisomes and the expression of peroxisomal Thiolase I were repressed. Cytosolic Thiolase I was virtually identical to peroxisomal Thiolase I in molecular mass, kinetic and immunochemical properties, and primary structure at the N-terminus. Amino acid sequence analysis revealed that cytosolic Thiolase I was the mixture of products of two genes (CT-T1A and CT-T1B), as in the case of the peroxisomal enzyme. CT-T1A and CT-T1B were expressed independently in the yeast Saccharomyces cerevisiae and the recombinant proteins were purified. Recombinant Thiolase IA and IB exhibited practically identical enzymatic properties to cytosolic and peroxisomal Thiolase Is from C. tropicalis. These results revealed that cytosolic Thiolase I and peroxisomal Thiolase I were encoded not by different genes, but by the same genes (CT-T1A and CT-T1B) and are present as a mixture of products expressed by both genes, although their subcellular localizations are different.

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  • Derepression of gene expression mediated by the 5' upstream region of the isocitrate lyase gene of Candida tropicalis is controlled by two distinct regulatory pathways in Saccharomyces cerevisiae 査読

    K Umemura, H Atomi, T Kanai, S Takeshita, N Kanayama, M Ueda, A Tanaka

    EUROPEAN JOURNAL OF BIOCHEMISTRY   243 ( 3 )   748 - 752   1997年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-BLACKWELL  

    The 5' upstream region of the gene encoding isocitrate lyase of Candida tropicalis (UPR-ICL) is functional as a promoter in Saccharomyces cerevisiae, and it is regulated by carbon source; the expression of the gene is repressed when cells are grown on glucose, while it increases to a higher level in acetate-grown cells. Therefore, we have investigated regions in UPR-ICL responsible for gene expression in glucose-grown and acetate-grown cells. In glucose-grown cells, a deletion of the region between -801 and -569 (region G1) significantly decreased gene expression compared with that observed with the complete UPR-ICL. The region from -421 to -379 (region G2) also repressed gene expression in glucose-grown cells. In acetate-grown cells, two regions were found to strongly enhance gene expression, one between -728 and -569 (region A1) and the other between -370 and -356 (region A2). Whereas region A2 contained a sequence motif similar to the carbon-source-responsive element (CSRE), which mediates regulation by carbon source of S. cerevisiae ICL1, region A1 did not show similarity to any reported cis-acting elements. Deletion mutants of UPR-ICL containing only one of these regions showed that each region could independently activate gene expression to a similar level when the cells were grown on acetate. The influences of null mutations in the MIG1, SNF1 and CAT8 genes on regulation of UPR-ICL-mediated gene expression were examined. Expression of the ICL gene with full-length UPR-ICL increased about tenfold in mig1 cells grown on glucose, while little difference was observed in acetate-grown cells. The effects of snf1 and cat8 mutations were different between region-Al-mediated and region-A2-mediated gene expression in acetate-grown cells. Region-A2-mediated expression decreased 95% and 86% in snf1 and cat8 cells, respectively, while region-A1-mediated expression decreased 72% in snf1 cells and was not affected by the cat8 mutation. This finding indicates that region-A1-mediated gene expression is regulated by a pathway independent of CAT8, which is necessary for derepression of CSRE-mediated gene expression in S. cerevisiae.

    DOI: 10.1111/j.1432-1033.1997.00748.x

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  • 3-KETOACYL COA THIOLASES OF A YEAST, CANDIDA-TROPICALIS - PROPERTIES AND FUNCTIONS 査読

    A TANAKA, T KURIHARA, N KANAYAMA, H ATOMI, M UEDA

    ENZYME ENGINEERING XII   750   39 - 43   1995年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NEW YORK ACAD SCIENCES  

    DOI: 10.1111/j.1749-6632.1995.tb19922.x

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  • COMPARISON OF MOLECULAR-STRUCTURES AND REGULATION OF BIOSYNTHESIS OF UNIQUE THIOLASE ISOZYMES LOCALIZED ONLY IN PEROXISOMES OF N-ALKANE-UTILIZABLE YEAST, CANDIDA-TROPICALIS 査読

    N KANAYAMA, M UEDA, H ATOMI, T KURIHARA, J KONDO, Y TERANISHI, A TANAKA

    JOURNAL OF FERMENTATION AND BIOENGINEERING   78 ( 4 )   273 - 278   1994年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SOC FERMENTATION BIOENGINEERING, JAPAN  

    Two transcribed genes encoding 3-ketoacyl-CoA thiolase [(EC 2.3.1.16) Thiolase III], one of the peroxisomal thiolase isozymes, of n-alkane-utilizable yeast, Candida tropicalis, were isolated. Restriction maps of these two genes were very similar. Each of the genes had only one open reading frame consisting of 1224 bp corresponding to 408 amino acid residues. The nucleotide sequences of another thiolase isozyme in peroxisomes of C. tropicalis, acetoacetyl-CoA thiolase (Thiolase IA and IB), have been already determined (Kurihara et al., fur. J. Biochem., 210, 999-1005, 1992). The amino acid sequences of these peroxisomal thiolase isozymes (Thiolases I and III) showed 35% homology. The regulation of biosynthesis of these thiolases in C. tropicalis was compared by RNA and Western blotting analyses. The results revealed that the biosynthesis of Thiolase III in C. tropicalis was strongly induced in a medium containing butyrate or alkanes as a carbon source and a peroxisome proliferator, while Thiolase I was constitutively expressed even in a medium with glucose or acetate, although a slight induction was observed at the levels of transcription and translation in the medium containing butyrate or alkanes. Thus, the regulations of biosyntheses of Thiolases I and III were found to be quite different, in spite of the enzymes in the final step of the peroxisomal fatty acid beta-oxidation system.

    DOI: 10.1016/0922-338X(94)90356-5

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  • MOLECULAR EVOLUTION OF YEAST THIOLASE ISOZYMES 査読

    N KANAYAMA, M UEDA, H ATOMI, T KURIHARA, A TANAKA

    JOURNAL OF FERMENTATION AND BIOENGINEERING   78 ( 4 )   279 - 282   1994年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SOC FERMENTATION BIOENGINEERING, JAPAN  

    The coexistence of two thiolase isozymes (acetoacetyl-CoA thiolase and 3-ketoacyl-CoA thiolase), essential for the complete degradation of fatty acids, only in peroxisomes of an n-alkane-utilizing yeast Candida tropicalis (Kurihara et al., J. Biochem., 106, 474-478, 1989) is unique in eukaryotic cells. As one of the methods of analysis of molecular information from these isozymes, the calculation of the evolutional distance among thiolases from various organisms suggested that yeast peroxisomal thiolase isozymes are important enzymes in examining the molecular evolution of the fatty acid metabolic pathway and the biogenesis of peroxisomes.

    DOI: 10.1016/0922-338X(94)90357-3

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  • Molecular cloning of the peroxisomal bifunctional enzyme gene from n-alkane-utilizable yeast, Candida tropicalis pK233(共著)

    Wei DZ, Kanayama N, Ueda M, Tanaka A

    The Annual Reports of ICBiotech(International Center of Cooperative Research in Biotechnology, Faculty of Engineering, Osaka University, Japan)   16   215 - 222   1993年

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    記述言語:英語   掲載種別:研究論文(大学,研究機関等紀要)  

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  • PEROXISOMAL ACETOACETYL-COA THIOLASE OF AN N-ALKANE-UTILIZING YEAST, CANDIDA-TROPICALIS 査読

    T KURIHARA, M UEDA, N KANAYAMA, J KONDO, Y TERANISHI, A TANAKA

    EUROPEAN JOURNAL OF BIOCHEMISTRY   210 ( 3 )   999 - 1005   1992年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:SPRINGER VERLAG  

    Two genes encoding acetoacetyl-CoA thiolase (thiolase I; EC 2.3.1.9), whose localization in peroxisomes was first found with an n-alkane-utilizing yeast, Candida tropicalis, were isolated from the lambdaEMBL3 genomic DNA library prepared from the yeast genomic DNA. Nucleotide sequence analysis revealed that both genes contained open reading frames of 1209 bp corresponding to 403 amino acid residues with methionine at the N-terminus, which were named as thiolase IA and thiolase IB. The calculated molecular masses were 41 898 Da for thiolase IA and 41 930 Da for thiolase IB. These values were in good agreement with the subunit mass of the enzyme purified from yeast peroxisomes (41 kDa). There was an extremely high similarity between these two genes (96% of nucleotides in the coding regions and 98% of amino acids deduced). From the amino acid sequence analysis of the purified peroxisomal enzyme, it was shown that thiolase IA and thiolase IB were expressed in peroxisomes at an almost equal level. Both showed similarity to other thiolases, especially to Saccharomyces uvarum cytosolic acetoacetyl-CoA thiolase (65% amino acids of thiolase IA and 64% of thiolase IB were identical with this thiolase). Considering the evolution of thiolases, the C. tropicalis thiolases and S. uvarum cytosolic acetoacetyl-CoA thiolase are supposed to have a common origin. It was noticeable that the carboxyl-terminal regions of thiolases IA and IB contained a putative peroxisomal targeting signal, -Ala-Lys-Leu-COOH, unlike those of other thiolases reported hitherto.

    DOI: 10.1111/j.1432-1033.1992.tb17505.x

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書籍等出版物

  • バイオ実験を安全に行うために

    (株)化学同人  2018年  ( ISBN:9784759819212

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  • ラボ必携 フローサイトメトリーQ&A〜正しいデータを出すための100箇条 (実験医学別冊)

    戸村 道夫

    羊土社  2017年11月  ( ISBN:4758122350

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    総ページ数:313  

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  • 生物工学よもやま話ー実験の基本原理から応用までー

    有限会社 学進出版  2013年  ( ISBN:9784907773052

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  • ひらく、ひらく「バイオの世界」

    化学同人  2012年  ( ISBN:9784759815382

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  • 次世代抗体医薬開発に向けた抗体工学の最前線

    シーエムシー出版  2012年  ( ISBN:9784781306735

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  • 改訂 細胞工学

    講談社  2010年  ( ISBN:9784061398306

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  • 未来をつくるバイオ 酒造りから再生医療まで60話 社団法人 日本生物工学会編 Part 5 生体素子とバイオテクノロジー

    学進出版  2008年 

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  • 生物工学実験書

    培風館(東京)  2002年 

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    EUROPEAN JOURNAL OF IMMUNOLOGY   46   1139 - 1139   2016年8月

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    その他リンク: http://search.jamas.or.jp/link/ui/2014247582

  • 3P-002 B細胞抗原レセプター依存性アポトーシスの制御によるニワトリB細胞株DT40-SWを用いた高親和性抗体の作製

    古賀 舞, 池田 美香, 小嶋 宏侑, 川上 夏奈江, 渡邊 康二, 徳光 浩, 曲 正樹, 大森 斉, 金山 直樹

    日本生物工学会大会講演要旨集   65   188 - 188   2013年

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    その他リンク: http://search.jamas.or.jp/link/ui/2014247581

  • 胚中心におけるB細胞のnegative selectionと濾胞樹状細胞

    臨床免疫・アレルギー科   58 ( 3 )   259 - 265   2012年

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  • 抗体遺伝子への突然変異能を有するニワトリB細胞株の機能拡張によるin vitroモノクローナル抗体作成・改良システム

    細胞工学   31 ( 10 )   1159 - 1165   2012年

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  • フローサイトメトリー〜「前にならえ」並べて順に数えます〜

    金山直樹

    日本生物工学会誌   90 ( 11 )   785 - 789   2012年

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  • DT40細胞株を用いたin vitro抗体作製システムにおける,抗原レセプターの刺激に依存した生存による抗原特異的抗体産生細胞の選択法の開発

    渡邊康二, 藤堂景史, 福岡純司, 曲正樹, 大森齊, 金山直樹

    日本分子生物学会年会プログラム・要旨集(Web)   35th   2012年

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  • 4Ep15 ニワトリB細胞株DT40-SWを用いた異種抗体改良システムの構築(抗体工学/有機化学,高分子化学,一般講演)

    佐井 燕, 小島 聡史, 藤井 忍, 北村 幸一, 鴨下 佳代子, 池田 美香, 曲 正樹, 大森 齊, 金山 直樹

    日本生物工学会大会講演要旨集   64   212 - 212   2012年

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    記述言語:日本語   出版者・発行元:日本生物工学会  

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    その他リンク: http://search.jamas.or.jp/link/ui/2013125691

  • 4Ep14 変異能力を有する培養B細胞株DT40を用いた新規なタンパク質ディスプレーシステムの開発(抗体工学/有機化学,高分子化学,一般講演)

    日笠 卓哉, 松田 修一, 植月 英智, 曲 正樹, 大森 斎, 金山 直樹

    日本生物工学会大会講演要旨集   64   212 - 212   2012年

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    記述言語:日本語   出版者・発行元:日本生物工学会  

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    その他リンク: http://search.jamas.or.jp/link/ui/2013125690

  • 4Ep16 ヒト型抗体発現ニワトリB細胞株DT40を用いた抗原特異的抗体の作製(抗体工学/有機化学,高分子化学,一般講演)

    川上 夏奈江, 井上 和恵, 岡山 展久, 金広 優一, 池田 美香, 曲 正樹, 大森 斉, 金山 直樹

    日本生物工学会大会講演要旨集   64   212 - 212   2012年

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    記述言語:日本語   出版者・発行元:日本生物工学会  

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  • B細胞免疫記憶 AIDに依存したIgV変異には、SRタンパク質SRSF1のスプライスアイソフォームが必要である(AID-dependent IgV hypermutation requires a splice isoform of the SR protein SRSF1)

    金山 直樹, 金広 優一, 藤堂 景史, 根岸 美咲, 福岡 純司, 曲 正樹, Li Xialu, Manley James L, 大森 斉

    日本免疫学会総会・学術集会記録   40   172 - 172   2011年11月

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    記述言語:英語   出版者・発行元:(NPO)日本免疫学会  

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  • AIDに依存した体細胞突然変異には、SRタンパク質SRSF1のスプライスアイソフォームが必要である

    金山 直樹, 金広 優一, 藤堂 景史, 根岸 美咲, 福岡 純司, 曲 正樹, Li Xialu, Manley James L, 大森 斉

    日本生化学会大会プログラム・講演要旨集   84回   3T15a - 15   2011年9月

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    記述言語:日本語   出版者・発行元:(公社)日本生化学会  

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  • 胚中心B細胞の分化と濾胞樹状細胞

    大森 斉, 曲 正樹, 金山直樹

    臨床免疫・アレルギー科   56 ( 2 )   116 - 122   2011年

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  • 変異能力を有するニワトリB細胞株DT40-SWを用いたマウスモノクローナル抗体の親和性成熟

    鴨下 佳代子, 小島 聡史, 藤井 忍, 北村 幸一, 井上 和恵, 岡山 展久, 松田 修一, 小嶋 宏侑, 池田 美香, 金広 優一, 藤堂 景史, 曲 正樹, 大森 齊, 金山 直樹

    日本生化学会大会・日本分子生物学会年会合同大会講演要旨集   83回・33回   2T6 - 10   2010年12月

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    記述言語:日本語   出版者・発行元:(公社)日本生化学会  

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  • 抗体遺伝子への体細胞突然変異におけるsplicing factor ASF/SF2の寄与

    金広 優一, 藤堂 景史, 根岸 美咲, 福岡 純司, 曲 正樹, 大森 齊, 金山 直樹

    日本生化学会大会・日本分子生物学会年会合同大会講演要旨集   83回・33回   2P - 0478   2010年12月

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    記述言語:日本語   出版者・発行元:(公社)日本生化学会  

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  • Efficient in vitro antibody generation system using an engineered chicken B cell line, DT40-SW

    Hitoshi Ohmori, Masaki Magari, Naoki Kanayama, Shuichi Matsuda, Yuichi Kanehiro

    JOURNAL OF BIOTECHNOLOGY   150   S440 - S441   2010年11月

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:ELSEVIER SCIENCE BV  

    DOI: 10.1016/j.jbiotec.2010.09.628

    Web of Science

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  • 変異能力を有するニワトリB細胞株DT40-SWを用いたマウスモノクローナル抗体の親和性成熟

    金山 直樹, 小島 聡史, 藤井 忍, 北村 幸一, 井上 知恵, 岡山 展久, 松田 修一, 鴨下 佳代子, 藤堂 景史, 池田 美香, 曲 正樹, 大森 齊

    日本生物工学会大会講演要旨集   平成22年度   81 - 81   2010年9月

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    記述言語:日本語   出版者・発行元:(公社)日本生物工学会  

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  • 変異能力を有するニワトリB細胞株を用いた抗体の親和性成熟:外来の抗体可変部遺伝子のDT40-SWにおける発現・改良系の構築

    小島聡史, 藤井忍, 北村幸一, 井上知恵, 岡山展久, 松田修一, 藤堂景史, 曲正樹, 金山直樹, 大森斉

    日本分子生物学会年会講演要旨集   32nd ( Vol.1 )   2009年

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  • 変異能力を有するニワトリB細胞株を用いた抗体の親和性成熟:変異様式の変換による親和性成熟の効率化

    梶田真道, 曲正樹, 池田美香, 藤堂景史, 金山直樹, 大森斉

    日本分子生物学会年会講演要旨集   32nd ( Vol.1 )   2009年

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  • 変異導入能力を有する培養B細胞株を用いたin vitro抗体作製システムの高機能化

    金山 直樹, 曲 正樹, 梶田 真道, 金広 優一, 藤堂 景史, 大森 斉

    日本生化学会大会・日本分子生物学会年会合同大会講演要旨集   81回・31回   1T9 - 7   2008年11月

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    記述言語:日本語   出版者・発行元:(公社)日本生化学会  

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  • ニワトリB細胞株を用いたin vitro抗体作製システムの高機能化 変異様式の転換の高性能抗体作製への応用

    梶田 真道, 藤堂 景史, 曲 正樹, 金山 直樹, 大森 斉

    日本生物工学会大会講演要旨集   平成20年度   109 - 109   2008年7月

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    記述言語:日本語   出版者・発行元:(公社)日本生物工学会  

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  • ニワトリB細胞株DT40-SWを用いたin vitro抗体作製システムの高機能化 抗体遺伝子への変異導入効率の増強

    金広 優一, 曲 正樹, 藤堂 景史, 金山 直樹, 大森 斉

    日本生物工学会大会講演要旨集   平成20年度   109 - 109   2008年7月

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    記述言語:日本語   出版者・発行元:(公社)日本生物工学会  

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  • 抗体医薬が切り拓く先端医療 培養B細胞株を用いるin vitro抗体作製システムによる有用抗体の創製

    金山 直樹, 藤堂 景史, 曲 正樹, 大森 齊

    日本薬学会年会要旨集   128年会 ( 1 )   134 - 134   2008年3月

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    記述言語:日本語   出版者・発行元:(公社)日本薬学会  

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  • 効率的in vitro抗体作製システムの開発

    大森 斉, 金山直樹

    BIO Clinica   23 ( 8 )   82 - 87   2008年

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  • ニワトリB細胞株DT40-SWを用いるin vitro抗体作製システムの開発 変異導入の促進による迅速な抗体ライブラリーの構築

    金広 優一, 曲 正樹, 藤堂 景史, 金山 直樹, 大森 斉

    日本生物工学会大会講演要旨集   平成19年度   148 - 148   2007年8月

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    記述言語:日本語   出版者・発行元:(公社)日本生物工学会  

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  • ニワトリB細胞株DT40-SWを用いるin vitro抗体作製システムの開発 Pax5発現抑制による抗体産生の増強

    曲 正樹, 綾 高宏, 藤堂 景史, 金山 直樹, 大森 斉

    日本生物工学会大会講演要旨集   平成19年度   149 - 149   2007年8月

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    記述言語:日本語   出版者・発行元:(公社)日本生物工学会  

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  • ニワトリB細胞株DT40-SWを用いるin vitro抗体作製システムの開発 抗原特異的クローンのセルソーターによる効率的単離

    岡澤 貴裕, 藤堂 景史, 梶田 真道, 曲 正樹, 金山 直樹, 大森 斉

    日本生物工学会大会講演要旨集   平成19年度   149 - 149   2007年8月

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    記述言語:日本語   出版者・発行元:(公社)日本生物工学会  

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  • 変異機能のON/OFF制御可能なB細胞株を用いた抗体および変異タンパク質の作製システム

    大森 斉, 藤堂景史, 金山直樹

    実験医学   124 (9), pp. 1331-1335   2006年

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  • 1K16-1 培養B細胞株を用いた新規in vitro抗体作製システムによるモノクローナル抗体の取得(プロセス工学/糖鎖工学/免疫工学,一般講演)

    藤堂 景史, 三宅 健司, 藤田 梨紗子, 岡澤 貴裕, 池田 美香, 曲 正樹, 金山 直樹, 大森 斉

    日本生物工学会大会講演要旨集   18   197 - 197   2006年

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    記述言語:日本語   出版者・発行元:日本生物工学会  

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  • 1K16-2 点突然変異と遺伝子変換の制御によるニワトリB細胞株DT40からの高性能抗体ライブラリの構築(プロセス工学/糖鎖工学/免疫工学,一般講演)

    梶田 真道, 藤堂 景史, 曲 正樹, 金山 直樹, 大森 斉

    日本生物工学会大会講演要旨集   18   197 - 197   2006年

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    記述言語:日本語   出版者・発行元:日本生物工学会  

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  • 抗体遺伝子変換機構を利用した非抗体タンパクのDNAシャッフリングによる機能改変

    藤堂 景史, 高橋 佐都子, 片岡 大輔, 曲 正樹, 金山 直樹, 大森 斉

    日本免疫学会総会・学術集会記録   35   80 - 80   2005年11月

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    記述言語:日本語   出版者・発行元:(NPO)日本免疫学会  

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  • 培養B細胞株の変異機能を利用する新規なin vitro抗体作製システム

    三宅 健司, 藤堂 景史, 曲 正樹, 金山 直樹, 大森 斉

    日本生物工学会大会講演要旨集   平成17年度   118 - 118   2005年9月

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    記述言語:日本語   出版者・発行元:(公社)日本生物工学会  

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  • 抗体の多様化メカニズムの不思議

    金山直樹

    日本生物工学会誌   83 (1), 33   2005年

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  • B細胞の遺伝子変換機構を利用する非抗体タンパクの機能改変

    藤堂景史, 高橋佐都子, 片岡大輔, 曲正樹, 金山直樹, 大森斉

    日本生物工学会大会講演要旨集   2005   2005年

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  • 高頻度変異機能のON/OFF制御可能なB細胞株を用いるin vitro抗体作製システム

    金山直樹, 藤堂景史, 三宅健司, 曲正樹, 大森斉

    日本分子生物学会年会講演要旨集   28th   2005年

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  • B細胞株の遺伝子改変能を利用したタンパク分子改変システムの構築

    藤堂 景史, 高橋 佐都子, 曲 正樹, 金山 直樹, 大森 斉

    日本免疫学会総会・学術集会記録   34   263 - 263   2004年11月

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    記述言語:日本語   出版者・発行元:(NPO)日本免疫学会  

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  • B細胞レパトアの限定されたマウスにおけるB細胞選択と親和性成熟の解析

    金山 直樹, 木本 崇文, 藤堂 景史, 曲 正樹, 大森 齊

    生化学   76 ( 3 )   306 - 306   2004年3月

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    記述言語:日本語   出版者・発行元:(公社)日本生化学会  

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  • ニワトリB細胞株を用いたタンパク分子改変系の構築:外来遺伝子の遺伝子変換

    高橋佐都子, 藤堂景史, 曲正樹, 金山直樹, 大森斉

    日本生物工学会大会講演要旨集   2004   2004年

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  • ニワトリB細胞株を用いたタンパク分子改変系の構築:変異導入機能のON/OFF制御

    藤堂景史, 曲正樹, 金山直樹, 大森斉

    日本生物工学会大会講演要旨集   2004   2004年

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  • B細胞レセプターを介するシグナルとB細胞応答

    曲正樹, 金山直樹, 大森斉

    臨床免疫   40, 454-457   2003年

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  • 平成13年度両備てい園記念財団研究助成金による研究報告 Qmasimonoclonal マウスにおける抗体親和性成熟

    金山直樹

    生物学に関する試験研究論叢   18, 57-67   2003年

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  • 辺縁帯B細胞の特性

    金山直樹, 大森斉

    臨床免疫   40, 450-453   2003年

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  • 2C09-1 ニワトリ B 細胞を用いる外来遺伝子の改変と発現

    藤堂 景史, 高橋 佐都子, 曲 正樹, 金山 直樹, 大森 斉

    日本生物工学会大会講演要旨集   15   87 - 87   2003年

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    記述言語:日本語   出版者・発行元:日本生物工学会  

    CiNii Article

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  • B細胞多様性の限定されたマウスにおけるクローン選択と親和性成熟の解析

    木本 崇文, 藤堂 景史, 疋田 正喜, 曲 正樹, 金山 直樹, 大森 斉

    日本免疫学会総会・学術集会記録   32   271 - 271   2002年10月

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    記述言語:日本語   出版者・発行元:(NPO)日本免疫学会  

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  • B細胞多様性の限定されたマウスにおける抗体の親和性成熟機構の解析

    藤堂 景史, 木本 崇文, 曲 正樹, 金山 直樹, 大森 斉

    日本生物工学会大会講演要旨集   平成14年度   206 - 206   2002年9月

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    記述言語:日本語   出版者・発行元:(公社)日本生物工学会  

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  • B細胞レパトアの限定されたマウスを用いるB細胞選択と親和性成熟の解析

    木本 崇文, 松浦 正明, 藤堂 景史, 曲 正樹, 疋田 正喜, 金山 直樹, 大森 齊

    生化学   74 ( 8 )   949 - 949   2002年8月

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    記述言語:日本語   出版者・発行元:(公社)日本生化学会  

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  • 末梢でのV(D)J遺伝子再構成の親和性成熟への寄与

    疋田正喜, 金山直樹, 大森 斉

    臨床免疫   36, 54-59   2001年

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  • 末梢リンパ組織における抗体遺伝子の再構成。 抗体遺伝子の構造はどこまで柔軟か?

    大森 斉, 疋田正喜, 金山直樹

    細胞工学   19, 482-487   2000年

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  • 胚中心B細胞におけるレセプターeditingとその意義。

    大森 斉, 疋田正喜, 金山直樹

    Molecular Midicne 「免疫1999-2000」   36, 20-28   1999年

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  • 内山勇三科学技術賞

    2013年   岡山工学振興会  

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    受賞国:日本国

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  • 岡山工学振興会科学技術賞

    2009年   岡山工学振興会  

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    受賞国:日本国

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  • 第44回生物工学奨励賞(斉藤賞)

    2008年   日本生物工学会  

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    受賞国:日本国

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  • 岡山工学振興会科学技術賞

    2004年   岡山工学振興会  

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担当授業科目

  • SDGs:生命科学 (2021年度) 第4学期  - 木7~8

  • ヘルスシステム統合科学専門英語 (2021年度) 後期  - その他

  • ヘルスシステム統合科学特別研究 (2021年度) 通年  - その他

  • 先進病院実習 (2021年度) 前期  - その他

  • 分析化学 (2021年度) 1・2学期  - 木3,木4

  • 分析化学1 (2021年度) 第1学期  - 木3,木4

  • 分析化学2 (2021年度) 第2学期  - 木3,木4

  • 工学基礎実験実習 (2021年度) 1・2学期  - 火5,火6,火7,火8

  • 工学基礎実験実習 (2021年度) 1・2学期  - 火5,火6,火7,火8

  • 工学基礎実験実習 (2021年度) 1・2学期  - 火5,火6,火7,火8

  • 技術表現発表学 (2021年度) 後期  - その他

  • 技術表現発表学 (2021年度) 後期  - その他

  • 放射線安全利用工学2 (2021年度) 第2学期  - 金5,金6

  • 放射線安全利用工学及び実験 (2021年度) 1・2学期  - 金5,金6

  • 生化学3 (2021年度) 第1学期  - 火3,火4,金3,金4

  • 細胞工学 (2021年度) 第4学期  - 月3,月4

  • 細胞工学 (2021年度) 第4学期  - 月3,月4

  • 細胞機能工学 (2021年度) 前期  - 月1~2

  • 細胞機能開発学 (2021年度) 後期  - その他

  • ヘルスシステム統合科学インターンシップ (2020年度) 通年  - その他

  • ヘルスシステム統合科学専門英語 (2020年度) 後期  - その他

  • ヘルスシステム統合科学特別研究 (2020年度) 通年  - その他

  • 分析化学 (2020年度) 1・2学期  - 水3,水4

  • 分析化学1 (2020年度) 第1学期  - 水3,水4

  • 分析化学2 (2020年度) 第2学期  - 水3,水4

  • 技術表現発表学 (2020年度) 後期  - その他

  • 放射線安全利用工学2 (2020年度) 第2学期  - 金5,金6

  • 放射線安全利用工学及び実験 (2020年度) 1・2学期  - 金5,金6

  • 教養生物学(バイオテクノロジー) (2020年度) 特別  - その他

  • 教養生物学(バイオテクノロジー) (2020年度) 第4学期  - 木3,木4

  • 生化学3 (2020年度) 第1学期  - 火3,火4,金3,金4

  • 細胞工学 (2020年度) 第4学期  - 月3,月4

  • 細胞工学 (2020年度) 第4学期  - 月3,月4

  • 細胞機能開発学 (2020年度) 後期  - その他

▼全件表示