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Mechanism of insulin exocytosis in pancreatic β cells by analyzing single insulin granule motion with evanescent wave microscopy

Research Project

Project/Area Number 14570130
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Pathological medical chemistry
Research InstitutionKyorin University

Principal Investigator

OHARA Mica  Kyorin University, School of Medicine, Lecturer, 医学部, 講師 (40201941)

Co-Investigator(Kenkyū-buntansha) NAGAMATSU Shinya  Kyorin University, School of Medicine, Professor, 医学部, 教授 (80231489)
Project Period (FY) 2002 – 2004
Project Status Completed (Fiscal Year 2004)
Budget Amount *help
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2004: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2003: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2002: ¥1,200,000 (Direct Cost: ¥1,200,000)
Keywordsinsulin / exocytosis / pancreatic beta cell / evanescentwave microscopy / type 2 diaetes / secretory granule / インスリン分泌 / エバネッセント顕微鏡 / 分泌顆粒 / 2型糖尿病
Research Abstract

It is essential to use primary β cell for revealing the molecular mechanism of insulin exocytosis and the machinery behind impaired insulin release in type 2 diabetes, nevertheless, technical difficulties prevented us to analyze such a process. We have recently succeeded in monitoring the dynamics of insulin granules in primary β cell prepared from rat pancreas using TIRF imaging system with high time resolution. In the 1^<st>-phase of insulin release, the fusing granules occurred mostly from previously docked granules, but fusion granules in 2^<nd>-phase originated from newcomers. TIRF images could reveal that the docking time of newcomers on the plasma membrane was extremely short within 50 ms, in contrast to relatively long time in insulinoma MIN6 cells (37±11s), thus the exocytotic mechanism in 1^<st>-phase is quite different from that in the 2^<nd>-phase in primary β cells. We have then addressed the question if insulin exocytotic site is associated with SNAREs or related proteins. To study the interaction between t-SNAREs and the docking and fusion of insulin granules, we labeled endogenous syntaxin 1A and SNAP-25 with Cy3-labeled anti-syntaxin 1A and anti SNAP-25 antibodies by conjugating antibodies with HIV-1 TAT, that was rapidly transduced into the living β cells. TIRF image showed that t-SNAREs are distributed in numerous separated clusters (<400 nm) on the plasma membrane. Insulin granules were preferentially docked (about 80%) to sites on syntaxin 1A clusters, colocalizing with SNAP-25 clusters. The fusion was seen at the syntaxin 1A clusters. Now, we are investigating the relationship between site of insulin exocytosis and CAZ (cytomatrix at the active zone) proteins. Thus, our data revealed the molecular mechanism of insulin exocytosis in primary β cells.

Report

(4 results)
  • 2004 Annual Research Report   Final Research Report Summary
  • 2003 Annual Research Report
  • 2002 Annual Research Report
  • Research Products

    (52 results)

All 2005 2004 2003 2002 Other

All Journal Article (40 results) Book (2 results) Publications (10 results)

  • [Journal Article] Docking and fusion of insulin secretory granules in SUR1 knock out mouse beta-cells observed by total internal reflection microscopy2005

    • Author(s)
      T.Kikuta, M.Ohara-Imaizumi et al.
    • Journal Title

      FEBS Lett. 579

      Pages: 1602-1606

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Rab27a mediates the tight docking of insulin granules onto the plasma membrane during glucose stimulation.2005

    • Author(s)
      K.Kasai, M.Ohara-Imaizumi et al.
    • Journal Title

      J.Clin.Invest. 115

      Pages: 388-396

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Annual Research Report 2004 Final Research Report Summary
  • [Journal Article] Rab27a mediates the tight docking of insulin granules onto the plasma membrane during glucose stimulation.2005

    • Author(s)
      Kasai, K., Ohara-Imaizumi, M., Takahashi, N., Mizutani, S., Zhao, S., Kikuta, T., Kasai, H., Nagamatsu, S., Gomi, H., Izumi, T.
    • Journal Title

      J.Clin.Invest. 115

      Pages: 388-396

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Docking and fusion of insulin secretory granules in SUR1 knock out mouse beta-cells observed by total internal reflection fluorescence microscopy.2005

    • Author(s)
      Kikuta, T., Ohara-Imaizumi, M., Nakazaki, M., Nishiwaki, C., Nakamichi, Y, Tei, C., Aguilar-Bryan, L., Bryan, J., Nagamatsu, S.
    • Journal Title

      FEBS Lett. 579

      Pages: 1602-1606

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Docking and fusion of insulin secretory granules in SURI knock out mouse beta-cells observed by total internal reflection microscopy2005

    • Author(s)
      T.Kikuta, M.Ohara-Imaizumi et al.
    • Journal Title

      FEBS Lett. 579

      Pages: 1602-22538

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Site of docking and fusion of insulin secretory granules in live MIN6 □ cells analyzed by TAT-conjugated anti-syntaxin 1 antibody and total internal reflection fluorescence microscopy.2004

    • Author(s)
      Ohara-Imaizumi M, Nagamatsu S et al.
    • Journal Title

      J Biol Chem 279

      Pages: 8403-8408

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] TIRF imaging of docking and fusion of single insulin granule motion in primary rat pancreatic 13 -cells : different behaviour of granule motion between normal and Goto-Kakizaki diabetic rat β -cells2004

    • Author(s)
      M.Ohara-Imaizurni et al.
    • Journal Title

      Biochem.J 381

      Pages: 13-18

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Corelation of syntaxin1 and SNAP-25 clusters with docking and fusion of insulin granules analyzed by total internal reflection microscopy.2004

    • Author(s)
      Mica Ohara-Imaizumi et al.
    • Journal Title

      Diabetologia 47

      Pages: 2200-2207

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] The cytokine IL-1 β reduces the docking and fusion of insulin granules in pancreatic β-cells, preferentially decreasting the first phase of exocytosis.2004

    • Author(s)
      Mica Ohara-Imaizumi et al.
    • Journal Title

      J.Biol.Chem 279

      Pages: 41271-41274

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Functions of pancreatic beta cells and adipocytes in bombesin receptor subtype-3-deficient mice.2004

    • Author(s)
      Y.Nakamich et al.
    • Journal Title

      Biochem, Biophys.Res.Commun. 318

      Pages: 698-703

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Spatio-temporal regulation of neurotransmitter release by PKC ; studies in adrenal chromaffin cells.2004

    • Author(s)
      Kumakura K., Sasakawa N., Murayama N., Ohara-Imaizumi M., et al.
    • Journal Title

      Crit Rev Neurobiol. 16

      Pages: 173-179

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Site of docking and fusion of insulin secretory granules in live MIN6 β cells analyzed by TAT-conjugated anti-syntaxin 1 antibody and total internal reflection fluorescence microscopy.2004

    • Author(s)
      Ohara-Imaizumi, M., Nishiwaki, C., Kikuta, T., Kumakura, K., Nakamichi, Y., Nagamatsu, S.
    • Journal Title

      J.Biol.Chem. 279

      Pages: 8403-8408

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] TIRF imaging of docking and fusion of single insulin granule motion in primary rat pancreatic beta-cells : different behaviour of granule motion between normal and Goto-Kakizaki diabetic rat beta-cells.2004

    • Author(s)
      Ohara-Imaizumi, M., Nishiwaki, C., Kikuta, T., Nagai, S., Nakamichi, Y., Nagamatsu, S.
    • Journal Title

      Biochem.J. 381

      Pages: 13-18

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Functions of pancreatic beta cells and adipocytes in bombesin receptor subtype-3-deficient mice.2004

    • Author(s)
      Nakamichi, Y., Wada, E., Aoki, K., Ohara-Imaizumi, M., Kikuta, T., Nishiwaki, C., Matsushima, S., Watanabe, T., Wada, K., Nagamatsu, S.
    • Journal Title

      Biochem.Biophys.Res.Commun. 318

      Pages: 698-703

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] The cytokine interleukin-1beta reduces the docking and fusion of insulin granules in pancreatic beta-cells, preferentially decreasing the first phase of exocytosis2004

    • Author(s)
      Ohara-Imaizumi, M., Cardozo, A.K., Kikuta, T., Eizirik, D.L., Nagamatsu, S.
    • Journal Title

      J.Biol.Chem. 279

      Pages: 41271-41274

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Spatio-temporal regulation of neurotransmitter release by PKC; studies in adrenal chromaffin cells.2004

    • Author(s)
      Kumakura, K., Sasakawa, N., Murayama, N., Ohara-Imaizumi, M.
    • Journal Title

      Crit.Rev.Neurobiol. 16

      Pages: 173-179

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Correlation of syntaxin-1 and SNAP-25 clusters with docking and fusion of insulin granules analyzed by total internal reflection fluorescence microscopy.2004

    • Author(s)
      Ohara-Imaizumi, M., Nishiwaki, C., Nakamichi, Y., Kikuta, T., Nagai, S., Nagamatsu, S.
    • Journal Title

      Diabetologia. 47(12)

      Pages: 2200-2207

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] TIRF imaging of docking and fusion of single insulin granule motion in primary rat pancreatic β-cells : different behavior of granule motion between normal and Goto-Kakizaki diabetic rat β-cells.2004

    • Author(s)
      M.Ohara-Imaizumi et al.
    • Journal Title

      Biochem.J 381

      Pages: 13-18

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Corelation of syntaxin 1 and SNAP-25 clusters with docking and fusion of insulin granules analyzed by total internal reflection microscopy.2004

    • Author(s)
      Mica Ohara-Imaizumi et al.
    • Journal Title

      Diabetologia 47

      Pages: 2200-2207

    • Related Report
      2004 Annual Research Report
  • [Journal Article] The cytokine IL-1β reduced the docking and fusion of insulin granules in pancreatic β-cells, preferentially decreasting the first phase of exocytosis.2004

    • Author(s)
      Mica Ohara-Imaizumi et al.
    • Journal Title

      J.Biol.Chem 279

      Pages: 41271-41274

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Functions of pancreatic beta cells and adipocytes in bombesin receptor subtype-3-deficient mice.2004

    • Author(s)
      Y.Nakamich et al.
    • Journal Title

      Biochem, Biphys.Res.Commun. 318

      Pages: 698-703

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Secretory granules are recaptured largely intact after stimulated exocytosis in cultured endocrine cells.2003

    • Author(s)
      Taraska J.W., Ohara-Imaizumi M(他3名)
    • Journal Title

      Proc.Natl.Acad.Sci.USA 100

      Pages: 2070-2075

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] An Insulin-related peptide expressed in response to exogenous insulin, which augments the muslin-stimulated glucose uptake.2003

    • Author(s)
      Nakamichi Y., Ohara-Imaizumi M., Ishida H., Nagamatsu S.
    • Journal Title

      J.Cell Sci. 116

      Pages: 73-79

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] インスリン開口放出の可視化(ミニレビュー)2003

    • Author(s)
      今泉美佳, 永松信哉
    • Journal Title

      生化学 75

      Pages: 49-54

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] PPAR-gamma overexpression suppresses glucose-induced proinsulin biosynthesis and insulin release synergistically with pioglitazone in MIN6 cells.2003

    • Author(s)
      Nakamichi Y, Kikuta T, Ito E, Ohara-Imaizumi M et al.
    • Journal Title

      Biochem Biophys Res Commun 306

      Pages: 832-836

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] An insulin-related peptide expressed in 3T3L1 adipocytes is localized in GLUT4 vesicles and secreted in response to exogenous insulin, which augments the insulin-stimulated glucose uptake.2003

    • Author(s)
      Nakamichi, Y., Ohara-Imaizumi, M., Ishida, H., Nagamatsu, S.
    • Journal Title

      J.Cell Sci. 116

      Pages: 73-79

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Secretory granules are recaptured largely intact after stimulated exocytosis in cultured endocrine cells.2003

    • Author(s)
      Taraska, J.W., Perrais, D., Ohara-Imaizumi, M., Nagamatsu, S., Almers, W.
    • Journal Title

      Proc.Natl.Acad.Sci. USA 100

      Pages: 2070-2075

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Visualizing exocytosis of insulin granules with evanescent wave microscopy.2003

    • Author(s)
      Ohara-Imaizumi, M., Nagamatsu, S.
    • Journal Title

      Seikagaku 75

      Pages: 49-54

    • NAID

      80015773647

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] PPAR-gamma overexpression suppresses glucose-induced proinsulin biosynthesis and insulin release synergistically with pioglitazone in MIN6 cells.2003

    • Author(s)
      Nakamichi, Y., Kikuta, T., Ito, E., Ohara-Imaizumi.M., Nishiwaki, C., Ishida, H., Nagamatsu, S.
    • Journal Title

      Biochem.Biophys.Res.Commun. 306

      Pages: 832-836

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Transduction of MIN6 β cell with TAT-Syntaxin SNARE Motif Inhibits Insulin Exocytosis in Biphasic Insulin Release in a Distinct Mechanism Analyzed by Evanescent Wave Microscopy.2002

    • Author(s)
      Ohara-Imaizumi M., Nakamichi Y., Nishiwaki C., Nagamatsu S
    • Journal Title

      J.Biol.Chem. 277

      Pages: 50805-50811

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Monitoring of exocytosis and endocytosis of insulin secretory granules in the pancreatic β cell line MIN6 using pH-sensitive green fluorescent protein (pHluorin)and confocal laser microscopy.2002

    • Author(s)
      Ohara-Imaizumi M., Nagamatsu S.(他4名)
    • Journal Title

      Biochem.J. 363

      Pages: 73-80

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Imaging exocytosis of single insulin secretory granules with evanescent wave microscopy.2002

    • Author(s)
      Ohara-Imaizumi M, Nagamatsu S.(他3名)
    • Journal Title

      J.Biol.Chem. 77

      Pages: 3805-3808

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] インスリン開口放出機構とSNARE蛋白質2002

    • Author(s)
      永松信哉, 今泉美佳
    • Journal Title

      日本臨床 60

      Pages: 213-218

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Decreased marble burying behavior in female mice lacking neuromedin-B receptor (NMB-R) implies the involvement of NMB/NMB-R in 5-HT neuron function.2002

    • Author(s)
      Yamada K., Wada E., Yamano M., Sun YJ., Ohara-Imaizumi M., (他2名)
    • Journal Title

      Brain Res. 942

      Pages: 71-78

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Roles of actin filaments and the actin-myosin interaction in the regulation of exocytosis in chromaffin cells.2002

    • Author(s)
      Sasakawa N., Ohara-Imaizumi M., (他4名)
    • Journal Title

      Ann N Y Acad Sci. 971

      Pages: 273-274

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Transduction of MIN6 beta cells with TAT-syntaxin SNARE motif inhibits insulin exocytosis in biphasic insulin release in a distinct mechanism analyzed by evanescent wave microscopy.2002

    • Author(s)
      Ohara-Imaizumi, M., Nakamichi, Y., Nishiwaki, C., Nagamatsu, S.
    • Journal Title

      J.Biol.Chem. 277

      Pages: 50805-50811

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Molecular mechanism of insulin exocytosis and the role of SNARE proteins.2002

    • Author(s)
      Nagamatsu, S., Ohara-Imaizumi, M
    • Journal Title

      Nihonrinsho 60

      Pages: 213-218

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Monitoring of exocytosis and endocytosis of insulin secretory granules in the pancreatic beta-cell line MIN6 using pH-sensitive green fluorescent protein (pHluorin) and confocal laser microscopy.2002

    • Author(s)
      Ohara-Imaizumi, M., Nakamichi, Y., Tanaka, T., Katsuta, H., Ishida, H., Nagamatsu, S.
    • Journal Title

      Biochem.J. 363

      Pages: 73-80

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Decreased marble burying behavior in female mice lacking neuromedin-B receptor (NMB-R) implies the involvement of NMB/NMB-R in 5-HT neuron function.2002

    • Author(s)
      Yamada, K., Wada, E., Yamano, M., Sun, Y.J., Ohara-Imaizumi, M., Nagamatsu, S., Wada, K.
    • Journal Title

      Brain Res. 942

      Pages: 71-78

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Journal Article] Roles of actin filaments and the actin-myosin interaction in the regulation of exocytosis in chromaffin cells.2002

    • Author(s)
      Sasakawa, N., Ohara-Imaizumi, M., Okubo, S., Hosaka, S., Hayashi, M., Kumakura, K.
    • Journal Title

      Ann N Y Acad.Sci. 971

      Pages: 273-274

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Book] Recent research development in biophysics & biochemistry2003

    • Author(s)
      Nagamatsu S, Ohara-Imaizumi M
    • Publisher
      SNARE-mediated insulin exocytosis.
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Book] ポスター発表はチャンスの宝庫!一歩進んだ発表のための計画・準備から当日のプレゼンまで2003

    • Author(s)
      今泉 美佳
    • Total Pages
      124
    • Publisher
      羊土社
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2004 Final Research Report Summary
  • [Publications] Ohara-Imaizumi M, Nagamatsu S, et al.: "Site of docking and fusion of insulin secretory granules in live MIN6 β cells analyzed by TAT-conjugated anti-syntaxin 1 antibody and total internal reflection fluorescence microscopy."J Biol Chem. 279. 8403-8408 (2004)

    • Related Report
      2003 Annual Research Report
  • [Publications] Nakamichi Y, Kikuta T, Ito E, Ohara-Imaizumi M et al.: "PPAR-gamma overexpression suppresses glucose-induced proinsulin biosynthesis and insulin release synergistically with pioglitazone in MIN6 cells."Biochem Biophys Res Commun. 306. 832-836 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Nagamatsu S, Ohara-Imaizumi M: "Recent research development in biophysics & biochemistry"SNARE-mediated insulin exocytosis.. 457-470 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Taraska J.W., Ohara-Imaizumi M: "Secretory granules are recaptured largely intact after stimulated exocytosis in cultured endocrine cells"Proc. Natl. Acad. Sci. USA. 100. 2070-2075 (2003)

    • Related Report
      2002 Annual Research Report
  • [Publications] Nakamichi Y., Ohara-Imaizumi M., Ishida H., Nagamatsu S.: "An Insulin-related peptide expressed in response to exogenous insulin, which augments the inuslin-stimulated glucose uptake"J. Cell Sci.. 116. 73-79 (2003)

    • Related Report
      2002 Annual Research Report
  • [Publications] Ohara-Imaizumi M., Nakamichi Y., Nishiwaki C., Nagamatsu S.: "Transduction of MIN6 β cell with TAT-Syntaxin SNARE Motif Inhibits Insulin Exocytosis in Biphasic Insulin Release in a Distinct Mechanism Analyzed by Evanescent Wave Microscopy"J. Biol. Chem.. 277. 50805-50811 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Ohara-Imaizumi M., Nagamatsu S.(他4名): "Monitoring of exocytosis and endocytosis of insulin secretory granules in the pancreatic β cell line MIN6 using pH-sensitive green fluorescent protein (pHluorin)and confocal laser microscopy"Biochem. J.. 363. 73-80 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Ohara-Imaizumi M, Nagamatsu S.(他3名): "Imaging exocytosis of single insulin secretory granules with evanescent wave microscopy"J. Biol. Chem.. 77. 3805-3808 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] 永松信哉, 今泉美佳: "インスリン開口放出機構とSNARE蛋白質"日本臨床. 60. 213-218 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] 今泉美佳, 永松信哉: "インスリン開口放出の可視化(ミニレビュー)"生化学. 75. 49-54 (2003)

    • Related Report
      2002 Annual Research Report

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Published: 2002-04-01   Modified: 2016-04-21  

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