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A novel method for fluorescence microscopic demonstration of caspase activity

Research Project

Project/Area Number 10557166
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section展開研究
Research Field Functional basic dentistry
Research InstitutionNagasaki University School of Dentistry

Principal Investigator

SAKAI Hideaki  Nagasaki University School of Dentistry, Department of Pharmacology, Associate Professor, 歯学部, 助教授 (40225769)

Co-Investigator(Kenkyū-buntansha) MATSUDA Naoki  Nagasaki University Radioisotope Center, Associate Professor, アイソトープ総合センター, 助教授 (00304973)
AWAYA Akira  Mitui Pharmaceutical Co., Product Planning Chief, 製品計画部, 主席部員
KATO Yuzo  Nagasaki University School of Dentistry, Department of Pharmacology, Professor, 歯学部, 教授 (20014128)
SAKAI Eiko  Nagasaki University School of Dentistry, Department of Pharmacology, Research Assistant, 歯学部, 教務職員 (10176612)
Project Period (FY) 1998 – 1999
Project Status Completed (Fiscal Year 1999)
Budget Amount *help
¥10,800,000 (Direct Cost: ¥10,800,000)
Fiscal Year 1999: ¥4,500,000 (Direct Cost: ¥4,500,000)
Fiscal Year 1998: ¥6,300,000 (Direct Cost: ¥6,300,000)
Keywordscaspase / apoptosis / fluorescence substrate / nitric oxide (NO) / osteoclast / MNA / プロテアーゼ
Research Abstract

We have developed a 4-methoxy-2-naphthylamide (MNA)-based substrate, Z-Asp(OME)-Glu(OME) -Val-Asp(OME) -MNA (DEVD-MNA), which is relatively specific for caspase-3, one of the major caspases that triggers multiple apoptotic cellular degeneration. MNA, released by caspase-3 showed fluorescence with an exitation wave length of 340nm and emission wave length of 425nm. Using this substrate, we first determined the time course of caspase-3-like enzyme activity in cell lysates of murine osteoclast-like cells treated with nitric oxide (NO)-releaser, NOC18. Caspase-3-like enzyme activity showed a transient peak at 8 h after NOC18 treatment and decreased thereafter. MNA forms a Schiff-base complex with 5-nitrosalycylaldehyde (NSA) under mild acidic condition (〜pH 6.0), and the complex grows as needle-like fluorescence crystals. When DEVD-MNA and NSA were applied to NOC18-treated murine osteoclast-like cells (8 h), small needle-like fluorescence crystals were formed in the cells. The crystal formation was completely inhibited with caspase inhibitor DEVD- fenylmethylketone (FMK). Thus, intracellular activation of caspase(s) was visualized with this fluorescence substrate. After 16 h of NOC18-treatment, the crystal formation in cells was markedly reduced, and typical apoptotic morphologies of nuclear condensation and cell shrinkage, were observed. The efficiency of the crystal formation correlated well with changes in caspase-3-like enzyme activity measured by DEVD-MNA in vitro. The visualized caspase activity preceded the apoptotic morphological changes. The advantages of the method we have developed are (1) stained cells with this fluorescence substrate were fixable and (2) among mixed cell populations, such as osteoclast culture, cells undergoing apoptotic degeneration could be specifically detected.

Report

(3 results)
  • 1999 Annual Research Report   Final Research Report Summary
  • 1998 Annual Research Report
  • Research Products

    (27 results)

All Other

All Publications (27 results)

  • [Publications] Y. Kobayashi: "Effects of local administration of osteocalcin on experimental tooth movement"Angle Orthod.. 68. 259-266 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] T. Kamiya: "Fluorescence microscopic demonstration of cathepsin K activity as the major lysosomal cysteine proteinase in osteoclasts"J. Biochem.. 123. 752-759 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] A. Kawakami: "insulin-like growth factor I stimulate proliferation and Fas-mediated apoptosis of human osteoblasts"Biochem. Biophys. Res. Commun.. 247. 46-51 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] T. Yamaza: "Study of immunoelectron microscopic localization of cathepsin k in osteoclasts and other bone cells in the mouse femar"Bone. 23. 499-509 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] I. Moller: "Unregulated exposure of the ribosomal M-site caused by NAC depletion results in delivery of non-secretory polypeptidesto the Secbl complex"FEBS Lett.. 441. 1-5 (1998)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] E.T. Kobayashi: "Force-induced rapid changes in all fate at midpalatal suture cartilage of growing rats"J. Dent. Res.. 78. 1495-1504 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] A. kawakami: "Regulation of synorial cell apoptosis by proteasome"Arthritis. Rheum.. 42. 2440-2448 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] H. Sakai: "cell adhesion is a prerequisite for osteoclast survival"Biochem. Biophys. Res. Commun.. (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Y. Kobayashi, H. Takagi, H. Sakai, F. Hashimoto, S. Mataki, K. Kobayashi and Y. Kato: "Effects of local administration of osteocalcin on experimental tooth movement."Angle Orthod.. 68(3). 259-266 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] T. Kamiya, Y. Kobayashi, K. Kanaoka, T. Nakashima, Y. Kato, A. Mizuno and H. Sakai: "Fluorescence microscopic demonstration of cathepsin K activity as the major lysosomal cysteine proteinase in osteoclasts."J. Biochem.. 123. 752-759 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] A. Kawakami, T. Nakashima, M. Tsuboi, S. Urayama, N. Matsuoka, H. Ida, Y. Kawabe, H. Sakai, K. Migita, T. Aoyagi. M. Nakashima. K. Maeda. And K. Eguchi: "Insulin-like growth factor I stimulate proliferation and Fas-mediated apoptosis of human osteoblasts."Biochem. Biophys. Res. Commun.. 247. 46-51 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] T. Yamaza, T. Goto, T. Kamiya, Y. Kobayashi, H. Sakai and T. Tanaka: "Study of immunoelectron microscopic localization of cathepsin K in osteoclasts and other bone cells i the mouse femur."Bone. 23(6). 499-509 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] I. Moller, B. Beatrix, G. Kreibich, H. Sakai, B. Lauring, M. Wiedmann: "Unregulated exposure of the ribosomal M-site caused by NAC depletion results in delivery of non-secretary polypeptides to the Sec61 complex."FEBS Lett. 441(1). 1-5 (1998)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] E.T. Kobayashi, F. Hashimoto, Y. Kobayashi, E. Sakai, M. Miyazaki, T. Kamiya, K. Kobayashi, Y. Kato and H. Sakai: "Force-induced rapid changes in cell fate at midpalatal suture cartilage of growing rats."J. Dent. Res.. 78(9). 1495-1504 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] A. Kawakami, T. Nakashima, H. Sakai, A. Ida, S. Urayama, S. Yamazaki, H. Nakamura, H. Ida, Y. Ichinose, T. Aoyagi, I. Furuichi, M. Nakashima, K. Migita, Y. Kawabe and K. Eguchi: "Regulation of synovial cell apoptosis by proteaseme inhibitor."Arthritis. Rheum. 42(11). 2440-2448 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] A. Kawakami, T. Nakashima, H. Sakai, S. Urayama, S. Yamazaki, A. Hida, M. Tsuboi, H. Nakamura, H. Ida, K.Migita, H.Ida, Y. Kawabe and K. Eguchi: "Inhibition of caspase cascade by HTLV -I Tax through inhibitor."Blood. 94(11). 3847-3854 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] H. Sakai, Y. Kobayashi, E. Sakai, M. Shibata and Y. Kato: "Cell adhesion is a prerequisite for osteoclast survival."Biochem. Biophys. Res. Commun.. (in press). (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1999 Final Research Report Summary
  • [Publications] Kobayashi E.T.et al.: "Force induced rapicl changes in cell fate at miclpalatal suture cartilage of growing rats"J.Dent.Res.. 78・9. 1495-1504 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Kawakami A.et al: "Regulation of synovicl cell apoptosis by proteasome inhibitor"Arthritis.Rheum.. 42・11. 2400-2448 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Kawakami A.et al: "Inhibition of caspase cascade by HTLV-l Tax through induction of NF-KlJ-nuclear Translocstion"Blood. 94・11. 1-9 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] 金岡和博: "破骨細胞のアポトーシス"The BONE. 13・2. 57-64 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Y.Kobayashi: "Effects of local administration of Osteocalcin on experimental tooth movement" Angle Orthodont.68・3. 259-266 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] T.Kamiya: "Fluorescence microscopic demonstration of CathepsinK activity as the major lysosomal cysteine proteinase in osteoclasts" J.Biochem.123. 752-759 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] A.Kawakami: "Insulin-like growth factorI stimulate proliferation and Fasmediated apoptosis of human osteoblasts." Biochem.Biophys.Res.Commun.247. 46-51 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] T.Yamaza: "Study of immunoelectron microscopic localization of Cathepsink in osteoclasts and other bone cells in the mouse femur" Bone. 23・6. 499-509 (1998)

    • Related Report
      1998 Annual Research Report
  • [Publications] I.Moller: "Specificity of co-translational protein targeting to the ER-membrane is controlled by SRP and NAC" FEBS Let.(in press). (1999)

    • Related Report
      1998 Annual Research Report
  • [Publications] 金岡和博: "破骨細胞におけるアポトーシス" The Bone. (in press). (1999)

    • Related Report
      1998 Annual Research Report

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Published: 1998-04-01   Modified: 2016-04-21  

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