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Isolation of the Cytocidal toxin (CCT) from periodontopathic bacteria and antibody preparation against CCT: with a view to clinical application.

Research Project

Project/Area Number 14571978
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Periodontal dentistry
Research InstitutionTokyo Medical and Dental University

Principal Investigator

ARAKAWA Shinichi  Tokyo Medical and Dental University, Dental Hospital Periodontal Clinic, Research Associate, 歯学部附属病院, 助手 (20302888)

Co-Investigator(Kenkyū-buntansha) ISHIKAWA Isao  Tokyo Medical and Dental University Graduate School, Department of Periodontology, Professor, 大学院・医歯学総合研究科, 教授 (10014151)
TSUCHIDA Nobuo  Tokyo Medical and Dental University Graduate School, Department of Molecular and Cellular Oncology and Microbiology, Professor, 大学院・医歯学総合研究科, 教授 (60089951)
NAKAJIMA Takuma  Tokyo Medical and Dental University Graduate School, Department of Molecular and Cellular Oncology and Microbiology, Professor, 大学院・医歯学総合研究科, 助教授 (90256678)
Project Period (FY) 2002 – 2003
Project Status Completed (Fiscal Year 2003)
Budget Amount *help
¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 2003: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2002: ¥1,500,000 (Direct Cost: ¥1,500,000)
Keywordscytocidal toxin (CCT) / periodontopathic bacteria / Tannerellaforsythensis / G2 arrest / apoptosis / periodontitis / cell cycle / CDT (cytolethal distending toxin). / Tonnerella forsythensis / 細胞死誘導因子(殺細胞因子:CCT) / G2 arrest / アポトーシス / CDT(cytolethal distending toxin)
Research Abstract

Tannetella forsythensis: one of the important periodontpathic bacteria which possesses the cytocidal toxin (TfCCT), lipoprotein, sialidase as a putative virulence factor. Among these, TfCCT might play an important role in initiation and progression of periodontitis and therefore it was of interest of further characterizing this factor.
1. Purification of the cytocidal toxin (TfCCT): TfCCT was purified from T.forsythensis extract by using DEAE Sepharose column and size-exclusion chromatography, and cytocidal activity was detected in two peak fractions corresponding to 28 and over 400kDa. Comparing the absorbance at 254 nm suggest that this toxin may interact with DNA. This toxin is a DNA binding protein, further purification was carried out by heparmn column.
2. Preparation of anti-cytocidal toxin monoclonal antibodies: Anti-cytocidal toxin monoclonal antibodies that are capable of removing cytocidal toxicity in Tforsythensis sonic extract with protein G-beads were established. As a resul … More t, anti-TfCCT antibody (Me-24) was identified.
3. Cloning of CCT-producing clone: The T.forsythensis genoniic library was constructed by using ZAP Express predigested Gigapack cloning kits. For screening, the oligonucleotide degenerative DNA based on the N-terminal amino acid sequence of purified CCT protein was used as a probe. Sequencing of this clone revealed that the size of the insert fragment was 2.1 kbp and there were putative four ORFs (CCT1, 2, 3, and 4). There were high homology in sequence between CCT and prtH
4. Evaluation of mechanisms of cytocidal activity: the effects to cell cycle: The recombinant proteins were isolated in vitro and were subjected to analyze the effect of cell cycle arrest or the levels of CyclinBi and p53 at each cell stage. Stained cells were subjected to flowcytometry. High levels of p53 and CyclinB1 were observed at G2 phase in cells treated with this toxin.
5. Application for clinical use: This bacterium which has been supposed to have the cytocidal toxin increased in periodontitis patients. We are attempt to analyze the relationship between the levels of antibody titer against CCT or those of CCT protein and the clinical status of periodontitis for the purpose of establishing the simple examination of periodontitis (ELISA, Less

Report

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

    (17 results)

All Other

All Publications (17 results)

  • [Publications] Hasebe, A.: "Biological activities of Bacteroides forsythus lipoproteins and their possible pathological roles in periodontal disease."Infect Immun. 72. 1318-1325 (2004)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Ishikura I.: "Cloning of the Tannerella forsythensis (Bacteroides forsythus) siaHI gene and purification of the sialidase enzyme."J Medical Microbiol. 52. 1101-1107 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Arakawa S.: "Isolation and functional analysis of Tannerella forsythensis Cytocidal toxin (CCT)"J Dental Research. 84. 268 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] 荒川 真一: "Tannerella forsythensis由来細胞致死毒素の精製及び機能解析"日本歯科保存学会雑誌. 46. 28 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] 中島 琢磨: "Bacteroides forsythusの産生する殺細胞因子の機能"歯科基礎医学会雑誌. 44・5. 92 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] 長谷部 明: "Bacteroides forsythus由来リポタンパク質の歯周病における病因的役割について"歯科基礎医学会雑誌. 44・5. 151 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] 石川 烈: "歯科医のためのスケーリング、ルート・プレーニング"クインテッセンス出版、佐々木一高. 134 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Hasebe, A., Yoshimura A., Kataoka H., Tanaka S., Arakawa S., Ishikura H., Golenbock D.T., Sugaya T., Tsuchida N., Kananii M., Hara Y., Shibata K.: "Biological activities of Bacteroidesforsythus lipoproteins and their possible pathological roles in periodontal disease."Infect Immun. 72. 1318-1325 (2004)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Ishikura H, Arakawa S, Nakajima T, Tsuchida N, Ishikawa I: "Cloning of the Tannerellaforsythensis (Bacteroidesforsythus) siaHl gene and purification of the sialidase enzyme."J Med Microbiol. 52. 1101-1107 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Hasebe, A.: "Biological activities of Bacteroides forsythus lipoproteins and their possible pathological roles in periodontal disease."Infect Immun. in press. (2004)

    • Related Report
      2003 Annual Research Report
  • [Publications] Ishikura I.: "Cloning of the Tannerella forsythensis(Bacteroides forsythus)siaHI gene and purification of the sialidase enzyme."J Medical Microbiol. 52. 1101-1107 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] 石川 烈: "歯科医のためのスケーリング、ルート・プレーニング"クインテッセンス出版. 134 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] 荒川 真一: "Tannerella forsythensis由来細胞致死毒素の精製及び機能解析"日本歯科保存学会雑誌. 46. 28 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] 中島 琢磨: "Bacteroides forsythusの産生する殺細胞因子の機能"歯科基礎医学会雑誌. 44・5. 92 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] 長谷部 明: "Bacteroides forsythus由来リポタンパク質の歯周病における病院的役割について"歯科基礎直学会雑誌. 44・5. 151 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] 中島 琢磨: "Bacteroides forsythusの産生する殺細胞因子の機能"歯科基礎医学会雑誌. 44・5. 93 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] 長谷部 晃: "Bacteroides forsythus由来リポタンパク質の歯周病における病因的役割について"歯科基礎医学会雑誌. 44・5. 151 (2002)

    • Related Report
      2002 Annual Research Report

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

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