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Analysis of cell death inducing factors from periodontopathic bacteria.

Research Project

Project/Area Number 12672031
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Periodontal dentistry
Research InstitutionTokyo Medical and DentaJ 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) 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, Associate Professor., 大学院・医歯学総合研究科, 助教授 (90256678)
Project Period (FY) 2000 – 2001
Project Status Completed (Fiscal Year 2001)
Budget Amount *help
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2001: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2000: ¥1,800,000 (Direct Cost: ¥1,800,000)
Keywordscytocidal toxin / periodontopathic bacteria / Bacteroides forsythus / G2 arrest / apoptosis / periodontitis / cell cycle / CDT (cytolethal distending toxin) / 殺細胞因子 / 細胞死誘導因子 / Actinobacillus actinomycetemcomitans / ネクローシス
Research Abstract

1. The effects of the cytocidal toxin
In squamous cell carcinoma, epithelial, and hematopoietic cell lines, cell death was induced dose-dependently by B. forsythus ultrasonic extract by the evaluation of WST-1 assay. These results suggest that this factor might induce dell death through a common receptor or non-specifically.
2. The ultrastructural change in HCT 116 cells treated with bacterial extract
Severe membrane ruffling was observed in SEM analysis (this is not a characteristic feature of apoptosis), Fragmentation and aggregation of nuclei, cytoplasmic vacuolization and swelling, and loss of plasma membrane integrity were demonstrated in TEM analysis.
3. Purification of the cytocidal toxin
B. forsythus extract was purified 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 bacterial DNA. Therefore, An … More apparent shift of cytocidal toxicity to 28 kDa fraction was observed following DNase I treatment. Since these results strongly suggest that this toxin is a DNA binding protein, further purification was carried out by heparin column.
4. Preparation of anti-cytocidal toxin monoclonal antibodies
A mouse was immunized with B. forsythus sonic extract, and the activated lymphocytes were collected from a swollen lymphatic node. The lymphocytes were immediately fused with mouse myeloma cell line. The clones with anti-cytocidal activity against MA1 cells were isolated from hybridomas.
5. The effects of cell cycle of MA1 cells
MA1 cells treated with purified toxin were incubated with anti-human p53 and mouse anti-human Cyclin B1 antibodies, and were stained with Cy-5 and FITC labeled antibodies, respectively. Stained cells were subjected to flowcytometry. High levels of p53 and Cyclin B1 were observed at G2 phase in cells treated with this toxin.
These results suggest that this toxin is a novel member of CDT family. Less

Report

(3 results)
  • 2001 Annual Research Report   Final Research Report Summary
  • 2000 Annual Research Report
  • Research Products

    (21 results)

All Other

All Publications (21 results)

  • [Publications] Shinichi Arakawa: "Novel apoptosis-inducing activity in Bacteroides forsythus : a comparative study with three serotypes of Actinobacillus actinomycetemcomitans"Infection and Immunity. 68・8. 4611-4615 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] 荒川 真一: "歯周病原性細菌の保有する細胞死誘導活性"口腔病学会雑誌. 68・1(発表予定). (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] 荒川 真一: "Bacteroides forsythusの保有するアポトーシス誘導活性について"歯科基礎医学会雑誌. 42・5. 111 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Shinichi Arakawa: "Induction of apoptosis by a novel factor isolated from Bacteroides forsythus"Journal of Dental Research. 79・special issue. 233 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] 中島 琢磨 他: "歯周病原性細菌の産生する細胞死誘導因子"歯科基礎医学会雑誌. 43・5. 88 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] 荒川 真一: "歯周病原性細菌の産生する細胞死誘導因子の解析"日本歯周病学会雑誌. 43. 45 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Shinichi Arakawa: "Analysis of cell death inducing factors isolated from periodontopathic bacteria"Journal of Dental Research. 80. 768 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Shinichi Arakawa, Takuma Nakajima, Hiroaki Ishikura, Isao Ishikawa, and Nobuo Tsuchida.: "Induction of apoptosis by a novel factor isolated from Bacteroides forsythus"Journal of Dental Research. 79. 233 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Shinichi Arakawa, Takuma Nakajima, Hiroaki Ishikura, Isao Ishikawa, and Nobuo Tsuchida.: "Analysis of cell death inducing factors isolated from periodontopathic bacteria"Journal of Dental Research. 80. 768 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Shinichi Arakawa, Takuma Nakajima, Hiroaki Ishikura, Shizuko Ichinose, Isao Ishikawa, and Nobuo Tsuchida.: "Novel Apoptosis-Inducing Activity in Bacteroides forsythus ; a Comparative Study with Three Serotypes of Actinobacillus actinomycetemcomftans"Infect Immun.. 68. 4611-4615 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2001 Final Research Report Summary
  • [Publications] Shinichi Arakawa: "Novel apoptosis-inducing activity in Bacteroides forsythus : a comparative study with three serotypes of Actinobacillus actinomycetemcomitans"Infection and Immunity. 68・8. 4611-4615 (2000)

    • Related Report
      2001 Annual Research Report
  • [Publications] 荒川 真一: "歯周病原性細菌の保有する細胞死誘導活性"口腔病学会雑誌. 68・1(発表予定). (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] 荒川 真一: "Bacteroides forsythusの保有するアポトーシス誘導活性について"歯科基礎医学会雑誌. 42・5. 111 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Shinichi Arakawa: "Induction of apoptosis by a novel factor isolated from Bacteroides forsythus"Journal of Dental Research. 79・(special issue). 233 (2000)

    • Related Report
      2001 Annual Research Report
  • [Publications] 中島 琢磨 他: "歯周病原性細菌の産生する細胞死誘導因子"歯科基礎医学会雑誌. 43・5. 88 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] 荒川 真一: "歯周病原性細菌の産生する細胞死誘導因子の解析"日本歯周病学会雑誌. 43. 45 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Shinichi Arakawa: "Analysis of cell death inducing factors isolated from periodontopathic bacteria"Journal of Dental Research. 80. 768 (2001)

    • Related Report
      2001 Annual Research Report
  • [Publications] Shinichi Arakawa: "Novel apoptosis-inducing activity in Bacteroides forsythus : a comparative study with three serotypes of Actinobacillus actinomycetemcomitans."Infection and Immunity. 68・8. 4611-4615 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] 荒川真一: "歯周病原性細菌の保有する細胞死誘導活性"口腔病学会雑誌. 68・1(発表予定). (2001)

    • Related Report
      2000 Annual Research Report
  • [Publications] 荒川真一: "Bacteroides forsythusの保有するアポトーシス誘導活性について"歯科基礎医学会雑誌. 42・5. 111 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Shinichi Arakawa: "Induction of apoptosis by a novel factor isolated from Bacteroides forsythus."Journal of Dental Research. 79・special issue. 233 (2000)

    • Related Report
      2000 Annual Research Report

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Published: 2000-04-01   Modified: 2016-04-21  

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