| Project/Area Number |
12672031
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Periodontal dentistry
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| Research Institution | Tokyo Medical and DentaJ University |
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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)
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| 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)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| 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)
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| Keywords | cytocidal 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 |
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| 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
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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
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