2003 Fiscal Year Final Research Report Summary
Isolation of the Cytocidal toxin (CCT) from periodontopathic bacteria and antibody preparation against CCT: with a view to clinical application.
| Project/Area Number |
14571978
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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 Dental 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) |
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)
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| Project Period (FY) |
2002 – 2003
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| Keywords | cytocidal toxin (CCT) / periodontopathic bacteria / Tannerellaforsythensis / G2 arrest / apoptosis / periodontitis / cell cycle / CDT (cytolethal distending toxin). |
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| 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
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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
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Research Products
(9 results)
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[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)
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