1998 Fiscal Year Final Research Report Summary
Analysis of the virulence factors from periodontopathic bacteria
| Project/Area Number |
08672095
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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 |
Morphological basic dentistry
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| Research Institution | TOKYO DENTAL COLLEGE |
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Principal Investigator |
KATO Tetsuo Tokyo Dental College, Faculty of Dentistry, Assocate Professor, 歯学部, 助教授 (00159253)
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| Co-Investigator(Kenkyū-buntansha) |
YAMANAKA Ayumi Tokyo Dental College, Faculty of Dentistry, Assistant, 歯学部, 助手 (40231667)
MIURA Tadashi Tokyo Dental College, Faculty of Dentistry, Assistant, 歯学部, 助手 (10266570)
ISHIHARA Kazuyuki Tokyo Dental College, Faculty of Dentistry, Assistant Professor, 歯学部, 講師 (00212910)
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| Project Period (FY) |
1996 – 1998
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| Keywords | periodontitis / cytokines / Actinobacillus actinomycetemcomitans / Porphyromonas gingivalis / hemolytic activity / cell surface polysaccharide / endotoxin / monoclonal antibody |
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| Research Abstract |
Actinobacillus actinomycetemcomitans, a gram-negative capnophlic rod, has been implicated in human periodontal diseases. This microorganism produces a number of virulence factors. Recently several investigators have been reported that strains of A.actinomycetemcomitans express hemolytic activity. In order to examine the potential role of the. hemolytic activity in the process of periodontal diseases, we have isolated a hemolytic gene from strain ATCC 43718. DNA hybridization analysis indicates that the hemolytic gene is conserved among all A.actinomycetemcomitans strains tested. The. complete nucleotide sequence of the gene has been determined. The deduced amino acid sequence. corresponds to a basic protein of 9.5 kDa. Sonicated extract from Escherichia coli clone possessed hemolytic activity on horse, sheep and human erythrocytes but not those of rabbit. To determine whether the antiserum against A.actinomycetemcomitans reacted with a protein of the sonic extract of the E.coli clone w
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as analyzed by Western blot analysis. Rabbit antiserum to A.actinomycetemcomitans ATCC 43718 whole cells reacted with sonic extract of E.coli clone at Ca. lOkDa which was dependent upon insert DNA of A.Actinobacillus actinomycetemcomitans wornztans on the plasmid. Also the rabbit antiserum against ATCC 43718 inhibited the hemolytic activity of the clone. These results indicate that a gene coding hemolytic activity has been isolated from Actinobacillus actinomycetemcomitans.
Two polysaccharide fractions, A and B, were prepared from autoclaved extract of A.actinomycetemcomitans ATCC 43718 (serotype. b) by DEAB anion-exchange chromatography. LPS was extracted by hot phenol- water procedure. Ouchterlony analysis demonstrated that LPS and fraction A reacted with rabbit antisera against A.actinomycetemcomitans serotypes a and b but not with antiserum to serotype c. Fraction B was found to react with antiserum to serotype b but not with antisera specific for other serotypes, suggesting that fraction consists of a serotype b specific polysaccharide antigen.
Porphyromanas gingivalis, a gram-negative anaerobic rod, has been implicated as a periodontal pathogen. Interleukin-6 (IL-6) is a multifunctional cytokine that is produced by numerous cell types, including macrophages, fibroblasts and endothelial cells, and play a central role in host defense mechanisms. In the present study, we investigated the effect of cell surface components of P.gingivalis on IL-6 production by human gingival fibroblasts. The release of IL-6 by these cells was measured using ELISA systems specific for IL-6 according to the manufacture's recommended procedures. Cell surface polysacebaride and outer membrane proteins upregulated the IL-6 production by human gingival fibroblast cell lines as well as LPS.These activities were dependent on the increase of the preparations dose. Less
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[Publications] Kato, T., Hirai, K., Okuda, K., Fournier, D., Menard, C.and Mouton, C.: "Animals are not reservoir for the human periodontal pathogen Porphyromonas gingivalis" Dentistry in Japan. 33. 27-30 (1997)
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[Publications] Okuda, K., Kigure, T., Yamada, S., Kaneko, T., Ishihara, K., Miura, T., Kato, T.and Takazoe, I.: "Role for the S-layer of Campylobacter rectus ATCC 33238 in complement mediated killing and phagocytic killing by leukocytes from guinea pig and human peripheral blood" Oral Diseases. 3. 113-120 (1997)
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[Publications] Honma, K., Ishii, N., Kato, T., Ishihara, K., Okuda, K.and Okuda, K.: "Genetic control of immune responses to a synthetic fimbrial antigen of Actinobacillus actinomycetemcomitans" Microbiol.Immunol.41. 609-614 (1997)
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