| Co-Investigator(Kenkyū-buntansha) |
YAMAMOTO Akihiro Applied Research Laboratories, Chugai Pharmaceutical Co. Ltd., Chier Research Wo, 主任研究員
KOKRGUCHI Susumu Okayama University Odental School, Assistant, 歯学部, 助手 (10144776)
FUKUI Kazuhiro Okayama University Dental School, Assistant Professor, 歯学部, 助教授 (70034171)
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| Budget Amount *help |
¥8,100,000 (Direct Cost: ¥8,100,000)
Fiscal Year 1987: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1986: ¥6,800,000 (Direct Cost: ¥6,800,000)
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| Research Abstract |
1. Lipoteichoic acid(LTA) capable of induction of fumor necrosis factor (TND) were obtained from Streptococcus oyogrnes SV cells which were cultured aerobically in TTY borth at 37 C for 8 h. TNF-inducing ability wa s assayed as describen(Yamamoto et al. Birt. J. Gancer 51, 739-742,1985).
2. Crude LTA was exteacted from S. pyogenes cells with a mixture of 95% phenol-water (1:1,v/v) at room temp -erature (Moskowitz's method) and was filltered through Sepharose 6B column. Crude LTA was purified by digestion with DNase, RNase and proteinase successively, and finally passed through Sepharose 6B column.
3. S. pyogened LTA was treated with 47% HF ad 4 C for 3 days, and glyceroglycolipid fractions were sepata ted-and isolated by Bligh Dyer's method followed by thin layer chromatography on Silica gel plates.
4. Cells or cell envelopes of various gram-positive bacteris other tha S. pyogenes were also treated with phenol-water mixture (Moskowitz'sor ito's method). LTA was obtained from lactobacillus plantarum, Eubacterium nodatum, Ebucaterium alactolyticum, Listeria monocytogenes and Bacillus subtilis, whereas amphipathic substances (= not containing glycerol phosphate) were obtained from cells of Cornebacterium diphtheriae, propionibacterium acnes and Mycobacterium tuberculosis.
5. It has proves the LTA derived from cells of s. pyogenes, L. plantarum, E. alactolyticum, and gigulucosy ldigly-ceredi (one of the blyceroglyocoilpid fraction), adn the amphipathic sybstance of %m. tuberculosis were active to incude TNF. 6. TNF induced by LTA (LTA-TNF) inhibited the growth of L-929 cells as well as TNF induced by S almonella abortusequi lipopolysaccharide (LPS-TNF). Growth inhibition of L-929 cells caused by LTA-and LPS-TNF were inhibited by the same anti LPS-TNF serum. it has proved that LTA-TNF and LPS-TNF were antigenically rela ted. LPS-TNF was highly cytotoxic, bu LTA-TNF was not cytotoxic, for P. acnes-prined and balactosamine-l oaded mice.
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