| Project/Area Number |
03670208
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
細菌学
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| Research Institution | Kanazawa University |
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Principal Investigator |
NAKAMURA Shinichi Kanazawa Univ., Dep., Med., Prof., 医学部, 教授 (90019620)
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| Co-Investigator(Kenkyū-buntansha) |
YAMAKAWA Kiyotaka Kanazawa Univ., Dep., Med., Asst. Prof., 医学部, 講師 (20110629)
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| Project Period (FY) |
1991 – 1992
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| Project Status |
Completed (Fiscal Year 1992)
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| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1992: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1991: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | C. difficile / Intracellular toxin A / Extracellular toxin A / Haemagglutination activity / Affinity chromatography / Pseudomembranous colitis / Antibiotic-associated diarrhea / Neutralisation / C,difficile / トキシンA / アフィニティクロマトグラフィ- |
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| Research Abstract |
After sonic disintegration of Clostridium difficile cells, intracellular toxin A was purified to homogeneity by thyroglobulin affinity chromatography (TGAC) followed by successive anion- exchange chromatography on Mono Q incorporated into fast protein liquid chromatography (FPLC) apparatus. High hemagglutination (HA) activity was detected in TGAC-unbound fractions (2^9), but not in TGAC thermal eluates (2^0). The low HA titer of the thermal eluates was markedly increased to 2^5 after dialysis against 0.02 M Tris-HCl (pH 7.5). A disparity in the position of the peak between cytotoxicity and HA activity was observed in the first Mono Q-FPLC. Intracellular toxin A without HA activity was obtained by second Mono Q-FPLC. The molecular weight of the intracellular toxin A was estimated by polyacrylamide gel electrophoresis to be 580 kDa in non-denaturing condition. The minimum doses of the toxin causing cytotoxicity, mouse lethality and enterotoxicity were 0.83 ng, 8.7 ng and 5 ug, respectively. Intracelluler toxin A and extracellular toxin A were compared serologically. In Ouchterlony test, anti-intracellular toxin A and anti-extracellular toxin A sera showed identity of the intracellular and extracellular toxin A. In neutralization tests, both anti-intracellular and anti-extracellular toxin A sera alternatively neutralized the heterologous toxins for cytotoxicity, mouse lethality and loop response at nearly the same titers (1:128 - 1:256) as did the homologous toxins. Although intracellular toxin A lacked HA activity, anti-intracellular toxin A neutralized HA activity of the extracellular toxin A at the same titer (1:16) as anti-extracellular toxin A serum. These findings suggest a possibility that toxin A is synthesized as HA-negative form in cells, which has a few binding sites to the erythrocytes, and converted to HA-positive form,when it is released from cells, or after release from cells.
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