| Project/Area Number |
08457087
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Bacteriology (including Mycology)
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| Research Institution | Koshien University (1997)
Osaka University (1996) |
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Principal Investigator |
MATSUDA Morihiro Koshien university, College of Nutrition, Professor, 栄養学部, 教授 (20029771)
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| Co-Investigator(Kenkyū-buntansha) |
SUGUMOTO Nakaba Osaka University, Res. Inst. Microb. Dis., Bact. Tox., Assoc. Prof., 微生物研究所, 助教授 (20142317)
片平 じゅん 大阪大学, 微生物病研究所, 助手 (30263312)
堀口 安彦 大阪大学, 微生物病研究所, 助手 (00183939)
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| Project Period (FY) |
1996 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥6,600,000 (Direct Cost: ¥6,600,000)
Fiscal Year 1997: ¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1996: ¥4,700,000 (Direct Cost: ¥4,700,000)
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| Keywords | Tetanus toxin / Diphtheria toxin / Toxin fragment / Recombined toxin fragments / Cytotoxicity / Toxic action / ヒトモノクロナール抗体 |
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| Research Abstract |
Tetanus and diphtheria toxins were highly purified by procedures includind HPLC.Two complementary polypeptide fragments from each toxin were separated and highly purified, using a Mono Q column in an FPLC system, from toxins dissociated by reduction with dithiothreitol (DTT) and treatment with 4M-2M urea. Complexes composed of an N-terminal enzymatic, active frament and a C-terminal cell-binding fragment from the different toxins were prepared by using CDI [1-ethyl-3- (3-L-dimethyl aminopropyl) carbodiimide-HCl] or SPDP [N-succinimidyl 3- (2-pyridyldithio) propionate] or by oxidation and removing urea by dialysis from a mixture of the dissociated toxins. Cytotoxicity on Vero and PC12 cells, dermonecrotizing activity and lethal, neurotoxic activities on guinea pigs of the complexes were examined, In contrast to the oridinal toxins and the respectivereconstituted toxins, these complexes composed of an enzymatic active fragment and a cell-binding fragment from different origins elicited only slight toxicity. But the complexes composed of the diphtheria toxin fragment A or diphrheria toxin and a nerve growth factor prepared by using CDI elicited cholinergic neuron-specfic cytotoxicity, indicating that the fragment A in the complex pepeared using CDI retained its enzymatic activity. The mixture of reconstituted complexes from the mixture of DTT-reduced, urea-dissociated toxins appeared to show an increase in cytotoxicity on Vero cells. So the cytotoxicity of the isolated and purified preparations of reconstituted and recombined materials are now being investigaed. The effects of the purified recombined toxins on noradrenaline release from neurosecretoy PC12 cell and lethal, neurotoxic activity of these materials on guinea pigs and diphtheria toxin-resistant rats and mice are being investigated together with the neutralizing effects of antibodies against each toxin-fragment.
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