| Project/Area Number |
11670282
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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 |
Bacteriology (including Mycology)
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| Research Institution | Suzuka National College of Technology |
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Principal Investigator |
IKIGAI Hajime Suzuka National College of Technology, Department of Chemistry and Biochemistry, Associate Professor, 生物応用化学科, 助教授 (60184389)
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| Project Period (FY) |
1999 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 2001: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2000: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1999: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Keywords | Vibrio cholerae / El Tor hemolysin / mouse intestinal mucus / Inhibitory factors / membrane damage / oligomer / cholesterol / protease / 溶血阻害因子 / トリプシン / 限定分解 / 細胞溶解 / 蛋白分解酵素 / マウス腸管粘膜 |
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| Research Abstract |
Vibrio cholerae 01 produces El Tor hemolysin (ETH ; 65 kDa), which expresses obvious strong cytotoxic effects. The ETH forms pore-forming transmembrane oligomers in lipid bilayer membranes that are permeable to smaller molecules less than 600 Da. We have shown that mouse small intestinal mucus (MSIM) inhibits hemolysis induced by ETH. Mechanism of the inhibition is not yet clear, but this leads to a speculation about a possible cytoprotective role of small intestinal mucus against toxin. Thus, we studied inhibitory factors of ETH in mouse small intestine and its inhibition mechanism.
We found that the toxin oligomers are formed by only MSIM under condition without membranes. Interestingly, we also found that ETH monomers were converted to oligomers in a suspension of cholesterol. Hemolysis of rabbit erythrocytes was not caused by oligomers isolated from artificial lipid bilayer membranes. These results suggest that hemolysis of rabbit erythrocytes was induced by ETH monomers, but not by the oligomers. The inhibition of hemolysis was induced by the formation of ETH oligomers by MSIM prior to encountering erythrocyte membranes, suggesting the possibility that cholesterol composed of mouse small intestine is an inhibitory factor.
We demonstrated that a 50-kDa truncated protein was yielded by the digestion of ETH monomers with trypsin or a-chymotrypsin, which are the major proteases in the mouse small intestines. These proteolytic enzymes inhibited ETH-induced hemolysis, suggesting that hemolytic activity of the 50-kDa truncated monomer was lower than that of the 65-kDa ETH monomer. It is clearly that proteases composed of MSIM are another inhibitory factor. Therefore, alternatively, hemolysis inhibition might be induced by proteolytic digestion of ETH monomers.
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