| Project/Area Number |
13470060
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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 | Kagawa Medical University |
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Principal Investigator |
OKABE akinobu Kagawa Medical University, Molecular Microbiology, Professor, 医学部, 教授 (20093677)
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| Co-Investigator(Kenkyū-buntansha) |
KOBAYASHI ryoji Kagawa Medical University, Signal Transduction Sciences, Professor, 医学部, 教授 (00020917)
MIYATA shigeru Kagawa Medical University, Molecular Microbiology, Assistant professor, 医学部, 助手 (90314913)
MATSUSHITA osamu Kagawa Medical University, Molecular Microbiology, Associated professor, 医学部, 助教授 (00209537)
TOKUDA masaki Kagawa Medical University, Physiology, Professor, 医学部, 教授 (10163974)
TOKUMITSU hiroshi Kagawa Medical University, Signal Transduction Sciences, Associated professor, 医学部, 助教授 (20237077)
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| Project Period (FY) |
2001 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥10,300,000 (Direct Cost: ¥10,300,000)
Fiscal Year 2002: ¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2001: ¥6,900,000 (Direct Cost: ¥6,900,000)
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| Keywords | Clostridium perfringens / Epsilon-toxin / Lipid rafts / Histopathology / Nephrotoxicity / Immunohistochemsitry / Receptor / MDCK cell / Histopathology / ウェルシュ菌 / 構造解析 / 腸性毒素血症 / ε毒素 / モノクローナル抗体 / 脂質ラフト / 膜孔形成型毒素 |
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| Research Abstract |
Clostridium perfringens epsilon-protoxin, in which His6 was N-terminally tagged and a factor Xa cleavage site was generated to cleave an N-terminal propeptide, was replaced with Se-methionine. The Se-methionine protoxin was purified, and then the N-terminal propeptide was cleaved off with factor Xa, followed by crystallization. Although the resulting crystal was shown to be twined, we are now attempting to solve the three-dimensional structure of the protoxin by computer analysis.
We showed that epsilon-toxin (e-toxin) assembles to a heptameric pore within the lipid rafts of the rat synaptosome and Madin-Darby canine kidney (MDCK) cell membranes. To assess how physicochemical properties of the lipid rafts affect e-toxin assembly, we change major lipid constituents, cholesterol and gangliosides of MDCK cells. The heptamerization of e-toxin and its cytotoxicity towards MDCKcells was decreased by depletion of cholesterol, and was adversely stimulated by inhibition of gangliosides synthesis
… More
, suggesting that alteration in a lipid rafts environment modulates the assembly and/or the insertion of the toxin therein.
In an attempt to study the molecular pathology of e-toxin enterotoxeamia, we examined the distribution of e-toxin by whole body autoradiography involving mice injected intravenously with 35S-labeled e-toxin. The toxin was most prominently distributed in the kidneys, and fairly abundantly in the brain, spinal cord, and nasal turbinates. Immunostaining of the kidneys showed that the toxin was detected mainly in the glomeruli and capillaries, and that it was also detectable in the distal tubules and collecting ducts. Although histological examination showed some pathological changes, e.g. shrinkage of glomeruli and degeneration of epithelial cells in the distal tubules and collecting ducts, they were not so severe as those found in the brain such as neuronal cell damage and perivascular edema. The biological relevance of the toxin accumulation in the kidneys was approached by examining an effect of nephrectomy on the lethal toxicity of e-toxin against mice. The nephrectomy shortened the time required for the toxin to kill mice. When mice was intoxicated with botulinus toxin or C. perfringens alpha-toxin, such an effect of nephrectomy was not observed. Based on these results, we propose that the kidneys contribute to the host defense by accumulating circulating e-toxin and thereby protecting the brain from a lethal damage.
A cDNA clone encoding for a portion of a putative e-toxin receptor has been isolated by a yeast two-hybrid system. Studies are currently under way to identify the corresponding whole receptor protein and also to characterize molecular mechanism of e-toxin cytotoxicity involving e-toxin-resistant clones isolated fromMDCK cells Less
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