| Co-Investigator(Kenkyū-buntansha) |
MIYATA Shigeru Kagawa Med. Univ. Assistant Professor, 医学部, 助手 (90314913)
KATAYAMA Seiichi Kagawa Med. Univ. Assistant Professor, 医学部, 助手 (70169473)
MATSUSHITA Osamu Kagawa Med. Univ. Associated Professor, 医学部, 助教授 (00209537)
TOKUDA Masaaki Kagawa Med. Univ. Professor, 医学部, 教授 (10163974)
KOBAYASHI Ryoji Kagawa Med. Univ. Professor, 医学部, 教授 (00020917)
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| Budget Amount *help |
¥14,300,000 (Direct Cost: ¥14,300,000)
Fiscal Year 2000: ¥4,700,000 (Direct Cost: ¥4,700,000)
Fiscal Year 1999: ¥9,600,000 (Direct Cost: ¥9,600,000)
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| Research Abstract |
In order to examine distribution of Clostridium perfringens epsilon-toxin (ε-toxin) in tissues of intoxicated animals, ^<125>I-labelled ε-toxin was injected intravenously into a mouse, and then subjected to whole body autoradiography. Although the toxin was detected mainly in the brain and kidneys, it was also in the thyroids, stomach, and salivary glands. Since iodolysis might have occurred after the intoxication, the whole body autoradiography was performed using ^<14>C-labelled ε-toxin. The labelled toxin was detected exclusively in the brain, spinal cord and kidneys. Analysis by immunohistochemistry revealed that the toxin was distributed mainly in the hippocampus and the glomerulus. However, other areas were also stained, and proteins in these tissues were shown to be cross-reactive with the antibody. An attempt has been made to increase the specificity of the immunoreactivity by using pre-adsorbed polyclonal or monoclonal antibodies. The action of ε-toxin on the rat synaptosomal
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membrane was investigated by using ^<125>I-labelled ε-toxin. While the ε-toxin monomer was initially detected in the membrane, it formed a large complex with incubation time. The large complex was shown to consist of a heptamer of ε-toxin. When the toxin and inactive protoxin were incubated with the membrane, the heptamerization of the monomer but not its association with the membrane was inhibited dose-dependently by the protoxin. These results suggest that a C-terminal propeptide, which was cleaved upon activation by tryptic digestion, does not affect the binding of ε-toxin to its receptor, while it masks a region necessary for the heptamerization. Several cultured neuronal cells were tested for ε-toxin cell toxicity. Either undifferentiated or differentiated cells were not sensitive to the toxin. A method for the isolation and identification of an ε-toxin binding receptor from the membrane fraction of the calf brain have been established : preparation of large quantities of the synaptosomal membrane, differential solubilization, immunological detection such as slot blotting and western blotting, and affinity chromatography. A few proteins including predominant 45 K protein, which seemed to possess a high affinity to the toxin, were isolated from the calf brain cell membrane and also from the calf kidney cell membrane. Identification and purification of these proteins are now in progress. Less
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