| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2004: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2003: ¥2,600,000 (Direct Cost: ¥2,600,000)
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| Research Abstract |
1.Monoclonal antibodies to epsilon toxin of Clostridum perfringens were prepared to determine the relation between the toxic activity of epsilon-toxin and its molecular structure. Epsilon-toxin was very toxic. Therefore, epsilon prototoxin was fixed with folmarin and the toxoid was used as antigen. Twelve clones of hybridoma cells producing monoclonal antibodies to epsilon toxin. were obtained by fuzing myeloma cells (SP2/0-Agl4) and spleen cells of the immunized BALB/c mouse. Seven lands of monoclonal antibodies neutralized the toxic activity of epsilon toxin. However, the amino acids sequences of their epitopes were not determined by phage peptide display cloning system because the higher-order structure of epsilon-toxin, but not the primary structure, was recognized by the monoclonal antibodies. In order to resolve this problem recombinant toxin fragments composedd of amino acids from Lys-14 to Glys-139, or from Met 77 to Lys 296 were used as antigen. Hybridoma cells producing monoclonal antibodies which react recombinant toxin fragments composed of amino acids from Ser-12 to Met-47, from Met-47 to Met 77, from Met 77 to Asn 109, from Gly-139 to Ale 173, or from IIe-206 to Lys-296 were obtained The purification of the monoclonal antibodies and analysis of the epitopes are presently in progress. Eleven of the nitones havebeen determined.
2.Epsilon toxin binds to and forms a heptameric pore within the microdomain of membranes, known as detergent resistant membranes (DRMs), of MDCK cells. Treatment of MDCK cells with fumonisin B1 and PDMP, inhibitors of sphingolipid and glucosphingolipid synthesis, respectively, increased the susceptibility, while D609, a sphingomyelin synthesis inhibitor, had the opposite effect. The exogenous addition of ganglioside GM1 dramatically decreased the epsilon toxin binding, heptamerization and cytotoxicity These results suggest that glycosphingolipid in DRMs interferes the cytotoxicity of epsilon-toxin.
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