| Research Abstract |
Long-term dialysis is very often complicated with amyloid deposits, predominantly in articular synovial membrane. The deposition is a serious complication of dialysis as it excessively deteriorates the quality of life of long-term dialysis patients. A major component of amyloid deposits was identified as β2-microglobulin (β2-m) having a molecular weight of 11,800 daltons. The details of amyloidogenesis of this type of amyloid remain unknown. In order to consider the pathogenesis of dialysis-related amyloidoses, it is essential to elucidate the general mechanisms of amyloid fibril formation in vitro. We first developed a fluorometric method to quantifiy amyloid fibrils in vitro, using the fluorescent dye, thioflavine T. Optimum fluorescence measurements of amyloid fibrils were obtained at the excitation and emission wavelengths of about 455 nm and 485 nm, respectively, and at pH 8.5-9.0. We focused our study on the extension phase of amyloid fibril formation in vitro. When amyloid fibri
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ls were incubated with their monomeric constituent, β2-m, the extension of amyloid fibrils was observed with electron microscopy. Quantitative fluorometry revealed that extension of amyloid fibrils proceeded by a pseudo-first-order exponential increase as measured by the fluorescence of thioflavin T and the net rate of extension was the sum of the rates of polymerization and depolymerization. Then we investigated the effect of advanced glycation end product (AGE) on fAβィイD22ィエD2-m extension in vitro, using the established first-order kinetic model of fAβィイD22ィエD2-m extension in vitro. During the incubation of fAβィイD22ィエD2-m with native βィイD22ィエD2-m at 37℃, the fluorescence of thioflavin T increased without a lag phase and proceeded to equilibrium. On the contrary, only a slight increase in fluorescence was observed during the incubation of fAβィイD22ィエD2-m with AGE-βィイD22ィエD2-m. Moreover, AGE-βィイD22ィエD2-m exhibited a dose-dependent inhibitory effect on the extension reaction of fAβィイD22ィエD2-m with nativeβィイD22ィエD2-m. These results may suggest that the modification ofβィイD22ィエD2-m with AGE does not play a promoting role in the formation of fAβィイD22ィエD2-m in vivo. Less
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