FIGURE 7.

Self-seeding effect and cytotoxicity of prefibrillar and mature amyloid fibrils. The prefibrillar intermediate and mature fibrils formed by heat treatment in the presence of 1.0 m NaCl were used as seeds. A, the time course for the seed-dependent extension reactions performed in a 25 mm HCl solution containing 0.1 m NaCl. In these reactions, 30 μg/ml of prefibrillar intermediate (●) and mature fibrils (Δ) were seeded. The time courses in the absence of seeds (open inverted triangles) and presence of conventional insulin fibrils obtained by repeated self-seeding at 0.1 m NaCl (■) were also plotted for negative and positive controls, respectively. B, cytotoxic effect in PC12 cells upon the addition of native insulin without heat treatment for prefibrillar intermediate, mature fibrils, and conventional insulin fibrils obtained by the repeated self-seeding of fibrils originally formed at 0.1 m NaCl. The concentration of fibrils added was 8.5 μm and the incubation time was 48 h. Fractions of surviving cells, which were calculated from the activity of lactate dehydrogenase released from dead cells, are represented in this figure. All measurements were performed three times and the error bars depict the S.D. ± mean. *, p < 0.0001.