The abnormal deposition and aggregation of beta-amyloid (Abeta) on brain tissues are considered to be one of the characteristic neuropathological features of Alzheimer's disease (AD). Environmental conditions such as metal ions, pH, and cell membranes are associated with Abeta deposition and plaque formation. According to the amyloid cascade hypothesis of AD, the deposition of Abeta42 oligomers as diffuse plaques in vivo is an important earliest event, leading to the formation of fibrillar amyloid plaques by the further accumulation of soluble Abeta under certain environmental conditions. In order to characterize the effect of metal ions on amyloid deposition and plaque growth on a solid surface, we prepared a synthetic template by immobilizing Abeta oligomers onto a N-hydroxysuccinimide ester-activated solid surface. According to our study using ex situ atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FT-IR), and thioflavin T (ThT) fluorescence spectroscopy, Cu2+ and Zn2+ ions accelerated both Abeta40 and Abeta42 deposition but resulted only in the formation of "amorphous" aggregates. In contrast, Fe3+ induced the deposition of "fibrillar" amyloid plaques at neutral pH. Under mildly acidic environments, the formation of fibrillar amyloid plaques was not induced by any metal ion tested in this work. Using secondary ion mass spectroscopy (SIMS) analysis, we found that binding Cu ions to Abeta deposits on a solid template occurred by the possible reduction of Cu ions during the interaction of Abeta with Cu2+. Our results may provide insights into the role of metal ions on the formation of fibrillar or amorphous amyloid plaques in AD.

Amyloidogenic proteins undergo an alternative folding pathway under stressful conditions leading to formation of ¢brils having cross L L-sheet structure, which is the hallmark of many neurodegenerative diseases. As a means of surviving against external stress, on the other hand, many microorganisms accumulate small stress molecules to prevent abnormal protein folding and to contribute to protein stability, which hints at the e⁄cacy of the solutes against amyloid formation. The current work demonstrates the e¡ectiveness of small stress molecules such as ectoine, betaine, trehalose, and citrulline on inhibition of insulin amyloid formation in vitro. The inhibitory e¡ects were analyzed by thio£avin T-induced £uorescence, circular dichroism, and atomic force microscopy. This report suggests that naturally occurring small molecules may serve a function that is typically ful¢lled by protein chaperones, and it provides a hint for designing inhibitors against amyloid formation associated with neurodegenerative disorders.

To investigate the folding behavior of amyloidogenic proteins under extreme temperatures, the kinetics of fibrillation and accompanying secondary structure transitions of bovine insulin were studied for temperatures ranging up to 140°C. The presence of extreme heat stress had traditionally been associated with irreversible denaturation of protein while the initial steps of such a denaturation process may be common with a fibril formation pathway of amyloidogenic proteins. The present work demonstrates the ability of insulin to form amyloid fibrils at above 100°C. Amyloid formation was gradually replaced by random coil generation after ∼80°C until no amyloid was detected at 140°C. The morphology of insulin amyloid fibrils underwent sharp changes with increasing the temperature. The dependence of amyloid formation rate on incubation temperature followed non-Arrhenius kinetics, which is explained by temperature-dependent enthalpy change for amyloid formation. The intermediate stage of amyloid formation and random coil generation consisted of a partially folded intermediate common to both pathways. The fully unfolded monomers in random coil conformation showed partial reversibility through this intermediate by reverting back to the amyloid pathway when formed at 140°C and incubated at 100°C. This study highlights the non-Arrhenius kinetics of amyloid fibrillation under extreme temperatures, and elucidates its intermediate stage common with random coil formation.Keywords: amyloid; insulin; extreme temperature; random coil; partially folded intermediate The boiling of protein solutions typically leads to irreversible and amorphous aggregation caused by breakage of hydrogen bonds, salt bridges, and disulfide bonds in a native protein structure (Hespenheide et al. 2002;Meersman and Heremans 2003). Thermal stress provides conformational freedom to polypeptide chains and rotational freedom to individual groups, thereby inducing the initial formation of partially folded intermediates (Makhatadze and Privalov 1995;Jayaraman et al. 1996). These intermediate structures with exposed hydrophobic patches coagulate together to form amorphous aggregates and precipitate out of the solution (Galani and Apenten 1999). In contrast, extremely thermostable proteins had been found in microorganisms isolated from habitats such as volcanoes, deep-sea hydrothermal vents, and hot springs (Stetter 1999;Huber et al. 2000). This extraordinary stability was attributed to a number of factors including increased rigidity, high packing efficiency, reduction of conformational strain, cooperative association, and absence of loose ends Robb and Maeder 1998). Despite their differences in thermostability, proteins from mesophilic or thermophilic sources are active only in a very narrow range of temperature, showing no activity outside this temperature range. The present work investigates the folding of bovine insulin that is functionally active under ambient conditions, but undergoes transformation to another "ordered" secondary Reprint requests...

The combinatorial approach: A library of fluorescent styryl dyes (320 compounds) was prepared by solid‐phase chemistry. The dyes were screened for their detection of amyloid aggregates, which are associated with diseases such as Alzheimer's, and two of the 320 compounds screened, 2C40 and 2E10, showed promise as brain‐imaging agents (see microscopy image).