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A study was conducted to examine the spontaneous formation of amyloid [beta] fibrils on the quartz surface. The results provide insight into nucleation process, fibril morphology which affect the cytotoxicity of fibril and taxonomy of amyloid supramolecular assemblies.

[[beta].sub.2]-microglobulin, a protein responsible for dialysis-related amyloidosis, is used to achieve heat-triggered conversion of the salt-induced thin and flexible protofibrils into well-organized thick and straight mature amyloid fibrils. The results have highlighted the importance of the change in heat capacity in addressing the biological significance of interactions between solvent water and amyloid fibrils.

Various methodologies including spectrophotometry, spectrofluorometry and surface plasmon resonance (SPR) are used to study the formation of Alzheimer's [beta]-amyloid fibrils (fA[beta]) by various anti-amyloidogenic compounds. The in vitro experiments reveal that flavonoids inhibit a preferential and reversible binding to the amyloid fibril structure of fA[beta], which leads to the amyloidogenic effect against the fibrils.

A cosolvent system is reported that induces the fibrous aggregation of human tau four-repeat domain. The flexibility of the present system allows to inspect the actual role of anions in forming the fibrous aggregates.

Research has been conducted on the beta-amyloid fibrils. The kinetic model of fibrils' extension and dissociation has been investigated via the use of surface plasmon resonance biosensor and the results are reported.

Research has been conducted on the amyloid fibril formation of the mouse V (sub)L domain. The effect of the destabilized conformation on the amyloid fibril formation is discussed.

Research has been conducted on the beta2-microglobulin-related (A(beta)2M) amyloidosis. The results of the fluorescence spectroscopic analysis with thioflavin T and electron microscopic study demonstrate that A(beta)2M amyloid fibrils depolymerize into the monomeric beta2-m at a neutral to basic pH.

Results from in vitro experiments indicate that beta-amyloid fibril formation is dependent on the fresh peptide components added to the reaction than on the preexisting fibrils. A model for the interaction between the peptides is presented.

The mechanisms of apolipoprotein E- and antioxidant-mediated inhibition of beta-amyloid fibril (fAbeta) formation in vitro were compared based on a nucleation-dependent polymerization model that uses fluorescence spectroscopy with thioflavin T. In addition, the effect of apolipoprotein and antioxidant on the extension reaction of (fAbeta) was examined. Results showed that apolipoprotein E and antioxidant inhibit (fAbeta) formation in vitro by different mechanisms, indicating the existence of multiple pharmacological targets for the prevention of fAbeta formation.

Fluorescence spectroscopy was utilized to determine the effects of human apolipoprotein E (apoE) on the formation of the beta-amyloid fibril (beta-Af) in vitro. Administration of apoE in freshly prepared betaAfs induced inhibited fibril formation in a dose-dependent manner. The differential effects of ApoE on beta-Af formation were also characterized by the inhibition of the nucleation phase via a mechanism that was similar to the attenuation of the elongation phase by the cholesterol transport protein.

The studies related to the lysophosphatidic acid (LPA), an in vivo occurring lysophospholipid mediator which promoted amyloid formation under physiological conditions through a complex mechanism is presented. The results obtained and the observed increase in blood LPA levels in dialysis patients suggested that the interaction of LPA with [beta]2m might contribute to the pathomechanism of dialysis-related amyloidosis.

Amyloid nucleation through agitation is analyzed with [[beta].sub.2]-microglobulin, which is responsible for dialysis-related amyloidosis, in the presence of salt under acid and neutral pH conditions. The agitation-treated aggregates of both proteins have a high propensity to produce an amyloid nucleus even at natural pH, thus showing that the aggregation pathway has involved amyloid nucleation under entirely native conditions.

The effects of heat on the acid-unfolded monomer at pH 2.5 were studied to understand the initial stages in the formation of amyloid fibrils of [beta.sub.2]-microglobulin ([beta]2-m), a protein responsible for dialysis-related amyloidosis. It was found that moderate heat treatment of [beta]2-m in the presence of seed fibrils results in an exothermic sigmoidal transition leading to a conformational state and may be interpreted in terms of the effects of heat associated with the formation, the association and unfolding of fibrils.

The role of electrostatic interactions and the effect of ionic cosolutes on amyloid formation are understood by investigating the effect of salts such as NaCl, NaI, NaClO4, and Na2SO4 on the amyloid fibril growth of beta2-microglobulin. The study conducted suggests that preferential interaction of sulfate ions with amyloidogenic proteins may have biological significance.

A study is carried out on beta2-microglobulin that is a major component of amyloid fibrils deposited in patients with dialysis-related amyloidosis. The results suggested that, the myofibrils derived from L-peptides and D-peptides form in a similar manner but with opposite stereochemistry, a cross-reaction between them is prevented because the geometry of the mixed sheet cannot satisfy dominant factors for beta-sheet stabilization.

The effects of sodium dodecyl sulfate (SDS) and other detergents on the conformation of beta2-m, the stability of Abeta2M amyloid fibrils, and the extension of amyloid fibrils at neutral pH, are investigated using fluorescence spectroscopy with thioflavin T (ThT), circular dichroism (CD) spectroscopy and electron microscopy. The findings display the sensitivity of Abeta2M fibril formation to solution conditions and suggest a possible role for lipid molecules in the development of Abeta2M amyloidosis.

To understand the initial stages in the formation of amyloid fibrils of β2-microglobulin, a protein responsible for dialysis-related amyloidosis, the effects of heat on the acid-unfolded monomer at pH 2.5 were studied. In the presence of a low concentration of seed fibrils, differential scanning calorimetric thermograms of acid-unfolded β2-microglobulin monomers showed a large decrease in heat capacity with a sigmoidal temperature-dependence, which was subsequently released at higher temperature. Measurements of circular dichroism, atomic force microscopy, ultracentrifugation, and repeated differential scanning calorimetry indicated that the exothermic sigmoidal transition is accompanied by the conversion of about 12% of the monomeric β2-microglobulin molecules into amyloid fibrils, which subsequently dissociate into monomers at high temperature. Interestingly, amyloid fibrils, formed partly after the sigmoidal transition, exhibited a heating rate-dependent, kinetically controlled thermal response, indicating that 12% of the total protein is enough to exhibit the unique thermal response. On the other hand, the salt-induced protofibrils did not show such a calorimetric response, indicating that the kinetic thermal response is unique to the particular structure of fibrils. Taken together, although the calorimetric behavior of amyloid fibrils remains elusive, it may be interpreted in terms of the effects of heat associated with the formation, the association, and the unfolding of fibrils, in which the interactions between specific β-sheet structures and water molecules play a crucial role and are sensitively reflected in the heat capacity change in protein solution. [ABSTRACT FROM AUTHOR]

Provides information on an experiment which compares the mechanisms of apolipoprotein E- and antioxidant- (AO-) mediated inhibition of beta-amyloid fibril formation in vitro, based on a nucleation-dependent polymerization model. Experimental procedures; Results; Discussion.

Amyloid formation in vivo occurs under complicated conditions in which various amyloidogenic and non-amyloidogenic components coexist, often under crowding. Controversy surrounds the role of additional components under complicated conditions. They have been suggested to accelerate amyloid formation because molecular crowding or interactions with additives increase effective concentrations and, thus, break the supersaturation of amyloidogenic proteins. On the other hand, cellular crowding conditions with various heterogeneous components may retard or prevent amyloid formation because they impede homologous amyloidogenic associations. To elucidate the roles of these additional components, we examined the amyloid formation of β 2 -microglobulin (β2m), a protein responsible for dialysis-related amyloidosis, with a simplified model system in which intact β2m and its proteolytic peptides coexist. Among the nine proteolytic peptides of β2m produced in vitro with lysyl endopeptidase, the 22-residue K3 peptide is highly amyloidogenic. The amyloid formation of the K3 peptide, which occurred with a lag time of 1 h at pH 2 and 37 °C, was significantly retarded by the coexistence of β2m or a mixture of the proteolytic digests. To identify the sites of inhibitory interactions, we performed paramagnetic relaxation enhancement measurements using spin-labeled K3 and uniformly 15 N-labeled β2m with nuclear magnetic resonance detection. The results revealed that K3 interacted weakly with a broad cluster of the hydrophobic residues of β2m, which accommodated the residues located in some distant sequence, leading to competitive inhibition. The results showed that relatively weak and broad interactions formed a nonproductive complex, implying a role for heterogeneous interactions under complicated conditions.

Investigation of factors that modulate amyloid formation of proteins is important to understand and mitigate amyloid-related diseases. To understand the role of electrostatic interactions and the effect of ionic cosolutes, especially anions, on amyloid formation, we have investigated the effect of salts such as NaCl, NaI, NaClO(4), and Na(2)SO(4) on the amyloid fibril growth of beta(2)-microglobulin, the protein involved in dialysis-related amyloidosis. Under acidic conditions, these salts exhibit characteristic optimal concentrations where the fibril growth is favored. The presence of salts leads to an increase in hydrophobicity of the protein as reported by 8-anilinonaphthalene-1-sulfonic acid, indicating that the anion interaction leads to the necessary electrostatic and hydrophobic balance critical for amyloid formation. However, high concentrations of salts tilt the balance to high hydrophobicity, leading to partitioning of the protein to amorphous aggregates. Such amorphous aggregates are not competent for fibril growth. The order of anions based on the lowest concentration at which fibril formation is favored is SO(4)(2)(-) > ClO(4)(-) > I(-) > Cl(-), consistent with the order of their electroselectivity series, suggesting that preferential anion binding, rather than general ionic strength effect, plays an important role in the amyloid fibril growth. Anion binding is also found to stabilize the amyloid fibrils under acidic condition. Interestingly, sulfate promotes amyloid growth of beta(2)-microglobulin at pH between 5 and 6, closer to its isoelectric point. Considering the earlier studies on the role of glycosaminoglycans and proteoglycans (i.e., sulfated polyanions) on amyloid formation, our study suggests that preferential interaction of sulfate ions with amyloidogenic proteins may have biological significance.

In beta(2)-microglobulin-related (Abeta2M) amyloidosis, partial unfolding of beta(2)-microglobulin (beta2-m) is believed to be prerequisite to its assembly into Abeta2M amyloid fibrils in vivo. Although low pH or 2,2,2-trifluoroethanol at a low concentration has been reported to induce partial unfolding of beta2-m and subsequent amyloid fibril formation in vitro, factors that induce them under near physiological conditions have not been determined. Using fluorescence spectroscopy with thioflavin T, circular dichroism spectroscopy, and electron microscopy, we here show that at low concentrations, sodium dodecyl sulfate (SDS) converts natively folded beta2-m monomers into partially folded, alpha-helix-containing conformers. Surprisingly, this results in the extension of Abeta2M amyloid fibrils at neutral pH, which could be explained basically by a first-order kinetic model. At low concentrations, SDS also stabilized the fibrils at neutral pH. These SDS effects were concentration-dependent and maximal at approximately 0.5 mM, around the critical micelle concentration of SDS (0.67 mM). As the concentration of SDS was increased above 1 mM, the alpha-helix content of beta2-m rose to approximately 10%, while the beta-sheet content decreased to approximately 20%, a change paralleled by a complete cessation of fibril extension and the destabilization of the fibrils. Detergents of other classes had no significant effect on the extension of fibrils. These findings are consistent with the hypothesis that in vivo, specific factors (e.g., phospholipids) that affect the conformation and stability of beta2-m and amyloid fibrils will have significant effects on the kinetics of Abeta2M fibril formation.