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The apomyoglobin mutant W7FW14F forms amyloidlike fibrils at physiological pH. We examined the kinetics of fibrillogenesis using three techniques: the time dependence of the fluorescence emission of thioflavin T and 1-anilino-8-naphthalenesulfonate, circular dichroism measurements, and electron microscopy. We found that in the early stage of fibril formation, non-native apomyoglobin molecules containing ␤-structure elements aggregate to form a nucleus. Subsequently, more molecules aggregate around the nucleus, thereby resulting in fibril elongation. We evaluated by MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) the cytotoxicity of these aggregates at the early stage of fibril elongation versus mature fibrils and the wild-type protein. Similar to other amyloid-forming proteins, cell toxicity was not due to insoluble mature fibrils but rather to early pre-fibrillar aggregates. Propidium iodide uptake showed that cell toxicity is the result of altered membrane permeability. Phalloidin staining showed that membrane damage is not associated to an altered cell shape caused by changes in the cytoskeleton.

Myoglobin is an ␣-helical globular protein that contains two highly conserved tryptophan residues located at positions 7 and 14 in the N-terminal region of the protein. Replacement of both indole residues with phenylalanine residues, i.e. W7F/W14F, results in the expression of an unstable, not correctly folded protein that does not bind the prosthetic group. Here we report data (Congo red and thioflavine T binding assay, birefringence, and electron microscopy) showing that the double Trp/Phe replacements render apomyoglobin molecules highly susceptible to aggregation and amyloid-like fibril formation under physiological conditions in which most of the wild-type protein is in the native state. In refolding experiments, like the wild-type protein, the W7F/W14F apomyoglobin mutant formed a soluble, partially folded helical state between pH 2.0 and pH 4.0. A pH increase from 4.0 to 7.0 restored the native structure only in the case of the wild-type protein and determined aggregation of W7F/W14F. The circular dichroism spectrum recorded immediately after neutralization showed that the polypeptide consists mainly of ␤-structures. In conclusion, under physiological pH conditions, some mutations that affect folding may cause protein aggregation and the formation of amyloid-like fibrils.Such chronic disorders as Alzheimer's disease, senile systemic amyloidosis, transmissible spongiform encephalopathies, and dialysis-related amyloidosis are characterized by the extracellular deposition of insoluble protein aggregates known as amyloid fibrils (1-6). About 20 proteins are now known to be involved in the generation of amyloid in vivo. Fibril formation is initiated in vitro under conditions that stabilize partially unfolded soluble intermediates of the native proteins either after the partial destabilization of physiologically folded proteins in the case of globular proteins (7) or after the partial stabilization (i.e. folding) of random coil polypeptide chains in the case of natively unfolded proteins (8). Despite substantial differences in both sequence and length (from 40 to 250 residues), all the proteins responsible for amyloid deposition form fibrils composed of ␤-strands oriented perpendicularly to the long axis of the fibril (9). Electron microscopy shows that the fibrils are straight and unbranched and are 40 -120 Å in diameter (9, 10). Also proteins not known to be associated with amyloid disease may form amyloid fibrils under in vitro conditions that favor partially folded states (11-15). These states are more prone to aggregation than the native state because hydrophobic residues, which are largely buried within the core of the native protein, become more exposed upon partial unfolding. The way in which proteins aggregate in the test tube is remarkably similar to how proteins form the so-called "amyloid" deposits. Even myoglobin, an ordinary all-␣ globular protein, can form fibrils containing ␤-strands under experimental conditions that favor the formation of partially folded states (15). Thus, amyloid formation d...

Atmospheric particulate matter (PM), an ingredient of urban pollution matter, is a mixture of solid and liquid particles differing in origin, dimension and composition. There is big concern about inhaled PM in urban areas, especially due to its adverse effects on the respiratory system. Diesel exhaust particulate (DEP), which constitutes the major part of PM, is characterized by a carbonic mixture composed of approximately 18,000 different high-molecular-weight organic compounds. Diesel engines release 10 times the amount of NO(2) aldehydes and breathable PM compared to unleaded gasoline engines and more than 100 times that produced by catalysed gasoline engines; these data gain great significance when taken into account the fact that diesel-powered vehicles are becoming more and more popular. DEP polyaromatic hydrocarbons (PAH), once deposited on airways mucous surfaces easily pass through epithelial cells (ECs) membranes, bind themselves to cytosolic receptors and then affect cell growth and differentiation. Human lung epithelial cells and macrophages engulf DEP, this resulting in increased proinflammatory cytokines release (IL-6, IL-8 and GM-CSF). We investigated the biological effects of DEP-PM on the human lung EC line A549. Light microscopy analysis suggested the presence of cell wall alterations, and provided evidence of PM internalization and cytoplasmic vacuolization. Following PM stimulation, nuclei also were seen undergo clear gross morphological modifications. Immunocytochemistry was used to detect intracytoplasmic IL-6 and IL-8 expression.

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