Folding of proteins features in Alzheimer's

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Alternative folding of proteins features in many neurodegenerative diseases, including Parkinson's, Huntington's, and Alzheimer's, as well as the prion diseases (1–3). In several of these diseases, the alternatively folded proteins can adopt β-sheet-rich conformations that facilitate polymerization into amyloid fibers. In prion diseases, amyloid deposition has been observed in both animals and humans (4–6) but is a nonobligatory feature of the disease (7, 8). Recombinant prion proteins (recPrP) with sequences derived from various species and composed of full-length and truncated segments of the protein have been shown to form amyloid fibers (9–11).

In vitro, the kinetics of amyloid formation usually exhibits an initial lag phase, in which no detectable amyloid forms, whereas monomers nucleate to form fibers (12). During this initial phase, monomeric protein molecules adopt partially denatured conformations, which assemble into multimeric species in a process with slow kinetics (13). Once the solution is nucleated, there is a period of amyloid fiber growth that eventually plateaus when the soluble protein pool has been depleted. When added to a fresh pool of soluble protein, fragmented amyloid fibrils can shorten the lag phase and initiate rapid amyloid formation, a phenomenon known as seeding. Therefore, amyloid fiber generation can be initiated through nucleation, which is a slow process, or by seeding the reaction, which leads to amyloid formation after a brief incubation.

Amyloid formation can be monitored in solution by using the dye Thioflavin T (ThT) (14). This dye undergoes a fluorescence shift upon binding to amyloid fibers, from 342/430 to 442/482 nm excitation/emission maxima. Because ThT does not fluoresce significantly at excitation/emission maxima of 442/482 nm in the absence of amyloid fibers, the background signal tends to be quite low, so the dye is a highly sensitive reporter. When used in conjunction with multiwell plates and automated plate readers that record fluorescence over time, ThT offers a facile means of detecting conformational changes of proteins in solution. Using the ThT assay, we find that many prion strains are capable of seeding the polymerization of recPrP into amyloid, and we demonstrate that this seeding property can be used as an assay for the detection of prions in biological samples. http://www.pnas.org

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