The Experts below are selected from a list of 8160 Experts worldwide ranked by ideXlab platform
Maurizio Leone - One of the best experts on this subject based on the ideXlab platform.
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secondary nucleation and accessible surface in insulin amyloid fibril formation
Journal of Physical Chemistry B, 2008Co-Authors: V Fodera, Minna Groenning, Fabio Librizzi, Marco Van De Weert, Maurizio LeoneAbstract:At low pH insulin is highly prone to self-assembly into amyloid fibrils. The process has been proposed to be affected by the existence of secondary nucleation pathways, in which already formed fibrils are able to catalyze the formation of new fibrils. In this work, we studied the fibrillation process of human insulin in a wide range of protein concentrations. Thioflavin T fluorescence was used for its ability to selectively detect amyloid fibrils, by mechanisms that involve the interaction between the dye and the accessible surface of the fibrils. Our results show that the rate of fibrillation and the Thioflavin T fluorescence intensity saturate at high protein concentration and that, surprisingly, the two parameters are proportional to each other. Because Thioflavin T fluorescence is likely to depend on the accessible surface of the fibrils, we suggest that the overall fibrillation kinetics is mainly governed by the accessible surface, through secondary nucleation mechanisms. Moreover, a statistical stud...
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secondary nucleation and accessible surface in insulin amyloid fibril formation
Journal of Physical Chemistry B, 2008Co-Authors: V Fodera, Minna Groenning, Fabio Librizzi, Marco Van De Weert, Maurizio LeoneAbstract:At low pH insulin is highly prone to self-assembly into amyloid fibrils. The process has been proposed to be affected by the existence of secondary nucleation pathways, in which already formed fibrils are able to catalyze the formation of new fibrils. In this work, we studied the fibrillation process of human insulin in a wide range of protein concentrations. Thioflavin T fluorescence was used for its ability to selectively detect amyloid fibrils, by mechanisms that involve the interaction between the dye and the accessible surface of the fibrils. Our results show that the rate of fibrillation and the Thioflavin T fluorescence intensity saturate at high protein concentration and that, surprisingly, the two parameters are proportional to each other. Because Thioflavin T fluorescence is likely to depend on the accessible surface of the fibrils, we suggest that the overall fibrillation kinetics is mainly governed by the accessible surface, through secondary nucleation mechanisms. Moreover, a statistical study of the fibrillation kinetics suggests that the early stages of the process are affected by stochastic nucleation events.
Yuji Goto - One of the best experts on this subject based on the ideXlab platform.
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distinguishing crystal like amyloid fibrils and glass like amorphous aggregates from their kinetics of formation
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Yuichi Yoshimura, Hironobu Naiki, Yuxi Lin, Hisashi Yagi, Youngho Lee, Hiroki Kitayama, Kazumasa Sakurai, Hirotsugu Ogi, Yuji GotoAbstract:Amyloid fibrils and amorphous aggregates are two types of aberrant aggregates associated with protein misfolding diseases. Although they differ in morphology, the two forms are often treated indiscriminately. β2-microglobulin (β2m), a protein responsible for dialysis-related amyloidosis, forms amyloid fibrils or amorphous aggregates depending on the NaCl concentration at pH 2.5. We compared the kinetics of their formation, which was monitored by measuring Thioflavin T fluorescence, light scattering, and 8-anilino-1-naphthalenesulfonate fluorescence. Thioflavin T fluorescence specifically monitors amyloid fibrillation, whereas light scattering and 8-anilino-1-naphthalenesulfonate fluorescence monitor both amyloid fibrillation and amorphous aggregation. The amyloid fibrils formed via a nucleation-dependent mechanism in a supersaturated solution, analogous to crystallization. The lag phase of fibrillation was reduced upon agitation with stirring or ultrasonic irradiation, and disappeared by seeding with preformed fibrils. In contrast, the glass-like amorphous aggregates formed rapidly without a lag phase. Neither agitation nor seeding accelerated the amorphous aggregation. Thus, by monitoring the kinetics, we can distinguish between crystal-like amyloid fibrils and glass-like amorphous aggregates. Solubility and supersaturation will be key factors for further understanding the aberrant aggregation of proteins.
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ultrasonication induced amyloid fibril formation of β2 microglobulin
Journal of Biological Chemistry, 2005Co-Authors: Yumiko Ohhashi, Hironobu Naiki, Miho Kihara, Yuji GotoAbstract:Abstract To obtain insight into the mechanism of fibril formation, we examined the effects of ultrasonication, a strong agitator, on β2 -microglobulin (β2-m), a protein responsible for dialysis-related amyloidosis. Upon sonication of an acid-unfolded β2-m solution at pH 2.5, Thioflavin T fluorescence increased markedly after a lag time of 1–2 h with a simultaneous increase of light scattering. Atomic force microscopy images showed the formation of a large number of short fibrils 3 nm in diameter. When the sonication-induced fibrils were used as seeds in the next seeding experiment at pH 2.5, a rapid and intense formation of long fibrils 3 nm in diameter was observed demonstrating seed-dependent fibril growth. We then examined the effects of sonication on the native β2-m at neutral pH, conditions under which amyloid deposits occur in patients. In the presence of 0.5 mm sodium dodecyl sulfate, a model compound of potential trigger and stabilizer of amyloid fibrils in patients, a marked increase of Thioflavin T fluorescence was observed after 1 day of sonication at pH 7.0. The products of sonication caused the accelerated fibril formation at pH 7.0. Atomic force microscopy images showed that the fibrils formed at pH 7.0 have a diameter of more than 7 nm, thicker than those prepared at pH 2.5. These results indicate that ultrasonication is one form of agitation triggering the formation of amyloid fibrils of β2-m, producing fibrils adapted to the respective pH.
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direct observation of amyloid fibril growth monitored by Thioflavin t fluorescence
Journal of Biological Chemistry, 2003Co-Authors: Tadato Ban, Daizo Hamada, Hironobu Naiki, Kazuhiro Hasegawa, Yuji GotoAbstract:Real-time monitoring of fibril growth is essential to clarify the mechanism of amyloid fibril formation. Thioflavin T (ThT) is a reagent known to become strongly fluorescent upon binding to amyloid fibrils. Here, we show that, by monitoring ThT fluorescence with total internal reflection fluorescence microscopy (TIRFM), amyloid fibrils of beta2-microgobulin (beta2-m) can be visualized without requiring covalent fluorescence labeling. One of the advantages of TIRFM would be that we selectively monitor fibrils lying along the slide glass, so that we can obtain the exact length of fibrils. This method was used to follow the kinetics of seed-dependent beta2-m fibril extension. The extension was unidirectional with various rates, suggesting the heterogeneity of the amyloid structures. Since ThT binding is common to all amyloid fibrils, the present method will have general applicability for the analysis of amyloid fibrils. We confirmed this with the octapeptide corresponding to the C terminus derived from human medin and the Alzheimer's amyloid beta-peptide.
V Fodera - One of the best experts on this subject based on the ideXlab platform.
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secondary nucleation and accessible surface in insulin amyloid fibril formation
Journal of Physical Chemistry B, 2008Co-Authors: V Fodera, Minna Groenning, Fabio Librizzi, Marco Van De Weert, Maurizio LeoneAbstract:At low pH insulin is highly prone to self-assembly into amyloid fibrils. The process has been proposed to be affected by the existence of secondary nucleation pathways, in which already formed fibrils are able to catalyze the formation of new fibrils. In this work, we studied the fibrillation process of human insulin in a wide range of protein concentrations. Thioflavin T fluorescence was used for its ability to selectively detect amyloid fibrils, by mechanisms that involve the interaction between the dye and the accessible surface of the fibrils. Our results show that the rate of fibrillation and the Thioflavin T fluorescence intensity saturate at high protein concentration and that, surprisingly, the two parameters are proportional to each other. Because Thioflavin T fluorescence is likely to depend on the accessible surface of the fibrils, we suggest that the overall fibrillation kinetics is mainly governed by the accessible surface, through secondary nucleation mechanisms. Moreover, a statistical stud...
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secondary nucleation and accessible surface in insulin amyloid fibril formation
Journal of Physical Chemistry B, 2008Co-Authors: V Fodera, Minna Groenning, Fabio Librizzi, Marco Van De Weert, Maurizio LeoneAbstract:At low pH insulin is highly prone to self-assembly into amyloid fibrils. The process has been proposed to be affected by the existence of secondary nucleation pathways, in which already formed fibrils are able to catalyze the formation of new fibrils. In this work, we studied the fibrillation process of human insulin in a wide range of protein concentrations. Thioflavin T fluorescence was used for its ability to selectively detect amyloid fibrils, by mechanisms that involve the interaction between the dye and the accessible surface of the fibrils. Our results show that the rate of fibrillation and the Thioflavin T fluorescence intensity saturate at high protein concentration and that, surprisingly, the two parameters are proportional to each other. Because Thioflavin T fluorescence is likely to depend on the accessible surface of the fibrils, we suggest that the overall fibrillation kinetics is mainly governed by the accessible surface, through secondary nucleation mechanisms. Moreover, a statistical study of the fibrillation kinetics suggests that the early stages of the process are affected by stochastic nucleation events.
Jeffrey Aubé - One of the best experts on this subject based on the ideXlab platform.
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Optimization of potent hepatitis C virus NS3 helicase inhibitors isolated from the yellow dyes Thioflavine S and primuline.
Journal of medicinal chemistry, 2012Co-Authors: Kevin J. Frankowski, Craig A. Belon, Benjamin Neuenswander, Jean Ndjomou, Alicia M. Hanson, Matthew A. Shanahan, Frank J. Schoenen, Brian S. J. Blagg, Jeffrey AubéAbstract:A screen for hepatitis C virus (HCV) NS3 helicase inhibitors revealed that the commercial dye Thioflavine S was the most potent inhibitor of NS3-catalyzed DNA and RNA unwinding in the 827-compound National Cancer Institute Mechanistic Set. Thioflavine S and the related dye primuline were separated here into their pure components, all of which were oligomers of substituted benzothiazoles. The most potent compound (P4), a benzothiazole tetramer, inhibited unwinding >50% at 2 ± 1 μM, inhibited the subgenomic HCV replicon at 10 μM, and was not toxic at 100 μM. Because P4 also interacted with DNA, more specific analogues were synthesized from the abundant dimeric component of primuline. Some of the 32 analogues prepared retained ability to inhibit HCV helicase but did not appear to interact with DNA. The most potent of these specific helicase inhibitors (compound 17) was active against the replicon and inhibited the helicase more than 50% at 2.6 ± 1 μM.
Guoying Bing - One of the best experts on this subject based on the ideXlab platform.
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comparative analysis of an improved Thioflavin s stain gallyas silver stain and immunohistochemistry for neurofibrillary tangle demonstration on the same sections
Journal of Histochemistry and Cytochemistry, 2002Co-Authors: Anyang Sun, Xuan V Nguyen, Guoying BingAbstract:An improved Thioflavin-S stain, Gallyas silver stain, and two immunostainings were quantitatively compared for demonstration of neurofibrillary tangles (NFTs) on the same sections. Sections of hippocampal formation from seven cases of Alzheimer's disease (AD) were immunofluorescently stained with a commercially available polyclonal NFT antibody or a PHF-1 monoclonal antibody, followed by an improved Thioflavin-S stain, and finally by Gallyas silver staining. The Thioflavin-S method was improved by using a combination quenching method that removes background autofluorescence without remarkable tissue damage and by post-treatment with concentrated phosphate buffer, which minimizes photobleaching. PHF-1 or NFT immunostaining is much less sensitive than the improved Thioflavin-S staining and Gallyas silver staining, particularly in the transentorhinal region. Moreover PHF-1 immunoreactivity varied greatly among AD individuals. Thioflavin-S staining and Gallyas silver staining show almost the same sensitivity in NFT demonstration, but only the former depends on the secondary protein structure of NFTs. This study suggests that the improved Thioflavin-S staining is a simple, sensitive, and consistent method for demonstration of neurofibrillary pathology.
