Fluorometric determination of amyloid fibrils in vitro using the fluorescent dye, thioflavine T

Systemic amyloidosis caused by extracellular deposition of insoluble fibrils derived from the pathological aggregation of circulating proteins, such as transthyretin, is a severe and usually fatal condition. Elucidation of the molecular pathogenic mechanism of the disease and discovery of effective therapies still represents a challenging medical issue. The in vitro preparation of amyloid fibrils that exhibit structural and biochemical properties closely similar to those of natural fibrils is central to improving our understanding of the biophysical basis of amyloid formation in vivo and may offer an important tool for drug discovery. Here, we compared the morphology and thermodynamic stability of natural transthyretin fibrils with those of fibrils generated in vitro either using the common acidification procedure or primed by limited selective cleavage by plasmin. The free energies for fibril formation were −12.36, −8.10, and −10.61 kcal mol−1, respectively. The fibrils generated via plasmin cleavage were more stable than those prepared at low pH and were thermodynamically and morphologically similar to natural fibrils extracted from human amyloidotic tissue. Determination of thermodynamic stability is an important tool that is complementary to other methods of structural comparison between ex vivo fibrils and fibrils generated in vitro. Our finding that fibrils created via an in vitro amyloidogenic pathway are structurally similar to ex vivo human amyloid fibrils does not necessarily establish that the fibrillogenic pathway is the same for both, but it narrows the current knowledge gap between in vitro models and in vivo pathophysiology.

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Spectroscopic characterization of diverse amyloid fibrils in vitro by the fluorescent dye Nile red

Molecular BioSystems ◽

10.1039/c0mb00236d ◽

2011 ◽

Vol 7 (4) ◽

pp. 1232 ◽

Cited By ~ 73

Author(s):

Rajesh Mishra ◽

Daniel Sjölander ◽

Per Hammarström

Keyword(s):

Amyloid Fibrils ◽

Nile Red ◽

Spectroscopic Characterization ◽

Fluorescent Dye

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5-Hydroxytryptamine (serotonin) in acute hypotensive shock

American Journal of Physiology-Legacy Content ◽

10.1152/ajplegacy.1964.207.1.215 ◽

1964 ◽

Vol 207 (1) ◽

pp. 215-222 ◽

Cited By ~ 24

Author(s):

Roy L. Swank ◽

Wolfgang Hissen ◽

J. H. Fellman

Keyword(s):

Blood Cell ◽

Blood Cells ◽

Cell Aggregation ◽

Glass Wool ◽

Fluorometric Determination ◽

Platelet Counts ◽

Blood Cell Aggregation

Alterations in the circulating 5-hydroxytryptamine (5-HT), platelet counts, and the degree of blood cell aggregation were studied before and during acute hypotensive "shock," induced either by bleeding or by histamine injections in anesthetized and heparinized dogs. Although marked aggregation of blood cells was observed during shock only slight increases in 5-HT and platelet counts were found. Since earlier studies in vitro had indicated a direct relationship between aggregation of blood cells and 5-HT the reliability of the fluorometric determination of 5-HT was questioned. Subsequent experiments with C14-labeled 5-HT showed increases up to 600% in circulating radioactivity during hypotension, representing 5-HT, or its metabolites. Radioactivity increased earlier and was greatest in portal blood. Glass-wool filtration experiments showed that adhesive platelets were important in the mechanism of blood cell aggregation. UML-491, a 5-HT and blood aggregation inhibitor in vitro, was shown to prevent or lower the tendency of blood cells to aggregate in vivo during hemorrhage. These experiments indicate the importance of 5-HT in the development of blood cell aggregation during shock, and its prevention by a 5-HT antagonist.

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