Prion domain initiation of amyloid formation in vitro from native Ure2p.
(1/365)
The [URE3] non-Mendelian genetic element of Saccharomyces cerevisiae is an infectious protein (prion) form of Ure2p, a regulator of nitrogen catabolism. Here, synthetic Ure2p1-65 were shown to polymerize to form filaments 40 to 45 angstroms in diameter with more than 60 percent beta sheet. Ure2p1-65 specifically induced full-length native Ure2p to copolymerize under conditions where native Ure2p alone did not polymerize. Like Ure2p in extracts of [URE3] strains, these 180- to 220-angstrom-diameter filaments were protease resistant. The Ure2p1-65-Ure2p cofilaments could seed polymerization of native Ure2p to form thicker, less regular filaments. All filaments stained with Congo Red to produce the green birefringence typical of amyloid. This self-propagating amyloid formation can explain the properties of [URE3]. (+info)
Designing conditions for in vitro formation of amyloid protofilaments and fibrils.
(2/365)
We have been able to convert a small alpha/beta protein, acylphosphatase, from its soluble and native form into insoluble amyloid fibrils of the type observed in a range of pathological conditions. This was achieved by allowing slow growth in a solution containing moderate concentrations of trifluoroethanol. When analyzed with electron microscopy, the protein aggregate present in the sample after long incubation times consisted of extended, unbranched filaments of 30-50 A in width that assemble subsequently into higher order structures. This fibrillar material possesses extensive beta-sheet structure as revealed by far-UV CD and IR spectroscopy. Furthermore, the fibrils exhibit Congo red birefringence, increased fluorescence with thioflavine T and cause a red-shift of the Congo red absorption spectrum. All of these characteristics are typical of amyloid fibrils. The results indicate that formation of amyloid occurs when the native fold of a protein is destabilized under conditions in which noncovalent interactions, and in particular hydrogen bonding, within the polypeptide chain remain favorable. We suggest that amyloid formation is not restricted to a small number of protein sequences but is a property common to many, if not all, natural polypeptide chains under appropriate conditions. (+info)
Amylopectinosis in fetal and neonatal Quarter Horses.
(3/365)
Three Quarter Horses, a stillborn filly (horse No. 1), a female fetus aborted at approximately 6 months of gestation (horse No. 2), and a 1-month-old colt that had been weak at birth (horse No. 3), had myopathy characterized histologically by large spherical or ovoid inclusions in skeletal and cardiac myofibers. Smaller inclusions were also found in brain and spinal cord and in some cells of all other tissues examined. These inclusions were basophilic, red-purple after staining with periodic acid-Schiff (both before and after digestion with diastase), and moderately dark blue after staining with toluidine blue. The inclusions did not react when stained with Congo red. Staining with iodine ranged from pale blue to black. Their ultrastructural appearance varied from amorphous to somewhat filamentous. On the basis of staining characteristics and diastase resistance, we concluded that these inclusions contained amylopectin. A distinctly different kind of inclusion material was also present in skeletal muscle and tongue of horse Nos. 1 and 3. These inclusions were crystalline with a sharply defined ultrastructural periodicity. The crystals were eosinophilic and very dark blue when stained with toluidine blue but did not stain with iodine. Crystals sometimes occurred freely within the myofibers but more often were encased by deposits of amylopectin. This combination of histologic and ultrastructural features characterizes a previously unreported storage disease in fetal and neonatal Quarter Horses, with findings similar to those of glycogen storage disease type IV. We speculate that a severe inherited loss of glycogen brancher enzyme activity may be responsible for these findings. The relation of amylopectinosis to the death of the foals is unknown. (+info)
A simple manganous chloride and Congo red disc method for differentiating Neisseria gonorrhoeae from Neisseria meningitidis.
(4/365)
Manganous chloride and Congo red incorporated into blotting paper discs have been used to differentiate gonococci from meningococci. The new technique is simple and reliable; the materials for the test are inexpensive. The method will increase the efficiency of distinguishing between the pathogenic Neisseria in any clinical bacteriology laboratory and especially in those in the tropical areas. (+info)
Inhibition of fibril formation in beta-amyloid peptide by a novel series of benzofurans.
(5/365)
A series of benzofuran derivatives have been identified as inhibitors of fibril formation in the beta-amyloid peptide. The activity of these compounds has been assessed by a novel fibril-formation-specific immunoassay and for their effects on the production of a biologically active fibril product. The inhibition afforded by the compounds seems to be associated with their binding to beta-amyloid, as identified by scintillation proximity binding assay. Binding assays and NMR studies also indicate that the inhibition is associated with self-aggregation of the compounds. There is a close correlation between the activity of the benzofurans as inhibitors of fibril formation and their ability to bind to beta-amyloid. Non-benzofuran inhibitors of the fibril formation process do not seem to bind to the same site on the beta-amyloid molecule as the benzofurans. Thus a specific recognition site might exist for benzofurans on beta-amyloid, binding to which seems to interfere with the ability of the peptide to form fibrils. (+info)
Role of the mitogen-activated protein kinase Hog1p in morphogenesis and virulence of Candida albicans.
(6/365)
The relevance of the mitogen-activated protein (MAP) kinase Hog1p in Candida albicans was addressed through the characterization of C. albicans strains without a functional HOG1 gene. Analysis of the phenotype of hog1 mutants under osmostressing conditions revealed that this mutant displays a set of morphological alterations as the result of a failure to complete the final stages of cytokinesis, with parallel defects in the budding pattern. Even under permissive conditions, hog1 mutants displayed a different susceptibility to some compounds such as nikkomycin Z or Congo red, which interfere with cell wall functionality. In addition, the hog1 mutant displayed a colony morphology different from that of the wild-type strain on some media which promote morphological transitions in C. albicans. We show that C. albicans hog1 mutants are derepressed in the serum-induced hyphal formation and, consistently with this behavior, that HOG1 overexpression in Saccharomyces cerevisiae represses the pseudodimorphic transition. Most interestingly, deletion of HOG1 resulted in a drastic increase in the mean survival time of systemically infected mice, supporting a role for this MAP kinase pathway in virulence of pathogenic fungi. This finding has potential implications in antifungal therapy. (+info)
Deletion of new covalently linked cell wall glycoproteins alters the electrophoretic mobility of phosphorylated wall components of Saccharomyces cerevisiae.
(7/365)
The incorporation of radioactive orthophosphate into the cell walls of Saccharomyces cerevisiae was studied. 33P-labeled cell walls were extensively extracted with hot sodium dodecyl sulfate (SDS). Of the remaining insoluble radioactivity more than 90% could be released by laminarinase. This radioactive material stayed in the stacking gel during SDS-polyacrylamide gel electrophoresis but entered the separating gel upon treatment with N-glycosidase F, indicating that phosphate was linked directly or indirectly to N-mannosylated glycoproteins. The phosphate was bound to covalently linked cell wall proteins as mannose-6-phosphate, the same type of linkage shown previously for soluble mannoproteins (L. Ballou, L. M. Hernandez, E. Alvarado, and C. E. Ballou, Proc. Natl. Acad. Sci. USA 87:3368-3372, 1990). From the phosphate-labeled glycoprotein fraction released by laminarinase, three cell wall mannoproteins, Ccw12p, Ccw13p and Ccw14p, were isolated and identified by N-terminal sequencing. For Ccw13p (encoded by DAN1 [also called TIR3]) and Ccw12p the association with the cell wall has not been described before; Ccw14p is identical with cell wall protein Icwp (I. Moukadiri, J. Armero, A. Abad, R. Sentandreu, and J. Zueco, J. Bacteriol. 179:2154-2162, 1997). In ccw12, ccw13, or ccw14 single or double mutants neither the amount of radioactive phosphate incorporated into cell wall proteins nor its position in the stacking gel was changed. However, the triple mutant brought about a shift of the 33P-labeled glycoprotein components from the stacking gel into the separating gel. The disruption of CCW12 results in a pronounced sensitivity of the cells to calcofluor white and Congo red. In addition, the ccw12 mutant shows a decrease in mating efficiency and a defect in agglutination. (+info)
Histochemically reactive zinc in plaques of the Swedish mutant beta-amyloid precursor protein transgenic mice.
(8/365)
Endogenous metals such as zinc may contribute to beta-amyloid (Abeta) aggregation and hence the plaque formation. In the present study, we examined brains of four Swedish mutant amyloid precursor protein (APP) transgenic mice at 12 months of age for histochemically reactive zinc in the plaques. Here, we report that all the Congo red (+) mature plaques contained chelatable zinc, as demonstrated by staining with the zinc-specific fluorescent dye 6-methoxy-8-quinolyl-para-toluenesulfonamide (TSQ). On the other hand, Congo red (-) preamyloid Abeta deposits were not stained with TSQ. Interestingly, although cerebellum contained similar degree of preamyloid Abeta deposits as cerebral cortex, it was completely devoid of Congo red- or TSQ-stained mature plaques. Although zinc from plaques was only slowly and partially removed by a specific zinc remover, dithizone, treatment of brain sections with heparinase-III, which degrades heparan sulfate proteoglycan (HSPG), another major constituent of plaques, greatly fastened the zinc removal with dithizone. The present study has demonstrated the presence of histochemically reactive zinc in plaques, but not preamyloid Abeta deposits, of the Swedish mutant APP transgenic mice. Because preamyloid Abeta deposits fail to develop into congophilic plaques in cerebellum where synaptic vesicle zinc is deficient, the synaptic zinc may be a necessary element in the plaque formation. In holding zinc inside plaques, HSPG may contribute in addition to Abeta. (+info)