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91.
F.M. Gomes I.B. Ramos C. Wendt W. Girard-Dias W. De Souza E.A. Machado E.A. K. Miranda 《European journal of histochemistry : EJH》2013,57(4)
Inorganic polyphosphate (PolyP) is a biological polymer that plays important roles in the cell physiology of both prokaryotic and eukaryotic organisms. Among the available methods for PolyP localization and quantification, a 4’,6-diamidino-2-phenylindole(DAPI)-based assay has been used for visualization of PolyP-rich organelles. Due to differences in DAPI permeability to different compartments and/or PolyP retention after fixation, a general protocol for DAPI-PolyP staining has not yet been established. Here, we tested different protocols for DAPI-PolyP detection in a range of samples with different levels of DAPI permeability, including subcellular fractions, free-living cells and cryosections of fixed tissues. Subcellular fractions of PolyP-rich organelles yielded DAPI-PolyP fluorescence, although those with a complex external layer usually required longer incubation times, previous aldehyde fixation and/or detergent permeabilization. DAPI-PolyP was also detected in cryosections of OCT-embedded tissues analyzed by multiphoton microscopy. In addition, a semi-quantitative fluorimetric analysis of DAPI-stained fractions showed PolyP mobilization in a similar fashion to what has been demonstrated with the use of enzyme-based quantitative protocols. Taken together, our results support the use of DAPI for both PolyP visualization and quantification, although specific steps are suggested as a general guideline for DAPI-PolyP staining in biological samples with different degrees of DAPI and PolyP permeability.Key words: DAPI, polyphosphate, fluorescence, fluorimetry 相似文献
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Benildo S. Cavada Socorro V. F. Madeira Juan J. Calvete Luiz A. G. Souza Liezelotte R. Bomfim Adilson R. Dantas 《Preparative biochemistry & biotechnology》2013,43(4):271-280
ABSTRACT A glucose/mannose-binding lectin was isolated from seeds of Parkia discolor (Mimosoideae) using affinity chromatography on Sephadex G-100 gel. The protein presented a unique component in SDS-PAGE corresponding to a molecular mass of 58,000 Da, which is very similar to that of a closely related lectin from Parkia platycephala. Among the simple sugars tested, mannose was the best inhibitor, but biantennary glycans, containing the trimannoside core, present in N-glycoproteins, also seem to be powerful inhibitors of the haemagglutinating activity induced by the purified lectin. The protein was characterised by high content of glycine and proline and absence of cysteine. Rabbit antibodies, anti-P. platycephala seed lectin, recognised the P.discolor lectin. However, no cross-reaction was observed when a set of other legume lectins from sub-family Papilionoideae and others from families Moraceae and Euphorbiaceae were assayed with the Parkia lectins. This suggests that Parkia lectins comprise a new group of legume lectins exhibiting distinct characteristics. 相似文献
94.
Lorena Souza e Silva Aline Mayrink de Miranda Cíntia Lopes de Brito Magalhães Rinaldo Cardoso dos Santos Maria Lúcia Pedrosa Marcelo Eustáquio Silva 《Journal of physiology and biochemistry》2013,69(4):811-820
The present study investigated the underlying mechanism associated with the hypocholesterolemic activity of beta-carotene by examining its effects on the serum lipid profile, fecal cholesterol excretion, and gene expression of the major receptors, enzymes, and transporters involved in cholesterol metabolism. Female Fischer rats were divided into three groups and were fed either a control or a hypercholesterolemic diet supplemented or not supplemented with 0.2 % beta-carotene. After 6 weeks of feeding, blood, livers, and feces were collected for analysis, and quantitative real-time polymerase chain reaction (qRT-PCR) was performed. Dietary supplementation with 0.2 % beta-carotene decreased serum total cholesterol, non-HDL cholesterol, the atherogenic index, and hepatic total lipid and cholesterol contents. These changes were accompanied by an increase in the total lipid and cholesterol contents excreted in the feces. The qRT-PCR analyses demonstrated that the hypercholesterolemic diet promoted a decrease in the gene expression of sterol regulatory element-binding protein 2, 3-hydroxy-3-methylglutaryl CoA reductase, and low-density lipoprotein receptor and an increase in the gene expression of peroxisome proliferator-activated receptor α and cholesterol-7a-hydroxylase. The expression of these genes and gene expression of ATP-binding cassette subfamily G transporters 5and 8 were unaffected by beta-carotene supplementation. In conclusion, the decrease in serum cholesterol and the elevation of fecal cholesterol obtained following beta-carotene administration indicate that this substance may decrease cholesterol absorption in the intestine and increase cholesterol excretion into the feces without a direct effect on the expression of cholesterol metabolism genes. 相似文献
95.
A. D. da Matta A. M. R. Bernardino G. A. Romeiro M. R. P. de Oliveira M. C. B. V. de Souza V. F. Ferreira 《Nucleosides, nucleotides & nucleic acids》2013,32(4):889-898
Abstract Ribosylation reactions of previously silylated 3-carbethoxy-8-methyl-1,4-dihydro-4-oxoquinoline (6a) and 3-carbethoxy-6-methyl-1,4-dihydro-4-oxoquinoline (6b) with 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose (7), under Lewis acid catalysis, were studied. The method using hexamethyldisilazane (HMDS)/trimethylchlorosilane (TMCS) mixture for silylation and anhydrous stannic chloride as catalyst for ribosylation failed to give any nucleoside product. On the other hand, the protected nucleoside 3-carbethoxy-6-methyl-1-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-1,4-dihydro-4-oxoquinoline (8b) was obtained in good yields using bis(trimethylsilyl)trifluoroacetamide (BSTFA) containing 1% of TMCS and the same catalyst. Compound 8b was more easily isolated in higher yields with an improvement of the later method by replacing stannic chloride with trimethylsilyl trifluoromethanesulfonate (TMSOTf). De-O-benzoylation of 8b with methanolic sodium hydroxide solution afforded the free riboside 3-carbomethoxy-6-methyl-1-β-D-ribofuranosyl-1,4-dihydro-4-oxoquinoline (9b). The structures of the obtained products were confirmed by their LTV, MS, IR, 1H and 13C-NMR data. 相似文献
96.
J.C. Souza E.C Vanzela R.A. Ribeiro L.F. Rezende C.A. de Oliveira E.M. Carneiro H.C.F. Oliveira A.C. Boschero 《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2013,1831(4):769-775
Aims/hypothesis
Changes in cellular cholesterol level may contribute to beta cell dysfunction. Islets from low density lipoprotein receptor knockout (LDLR−/−) mice have higher cholesterol content and secrete less insulin than wild-type (WT) mice. Here, we investigated the association between cholesterol content, insulin secretion and Ca2 + handling in these islets.Methods
Isolated islets from both LDLR−/− and WT mice were used for measurements of insulin secretion (radioimmunoassay), cholesterol content (fluorimetric assay), cytosolic Ca2 + level (fura-2AM) and SNARE protein expression (VAMP-2, SNAP-25 and syntaxin-1A). Cholesterol was depleted by incubating the islets with increasing concentrations (0–10 mmol/l) of methyl-beta-cyclodextrin (MβCD).Results
The first and second phases of glucose-stimulated insulin secretion (GSIS) were lower in LDLR−/− than in WT islets, paralleled by an impairment of Ca2 + handling in the former. SNAP-25 and VAMP-2, but not syntaxin-1A, were reduced in LDLR−/− compared with WT islets. Removal of excess cholesterol from LDLR−/− islets normalized glucose- and tolbutamide-induced insulin release. Glucose-stimulated Ca2 + handling was also normalized in cholesterol-depleted LDLR−/− islets. Cholesterol removal from WT islets by 0.1 and 1.0 mmol/l MβCD impaired both GSIS and Ca2 + handling. In addition, at 10 mmol/l MβCD WT islet showed a loss of membrane integrity and higher DNA fragmentation.Conclusion
Abnormally high (LDLR−/− islets) or low cholesterol content (WT islets treated with MβCD) alters both GSIS and Ca2 + handling. Normalization of cholesterol improves Ca2 + handling and insulin secretion in LDLR−/− islets. 相似文献97.
98.
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100.
Thiago Moreno L. Souza Paola C. Resende Natalia Fintelman-Rodrigues Tatiana Schaffer Gregianini Nilo Ikuta Sandra Bianchini Fernandes Ana Luisa Furtado Cury Maria do Carmo Debur Rosa Marilda M. Siqueira 《PloS one》2013,8(11)
Although surveillance efforts that monitor the emergence of drug-resistant strains of influenza are critical, systematic analysis is overlooked in most developing countries. We report on the occurrence of strains of pandemic influenza A(H1N1)pdm09 with resistance and decreased susceptibility to oseltamivir (OST) in Brazil in 2009, 2011 and 2012. We found 7 mutant viruses, 2 with the mutation S247N and other 5 with the mutation H275Y. Most of these viruses were from samples concentrated in the southern region of Brazil. Some of these resistant viruses were detected prior to the initiation of OST treatment, suggesting that community transmission of mutant viruses may exist. Moreover, we show that one of these OST-resistant (H275Y) strains of A(H1N1)pdm09 was discovered in the tri-border region between Brazil, Argentina and Paraguay, highlighting that this strain could also be found in other Latin American countries. Our findings reinforce the importance of enhanced antiviral resistance surveillance in Brazil and in other Latin American countries to confirm or rule out the community transmission of OST-resistant strains of A(H1N1)pdm09. 相似文献