The Mycobacterium tuberculosis Rv2540c DNA sequence encodes a bifunctional chorismate synthase

Background. The emergence of multi- and extensively-drug resistant Mycobacterium tuberculosis strains has created an urgent need for new agents to treat tuberculosis (TB). The enzymes of shikimate pathway are attractive targets to the development of antitubercular agents because it is essential for M. tuberculosis and is absent from humans. Chorismate synthase (CS) is the seventh enzyme of this route and catalyzes the NADH- and FMN-dependent synthesis of chorismate, a precursor of aromatic amino acids, naphthoquinones, menaquinones, and mycobactins. Although the M. tuberculosis Rv2540c (aroF) sequence has been annotated to encode a chorismate synthase, there has been no report on its correct assignment and functional characterization of its protein product. Results. In the present work, we describe DNA amplification of aroF-encoded CS from M. tuberculosis (MtCS), molecular cloning, protein expression, and purification to homogeneity. N-terminal amino acid sequencing, mass spectrometry and gel filtration chromatography were employed to determine identity, subunit molecular weight and oligomeric state in solution of homogeneous recombinant MtCS. The bifunctionality of MtCS was determined by measurements of both chorismate synthase and NADH:FMN oxidoreductase activities. The flavin reductase activity was characterized, showing the existence of a complex between FMN ox and MtCS. FMNox and NADH equilibrium binding was measured. Primary deuterium, solvent and multiple kinetic isotope effects are described and suggest distinct steps for hydride and proton transfers, with the former being more rate-limiting. Conclusion. This is the first report showing that a bacterial CS is bifunctional. Primary deuterium kinetic isotope effects show that C4-proS hydrogen is being transferred during the reduction of FMNox by NADH and that hydride transfer contributes significantly to the rate-limiting step of FMN reduction reaction. Solvent kinetic isotope effects and proton inventory results indicate that proton transfer from solvent partially limits the rate of FMN reduction and that a single proton transfer gives rise to the observed solvent isotope effect. Multiple isotope effects suggest a stepwise mechanism for the reduction of FMNox. The results on enzyme kinetics described here provide evidence for the mode of action of MtCS and should thus pave the way for the rational design of antitubercular agents. © 2008 Ely et al; licensee BioMed Central Ltd.

Glucocorticoids in vivo induce both insulin hypersecretion and enhanced glucose sensitivity of stimulus-secretion coupling in isolated rat islets

Although glucocorticoids are widely used as antiinflammatory agents in clinical therapies, they may cause serious side effects that include insulin resistance and hyperinsulinemia. To study the potential functional adaptations of the islet of Langerhans to in vivo glucocorticoid treatment, adult Wistar rats received dexamethasone (DEX) for 5 consecutive days, whereas controls (CTL) received only saline. The analysis of insulin release in freshly isolated islets showed an enhanced secretion in response to glucose in DEX-treated rats. The study of Ca2 2+ signals by fluorescence microscopy also demonstrated a higher response to glucose in islets from DEX-treated animals. However, no differences in Ca2 2+signals were found between both groups with tolbutamide or KCl, indicating that the alterations were probably related to metabolism. Thus, mitochondrial function was explored by monitoring oxidation of nicotinamide dinucleotide phosphate autofluorescence and mitochondrial membrane potential. Both parameters revealed a higher response to glucose in islets from DEX-treated rats. The mRNA and protein content of glucose transporter-2, glucokinase, and pyruvate kinase was similar in both groups, indicating that changes in these proteins were probably not involved in the increased mitochondrial function. Additionally,weexplored the status of Ca2 2+-dependent signaling kinases. Unlike calmodulin kinase II, we found an augmented phosphorylation level of protein kinase Cα as well as an increased response of the phospholipase C/inositol 1,4,5-triphosphate pathway in DEX-treated rats. Finally, an increased number of docked secretory granules were observed in the β-cells of DEX animals using transmission electron microscopy. Thus, these results demonstrate that islets from glucocorticoid-treated rats develop several adaptations that lead to an enhanced stimulus-secretion coupling and secretory capacity. Copyright © 2010 by The Endocrine Society.

Role of nitric oxide in the remodelling of extracellular matrix in myxomatous mitral valve degeneration of dogs and pigs

Myxomatous mitral valve degeneration (MMVD) or endocardiosis is a heart valve disease that occurs in many mammalian species, especially in humans, dogs and pigs. Nitric oxide (NO) plays an important role in the MMVD development. NO can be indirectly evaluated by the nitric-oxide synthase (NOS) expression and by the histochemical reaction of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d). The aim of this study was to evaluate NOS activity, by NADPH-d reaction, in the anterior leaflet of dogs with regular mitral valves and in those with MMVD, as well as in young swine and old females, comparing the reaction level with the degree of endocardiosis disease and also the histological alterations. Twelve mitral valves of dogs and 22 of swine were used for the research. All the valves were macroscopically analyzed for the occurrence or not of endocardiosis. They were fixed in a 4% paraformaldehyde, exposed to NADPH-d reaction, routinely processed and microscopically evaluated for the detection of mucopolysaccharides (MPS) deposition, collagen degeneration, fibrosis and level of endocardiosis. In dogs, relation was observed between higher intensity of the NADPH-d reaction, higher endocardiosis degree, MPS deposition as well as the collagen degeneration. No alteration in color was observed in pigs ́ valves during NADPH-d reaction. In conclusion, NO works in canine mitral valve remodeling extracellular matrix and plays an important role in endocardiosis disease. In swine, the lack of reaction reinforces the absence of macroscopical endocardiosis lesions, suggesting restrict NO action or major differences in the structures of swine valves.

Apocynin: Chemical and biophysical properties of a NADPH oxidase inhibitor

Apocynin is the most employed inhibitor of NADPH oxidase (NOX), a multienzymatic complex capable of catalyzing the one-electron reduction of molecular oxygen to the superoxide anion. Despite controversies about its selectivity, apocynin has been used as one of the most promising drugs in experimental models of inflammatory and neurodegenerative diseases. Here, we aimed to study the chemical and biophysical properties of apocynin. The oxidation potential was determined by cyclic voltammetry (Epa = 0.76V), the hydrophobicity index was calculated (logP = 0.83) and the molar absorption coefficient was determined (ε275nm = 1.1 × 104 M-1 cm-1). Apocynin was a weak free radical scavenger (as measured using the DPPH, peroxyl radical and nitric oxide assays) when compared to protocatechuic acid, used here as a reference antioxidant. On the other hand, apocynin was more effective than protocatechuic acid as scavenger of the non-radical species hypochlorous acid. Apocynin reacted promptly with the non-radical reactive species H2O2 only in the presence of peroxidase. This finding is relevant, since it represents a new pathway for depleting H2O2 in cellular experimental models, besides the direct inhibition of NADPH oxidase. This could be relevant for its application as an inhibitor of NOX4, since this isoform produces H 2O2 and not superoxide anion. The binding parameters calculated by fluorescence quenching showed that apocynin binds to human serum albumin (HSA) with a binding affinity of 2.19 × 104 M -1. The association did not alter the secondary and tertiary structure of HSA, as verified by synchronous fluorescence and circular dichroism. The displacement of fluorescent probes suggested that apocynin binds to site I and site II of HSA. Considering the current biomedical applications of this phytochemical, the dissemination of these chemical and biophysical properties can be very helpful for scientists and physicians interested in the use of apocynin.

Estudo da eficiência de produção de peróxido de hidrogênio por naftoquinonas em associação com ácido ascórbico: aplicações como agentes citotóxicos em linhagem de células normal e tumoral

Pós-graduação em Biociências e Biotecnologia Aplicadas à Farmácia - FCFAR

Ácido protocatecúico e seus ésteres alquílicos: propriedades antioxidantes e seus efeitos no metabolismo oxidativo de leucócitos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Standardization of metachromatic staining method of myofibrillar ATPase activity of myosin to skeletal striated muscle of mules and donkeys

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Determination of salicylate in blood serum using an amperometric biosensor based on salicylate hydroxylase immobilized in a polypyrrole-glutaraldehyde matrix

The use of an amperometric biosensor for the salicylate determination in blood serum is described. The biosensor is based on salicylate hydroxylase (EC 1.14.13.1) electropolymerized onto a glassy carbon-working electrode with polypyrrole and glutaraldehyde, to improve the biosensor lifetime. The hexacyanoferrate (II) was also incorporated to work as a redox mediator to minimize possible interferences. The salicylate is enzymatically converted to catechol, which is monitored amperometrically by its electrooxidation at +0.170 V versus SCE (saturated calomel electrode). Salicylate determination was carried out maintaining the ratio between β-NADH and salicylate at 4:1 (30°C). The amperometric response of the biosensor was linearly proportional to the salicylate concentration between 2.3 x 10-6 and 1.4 x 10-5 mol l- 1, in 0.1 mol l-1 phosphate buffer (pH 7.8), containing 0.1 mol l-1 KCl and 5.0 x 10-4 mol l-1 Na2H2EDTA, as supporting electrolyte. The recovery studies, in the presence of several interfering compounds, showed recoveries between 96.4 and 104.8%. The useful lifetime of the biosensor in the concentration range evaluated was at least 40 days, in continuous use. Blood serum samples analyzed by this biosensor showed a good correlation compared to the spectrophotometric method (Trinder) used as reference, presenting relative deviations lower than 7.0%. (C) 2000 Elsevier Science B.V.

Inibição sistêmica da NADPH oxidase: estudo do coração de ratos espontaneamente hipertensos com diabetes mellitus

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

The antibiotics roseoflavin and 8-demethyl-8-amino-riboflavin from Streptomyces davawensis are metabolized by human flavokinase and human FAD synthetase

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Kinetic aspects of rat (Rattus-norvegicus albinus) submandibular glands lactic-dehydrogenase

Submandibular glands of male rats were homogenized with 33 mM sodium potassium phosphate buffer, pH 6.5, containing 1 mM MgCl2 and 0.1 mM DTT and purified with ammonium sulphate, phosphocellulose chromatography, eluted with KC1 0.5 M, followed by Blue Sepharose CL-6B chromatography, eluted with NADH 0.5 mM. The enzyme kepts stable for 60 days when stored at -15-degrees-C in 33 mM phosphate buffer. In other experiment the enzyme was purified by oxamate-agarose chromatography from a crude extract of submandibular gland and the results obtained were better than by phosphocellulose and Sepharose CL-6B chromatography. The Km values for pyruvate. NADH, lactate and NAD+ were established. Sodium oxamate at 0.1 and 0.9 mM concentrations inhibited the LDH activity by 40 and 85%, respectively (competitive); with sodium oxalate the inhibition was of 30% (uncompetitive) and with 3-acetyl pyridine adenine dinucleotide was 80%.

Structural insights into selectivity and cofactor binding in snake venom l-amino acid oxidases

l-Amino acid oxidases (LAAOs) are flavoenzymes that catalytically deaminate l-amino acids to corresponding α-keto acids with the concomitant production of ammonia (NH 3) and hydrogen peroxide (H 2O 2). Particularly, snake venom LAAOs have been attracted much attention due to their diverse clinical and biological effects, interfering on human coagulation factors and being cytotoxic against some pathogenic bacteria and Leishmania ssp. In this work, a new LAAO from Bothrops jararacussu venom (BjsuLAAO) was purified, functionally characterized and its structure determined by X-ray crystallography at 3.1å resolution. BjsuLAAO showed high catalytic specificity for aromatic and aliphatic large side-chain amino acids. Comparative structural analysis with prokaryotic LAAOs, which exhibit low specificity, indicates the importance of the active-site volume in modulating enzyme selectivity. Surprisingly, the flavin adenine dinucleotide (FAD) cofactor was found in a different orientation canonically described for both prokaryotic and eukaryotic LAAOs. In this new conformational state, the adenosyl group is flipped towards the 62-71 loop, being stabilized by several hydrogen-bond interactions, which is equally stable to the classical binding mode. © 2012 Elsevier Inc.

Glyceraldehyde 3-phosphate dehydrogenase-telomere association correlates with redox status in trypanosoma cruzi

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Preliminary study on age- and sex-dependent alterations in the composition of skeletal muscle fibers of Brasileiro de Hipismo horses

The aim of this work was to characterize the distribution of myofibers in the gluteus medius muscle of inactive horses of the Brasileiro de Hipismo (BH) breed at different ages by means of histochemical analyses, according to sex and depth of the biopsy. A total of 78 inactive horses (9 castrated males, 35 stallions, and 34 females) of the BH breed, aged 1 to 4 years, were used. A percutaneous muscle biopsy was obtained with a 6.0-mm Bergstrom-type needle, which allowed the removal of muscle fragments at depths of 20 and 60 mm. Myofiber types were determined based on myofibrillar adenosine triphosphatase (mATPase) and nicotinamide dinucleotide tetrazolium reductase (NADH-TR) techniques. Morphometry of the fibers was determined based on cross-sectional area (CSA), mean frequency (F), and relative cross-sectional area (RCSA). The current study demonstrated that BH horses 3 and 4 years of age show a greater percentage of, and area occupied by, type IIA fibers and lower percentage of type IIX fibers in the gluteus medius muscle compared with horses 1 and 2 years of age. No difference was found between sexes in the frequency of and area occupied by the different fiber types at any of the depths and ages studied. In this study, females showed a greater CSA for all fibers in comparison with males, at 1 year of age. The results of the current study indicate that the gluteus medius muscle of inactive BH horses shows modifications in its structural and biochemical composition during the growth of the animals, leading to a better oxidative capacity.

EVOLUTION OF ADENINE CLUSTERING IN 5S RIBOSOMAL-RNA

The frequency of adenine mononucleotides (A), dinucleotides (AA) and clusters, and the positions of clusters, were studied in 502 molecules of the 5S rRNA.All frequencies were reduced in the evolutive lines of vertebrates, plants and fungi, in parallel with increasing organismic complexity. No change was observed in invertebrates. All frequencies were increased in mitochondria, plastids and mycoplasmas. The presumed relatives to the ancestors of the organelles, Rhodobacteria alfa and Cyanobacteria, showed intermediate values, relative to the eubacterial averages. Firmibacterid showed very high number of cluster sites.Clusters were more frequent in single-stranded regions in all organisms. The routes of organelles and mycoplasmas accummulated clusters at faster rates in double-stranded regions. Rates of change were higher for AA and clusters than for A in plants, vertebrates and organeltes, higher for cluster sites and A in mycoplasmas, and higher for AA and A in fungi. These data indicated that selection pressures acted more strongly on adenine clustering than on adenine frequency.It is proposed that AA and clusters, as sites of lower informational content. have the property of tolerating positional variation in the sites of other molecules (or other regions of the same molecule) that interact with the adenines. This reasoning was consistent with the degrees of genic polymorphism. low in plants and vertebrates and high in invertebrates. In the eubacteria endosymbiontic or parasitic to eukaryotes, the more tolerant RNA would be better adapted to interactions with the homologous nucleus-derived ribosomal proteins: the intermediate values observed in their precursors were interpreted as preadaptive.Among other groups, only the Deinococcus-Thermus eubacteria showed excessive AA and cluster contents, possibly related to their peculiar tolerance to mutagens, and the Ciliates showed excessive AA contents, indicative of retention of primitive characters.