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.

Pectin and pectinases: Production, characterization and industrial application of microbial pectinolytic enzymes

Pectinases are a big group of enzymes that break down pectic polysaccharides of plant tissues into simpler molecules like galacturonic acids. It has long been used to increase yields and clarity of fruit juices. Since pectic substances are a very complex macromolecule group, various pectinolytic enzymes are required to degrade it completely. These enzymes present differences in their cleavage mode and specificity being basically classified into two main groups that act on pectin smooth regions or on pectin hairy regions. Pectinases are one of the most widely distributed enzymes in bacteria, fungi and plants. This review describes the pectinolytic enzymes and their substrates, the microbial pectinase production and characterization, and the industrial application of these enzymes. © Pedrolli et al.; Licensee Bentham Open.

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.

Thiamin Requirement of Nile Tilapia, Oreochromis niloticus

Juvenile Nile tilapia, Oreochromis niloticus, were fed to apparent satiation twice daily with purified diets containing 0, 1.0, 2.0, 4.0, 8.0, and 16.0 mg/kg and 0, 2.0, 4.0, 8.0, 16.0, and 32.0 mg/kg of thiamin in separate 14- and 8-wk trials (Experiments 1 and 2, respectively). Fish fed the diet devoid of thiamin developed neurological disorders, anorexia, reduced growth, and feed efficiency and increased mortality (Experiment 2 only) within 4-6 and 8-10 wk for Experiments 2 and 1, respectively. Low red blood cell count (RBC) and hematocrit (Ht) were observed in fish fed the thiamin-deficient diet. Serum pyruvate was elevated in fish fed the thiamin unsupplemented diet. Serum lactate was not affected by dietary thiamin levels. Whole body protein was unaffected by dietary levels of thiamin. Body moisture and ash increased whereas body lipid decreased in fish fed the thiamin unsupplemented diets. None of these abnormalities were observed in fish fed the thiamin-supplemented diets. Using the response curves determined by PROC NLMIXED to estimate dietary thiamin levels required for various response variables, a dietary thiamin level of 3.5 mg/kg diet was adequate for optimum growth, feed intake and efficiency, survival, prevention of neurological symptoms, and maintaining normal levels of RBC, Ht, serum pyruvate, and proximate body composition. © 2011.

Efeito de produtos químicos sobre a mancha bacteriana (xanthomonas perforans) e na ativação de proteínas relacionadas à patogênese em tomateiro

The present study aims to evaluate the effect of fungicides and antibiotics to control bacterial spot (Xanthomonas perforans) in tomato, and the activation of pathogenesis-related proteins. Hybrid tomato AP 529 was used to assess the severity of disease. The treatments consisted of spraying with acibenzolar-S-methyl, fluazinam, pyraclostrobin, pyraclostrobin + methiran, copper oxychloride, copper oxychloride and mancozeb + oxytetracycline, and inoculated and non-inoculated controls. After three days of treatment, all plants were inoculated with X. perforans (10 6 CFU / mL). Leaf discs were collected for assessment of peroxidase, polyphenol oxidase, β-1,3 glucanase, phenylalanine ammonia lyase and protease. The area under the disease progress curve (AUDPC) was calculated with the data of severity. All treatments had reduced AUDPC compared to the inoculated control. Fungicides acibenzolar-S-methyl, pyraclostrobin, and pyraclostrobin + methiran had more satisfactory results in reducing the severity of bacterial spot on tomato. The products based on pyraclostrobin together with acibenzolar-S-methyl induced enzymatic activities of peroxidase, polyphenoloxidase and β-1,3 glucanase, indicating that these products may be related to the induction of resistance to bacterial spot on tomato plants.

Low dose of dichloroacetate infusion reduces blood lactate after submaximal exercise in horses

The acute administration of an indirect activator of the enzyme pyruvate dehydroge-nase (PDH) in human athletes causes a reduction in blood lactate level during and after exercise. A single IV dose (2.5m.kg-1) of dichloroacetate (DCA) was administered before a submaximal incremental exercise test (IET) with five velocity steps, from 5.0 m.s-1 for 1 min to 6.0, 6.5, 7.0 and 7.5m.s-1 every 30s in four untrained mares. The blood collections were done in the period after exercise, at times 1, 3, 5, 10, 15 and 20 min. Blood lactate and glucose (mM) were determined electro-enzymatically utilizing a YSI 2300 automated analyzer. There was a 15.3% decrease in mean total blood lactate determined from the values obtained at all assessment times in both trials after the exercise. There was a decrease in blood lactate 1, 3, 5, 10, 15 and 20 min after exercise for the mares that received prior DCA treatment, with respective mean values of 6.31±0.90 vs 5.81±0.50, 6.45±1.19 vs 5.58±1.06, 6.07±1.56 vs 5.26±1.12, 4.88±1.61 vs 3.95±1.00, 3.66±1.41 vs 2.86±0.75 and 2.75±0.51 vs 2.04±0.30. There was no difference in glucose concentrations. By means of linear regression analysis, V140, V160, V180 and V200 were determined (velocity at which the rate heart is 140, 160, 180, and 200 beats/minute, respectively). The velocities related to heart rate did not differ, indicating that there was no ergogenic effect, but prior administration of a relatively low dose of DCA in mares reduced lactatemia after an IET.

Combined effects of age and diet-induced obesity on biochemical parameters and cardiac energy metabolism in rats

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

Qualidade de mangas cv. palmer após armazenamento sob baixas temperaturas

This study aimed to evaluate the quality of 'Palmer' mangoes previously stored at low temperatures, after their transference to the environmental condition. Fruits harvested at physiological maturity were carefully transported to the Laboratory where they were selected, standardized as the color, size and absence of injuries and treated with fungicide before they were stored at 2°C (75.7% RH), 5°C (73.8% RH) e 12°C (82% RH) for 7, 14 and 21 days. At the end of each period, the fruits were transferred to environmental condition (22.9°C; 62.3% RH), where they were kept for 1, 3, 5 and 7 days, simulating the trading period, and evaluated for the occurrence of injuries and rottenness; peel and pulp color; firmness; contents of soluble solids, titratable acidity and ascorbic acid, as well as, the activities of the enzymes peroxidase, polyphenoloxidase and phenylalanine ammonia-lyase. The results indicated that 'Palmer' mangoes can be stored at 12°C for 21 days without damage to ripening, but with limitations due to the occurrence of decay. The storage at 2°C and 5°C was limited by the occurrence of injuries in the peel, but at the temperature of 2°C these symptoms were more severe and compromised the development of the characteristic color of the peel. However, the ripening of the pulp was not harmed, but this process occurred with less intensity than in mangoes maintained at 12°C.

Caracterização estrutural de polissacarídeos produzidos por bactérias dos gêneros Rhizobium e Mesorhizobium

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estudo de parâmetros de fermentação na produção de biopolímeros por bactérias isoladas do solo e caracterização química dos grupamentos acetila e piruvato nos biopolímeros obtidos

Pós-graduação em Engenharia e Ciência de Alimentos - IBILCE

Influência da oxidação de glicose sobre a função miocárdica de ratos obesos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Caracterização fisiológica e produção de metabólitos secundários em Achillea millefolium L. após aplicação de quitosana

Elicitation with chitosan is a tool used to improve the responses of plant defense by inducing secondary metabolism routs. In addition, the adequate application of this elicitor on medicinal plants can promote the increase of major components in the composition of the oil. However, we lack information concerning which are the main physiological processes responsible for the changes in the composition of the oil. Thus, we aimed at evaluating the action of chitosan and determine an ideal concentration for optimizing the production of essential oil in Achillea millefolium L. and the changes in the physiological processes responsible for this increase. The research was conducted in greenhouse of the Plant Physiology sector of the Universidade Federal de Lavras (UFLA) with A. millefolium plants. The treatments consisted of control (water); acetic acid solvent (with no elicitor); and chitosan in the concentrations of 2, 4 and 6 g L -1 . The measurements of growth, gas exchange, enzyme activity of the antioxidant system and phenylalanine ammonia lyase (PAL), in addition to the production and composition of the essential oil. We verified that the application of chitosan promoted decrease in plant growth. However, the concentration of 4 g L -1 of chitosan induced an increase in the content and yield of the essential oil. In the oil, there was predominance of sesquiterpenic compounds, including the major compounds borneol, β-caryophyllene, β-cubebene, α-farnesene and chamazulene. The elicitation with chitosan at 4 g L -1 promoted an increase of the photosynthetic rate, activity of the antioxidant system and of PAL, however, this increase occurred in short-term, only in the first days after elicitation.

Maturação in vitro de oócitos ovinos com o uso da roscovitina e ciclohexemida

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

Obtenção, manutenção e caracterização de células-tronco embrionárias equinas

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

Vitrificação de embriões bovinos produzidos in vivo e in vitro após lipólise química

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