Purification, characterization, crystallization and theoretical molecular modeling of gyroxin fraction from Crotalus durissus terrificus venom (Laurenti, 1768)

The venom of Crotalus durissus terrificus snakes presents various substances, including a serine protease with thrombin-like activity, called gyroxin, that clots plasmatic fibrinogen and promote the fibrin formation. The aim of this study was to purify and structurally characterize the gyroxin enzyme from Crotalus durissus terrificus venom. For isolation and purification, the following methods were employed: gel filtration on Sephadex G75 column and affinity chromatography on benzamidine Sepharose 6B; 12% SDS-PAGE under reducing conditions; N-terminal sequence analysis; cDNA cloning and expression through RT-PCR and crystallization tests. Theoretical molecular modeling was performed using bioinformatics tools based on comparative analysis of other serine proteases deposited in the NCBI (National Center for Biotechnology Information) database. Protein N-terminal sequencing produced a single chain with a molecular mass of similar to 30 kDa while its full-length cDNA had 714 bp which encoded a mature protein containing 238 amino acids. Crystals were obtained from the solutions 2 and 5 of the Crystal Screen Kit (R), two and one respectively, that reveal the protein constitution of the sample. For multiple sequence alignments of gyroxin-like B2.1 with six other serine proteases obtained from snake venoms (SVSPs), the preservation of cysteine residues and their main structural elements (alpha-helices, beta-barrel and loops) was indicated. The localization of the catalytic triad in His57, Asp102 and Ser198 as well as S1 and S2 specific activity sites in Thr193 and Gli215 amino acids was pointed. The area of recognition and cleavage of fibrinogen in SVSPs for modeling gyroxin B2.1 sequence was located at Arg60, Arg72, Gln75, Arg81, Arg82, Lis85, Glu86 and Lis87 residues. Theoretical modeling of gyroxin fraction generated a classical structure consisting of two alpha-helices, two beta-barrel structures, five disulfide bridges and loops in positions 37, 60, 70, 99, 148, 174 and 218. These results provided information about the functional structure of gyroxin allowing its application in the design of new drugs.

Biochemical and functional properties of a thrombin-like enzyme isolated from Bothrops pauloensis snake venom

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

Levels of digestible threonine for male broilers from 1 to 7 days of age

The influence of threonine has been studied in broilers; however, information concerning the phase from 1 to 7 d is lacking. In this study, we evaluated the effects on performance and body composition and the relationship with other amino acids with respect to digestible lysine and total glycine + glycine. Research on the initial 7-d phase of broiler growth is still poorly studied, even though it is important, especially in nutrition. It was found that threonine had a great influence on the broilers at that phase and nutritional recommendations need constant evaluation. In conclusion, the digestible threonine: digestible lysine ratio and digestible threonine: total glycine + serine ratio recommended for better performance and body composition are 0.68 and 0.40, respectively. The levels of digestible threonine and total glycine + serine that provide the best results are 0.864 and 2.192%, respectively.

Genomics-based design of defined growth media for the plant pathogen Xylella fastidiosa

Based on the genetic analysis of the phytopathogen Xylella fastidiosa genome, five media with defined composition were developed and the growth abilities of this fastidious prokaryote were evaluated in liquid media and on solid plates. All media had a common salt composition and included the same amounts of glucose and vitamins but differed in their amino acid content. XDM1 medium contained amino acids threonine, serine, glycine, alanine, aspartic acid and glutamic acid, for which complete degradation pathways occur in X fastidiosa; XDM2 included serine and methionine, amino acids for which biosynthetic enzymes are absent, plus asparagine and glutamine, which are abundant in the xylem sap; XDM3 had the same composition as XDM2 but with asparagine replaced by aspartic acid due to the presence of complete degradation pathway for aspartic acid; XDM4 was a minimal medium with glutamine as a sole nitrogen source; XDM5 had the same composition as XDM4, plus methionine. The liquid and solidified XDM2 and XDM3 media were the most effective for the growth of X. fastidiosa. This work opens the opportunity for the in silico design of bacterial defined media once their genome is sequenced. (C) 2002 Federation of European Microbiological Societies. Published by Elsevier B.V. B.V. All rights reserved.

Bacterial functional redundancy along a soil reclamation gradient

A strategy to measure bacterial functional redundancy was developed and tested with soils collected along a soil reclamation gradient by determining the richness and diversity of bacterial groups capable of in situ growth on selected carbon substrates. Soil cores were collected from four sites along a transect from the Jamari tin mine site in the Jamari National Forest, Rondonia, RO, Brazil: denuded mine spoil, soil from below the canopy of invading pioneer trees, revegetated soil under new growth on the forest edge, and the forest floor of an adjacent preserved forest. Bacterial population responses were analyzed by amending these soil samples with individual carbon substrates in the presence of bromodeoxyuridine (BrdU), BrdU-labeled DNA was then subjected to a 16S-23S rRNA intergenic analysis to depict the actively growing bacteria from each site, the number and diversity of bacterial groups responding to four carbon substrates (L-serine, L-threonine, sodium citrate, and or-lactose hydrate) increased along the reclamation-vegetation gradient such that the preserved forest soil samples contained the highest functional redundancy for each substrate. These data suggest that bacterial functional redundancy increases in relation to the regrowth of plant communities and may therefore represent an important aspect of the restoration of soil biological functionality to reclaimed mine spoils. They also suggest that bacterial functional redundancy may be a useful indicator of soil quality and ecosystem functioning.

Cellular and molecular characterization of an embryonic cell line (BME26) from the tick Rhipicephalus (Boophilus) microplus

The cellular and molecular characteristics of a cell line (BME26) derived from embryos of the cattle tick Rhipicephalus (Boophilus) microplus were studied. The cells contained glycogen inclusions, numerous mitochondria, and vesicles with heterogeneous electron densities dispersed throughout the cytoplasm. Vesicles contained lipids and sequestered palladium meso-porphyrin (Pd-mP) and rhodamine-hemoglobin, suggesting their involvement in the autophagic and endocytic pathways. The cells phagocytosed yeast and expressed genes encoding the antimicrobial peptides (microplusin and defensin). A cDNA library was made and 898 unique mRNA sequences were obtained. Among them, 556 sequences were not significantly similar to any sequence found in public databases. Annotation using Gene Ontology revealed transcripts related to several different functional classes. We identified transcripts involved in immune response such as ferritin, serine proteases, protease inhibitors,. antimicrobial peptides, heat shock protein, glutathione S-transferase, peroxidase, and NADPH oxidase. BME26 cells transfected with a plasmid carrying a red fluorescent protein reporter gene (DsRed2) transiently expressed DsRed2 for up to 5 weeks. We conclude that BME26 can be used to experimentally analyze diverse biological processes that occur in R. (B.) microplus such as the innate immune response to tick-borne pathogens. (C) 2008 Elsevier Ltd. All rights reserved.

Digestibilidade do extrato de leveduras em frangos de corte

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

Signal transducer and activator of transcription 3-regulated sarcoendoplasmic reticulurn Ca2+-ATPase 2 expression by prolactin and glucocorticoids is involved in the adaptation of insulin secretory response during the peripartum period

During pregnancy, the maternal endocrine pancreas undergoes, as a consequence of placental lactogens and prolactin (PR,L) action, functional changes that are characterized by increased glucose-induced insulin secretion. After delivery, the maternal endocrine pancreas rapidly returns to nonpregnant state, which is mainly attributed to the increased serum levels of glucocorticoids (GCs). Although GCs are known to decrease insulin secretion and counteract PRL action, the mechanisms for these effects are poorly understood. We have previously demonstrated that signal transducer and activator of transcription 3 (STAT3) is increased in islets treated with PRL. In the present study, we show that STAT3 expression and serine phosphorylation are increased in pancreatic islets at the end of pregnancy (P19). STAT3 serine phosphorylation rapidly returned to basal levels 3 days after delivery (U). The expression of the sarcoendoplasmic reticulum Ca2+-ATPase 2 (SERCA2), a crucial protein involved in the regulation of calcium handling in P-cells, was also increased in P19, returning to basal levels at L3. PRL increased SERCA2 and STAT3 expressions and STAT3 serine phosphorylation in RINm5F cells. The upregulation of SERCA2 by PRL was abolished after STAT3 knockdown. Moreover, PRL-induced STAT3 serine phosphorylation and SERCA2 expression were inhibited by dexamethasone (DEX). Insulin secretion from islets of PI 9 rats pre-incubated with thapsigargin and L3 rats showed a dramatic suppression of first phase of insulin release. The present results indicate that PRL regulates SERCA2 expression by a STAT3-dependent mechanism. PRL effect is counteracted by DEX and might contribute to the adaptation of maternal endocrine pancreas during the peripartum period.

Reduced pancreatic β-cell mass is associated with decreased FoxO1 and Erk1/2 protein phosphorylation in low-protein malnourished rats

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

Variability and resistance mutations in the hepatitis C virus NS3 protease in patients not treated with protease inhibitors

The goal of treatment of chronic hepatitis C is to achieve a sustained virological response, which is defined as exhibiting undetectable hepatitis C virus (HCV) RNA levels in serum following therapy for at least six months. However, the current treatment is only effective in 50% of patients infected with HCV genotype 1, the most prevalent genotype in Brazil. Inhibitors of the serine protease non-structural protein 3 (NS3) have therefore been developed to improve the responses of HCV-infected patients. However, the emergence of drug-resistant variants has been the major obstacle to therapeutic success. The goal of this study was to evaluate the presence of resistance mutations and genetic polymorphisms in the NS3 genomic region of HCV from 37 patients infected with HCV genotype 1 had not been treated with protease inhibitors. Plasma viral RNA was used to amplify and sequence the HCV NS3 gene. The results indicate that the catalytic triad is conserved. A large number of substitutions were observed in codons 153, 40 and 91; the resistant variants T54A, T54S, V55A, R155K and A156T were also detected. This study shows that resistance mutations and genetic polymorphisms are present in the NS3 region of HCV in patients who have not been treated with protease inhibitors, data that are important in determining the efficiency of this new class of drugs in Brazil.

Biochemical and biological evaluation of gyroxin isolated from Crotalus durissus terrificus venom

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

A NEW FIBRIN SEALANT FROM Crotalus durissus terrificus VENOM: APPLICATIONS IN MEDICINE

Fibrin sealant, a widely available tissue adhesive, has been used since 1940 in a variety of clinical applications. Commercially available fibrin sealant products are synthesized from bovine thrombin and human fibrinogen, which may transmit infectious diseases, and recipients may also develop antibodies against bovine thrombin. Bearing these disadvantages in mind, a new fibrin sealant was developed in 1989 by a group of researchers from the Center for the Study of Venoms and Venomous Animals, in São Paulo State, Brazil. The main purpose was to produce an adhesive fibrin without using human blood, to avoid transmitting infectious diseases. The components of this novel sealant were extracted from large animals and a serine proteinase extracted from Crotalus durissus terrificus snake venom. The applicability of this sealant was tested in animals and humans with beneficial results. The new fibrin sealant can be a useful tool clinically due to its flexibility and diversity of applications. This sealant is a biological and biodegradable product that ( 1) does not produce adverse reactions, ( 1) contains no human blood, ( 3) has a good adhesive capacity, ( 4) gives no transmission of infectious diseases, and ( 5) may be used as an adjuvant in conventional suture procedures. The effectiveness of this new fibrin sealant is reviewed and its development and employment are described.

Plasma glutathione of HIV+ patients responded positively and differently to dietary supplementation with cysteine or glutamine

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

Digestibilidade aparente do triticale para a tilápia-do-nilo

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

NaF treatment increases TNF-alpha and resistin concentrations and reduces insulin signal in rats

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