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.

Evaluation of the interactions of DNA with the textile dyes Disperse Orange 1 and Disperse Red 1 and their electrolysis products using an electrochemical biosensor

A disposable pencil graphite electrode modified with dsDNA was used in combination with square wave voltammetry in order to evaluate the interaction of DNA with the textile dyes Disperse Orange 1 (DO1) and Disperse Red 1 (DR1), and with the products of their electrolysis. Significant changes in the characteristic oxidation peaks of the guanine and adenine moieties of immobilized dsDNA were observed after incubation of the modified electrode for 180 s in solutions of the dyes in their original forms. The same was observed using the electrolysis products obtained by oxidation and reduction conversions. The oxidation peak currents of the guanine and adenine moieties decreased when the concentrations of DO1 and DR1 were increased up to 5.0 × 10 -6 and 1.0 × 10-6 mol L-1, respectively; the signal decreases were more pronounced after interaction with the oxidized dyes, compared to the reduced compounds. The interactions between DNA and DO1, DR1, and the electrolyzed dyes were further investigated by UV-vis spectrophotometry in solution, and different effects such as hypochromism and hyperchromism were observed in the resulting DNA spectra. The investigated interactions showed clear evidence of changes in the DNA structure, and suggested a predominant intercalation mode leading to damage in the biomolecule. © 2013 Elsevier B.V.

Phylogenetic and structural studies of a novel equine papillomavirus identified from aural plaques

Papillomaviruses (PVs) infect a wide range of animal species and show great genetic diversity. To date, excluding equine sarcoids, only three species of PVs were identified associated with lesions in horses: Equus caballus papillomavirus 1 (EcPV1-cutaneous), EcPV2 (genital) and EcPV3 (aural plaques). In this study, we identified a novel equine PV from aural plaques, which we designated EcPV4. Cutaneous samples from horses with lesions that were microscopically diagnosed as aural plaques were subjected to DNA extraction, amplification and sequencing. Rolling circle amplification and inverse PCR with specific primers confirmed the presence of an approximately 8. kb circular genome. The full-length EcPV4 L1 major capsid protein sequence has 1488 nucleotides (495 amino acids). EcPV4 had a sequence identity of only 53.3%, 60.2% and 51.7% when compared with the published sequences for EcPV1, EcPV2 and EcPV3, respectively. A Bayesian phylogenetic analysis indicated that EcPV4 clusters with EcPV2, but not with EcPV1 and EcPV3. Using the current PV classification system that is based on the nucleotide sequence of L1, we could not define the genus of the newly identified virus. Therefore, a structural analysis of the L1 protein was carried out to aid in this classification because EcPV4 cause lesion similar to the lesion caused by EcPV3. A comparison of the superficial loops demonstrated a distinct amino acid conservation pattern between EcPV4/EcPV2 and EcPV4/EcPV3. These results demonstrate the presence of a new equine PV species and that structural studies could be useful in the classification of PVs. © 2012 Elsevier B.V.

Clinical and molecular study of a new form of hereditary myotonia in Murrah water buffalo

Hereditary myotonia caused by mutations in CLCN1 has been previously described in humans, goats, dogs, mice and horses. The goal of this study was to characterize the clinical, morphological and genetic features of hereditary myotonia in Murrah buffalo. Clinical and laboratory evaluations were performed on affected and normal animals. CLCN1 cDNA and the relevant genomic region from normal and affected animals were sequenced. The affected animals exhibited muscle hypertrophy and stiffness. Myotonic discharges were observed during EMG, and dystrophic changes were not present in skeletal muscle biopsies; the last 43 nucleotides of exon-3 of the CLCN1 mRNA were deleted. Cloning of the genomic fragment revealed that the exclusion of this exonic sequence was caused by aberrant splicing, which was associated with the presence of a synonymous SNP in exon-3 (c.396C>T). The mutant allele triggered the efficient use of an ectopic 5' splice donor site located at nucleotides 90-91 of exon-3. The predicted impact of this aberrant splicing event is the alteration of the CLCN1 translational reading frame, which results in the incorporation of 24 unrelated amino acids followed by a premature stop codon. Copyright © 2012 Elsevier B.V. All rights reserved.

The role of mitochondria and biotransformation in abamectin-induced cytotoxicity in isolated rat hepatocytes

Abamectin (ABA), which belongs to the family of avermectins, is used as a parasiticide; however, ABA poisoning can impair liver function. In a previous study using isolated rat liver mitochondria, we observed that ABA inhibited the activity of adenine nucleotide translocator and FoF1-ATPase. The aim of this study was to characterize the mechanism of ABA toxicity in isolated rat hepatocytes and to evaluate whether this effect is dependent on its metabolism. The toxicity of ABA was assessed by monitoring oxygen consumption and mitochondrial membrane potential, intracellular ATP concentration, cell viability, intracellular Ca2+ homeostasis, release of cytochrome c, caspase 3 activity and necrotic cell death. ABA reduces cellular respiration in cells energized with glutamate and malate or succinate. The hepatocytes that were previously incubated with proadifen, a cytochrome P450 inhibitor, are more sensitive to the compound as observed by a rapid decrease in the mitochondrial membrane potential accompanied by reductions in ATP concentration and cell viability and a disruption of intracellular Ca2+ homeostasis followed by necrosis. Our results indicate that ABA biotransformation reduces its toxicity, and its toxic action is related to the inhibition of mitochondrial activity, which leads to decreased synthesis of ATP followed by cell death. © 2012 Elsevier Ltd.

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.

Estudos estruturais da Purina Nucleosídeo Fosforilase do Mycobacterium tuberculosis complexada com ligantes

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Clonagem e expressão da proteína E2 no vírus da hepatite C Humana: estudo da interação molecular E2-rLDL in vitro

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Papel funcional de microRNAs na arquitetura vegetativa e radicular de plantas

Pós-graduação em Ciências Biológicas (Genética) - IBB

Estudo do papel das proteínas mitocondriais desacopladas na tolerância aos estresses abióticos empregando diferentes abordagens

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Complementação do seqüenciamento do genoma do Southern bean mosaic virus, isolado São Paulo, expressão da porção C-terminal da polimerase e produção de anti-soro policlonal

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Perfil eletroforético e cromatográfico das hemoglobinas S-like

Pós-graduação em Genética - IBILCE

Marcadores moleculares associados à qualidade de carne em bovinos Nelore

Pós-graduação em Genética e Melhoramento Animal - FCAV

Identificação de estirpes de rizóbios por seqüenciamento parcial dos genes 16S rDNA e nifH

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)