Crystallization, preliminary X-ray analysis and molecular-replacement solution of haemoglobin-II from the fish matrinxa (Brycon cephalus)

Haemoglobins constitute a set of proteins with interesting structural and functional properties, especially when the two large animal groups reptiles and fishes are focused on. Here, the crystallization and preliminary X-ray analysis of haemoglobin-II from the South American fish matrinxa (Brycon cephalus) is reported. X-ray diffraction data have been collected to 3.0 Angstrom resolution using synchrotron radiation (LNLS). Crystals were determined to belong to space group P2(1) and preliminary structural analysis revealed the presence of two tetramers in the asymmetric unit. The structure was determined using the standard molecular-replacement technique.

Crystallization and preliminary X-ray diffraction analysis of a eumenine mastoparan toxin: a new class of mast-cell degranulating peptide in the wasp venom

Mastoparans are tetradecapeptides found to be the major component of vespid venoms. A mastoparan toxin isolated from the venom of Anterhynchium flavomarginatum micado has been crystallized and X-ray diffraction data collected to 2.7 Angstrom resolution using a synchrotron-radiation source. Crystals were determined to belong to the space group P6(2)22 (P6(4)22). This is the first mastoparan to be crystallized and will provide further insights into the conformational significance of mastoparan toxins with respect to their potency and activity in G-protein regulation.

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.

Detection of Leptospira spp. in the semen and urine of bulls serologically reactive to Leptospira interrogans serovar hardjo

O presente trabalho avaliou a PCR na detecção de leptospiras em sêmen e urina de dez touros sorologicamente reagentes, comparando seus resultados com aqueles obtidos por outras técnicas de diagnóstico. Foram realizadas duas colheitas de materiais em dias alternados. As amostras de sêmen e de urina foram separadas em alíquotas para visualização direta em microscopia de campo escuro, inoculação em hamsters (apenas para o sêmen), isolamento em meio de cultura e PCR. Nenhum hamster apresentou positividade na prova de soroaglutinação microscópica (SAM); fragmentos de rins e fígado desses animais foram utilizados para a tentativa de isolamento em meio de cultura, sendo positivo o cultivo a partir do rim de hamster inoculado com semen de um touro, e do fígado de hamsters inoculados com o semen de três touros. O isolamento em meio de cultura foi negativo para todas as amostras de sêmen, mas foi positivo para cinco amostras de urina. Na PCR não houve resultado positivo para as amostras de sêmen, e apenas uma amostra de urina apresentou resultado positivo, sendo coincidente com uma das culturas positivas. Não foi possível visualizar leptospiras em nenhuma das amostras por exame direto em microscopia de campo escuro.

Crystallization and preliminary X-ray diffraction analysis of a glutathione S-transferase from Xylella fastidiosa

Glutathione S-transferases (GSTs) form a group of multifunctional isoenzymes that catalyze the glutathione-dependent conjugation and reduction reactions involved in the cellular detoxification of xenobiotic and endobiotic compounds. GST from Xylella fastidiosa (xfGST) was overexpressed in Escherichia coli and purified by conventional affinity chromatography. In this study, the crystallization and preliminary X-ray analysis of xfGST is described. The purified protein was crystallized by the vapour-diffusion method, producing crystals that belonged to the triclinic space group P1. The unit-cell parameters were a = 47.73, b = 87.73, c = 90.74 angstrom, alpha = 63.45, beta = 80.66, gamma = 94.55 degrees. xfGST crystals diffracted to 2.23 angstrom resolution on a rotating-anode X-ray source.

Crystallization of SiO2-CaO-Na2O Glass Using Sugarcane Bagasse Ash as Silica Source

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

Understanding the Crystallization Mechanism of a Wollastonite Base Glass Using Isoconversional, IKP Methods and Master Plots

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

Effect of therapeutic plasma concentrations of non-steroidal anti-inflammatory drugs on the production of reactive oxygen species by activated rat neutrophils

The release of reactive oxygen specie (ROS) by activated neutrophil is involved in both the antimicrobial and deleterious effects in chronic inflammation. The objective of the present investigation was to determine the effect of therapeutic plasma concentrations of non-steroidal anti-inflammatory drugs (NSAIDs) on the production of ROS by stimulated rat neutrophils. Diclofenac (3.6 µM), indomethacin (12 µM), naproxen (160 µM), piroxicam (13 µM), and tenoxicam (30 µM) were incubated at 37ºC in PBS (10 mM), pH 7.4, for 30 min with rat neutrophils (1 x 10(6) cells/ml) stimulated by phorbol-12-myristate-13-acetate (100 nM). The ROS production was measured by luminol and lucigenin-dependent chemiluminescence. Except for naproxen, NSAIDs reduced ROS production: 58 ± 2% diclofenac, 90 ± 2% indomethacin, 33 ± 3% piroxicam, and 45 ± 6% tenoxicam (N = 6). For the lucigenin assay, naproxen, piroxicam and tenoxicam were ineffective. For indomethacin the inhibition was 52 ± 5% and diclofenac showed amplification in the light emission of 181 ± 60% (N = 6). Using the myeloperoxidase (MPO)/H2O2/luminol system, the effects of NSAIDs on MPO activity were also screened. We found that NSAIDs inhibited both the peroxidation and chlorinating activity of MPO as follows: diclofenac (36 ± 10, 45 ± 3%), indomethacin (97 ± 2, 100 ± 1%), naproxen (56 ± 8, 76 ± 3%), piroxicam (77 ± 5, 99 ± 1%), and tenoxicam (90 ± 2, 100 ± 1%), respectively (N = 3). These results show that therapeutic levels of NSAIDs are able to suppress the oxygen-dependent antimicrobial or oxidative functions of neutrophils by inhibiting the generation of hypochlorous acid.

Inhibition of peroxidase activity and scavenging of reactive oxygen species by astilbin isolated from Dimorphandra mollis (Fabaceae, Caesalpinioideae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Intermediate reactive oxygen and nitrogen from macrophages induced by Brazilian lichens

The activity of ten compounds isolated from Brazilian lichen over the release of hydrogen peroxide and nitric oxide was evaluated in the culture of peritoneal macrophage cells from mice. Salazinic, secalonic A and fumarprotocetraric acids were the compounds that induced the greatest release of H2O2, whereas 12R-usnic and diffractaic acids induced the release of NO. These results indicate that lichen products have potential immunological modulating activities. (C) 2004 Elsevier B.V. All rights reserved.

Yersinia enterocolitica O : 3 isolated from patients with or without reactive arthritis induces polyclonal activation of B cells and autoantibody production in vivo

The mechanisms by which arthritis-provoking pathogens such as Yersinia enterocolitica interact with the human immune system to produce inflammatory synovitis are not well known. One of the immunomodulating mechanisms used against these pathogens is the polyclonal activation of lymphocytes. In this study, we investigated the extent of the B-lymphocyte activation induced in mice by a strain of Y. enterocolitica O:3 (FCF 526) isolated from a patient with arthritis, and compared it with two other strains, a virulent one (FCF 397[+]) isolated from a patient without arthritis and its plasmidless isogenic pair (FCF397[-]). Also we investigated the production of autoantibodies in mice infected with these different strains. SPF Swiss mice were infected intravenously with a suspension of Y. enterocolitica . Spleen cells were taken on days 7, 14, 21 and 28 after infection and the number of cells secreting nonspecific and specific antibodies of IgG 1 , IgG 2a , IgG 2b , IgG 3 , IgM and IgA isotypes were determined by the ELISPOT technique. The presence of autoantibodies in mouse serum was investigated by the dot-blot assay. The pattern of infection of the three bacterial strains were almost the same. We observed a general increase in the number of nonspecific Ig-secreting cells with all three strains, and the greatest increases observed were in the IgG 2a and IgG 3 isotypes. Only a small fraction of the immunoglobulins detected were antibacterial, suggesting that the rest resulted from polyclonal B cell activation. The strain isolated from the patient with arthritis (FCF526) induced the greatest production of autoantibodies, coinciding with the period in which the greatest activation of nonspecific B lymphocytes was seen. There were no signs of arthritis or inflammation in the joints of the infected animals. Based on our results, we were unable to determine whether there is an association between the arthritogenic capability of Y. enterocolitica and polyclonal activation of B cells.

Crystallization study of the (1-x)Sb2O3-(x)SbPO4 glass system

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Columnar microstructure of nanocrystalline Ga1-xMnxN films deposited by reactive sputtering

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

Effect of pH of colloidal suspension on crystallization and activation energy of deep levels in SnO2 thin films obtained via sol-gel

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)