Leisure time behaviors: Prevalence, correlates and associations with overweight in Brazilian adults. A cross-sectional analysis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Crystallization and preliminary diffraction data of BaP1, a haemorrhagic metalloproteinase from Bothrops asper snake venom

BaP1 is a metalloproteinase isolated from the venom of the Central American snake Bothrops asper (terciopelo). It is a 24 kDa protein consisting of a single chain which includes the metalloproteinase domain only, therefore being classified as a class P-I snake-venom metalloproteinase. BaP1 induces prominent local tissue damage, such as haemorrhage, myonecrosis, blistering, dermonecrosis and oedema. In order to elucidate its structure, BaP1 was crystallized by the hanging-drop vapour-diffusion technique in 0.1 M bicine pH 9.0, 10% PEG 20 000 and 2%(v/v) dioxane. Diffraction data were observed to a resolution of 2.7 Angstrom. Crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 38.22, b = 60.17, c = 86.09 Angstrom.

Crystallization of bothrombin, a fibrinogen-converting serine protease isolated from the venom of Bothrops jararaca

Bothrombin, a snake-venom serine protease, specifically cleaves fibrinogen, releasing fibrinopeptide A to form non-crosslinked soft clots, aggregates platelets in the presence of exogeneous fibrinogen and activates blood coagulation factor VIII. Bothrombin shares high sequence homology with other snake-venom proteases such as batroxobin (94% identity), but only 30 and 34% identity with human alpha-thrombin and trypsin, respectively. Single crystals of bothrombin have been obtained and X-ray diffraction data have been collected at the Laboratorio Nacional de Luz Sincrotron to a resolution of 2.8 Angstrom. The crystals belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 94.81, b = 115.68, c = 155.97 Angstrom.

Crystallization and preliminary X-ray analysis of bucain, a novel toxin from the Malayan krait Bungarus candidus

Bucain is a three-finger toxin, structurally homologous to snake-venom muscarinic toxins, from the venom of the Malayan krait Bungarus candidus. These proteins have molecular masses of approximately 6000-8000 da and encompass the potent curaremimetic neurotoxins which confer lethality to Elapidae and Hydrophidae venoms. Bucain was crystallized in two crystal forms by the hanging-drop vapour-diffusion technique in 0.1 M sodium citrate pH 5.6, 15% PEG 4000 and 0.15 M ammonium acetate. Form I crystals belong to the monoclinic system space group C2, with unit-cell parameters a = 93.73, b = 49.02, c = 74.09 Angstrom, beta = 111.32degrees, and diffract to a nominal resolution of 1.61 Angstrom. Form II crystals also belong to the space group C2, with unit-cell parameters a = 165.04, b = 49.44, c = 127.60 Angstrom, beta = 125.55degrees, and diffract to a nominal resolution of 2.78 Angstrom. The self-rotation function indicates the presence of four and eight molecules in the crystallographic asymmetric unit of the form I and form II crystals, respectively. Attempts to solve these structures by molecular-replacement methods have not been successful and a heavy-atom derivative search has been initiated.

Purification, characterization and crystallization of Jararacussin-I, a fibrinogen-clotting enzyme isolated from the venom of Bothrops jararacussu

A fibrinogen-clotting enzyme, Jararacussin-I, was purified from the venom of Bothrops jararacussu by a combination of ion exchange chromatography using Resource 15S resin and affinity chromatography using Benzamidine Sepharose 6B resin. Jararacussin-I displays a molecular mass of 28 kDa as estimated by sodium dodecyl sulphate-PAGE and possesses an isoetectric point of 5.0. The coagulant specific activity of the enzyme was determined to be 45.8 NIH U/mg using bovine fibrinogen as the substrate and the esterase specific activity was determined to be 258.7 U/mg. The protease inhibitors, benzamidine and DTT inhibited the esterase specific activity by 72.4 and 69.7%, respectively. The optimal temperature and pH for the degradation of both chains of fibrinogen and esterase specific activity were determined to be 37 degreesC and 7.4-8.0, respectively. The enzyme was inactivated at both 4 and 75 T. Single crystals of Jararacussin-I were obtained and complete three-dimensional X-ray diffraction data was collected at the Brazilian National Synchrotron Source (LNLS) to a resolution of 2.4 Angstrom. (C) 2002 Published by Elsevier B.V. Ltd.

Crystallization and preliminary diffraction data of a platelet-aggregation inhibitor from the venom of Agkistrodon piscivorus piscivorus (North American water moccasin)

Applaggin (Agkistrodon piscivorus piscivorus platelet-aggregation inhibitor) is a potent inhibitor of blood platelet aggregation derived from the venom of the North American water moccasin, the protein consists of 71 amino acids, is rich in cysteines, contains the sequence-recognition site of adhesion proteins at positions 50-52 (Arg-Gly-Asp) and shares high sequence homology with other snake-venom disintegrins such as echistatin, kistrin and trigramin, Single crystals of applaggin have been grown and X-ray diffraction data have been collected to a resolution of 3.2 Angstrom. The crystals belong to space group P4(1)2(1)2 (or its enantiomorph), with unit-cell dimensions a = b = 63.35, c = 74.18 Angstrom and two molecules per asymmetric unit. Molecular replacement using models constructed from the NMR structures of echistatin and kistrin has not been successful in producing a trial structure for applaggin.

Crystallization and preliminary X-ray diffraction analysis of a Lys49-PLA(2) homologue from Cerrophidion godmani venom.

Lys49-phospholipase A(2) (Lys49-PLA(2)) homologues damage membranes by a Ca2+-independent mechanism which does not involve catalytic activity. The myotoxic Lys-49 phospholipase myotoxin II from Cerrophidion (Bothrops) godmani has been crystallized, and X-ray diffraction data were collected to 2.8 Angstrom resolution. Preliminary analysis reveals the presence of one molecule in the asymmetric unit.

Crystallization of the Lys49 PLA(2) homologue, myotoxin II, from the venom of Atropoides nummifer

Myotoxin H, a Lys49 catalytically inactive phospholipase A(2) homologue from Atropoides nummifer venom, was purified, characterized and crystallized. The crystals belongs to the tetragonal system, space group P4(3)2(1)2, with unit cell parameters (a=b=68.66 and c=63.87 Angstrom). Diffraction data were collected to a resolution of 2.32 Angstrom. The crystal structure is currently being determined using molecular replacement techniques. (C) 2004 Elsevier B.V. All rights reserved.

Crystallization and preliminary X-ray crystallographic studies of Protac (R), a commercial protein C activator isolated from Agkistrodon contortrix contortrix venom

The protein C pathway plays an important role in the control and regulation of the blood coagulation cascade and prevents the propagation of the clotting process on the endothelium surface. In physiological systems, protein C activation is catalyzed by thrombin, which requires thrombomodulin as a cofactor. The protein C activator from Agkistrodon contortrix contortrix acts directly on the zymogen of protein C converting it into the active form, independently of thrombomodulin. Suitable crystals of the protein C activator from Agkistrodon contortrix contortrix were obtained from a solution containing 2 M ammonium sulfate as the precipitant and these crystals diffracted to 1.95 angstrom resolution at a synchrotron beamline. The crystalline array belongs to the monoclinic space group C2 with unit cell dimensions a=80.4, b = 63.3 and c = 48.2 angstrom, alpha = gamma = 90.0 degrees and beta = 90.8 degrees. (C) 2005 Elsevier B.V. All rights reserved.

Crystallization and preliminary X-ray diffraction analysis of the catalytic subunit of ADP-glucose pyrophosphorylase from potato tuber

ADP-glucose pyrophosphorylase is the key regulatory enzyme in the biosynthesis of starch in plants and glycogen in bacteria. The enzyme from potato tuber is comprised of a regulatory subunit and a catalytic subunit and is present as a heterotetramer (alpha(2)beta(2)) the catalytic subunit from potato tuber (50 kDa) was crystallized in four different forms, two of which are suitable for structural studies. A tetragonal crystal form obtained in the presence of the substrate analog Cr-ATP diffracted to 2.2 Angstrom and belongs to space group P4(1) (or its enantiomorph), with unit-cell parameters a = b = 110.57, c = 190.14 Angstrom. A second crystal form obtained diffracted to 2.8 Angstrom and belongs to space group PZ, with unit-eel parameters a = 80.06, b = 138.84, c = 92.20 Angstrom, beta = 112.40 degrees. As this protein displays no significant homology to any currently known protein structure, a search for heavy-atom derivatives has been initiated.

Crystallization and preliminary X-ray crystallographic analysis of the heterodimeric crotoxin complex and the isolated subunits crotapotin and phospholipase A(2)

Crotoxin, a potent neurotoxin from the venom of the South American rattlesnake Crotalus durissus terrificus, exists as a heterodimer formed between a phospholipase A(2) and a catalytically inactive acidic phospholipase A(2) analogue (crotapotin). Large single crystals of the crotoxin complex and of the isolated subunits have been obtained. The crotoxin complex crystal belongs to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 38.2, b = 68.7, c = 84.2 angstrom, and diffracted to 1.75 angstrom resolution. The crystal of the phospholipase A(2) domain belongs to the hexagonal space group P6(1)22 (or its enantiomorph P6(5)22), with unit-cell parameters a = b = 38.7, c = 286.7 angstrom, and diffracted to 2.6 angstrom resolution. The crotapotin crystal diffracted to 2.3 angstrom resolution; however, the highly diffuse diffraction pattern did not permit unambiguous assignment of the unit-cell parameters.

Crystallization and preliminary crystallographic analysis of SMase I, a sphingomyelinase from Loxosceles laeta spider venom

SMase I, a 32 kDa sphingomyelinase found in Loxosceles laeta venom, is responsible for the major pathological effects of spider envenomation. This toxin has been cloned and functionally expressed as a fusion protein containing a 6 x His tag at its N-terminus to yield a 33 kDa protein [Fernandes-Pedrosa et al. (2002), Biochem. Biophys. Res. Commun. 298, 638 - 645]. The recombinant protein possesses all the biological properties ascribed to the whole L. laeta venom, including dermonecrotic and complement-dependent haemolytic activities. Dynamic light-scattering experiments conducted at 291 K demonstrate that the sample possesses a monomodal distribution, with a hydrodynamic radius of 3.57 nm. L. laeta SMase I was crystallized by the hanging-drop vapour-diffusion technique using the sparse-matrix method. Single crystals were obtained using a buffer solution consisting of 0.08 M HEPES and 0.9 M trisodium citrate, which was titrated to pH 7.5 using 0.25 M sodium hydroxide. Complete three-dimensional diffraction data were collected to 1.8 Angstrom at the Laboratorio Nacional de Luz Sincrotron (LNLS, Campinas, Brazil). The crystals belong to the hexagonal system ( space group P6(1) or P6(5)), with unit-cell parameters a = b = 140.6, c = 113.6 Angstrom. A search for heavy-atom derivatives has been initiated and elucidation of the crystal structure is currently in progress.

Crystallization and preliminary X-ray diffraction analysis of a class II phospholipase D from Loxosceles intermedia venom

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

Production, purification, crystallization and preliminary X-ray diffraction studies of the nucleoside diphosphate kinase b from Leishmania major

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