Antitumoural effect of an L-amino acid oxidase isolated from Bothrops jararaca snake venom

An L-amino acid oxidase (BjarLAAO-I) from Bothrops jararaca snake venom was highly purified using a stepwise sequential chromatography on Sephadex G-75, Benzamidine Sepharose and Phenyl Sepharose. Purified BjarLAAO-I showed a molecular weight around 60,000 under reducing conditions and about 125,000 in the native form, when analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively. BjarLAAO-I is a homodimeric acidic glycoprotein, pI similar to 5.0, and N-terminal sequence showing close structural homology with other snake venom LAAOs. The purified enzyme catalysed the oxidative deamination of L-amino acids, the most specific substrate being L-Phe. Five amino acids, L-Ser, L-Pro, L-Gly, L-Thr and L-Cys were not oxidized, clearly indicating a significant specificity. BjarLAAO-I significantly inhibited Ehrlich ascites tumour growth and induced an influx of polymorphonuclear cells, as well as spontaneous liberation of H(2)O(2) from peritoneal macrophages. Later, BjarLAAO-I induced mononuclear influx and peritoneal macrophage spreading. Animals treated with BjarLAAO-I showed higher survival time.

Amino acid availability and protein digestibility of several protein sources for Nile tilapia, Oreochromis niloticus

Apparent amino acid availability coefficients and protein digestibility of four animal products [fish meal (FM), meat and bone meal (MBM), poultry by-product and feather meal] and four plant protein-rich products [soybean meal (SBM), cottonseed meal-28, cottonseed meal-38 and corn gluten meal (CGM)] were determined for Nile tilapia, Oreochromis niloticus. Ingredients were incorporated to a practical reference diet at a 7 : 3 ratio (70% of reference diet and 30% of test ingredient). Chromic oxide was used as external digestibility marker. Among animal products poultry by-product meal (PBM; 89.7%) and FM (88.6%) presented the highest apparent protein digestibility (APD) while MBM (78.4%) and feather meal (78.5%) presented the lowest APD. Among plant protein-rich products CGM (91.4%) and SBM (92.4%) presented the highest APD values while cottonseed meal-28 presented the lowest APD (78.6%). Average apparent amino acid availability of feed ingredients was similar to protein digestibility with 92.3%, 89.6%, 73.4%, 80.7%, 88.9%, 84.4%, 91.2% and 79.7% values for SBM, CGM, cottonseed meal-28 and 38, FM, MBM, PBM and feather meal respectively. These results indicate that O. niloticus is able to utilize efficiently different feedstuffs.

Energy and amino acid content in phase 1 nursery diet: piglet performance and body chemical composition

Avaliaram-se os efeitos da densidade de energia metabolizável (EM) e lisina digestível (LIS) sobre o desempenho e a composição corporal de leitões após o desmame. Utilizaram-se 114 animais desmamados aos 7,4 ± 0,80 kg; desses animais, 108 foram alojados na unidade de creche e 6 foram abatidos no dia do desmame para determinação dos dados comparativos da composição química corporal. Seis densidades de nutrientes foram estipuladas a partir de estudo anterior, com base na maior retenção de nitrogênio, mantendo-se as seguintes relações EM:LIS nas dietas experimentais: 3.390:1,291; 3.450:1,409; 3.650:1,411; 3.780:1,461; 3.940:1,507; e 4.109 kcal/kg EM:1,564% LIS. As dietas experimentais foram oferecidas durante 13 dias, quando os leitões atingiram o peso de 12,986 ± 1,449 kg. Avaliaram-se os prováveis efeitos residuais da densidade nutricional no desempenho subseqüente dos leitões. Ao término da fase inicial-1, seis leitões de cada densidade foram abatidos para determinação da composição química nas frações corporais e no corpo vazio. Não houve influência significativa dos níveis nutricionais no desempenho dos leitões ao término da avaliação. Os resultados de conversão alimentar e composição corporal ratificam o nível indicado em estudo anterior, de 4 g LIS/Mcal. O aumento da densidade de energia e lisina confirma a necessidade da correta relação entre ambos para assegurar o melhor desempenho dos leitões na fase inicial de crescimento.

Molecular approaches for structural characterization of Bothrops L-amino acid oxidases with antiprotozoal activity: cDNA cloning, comparative sequence analysis, and molecular modeling

Two L-amino acid oxidases (LAAOs) were identified by random sequencing of cDNA libraries from the venom glands of Bothrops moojeni (BmooLAAO) and Bothrops jararacussu (Bjussu LAAO). Phylogenetic analysis involving other SV-LAAOs showed sequence identities within the range 83-87% being closely related to those from Agkistrodon and Trimeresurus. Molecular modeling experiments indicated the FAD-binding, substrate-binding, and helical domains of Bmoo and Bjussu LAAOs. The RMS deviations obtained by the superposition of those domains and that from Calloselasma rhodostoma LAAO crystal structure confirm the high degree of structural similarity between these enzymes. Purified BjussuLAAO-I and BmooLAAO-I exhibited antiprotozoal activities which were demonstrated to be hydrogen-peroxide mediated. This is the first report on the isolation and identification of cDNAs encoding LAAOs from Bothrops venom. The findings here reported contribute to the overall structural elucidation of SV-LAAOs and will advance the understanding on their mode of action. (c) 2006 Elsevier B.V. All rights reserved.

Structural and functional properties of Bp-LAAO, a new L-amino acid oxidase isolated from Bothrops pauloensis snake venom

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

Using Amino Acid Correlation and Community Detection Algorithms to Identify Functional Determinants in Protein Families

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

Amino acid sequence of a myotoxic Lys49-phospholipase A(2) homologue from the venom of Cerrophidion (Bothrops) godmani

The complete amino acid sequence of myotoxin II (godMT-II), a myotoxic phospholipase A( 2 )(PLA(2)) homologue from the venom of the Central American crotaline snake Cerrophidion (Bothrops) godmani, was determined by direct protein sequencing methods. GodMT-II is a class II PLA, showing a Lys instead of Asp at position 49. An additional substitution in the calcium binding loop region (Asn instead of Tyr at position 28) suggests the lack of enzymatic activity observed in this toxin is due to loss of its ability to bind the co-factor Ca2+, since the residues involved in forming the catalytic network of PLA(2)s (His-48, Tyr-52 and Asp-99) an conserved in godMT-II. This myotoxin shows highest sequence homology with other Lys-49 PLA(2)s from Bothrops, Agkistrodon and Trimeresurus species, suggesting that they constitute a conserved family of proteins, yet in contrast presents lower homology with Bothrops asper myotoxin III, a catalytically-active PLA(2). The C-terminal region of godMT-II, which is rich in cationic and hydrophobic residues, shares high sequence homology to the corresponding region in the myotoxin II from B. asper, which has been proposed to play an important role in the Ca2+-independent membrane damaging activity. (C) 1998 Elsevier B.V. B.V. All rights reserved.

Amino acid sequence and crystal structure of BaP1, a metalloproteinase from Bothrops asper snake venom that exerts multiple tissue-damaging activities

BaP1 is a 22.7-kD P-I-type zinc-dependent metalloproteinase isolated from the venom of the snake Bothrops asper, a medically relevant species in Central America. This enzyme exerts multiple tissue-damaging activities, including hemorrhage, myonecrosis, dermonecrosis, blistering, and edema. BaP1 is a single chain of 202 amino acids that shows highest sequence identity with metalloproteinases isolated front the venoms of snakes of the subfamily Crotalinae. It has six Cys residues involved in three disulfide bridges (Cys 117-Cys 197, Cys 159-Cys 181, Cys 157-Cys 164). It has the consensus sequence H(142)E(143)XXH(146)XXGXXH(152), as well as the sequence C164I165M166, which characterize the metzincin superfamily of metalloproteinases. The active-site cleft separates a major subdomain (residues 1-152), comprising four a-helices and a five-stranded beta-sheet, from the minor subdomain, which is formed by a single a-helix and several loops. The catalytic zinc ion is coordinated by the N-epsilon2 nitrogen atoms of His 142, His 146, and His 152, in addition to a solvent water molecule, which in turn is bound to Glu 143. Several conserved residues contribute to the formation of the hydrophobic pocket, and Met 166 serves as a hydrophobic base for the active-site groups. Sequence and structural comparisons of hemorrhagic and nonhemorrhagic P-I metalloproteinases from snake venoms revealed differences in several regions. In particular, the loop comprising residues 153 to 176 has marked structural differences between metalloproteinases with very different hemorrhagic activities. Because this region lies in close proximity to the active-site microenvironment, it may influence the interaction of these enzymes with physiologically relevant substrates in the extracellular matrix.

Antimicrobial activity of an L-amino acid oxidase isolated from Bothrops leucurus snake venom

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The structure of a native L-amino acid oxidase, the major component of the Vipera ammodytes ammodytes venomic, reveals dynamic active site and quaternary structure stabilization by divalent ions

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

Crystallization and preliminary X-ray diffraction analysis of an l-amino-acid oxidase from Bothrops jararacussu venom

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

Amino acid, Antioxidant and Ion Profiles of Carpolobia lutea Leaf (Polygalaceae)

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

X-ray powder diffraction analysis of a new palladium(II) amino acid complex

Powder X-ray diffraction data for a new palladium(II) amino acid complex, of composition PdC12H2ON2O4S2, are presented in this paper. Orthorhombic cell parameters are: a = 10.740 angstrom, b = 19.999 angstrom, and c = 5.2470 angstrom. (c) 2004 International Centre for Diffraction Data.

Molecular orbital calculation for the model compounds of kainoid amino acids, agonists of excitatory amino acid receptors. Does the kainoid C4-substituent directly interact with the receptors?

Kainoid amino acids are agonists of the AMPA/kainate receptors and exhibit highly potent neuroexcitatory activity. From the results of extensive structure-activity relationship studies, we previously postulated that the C4-substituent of the kainoid amino acids interacts with an allosteric site of the glutamate receptor with electron-donating character. In order to investigate the mode of action in more detail, molecular orbital calculation for model compounds of the kainoid were performed. The results indicated that the HOMO energy level of the C4-substituent is involved in the potent neuroexcitatory activity, thus supporting our hypothesis. (C) 2002 Elsevier B.V. Ltd. All rights reserved.

Isolation of a new L-amino acid oxidase from Crotalus durissus cascavella venom

A novel L-amino acid oxidase (LAO) (Casca LAO) from Crotalus durissus cascavella venom was purified to a high degree of molecular homogeneity using a combination of molecular exclusion and ion-exchange chromatography system. The purified monomer of LAO presented a molecular mass of 68 kDa and pI estimated in 5.43, which were determined by two-dimensional electrophoresis. The 71st N-terminal amino acid sequence of the LAO from Crotalus durissus cascavella presented a high amino acid sequence similarities with other LAOs from Colloselasma rhosostoma, Crotalus adamanteus, Agkistrodon h. blomhoffi, Agkistrodon h. halys and Trimeresurus stejnegeri. LAO displayed a Michaelis-Menten behavior with a kilometer of 46.7 mu M and an optimum pH for enzymatic activity of 6.5. Casca LAO induced a dose-dependent platelet aggregation, which was abolished by catalase and inhibited by indomethacin and aspirin. These results suggest that the production of H2O2 is involved in subsequent activation of inflammatory enzymes, such as thromboxane. Casca LAO also inhibited the bacterial Growth of Gram-negative (Xanthomonas axonopodis pv passiflorae) and Gram-positive (S. mutans) strains. Electron microscopy assessments of both bacterial strains suggest that the hydrogen peroxide produced by LAO induce bacterial membrane rupture and consequently loss of cytoplasmatic content. This LAO exhibited a high antileishmanic activity against the promastigote of Leishmania amazonensis in vitro, its activity was dependent on the production of hydrogen peroxide, and the 50% inhibitory concentration was estimated in 2.39 mu g/ml. (C) 2005 Elsevier Ltd. All rights reserved.