BioVenom - Banco de Dados de Venenos de Serpentes Brasileiras - Bactericida

Biochemical and functional characterization of an l-amino acid oxidase isolated from Bothrops pirajai snake venom.

Tue, 12 Sep 2006 12:22:47 -0300

Autores:

Izidoro LF, Ribeiro MC, Souza GR, Sant'ana CD, Hamaguchi A, Homsi-Brandeburgo MI, Goulart LR, Beleboni RO, Nomizo A, Sampaio SV, Soares AM, Rodrigues VM.

Resumo:

In this work we describe the isolation of a new l-amino acid oxidase (LAAO) referred to as BpirLAAO-I from Bothrops pirajai snake venom, which was highly purified using a combination of molecular exclusion, affinity, and hydrophobic chromatography steps. BpirLAAO-I homodimeric acid glycoprotein (approximate Mr and pI of 130,000 and 4.9, respectively) displays high specificity toward hydrophobic/aromatic amino acids, while deglycosylation does not alter its enzymatic activity. The N-terminal LAAO sequence of its first 49 amino acids presented a high similarity between a amino acid sequence with other LAAOs from: Bothrops spp., Crotalus spp., Calloselasma rhodostoma, Agkistrodon spp., Trimeresurus spp., Pseudechis australis, Oxyuranus scutellatus, and Notechis scutatus. BpirLAAO-I induces time-dependent platelet aggregation, mouse paw edema, cytotoxic activity against Escherichia coli, Pseudomonas aeruginosa, Leishmania sp., and tumor cells, and also a typical fago (M13mp18) DNA fragmentation. Platelet aggregation, leishmanicidal and antitumoral activities were reduced by catalase. Thus, BpirLAAO-I is a multifunctional protein with promising biotechnological and medical applications.

Dados de Publicação:
Bioorg Med Chem. 2006 Jun 27; [Epub ahead of print]

Palavras-Chave:
l-amino acid oxidase, snake venom, biochemical and functional characterization

In vitro antimicrobial activity of natural toxins and animal venoms tested against Burkholderia pseudomallei.

Tue, 12 Sep 2006 14:10:40 -0300

Autores:

Perumal Samy RR, Gopalakrishnakone PP, Pachiappan AA, Thwin MM, Hian YE, Bow H, Chow VT, Tuck Weng JT.

Resumo:

ABSTRACT: BACKGROUND: Burkholderia pseudomallei are the causative agent of melioidosis. Increasing resistance of the disease to antibiotics is a severe problem in treatment regime and has led to intensification of the search for new drugs. Antimicrobial peptides are the most ubiquitous in nature as part of the innate immune system and host defense mechanism. METHODS: Here, we investigated a group of venoms (snakes, scorpions and honey bee venoms) for antimicrobial properties against two strains of Gram-negative bacteria Burkholderia pseudomallei by using disc diffusion assay for in vitro susceptibility testing. The antibacterial activities of the venoms were compared with that of the isolated L-amino acid oxidase (LAAO) and phospholipase A2 (PLA2s) enzymes. MICs were determined using broth dilution method. Bacterial growth was assessed by measurement of optical density at the lowest dilutions (MIC 0.25 mg/mL). The cell viability was measured using tetrazolium salts (XTT) based cytotoxic assay. RESULTS: The studied venoms showed high antimicrobial activity. The venoms of C. adamanteus, Daboia russelli russelli, A. halys, P. australis, B. candidus and P. guttata were equally as effective as Chloramphenicol and Cefazidime (30 ug/disc). Among those tested, phospholipase A2 enzymes (crotoxin B and daboiatoxin), showed the most potent antibacterial activity against Gram-negative (TES) bacteria. Naturally occurring venom peptides and phospholipase A2 proved to possess highly potent antimicrobial activity against Burkholderia pseudomallei. The XTT-assay results showed that the cell survival decreased with increasing concentrations (0.05-10 mg/mL) of Crotalus adamanteus venom, with no effect on the cell viability evident at 0.5 mg/mL. CONCLUSION: This antibacterial profile of snake venoms reported herein will be useful in the search for potential antibacterial agents against drug resistant microorganisms like B. pseudomallei.

Dados de Publicação:
BMC Infect Dis. 2006 Jun 20;6(1):100 [Epub ahead of print]

Palavras-Chave:
l-amino acid oxidase, snake venom, antimicrobial activity

Bothrops moojeni myotoxin-II, a Lys49-phospholipase A2 homologue: an example of function versatility of snake venom proteins

Mon, 14 Aug 2006 09:15:08 -0300

Autores:

Stabeli RG, Amui SF, Sant'Ana CD, Pires MG, Nomizo A, Monteiro MC, Romao PR, Guerra-Sa R, Vieira CA, Giglio JR, Fontes MR, Soares AM.

Resumo:

MjTX-II, a myotoxic phospholipase A(2) (PLA(2)) homologue from Bothrops moojeni venom, was functionally and structurally characterized. The MjTX-II characterization included: (i) functional characterization (antitumoral, antimicrobial and antiparasitic effects); (ii) effects of structural modifications by 4-bromophenacyl bromide (BPB), cyanogen bromide (CNBr), acetic anhydride and 2-nitrobenzenesulphonyl fluoride (NBSF); (iii) enzymatic characterization: inhibition by low molecular weight heparin and EDTA; and (iv) molecular characterization: cDNA sequence and molecular structure prediction. The results demonstrated that MjTX-II displayed antimicrobial activity by growth inhibition against Escherichia coli and Candida albicans, antitumoral activity against Erlich ascitic tumor (EAT), human breast adenocarcinoma (SK-BR-3) and human T leukemia cells (JURKAT) and antiparasitic effects against Schistosoma mansoni and Leishmania spp., which makes MjTX-II a promising molecular model for future therapeutic applications, as well as other multifunctional homologous Lys49-PLA(2)s or even derived peptides. This work provides useful insights into the structural determinants of the action of Lys49-PLA(2) homologues and, together with additional strategies, supports the concept of the presence of others "bioactive sites" distinct from the catalytic site in snake venom myotoxic PLA(2)s.

Dados de Publicação:
Comp Biochem Physiol C Toxicol Pharmacol. 2006 Mar-Apr;142(3-4):371-81. Epub 2006 Jan 24

Atividades Biológicas induzidas pela MjTX-II isolada do veneno de Bothrops moojeni

Tue, 26 Sep 2006 12:10:00 -0300

Pesquisadores envolvidos:
Stabeli RG, Amui SF, Sant'Ana CD, Pires MG, Nomizo A, Monteiro MC, Romao PR, Guerra-Sa R, Vieira CA, Giglio JR, Fontes MR, Soares AM.

Local:
Depto. Análises Clínicas, Toxicológicas e Bromatológicas-FCFRP-USP, Ribeirão Preto-SP.

Ensaios realizados:
Atividade Miotóxica, indução de edema, citotoxicidade, neurotoxicidade

Dados Publicados em:
Artigos Científicos (Periódicos)

Referências de Publicação:

Comp Biochem Physiol C Toxicol Pharmacol. 2006 Mar-Apr;142(3-4):371-81. Epub 2006 Jan 24

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

Wed, 13 Sep 2006 12:29:36 -0300

Autores:

Toyama MH, Toyama Dde O, Passero LF, Laurenti MD, Corbett CE, Tomokane TY, Fonseca FV, Antunes E, Joazeiro PP, Beriam LO, Martins MA, Monteiro HS, Fonteles MC.

Resumo:

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 microM 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 microg/ml.

Dados de Publicação:
Toxicon. 2006 Jan;47(1):47-57. Epub 2005 Nov 22.

Palavras-Chave:
l-amino acid oxidase, Crotalus durissus cascavella;antibacterial; antimicrobial; hydrogen peroxide; gyroxin; leishmanicidal

Cloning and identification of a complete cDNA coding for a bactericidal and antitumoral acidic phospholipase A2 from Bothrops ja

Tue, 06 Dec 2005 16:28:35 -0200

Autores:

Roberto PG, Kashima S, Marcussi S, Pereira JO, Astolfi-Filho S, Nomizo A, Giglio JR, Fontes MR, Soares AM, Franca SC.

Resumo:

Cloning and identification of a complete cDNA coding for a bactericidal and antitumoral acidic phospholipase A2 from Bothrops jararacussu venom.

In order to better understand the function of acidic phospholipases A2 (PLA2s) from snake venoms, expressed sequence tags (ESTs) that code for acidic PLA2s were isolated from a cDNA library prepared from the poly(A)+ RNA of venomous glands of Bothrops jararacussu. The complete nucleotide sequence (366 bp), named BOJU-III, encodes the BthA-I-PLA2 precursor, which includes a signal peptide and the mature protein with 16 and 122 amino acid residues, respectively. Multiple comparison of both the nucleotide and respective deduced amino acid sequence with EST and protein sequences from databases revealed that the full-length cDNA identified (BOJU III--AY145836) is related to an acidic PLA2 sharing similarity, within the range 55-81%, with acidic phospholipases from snake venoms. Moreover, phylogenetic analysis of amino acid sequences of acidic PLA2s from several pit viper genera showed close evolutionary relationships among acidic PLA2s from Bothrops, Crotalus, and Trimeresurus. The molecular modeling showed structural similarity with other dimeric class II PLA2s from snake venoms. The native protein BthA-I-PLA2, a nontoxic acidic PLA2 directly isolated from Bothrops jararacussu snake venom, was purified and submitted to various bioassays. BthA-I-PLA2 displayed high catalytic activity and induced Ca2+-dependent liposome disruption. Edema induced by this PLA2 was inhibited by indomethacin and dexamethasone, thus suggesting involvement of the cyclo-oxygenase pathway. BthA-I-PLA2 showed anticoagulant activity upon human plasma and inhibited phospholipid-dependent platelet aggregation induced by collagen or ADP. In addition, it displayed bactericidal activity against Escherichia coli and Staphylococcus aureus and antitumoral effect upon breast adrenocarcinoma as well as upon human leukemia T and Erlich ascitic tumor. Following chemical modification with p-bromophenacyl bromide, total loss of the enzymatic and pharmacological activities were observed. This is the first report on the isolation and identification of a cDNA encoding a complete acidic PLA2 from Bothrops venom, exhibiting bactericidal and antitumoral effects.

Dados de Publicação:
Protein J. 2004 (4):273-85.

Isolamento e Caracterização Funcional de uma L-Aminoácido Oxidase do veneno de Bothrops moojeni com atividades bactericida

Mon, 14 Aug 2006 09:25:04 -0300

Autores:

Marcussi, S.*; Stábeli, R.G.**; Monteiro, M.C.***; Romão, P.R.T.***; Oliveira, E.B.**; França, S.C.*; Soares, A. M.*

Resumo:

A L-aminoácido oxidase (LAAO) é uma enzima glicoprotéica que catalisa a deaminação oxidativa de L-aminoácidos, produzindo ?-cetoácidos, peróxido de hidrogênio e amônia. Estas enzimas encontram-se presentes nos venenos de serpentes e participam do envenenamento atuando sobre plaquetas, induzindo citotoxicidade e apoptose celular.

Atividades Bactericida e Neurotóxica de duas Fosfolipases A2 Miotóxicas isoladas do veneno de Bothrops neuwiedi pauloensis

Thu, 15 Sep 2005 18:14:55 -0300

Autores:

1CAMBRAIA, R.S.; 2RODRIGUES, V.M.; 1MALTA-NETO., N.R.; 1,3MARCUSSI, S.; 4SANT´ANA, C.D.; 1ARAÚJO, A.L.; 1SILVEIRA, L.B.; 2FERRO, E.A.V.; 3GIGLIO, J.R.; 2HOMSI-BRANDEBURGO, M.I.; 1,*SOARES, A.M.

Resumo:

Proteínas de venenos de serpentes têm sido utilizadas como ferramentas em laboratórios, como modelos na elaboração de novos fármacos e até mesmo diretamente na clínica médica. As PLA2s desempenham papéis importantes no catabolismo de lipídios da dieta e no metabolismo geral de lipídios estruturais de membranas celulares, estando envolvidas em uma grande variedade de doenças inflamatórias humanas, nos envenenamentos ofídicos e por abelhas, sendo de grande interesse médico-científico. O estudo destas proteínas podem responder questões sobre os mecanismos de ação durante o envenenamento ou resultar na cura para algumas doenças.

Anti-protozoary, Anti-bacterial and antitumor activity of L-Amino Acid Oxidase from Bothrops moojeni “in vitro”

Fri, 15 Sep 2006 14:54:53 -0300

Pesquisadores envolvidos:
Malta-Neto, N.R.1; Stabeli, R.G.2; Marcussi, S.1; Monteiro M.C.2; Romão P.R.T.2;3; Nomizo, A.4;

Local:
1Unidade de Biotecnologia UNAERP, Ribeirão Preto-SP; 2Departamento de Bioquímica e Imunologia and Departamento de Farmacologia, FMRP/USP, Ribeirão Preto-SP; 3Departamento de Imunologia, UNISUL-Tuba

Dados Publicados em:
Eventos (Congressos, Simpósios, etc)

Referências de Publicação:

SBTX 2004

Atividades bactericida e neurotóxica de duas fosfolipases A2 miotóxicas isoladas do veneno de Bothrops neuwiedi pauloensis.

Fri, 15 Sep 2006 15:14:14 -0300

Pesquisadores envolvidos:
1CAMBRAIA, R.S.; 2RODRIGUES, V.M.; 1MALTA-NETO., N.R.; 1,3MARCUSSI, S.; 4SANT´ANA, C.D.; 1ARAÚJO, A.L.; 1SILVEIRA, L.B.; 2FERRO, E.A.V.; 3GIGLIO, J.R.; 2HOMSI-BRANDEBURGO, M.I.; 1SOARES, A.M.

Local:
1 Instituto de Genética e Bioquímica e Instituto de Ciências Biomédicas, UFU, Uberlândia-MG; 2 Unidade de Biotecnologia, UNAERP, 3Depto Bioquímica e Imunologia, FMRP-USP e 4Depto. Análises Clí

Dados Publicados em:
Eventos (Congressos, Simpósios, etc)

Referências de Publicação:

XII Congresso de Iniciação Científica da Universidade Federal de São Carlos - UFSCar

Platelet aggregation and antibacterial effects of an l-amino acid oxidase purified from Bothrops alternatus snake venom.

Wed, 13 Sep 2006 12:53:41 -0300

Autores:

Stabeli RG, Marcussi S, Carlos GB, Pietro RC, Selistre-de-Araujo HS, Giglio JR, Oliveira EB, Soares AM.

Resumo:

The isolation and biochemical/enzymatic characterization of an L-amino acid oxidase, Balt-LAAO-I, from Bothrops alternatus snake venom, is described. Balt-LAAO-I is an acidic glycoprotein, pI approximately 5.37, homodimeric, Mr approximately 123,000, whose N-terminal sequence is ADVRNPLE EFRETDYEVL. It displays a high specificity toward hydrophobic and basic amino acids, while deglycosylation does not alter its enzymatic activity. Balt-LAAO-I induces platelet aggregation and shows bactericidal activity against Escherichia coli and Staphylococcus aureus. In addition, this enzyme is slightly hemorrhagic and induces edema in the mouse paw. Balt-LAAO-I is a multifunctional enzyme with promising relevant biotechnological and medical applications.

Dados de Publicação:
Bioorg Med Chem. 2004 Jun 1;12(11):2881-6.

Palavras-Chave:
Snake venom; -Amino acid oxidase; Bothrops alternatus; Bactericidal effect; Platelet aggregation; Biotechnological application

Purification, characterization and biological activity of an L-amino acid oxidase from Trimeresurus mucrosquamatus venom.

Thu, 14 Sep 2006 12:38:45 -0300

Autores:

Wei JF, Wei Q, Lu QM, Tai H, Jin Y, Wang WY, Xiong YL.

Resumo:

An L-amino acid oxidase (TM-LAO) from the venom of Hunan Trimeresurus mucrosquamatus was purified to homogeneity by three steps including DEAE Sephadex A-50 ion-exchange chromatography, Sephadex G-75 gel filtration and Resource Q ion-exchange chromatography. TM-LAO is composed of two identical subunits with a molecular weight of 55 kD by SDS-polyacrylamide gel electrophoresis. The molecular weight was different with that of LAO purified from the same species distributed in Taiwan that was 70 kD. The 24 N-terminal amino acid sequence of TM-LAO is ADNKNPLEECFRETNYEEFLEIAR, which shares high similarity with other Viperid snake venom LAOs and has moderate similarity with Elapid snake venom LAOs. Further studies found that TM-LAO inhibited the growth of E. coli, S. aurues and B. dysenteriae. TM-LAO also showed cytotoxicity and platelet aggregation activity. All the biological activities were eliminated by catalase, a H(2)O(2) scavenger. It was shown that these biological effects were possibly due to the formation of H(2)O(2) produced by TM-LAO.

Dados de Publicação:
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai). 2003 Mar;35(3):219-24.

Palavras-Chave:
L-amino acid oxidase; Trimeresurus mucrosquamatus; antibacterial activity; cytotoxicity; platelet aggregation

L-amino acid oxidase from Trimeresurus jerdonii snake venom: purification, characterization, platelet aggregation-inducing and a

Thu, 14 Sep 2006 12:55:54 -0300

Autores:

Lu QM, Wei Q, Jin Y, Wei JF, Wang WY, Xiong YL.

Resumo:

An L-amino acid oxidase (LAO), designated as TJ-LAO, was purified to homogeneity from the venom of Trimeresurus jerdonii by Sephadex G-100 and Q Sepharose HP chromatography. The molecular weight of this enzyme was 110 kD as estimated by analytical gel filtration and was 55 kD by SDS-polyacrylamide gel electrophoresis, suggesting that the enzyme is composed of two subunits. The enzyme has an absorption spectrum characteristic of flavoproteins, containing 2 moles of FMN per mole of enzyme. The N-terminal sequence of TJ-LAO shares high homology with other viperid snake venom LAOs. Homology with elapid venom LAO is lower. TJ-LAO inhibited the growth of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus megaterium. The antibacterial effect associated with LAO activity was elminated with the addition of catalase. Platelets in platelet-rich plasma aggregated upon the addition of TJ-LAO. The enzyme-induced aggregation was inhibited by catalase, suggesting formation of H2O2 was essential for TJ-LAO to induce platelet aggregation. These results showed H2O2 formation is important for the biological effects of LAO.

Dados de Publicação:
J Nat Toxins. 2002 Dec;11(4):345-52.

Palavras-Chave:
l-amino acid oxidase, snake venom, characterization, antimicrobial

Purification and characterization of L-amino acid oxidase from Agkistrodon halys pallas venom

Thu, 14 Sep 2006 13:50:51 -0300

Autores:

Liu JW, Chai MQ, Du XY, Song JG, Zhou YC.

Resumo:

L-amino acid oxidase (LAO, EC 1.4.3.2) is widely found in snake venoms and is thought to contribute to the toxicity in envenoming. By using of Sephadex G-150, DEAE-Sepharose CL-6B and FPLC Superose 12 chromatography, a protein with L-amino acid oxidase activity was purified and characterized from Agkistrodon haly Pallas venom. Its molecular mass was 57 kD as determined by SDS-PAGE analysis under both reducing and non-reducing conditions, and its pI was about 4.9.The protein catalysed the stereospecific oxidative deamination of L-amino acid substrate. It inhibited the platelet aggregation induced by ADP and collagen dose-dependently, even at low concentrations of 0.2 micromol/L and 0.08 micromol/L, respectively. The LAO had antibacterial effect to E.coli K12D31, and the effective concentration was as low as 0.03 g/L. Furthermore, the LAO showed cytotoxicity in crystal violet assay and apoptosis-inducing activity in the A549 cells. After 24h treatment with 5 mg/L LAO, the typical DNA fragmentation pattern of apoptotic cells was observed by using of agrose gel electrophoresis.

Dados de Publicação:
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai). 2002 May;34(3):305-10.

Palavras-Chave:
l-amino acid oxidase, snake venom, purification, characterization, antimicrobial

Antibacterial and antifungal effects of Agkistrodon halys Pallas: purification of its antibacterial protein--LAO

Thu, 14 Sep 2006 12:45:05 -0300

Autores:

Yan XM, Zhang SQ, Chang Q, Liu P, Xu JS.

Resumo:

This paper reports the venom from Agkistrodon halys Pallas have inhibitory activity against fungi and E. coli by tested in a disc diffusion assay. An antibacterial component--LAO from the venom were purified to homogeneous. It had not only antibacterial effect, but L-amino acid oxidase activity. And its enzymatic specific activity was 808 U/mg. The venom had at least 3 antibacterial components (I, II, III) as determined by acid polyacrilamide gel electrophoresis, LAO is the antibaterial components II.

Dados de Publicação:
Shi Yan Sheng Wu Xue Bao. 2000 Dec;33(4):309-16.

Palavras-Chave:
l-amino acid oxidase, snake venom, antimicrobial activity