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

Estudo Funcional comparativo entre as Fosfolipases A2 ácida e básicas isoladas do veneno de Bothrops jararacussu

Thu, 24 Aug 2006 13:51:59 -0300

Pesquisadores envolvidos:
Marcussi, S; Urzeda, MA; Mazzi, MV; Amui, SF; Fernandes, VC; Cambraia, RS; Silveira, LB; França, SC; Soares, AM.

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

Ensaios realizados:
Atividade Fosfolipásica, atividades miotóxica, edematogênica, citotóxica sobre células endoteliais, tumorais, hipotensora, efeitos sobre a agregação plaquetária e ruptura de lipossomos.

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

Referências de Publicação:

CONIC 2003

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

Influence of phospholipases A2 from snake venoms on survival and neurite outgrowth in pheochromocytoma cell line PC12.

Tue, 19 Sep 2006 11:10:49 -0300

Autores:

Makarova YV, Osipov AV, Tsetlin VI, Utkin YN.

Resumo:

To determine whether the ability to induce neurite outgrowth in rat pheochromocytoma cell line PC12 is characteristic of phospholipases of different types, we have studied the influence of phospholipase A(2) (PLA2) from cobra Naja kaouthia venom and two PLA2s from viper Vipera nikolskii venom on PC12 cells. Phospholipases from the viper venom are heterodimers in which only one of the subunits is enzymatically active, while PLA2 from the cobra venom is a monomer. It was found that all three PLA2s induce neurite outgrowth in PC12. The PLA2 from cobra venom exhibits this effect at higher concentrations as compared to the viper enzymes. We have not observed such an activity for isolated subunits of viper PLA2s, since the enzymatically active subunits have very high cytotoxicity, while the other subunits are not active at all. However, co-incubation of active and inactive subunits before addition to the cells leads to a marked decrease in cytotoxicity and to restoration of the neurite-inducing activity. It has also been shown that all enzymatically active PLA2s are cytotoxic, the PLA2 from cobra venom being the least active. Thus, for the first time we have shown that PLA2s from snake venoms can induce neurite outgrowth in PC12 cells.

Dados de Publicação:
Biochemistry (Mosc). 2006 Jun;71(6):678-84.

Palavras-Chave:
phospholipase A2, cytotoxicity, snake venom, PC12 cells, neurite outgrowth

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

Inibição de PLA2s isoladas de veneno de serpente Bothrops jararacussu, por substâncias naturais e artificiais.

Wed, 07 Dec 2005 14:09:25 -0200

Autores:

Marcussi, S(1); Mazzi, MV(3); Fernandes, VC(1); Sant'Ana, CD(3); Ticli, FK(3); França, SC(1); Giglio, JR(2); Soares, AM(1)

Resumo:

Inibição de PLA2s isoladas de veneno de serpente Bothrops jararacussu, por substâncias naturais e artificiais.

O grande interesse médico-científico despertado pelo envolvimento destas proteínas em diferentes processos fisiopatológicos levou a uma crescente busca por ligação destas com inibidores, substratos naturais e artificiais, visando sua neutralização. Este trabalho tem por objetivo verificar o efeito inibitório de diferentes substâncias sobre as PLA_2s de B. jararacussu. Três PLA_2s (BthTX-I, Lys49; BthTX-II, Asp49; e BthA-I-PLA2s, Asp49) foram isoladas do veneno de Bothrops jararacussu e testadas contra diferentes inibidores naturais e artificiais. Estudos de mecanismos de ação foram desenvolvidos através de ensaios com diferentes inibidores sintéticos (EDTA, BPB, manoalide B) e naturais (heparina, BmjMIP, manoalide A). A modificação química induzida pelo BPB resultou em inibição total das atividades, fosfolipásica e anticoagulante, induzidas pelas BthTX-II e BthA-I-PLA2. As atividades de ruptura de lipossomos e edema induzidas pelas Asp49 foram parcialmente inibidas, assim como os efeitos miotóxico, citotóxico e indução de edema causados pela BthTX-I. A interação com BPB também alterou o efeito das Asp49 sobre plaquetas, da BthA-I-PL_A2 sobre a pressão arterial e das BthTX-I e II em induzir letalidade. A incubação com EDTA resultou em inibição total da atividade fosfolipásica das Asp49, diminuindo os efeitos de ruptura de lipossomos, miotoxicidade, citotoxicidade e edematogênico da BthTX-II, e de ruptura de lipossomos da BthA-I-PL_A2. A heparina, BmjMIP e os Manoalide A e B também inibiram a citotoxicidade, a miotoxicidade, o edema e a ruptura de liposssomos, em diferentes intensidades, agindo particularmente sobre o efeito de cada proteína. As PLA₂ de venenos são algumas das substâncias naturais com atividades farmacológicas de interesse médico-científico que têm sido amplamente pesquisadas.

Dados de Publicação:
SBTox 2004

Activation of cellular functions in macrophages by venom secretory Asp-49 and Lys-49 phospholipases A2

Thu, 24 Aug 2006 23:00:38 -0300

Autores:

Zuliani JP, Gutierrez JM, Casais e Silva LL, Coccuzzo Sampaio S, Lomonte B, Pereira Teixeira Cde F

Resumo:

The in vitro effects of myotoxin III (MT-III), an Asp-49 catalytically-active phospholipase A2, and myotoxin II (MT-II), a catalytically-inactive Lys-49 variant, isolated from Bothrops asper snake venom, on phagocytosis and production of hydrogen peroxide (H2O2) by thioglycollate-elicited macrophages were investigated. MT-II and MT-III were cytotoxic to mouse peritoneal macrophages at concentrations higher than 25 μg/ml. At non-cytotoxic concentrations, MT-II stimulated Fcγ, complement, mannose and β-glucan receptors-mediated phagocytosis, whereas MT-III stimulated only the mannose and β-glucan receptors-mediated phagocytosis. Moreover, both myotoxins induced the release of H2O2 by thioglycollate-elicited macrophages, MT-III being the most potent stimulator. MT-II induced the release of H2O2 only at a concentration of 3.2 μg/ml (130% increment) while MT-III induced this effect at all concentrations tested (0.5–2.5 μg/ml; average of 206% increment). It is concluded that, at non-cytotoxic concentrations, MT-II and MT-III activate defense mechanisms in macrophages upregulating phagocytosis, mainly via mannose and β-glucan receptors, and the respiratory burst.

Dados de Publicação:
Toxicon. 2005 Oct;46(5):523-32.

Palavras-Chave:
Venom myotoxic PLA2; Macrophages; Phagocytosis; Hydrogen peroxide; Inflammation

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

Natural and Synthetic Phospholipases A2 Inhibitors (PLIs): Inhibition of PLA2s isolated from Bothrops jararacussu Snake Venom.

Fri, 15 Sep 2006 14:30:22 -0300

Pesquisadores envolvidos:
1,2Marcussi, S.; 1Fernandes, V. C.; 1Silveira, L. B.; 1Cambraia, R. S.; 3Ticli, F. K.; 3Sant`Ana, C. D.; 3Mazzi, M. V.; 1França, S. C.; 3Sampaio, S. V.; 2Giglio, J. R. and 1Soares, A. M.

Local:
1Unidade de Biotecnologia-UNAERP, Ribeirão Preto-SP; 2Depto Bioquímica e Imunologia, FMRP-USP, Ribeirao Preto-SP and 3Depto. Análises Clínicas, Toxicológicas e Bromatológicas-FCFRP-USP, Ribeirã

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

Referências de Publicação:

SBTX 2004

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

Snakebites and ethnobotany in the Colombia Part II: Neutralization of lethal and enzymatic effects of Bothrops atrox venom

Tue, 04 Apr 2006 16:59:41 -0300

Autores:

Otero R^a, Nunez V^a, Jimenez SL^a, Fonnegra Ra, Osorio RG^a, Garcia ME^a, Diaz A^a

Resumo:

Twelve of 74 ethanolic extracts of plants used by traditional healers for snakebites in the northwest region of Colombia, were active against lethal effect of Bothrops atrox venom when they were i.p. injected into mice (18–20 g). After preincubation of sublethal doses of every extract (0.5–4.0 mg/mouse) with 1.5 i.p. lethal dose 50% (LD₅₀) (99.3 μg) of venom, seven of them demonstrated 100% neutralizing capacity within 48 h. These were the stem barks of Brownea rosademonte (Caesalpiniaceae) and Tabebuia rosea (Bignoniaceae); rhizomes of Renealmia alpinia ( Zingiberaceae) and Heliconia curtispatha (Heliconiaceae); the whole plants of Pleopeltis percussa (Polypodiaceae) and Trichomanes elegans (Hymenophyllaceae); and the ripe fruits of Citrus limon (Rutaceae). The other five extracts showing partial neutralization (45–80%; 10–30% survival rate in the control group receiving the venom alone; P<0.05) were: leaves, branches and stem of Costus lasius (Costaceae); the whole plant of Sida acuta (Malvaceae); rhizomes of Dracontium croatii (Araceae); leaves and branches of Bixa orellana (Bixaceae) and Struthanthus orbicularis (Loranthaceae). When the extracts were independently administered per oral or i.p. route 60 min before an i.m. venom injection (204 μg=1.5 i.m. LD₅₀), C. limon, T. elegans, B. orellana and T. rosea extracts had partial and significant neutralizing capacity against B. atrox venom lethal effect. C. limon extract was also partially effective when it was administered either i.v. 15 min before or i.p. 5 min after an i.m. venom injection. Three of the 12 extracts with anti-lethal effect (C. limon, D. croatii and S. acuta) were devoid of antiphospholipase A₂ activity, when they were tested against one minimum indirect hemolytic dose of B. atrox venom (2 μg) in agarose-erythrocyte-egg yolk gels.

Dados de Publicação:
Journal of Ethnopharmacology 71 (2000) 505–511

Palavras-Chave:
Neutralization; B. atrox venom; Plant extracts; Colombia

Myotoxic phospholipases A(2) in bothrops snake venoms: effect of chemical modifications on the enzymatic and pharmacological pro

Mon, 21 Aug 2006 14:06:15 -0300

Autores:

^aAndriao-Escarso SH, ^a,bSoares AM, ^aRodrigues VM,^bAngulo Y, ^bDiaz C, ^bLomonte B, ^bGutierrez JM,^aGiglio JR

Resumo:

Venoms from eight Bothrops spp. were fractionated by ion-exchange chromatography on CM-Sepharose at pH 8.0 for the purification of myotoxins. Chromatographic profiles showed differences regarding myotoxic components among these venoms. B. alternatus, B. atrox and B. jararaca venoms did not show the major basic myotoxic fractions identified in the other venoms. Polyacrylamide gel electrophoresis for basic proteins also showed distinct patterns for these toxins. In vivo, all the isolated myotoxins induced release of creatine kinase due to necrosis of muscle fibers, accompanied by polymorphonuclear cell infiltration, and edema in the mouse paw. In addition, the toxins showed cytotoxic and liposome-disrupting activities in vitro. B. jararacussu bothropstoxins-I (BthTX-I) and II (BthTX-II) were submitted to chemical modifications of: His, by 4-bromophenacyl bromide (BPB) or photooxidation by Rose Bengal (RB); Tyr, by 2-nitrobenzenesulphonyl fluoride (NBSF); and Trp, by o-nitrophenylsulphenyl chloride (NPSC). The myotoxic and cytotoxic activities of BthTX-I, a Lys49 PLA₂ homologue, after modification by BPB, RB, NBSF and NPSC, were reduced to 50%, 20%, 75%, 65% and 13%, 0.5%, 76%, 58%, respectively. However, the edema-inducing and liposome-disrupting activities were not significantly reduced by the above modifications. BPB-treated BthTX-II, an Asp49 PLA₂ homologue, lost most of its catalytic, indirect hemolytic, anticoagulant, myotoxic and cytotoxic activities. The edema-inducing and liposome-disrupting activities were reduced to 50% and 80%, respectively. Lethality caused by BthTX-I and -II was strongly reduced after treatment with BPB or RB, but only partially with NBSF or NPSC. BthTX-I and -II, both native or modified, migrated similarly in a charge-shift electrophoresis. Antibodies raised against BthTX-I or -II, B. asper Basp-II and the C-terminal 115-129 peptide from Basp-II did not show significant differences in their cross-reactivity with the modified toxins, except with RB photooxidized toxins.

Dados de Publicação:
Biochimie 82 (2000) 755−763

Isolation and structural characterization of a cytotoxic L-amino acid oxidase from Agkistrodon contortrix laticinctus snake veno

Tue, 12 Sep 2006 03:22:25 -0300

Autores:

Souza DH, Eugenio LM, Fletcher JE, Jiang MS, Garratt RC, Oliva G, Selistre-de-Araujo HS.

Resumo:

We have purified a cytotoxic L-amino acid oxidase (LAO) from Agkistrodon contortrix laticinctus snake venom by means of Superdex-200 gel filtration, followed by phenyl-Sepharose CL-4B chromatography. The purified enzyme (ACL LAO) is a dimer on gel filtration, with a M(r) of 60,000 for the monomer as estimated by SDS-PAGE. LAO activity was tested against 15 amino acids, but only 9 were oxidized by the enzyme, suggesting that it presents some degree of specificity. ACL LAO has apoptosis-inducing activity in an HL-60 cell culture assay. After 24 h treatment with 25 micrograms/ml of ACL LAO, the typical DNA fragmentation pattern of apoptotic cells was observed on agarose gel electrophoresis. NMR analysis showed the presence of a flavin mononucleotide prosthetic group. To solve its 3-D structure, crystals of the purified protein were grown in 0.1 M Tris-HCl, pH 8.5, and 2 M (NH(4))(2)SO(4). Diffraction data collected to 3.5 A showed that the protein crystallized in the tetragonal system, with unit cell a = b = 103.22 A, c = 183.45 A. This is the first report of preliminary crystallization data for a snake venom L-amino acid oxidase. Copyright 1999 Academic Press.

Dados de Publicação:
Arch Biochem Biophys. 1999 Aug 15;368(2):285-90.

Characterization and cytotoxicity of L-amino acid oxidase from the venom of king cobra (Ophiophagus hannah).

Fri, 01 Sep 2006 01:38:16 -0300

Autores:

Ahn MY, Lee BM, Kim YS.

Resumo:

The aim of this project was to determine the cytotoxic components from the venom of king cobra, Ophiophagus hannah. Venom was purified by a combination of gel-filtration, ion-exchange and reversed-phase chromatographic steps. The biochemical properties of the cytotoxic component were consistent with those of L-amino acid oxidase. The molecular weight of the enzyme was estimated to be 150,000 by gel filtration and 70,000 under the denaturing conditions of SDS-PAGE, indicating a dimer. It has an isoelectric point of 4.5 and is a glycoprotein. The N-terminal sequence of L-amino acid oxidase from the king cobra venom was determined to be SVINLEESFQEPEYE. The cytotoxicity of L-amino acid oxidase was observed in stomach cancer, murine melanoma, fibrosarcoma, colorectal cancer and Chinese hamster ovary cell lines. Cytotoxicity resulted in the loss of ability in attachment and inhibition of cell proliferation. The cytotoxic protein decreased the level of cell proliferation by 74% according to [3H]thymidine uptake assay. The mechanism of enzyme action may be related to the inhibition of thymidine incorporation and an interaction with DNA.

Dados de Publicação:
Int J Biochem Cell Biol. 1997 Jun;29(6):911-9.

Palavras-Chave:
snake venom, L-amino acid oxidase, cytotoxicity

Identification of the snake venom substance that induces apoptosis.

Fri, 01 Sep 2006 01:34:33 -0300

Autores:

Suhr SM, Kim DS.

Resumo:

Hemorrhagic snake venom induces apoptosis of vascular endothelial cells [S. Araki, T. Ishida, T. Yamamoto, K. Kaji, and H. Hayashi (1993) Biochem. Biophys. Res. Comm. 190 , 148-153]. We have identified that a cytotoxic substance of Korean snake venom which is responsible for the apoptosis is L-amino acid oxidase (LAO). The purified enzyme is a homodimeric glycoprotein of 110,000 and is capable of generating H2O2 by catalyzing oxidation of L-amino acid. In the presence of the enzyme, cultured L1210 cell nuclei were splitted and showed the characteristic ladder-like pattern of DNA fragmentation. The enzyme binds directly to the cell surface, thereby increasing local concentration of H2O2. However, experimental evidence suggests that the LAO-induced apoptotic mechanism is distinguished from the one caused by exogenous H2O2.

Dados de Publicação:
Biochem Biophys Res Commun. 1996 Jul 5;224(1):134-9.

Palavras-Chave:
snake venom, L-amino acid oxidase