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Abstract: Phospholipase A2 (PLA2, EC 3.1.1.4), a major component of snake venoms, specifically catalyzes the hydrolysis of fatty acid ester bonds at position 2 of 1,2-diacyl-sn-3-phosphoglycerides in the presence of calcium. This article reports the purification and biochemical/functional characterization of BmooTX-I, a new myotoxic acidic phospholipase A2 from Bothrops moojeni snake venom. The purification of the enzyme was carried out through three chromatographic steps (ion-exchange on DEAE–Sepharose, molecular exclusion on Sephadex G-75 and hydrophobic chromatography on Phenyl–Sepharose). BmooTX-I was found to be a single-chain protein of 15,000Da and pI 4.2. The N-terminal sequence revealed a high homology with other acidic Asp49 PLA2s from Bothrops snake venoms. It displayed a high phospholipase activity and platelet aggregation inhibition induced by collagen or ADP. Edema and myotoxicity in vivo were also induced by BmooTX-I. Analysis of myotoxic activity was carried out by optical and ultrastructural microscopy, demonstrating high levels of leukocytary infiltrate. Previous treatment of BmooTX-I with BPB reduced its enzymatic and myotoxic activities, as well as the effect on platelet aggregation. Acidic myotoxic PLA2s from Bothrops snake venoms have been little explored and the knowledge of its structural and functional features will be able to contribute for a better understanding of their action mechanism regarding enzymatic and toxic activities. [Copyright &y& Elsevier]

Abstract: This paper reports the purification and biochemical/pharmacological characterization of two myotoxic phospholipases A2 (PLA2s) from Bothrops brazili venom, a native snake from Brazil. Both myotoxins (MTX-I and II) were purified by a single chromatographic step on a CM-Sepharose ion-exchange column up to a high purity level, showing M r ∼14,000 for the monomer and 28,000Da for the dimer. The N-terminal and internal peptide amino acid sequences showed similarity with other myotoxic PLA2s from snake venoms, MTX-I belonging to Asp49 PLA2 class, enzymatically active, and MTX-II to Lys49 PLA2s, catalytically inactive. Treatment of MTX-I with BPB and EDTA reduced drastically its PLA2 and anticoagulant activities, corroborating the importance of residue His48 and Ca2+ ions for the enzymatic catalysis. Both PLA2s induced myotoxic activity and dose–time dependent edema similar to other isolated snake venom toxins from Bothrops and Crotalus genus. The results also demonstrated that MTXs and cationic synthetic peptides derived from their 115–129 C-terminal region displayed cytotoxic activity on human T-cell leukemia (JURKAT) lines and microbicidal effects against Escherichia coli, Candida albicans and Leishmania sp. Thus, these PLA2 proteins and C-terminal synthetic peptides present multifunctional properties that might be of interest in the development of therapeutic strategies against parasites, bacteria and cancer. [Copyright &y& Elsevier]

Abstract: l-Amino acid oxidases (LAAOs, EC 1.4.3.2) are flavoenzymes that catalyze the stereospecific oxidative deamination of an l-amino acid substrate to the corresponding α-ketoacid with hydrogen peroxide and ammonia production. The present work describes the first report on the antiviral (Dengue virus) and antiprotozoal (trypanocidal and leishmanicide) activities of a Bothrops jararaca l-amino acid oxidase (BjarLAAO-I) and identify its cDNA sequence. Antiparasite effects were inhibited by catalase, suggesting that they are mediated by H2O2 production. Cells infected with DENV-3 virus previously treated with BjarLAAO-I, showed a decrease in viral titer (13–83-fold) when compared with cells infected with untreated viruses. Untreated and treated promastigotes (T. cruzi and L. amazonensis) were observed by transmission electron microscopy with different degrees of damage. Its complete cDNA sequence, with 1452bp, encoded an open reading frame of 484 amino acid residues with a theoretical molecular weight and pI of 54,771.8 and 5.7, respectively. The cDNA-deduced amino acid sequence of BjarLAAO shows high identity to LAAOs from other snake venoms. Further investigations will be focused on the related molecular and functional correlation of these enzymes. Such a study should provide valuable information for the therapeutic development of new generations of microbicidal drugs. [Copyright &y& Elsevier]

Abstract: A myotoxic Asp49-phospholipase A2 (Asp49-PLA2) with low catalytic activity (BthTX-II from Bothrops jararacussu venom) was crystallized and the molecular-replacement solution has been obtained with a dimer in the asymmetric unit. The quaternary structure of BthTX-II resembles the myotoxic Asp49-PLA2 PrTX-III (piratoxin III from B. pirajai venom) and all non-catalytic and myotoxic dimeric Lys49-PLA2s. Despite of this, BthTX-II is different from the highly catalytic and non-myotoxic BthA-I (acidic PLA2 from B. jararacussu) and other Asp49-PLA2s. BthTX-II structure showed a severe distortion of calcium-binding loop leading to displacement of the C-terminal region. Tyr28 side chain, present in this region, is in an opposite position in relation to the same residue in the catalytic activity Asp49-PLA2s, making a hydrogen bond with the atom Oδ2 of the catalytically active Asp49, which should coordinate the calcium. This high distortion may also be confirmed by the inability of BthTX-II to bind Na+ ions at the Ca2+-binding loop, despite of the crystallization to have occurred in the presence of this ion. In contrast, other Asp49-PLA2s which are able to bind Ca2+ ions are also able to bind Na+ ions at this loop. The comparison with other catalytic, non-catalytic and inhibited PLA2s indicates that the BthTX-II is not able to bind calcium ions; consequently, we suggest that its low catalytic function is based on an alternative way compared with other PLA2s. [Copyright &y& Elsevier]

Abstract: Many plants are used in traditional medicine as active agents against various effects induced by snakebite. The methanolic extract from Cordia verbenacea (Cv) significantly inhibited paw edema induced by Bothrops jararacussu snake venom and by its main basic phospholipase A2 homologs, namely bothropstoxins I and II (BthTXs). The active component was isolated by chromatography on Sephadex LH-20 and by RP-HPLC on a C18 column and identified as rosmarinic acid (Cv-RA). Rosmarinic acid is an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid [2-O-cafeoil-3-(3,4-di-hydroxy-phenyl)-R-lactic acid]. This is the first report of RA in the species C. verbenacea (‘baleeira’, ‘whaler’) and of its anti-inflammatory and antimyotoxic properties against snake venoms and isolated toxins. RA inhibited the edema and myotoxic activity induced by the basic PLA2s BthTX-I and BthTX-II. It was, however, less efficient to inhibit the PLA2 activity of BthTX-II and, still less, the PLA2 and edema-inducing activities of the acidic isoform BthA-I-PLA2 from the same venom, showing therefore a higher inhibitory activity upon basic PLA2s. RA also inhibited most of the myotoxic and partially the edema-inducing effects of both basic PLA2s, thus reinforcing the idea of dissociation between the catalytic and pharmacological domains. The pure compound potentiated the ability of the commercial equine polyvalent antivenom in neutralizing lethal and myotoxic effects of the crude venom and of isolated PLA2s in experimental models. CD data presented here suggest that, after binding, no significant conformation changes occur either in the Cv-RA or in the target PLA2. A possible model for the interaction of rosmarinic acid with Lys49-PLA2 BthTX-I is proposed. [Copyright &y& Elsevier]

Many plants are used in traditional medicine as active agents against various effects of snake bites. Phospholipase A2 enzymes are commonly found in venoms of snakes of the Viperidae and Elaphidae families, which are their main components. This article presents an overview of inhibitors isolated from plants, which show antiophidian properties.

Phospholipases A(2) (PLA(2)) are enzymes commonly found in snake venoms from Viperidae and Elaphidae families, which are major components thereof. Many plants are used in traditional medicine as active agents against various effects induced by snakebite. This article presents the PLA(2) BthTX-I structure prediction based on homology modeling. In addition, we have performed virtual screening in a large database yielding a set of potential bioactive inhibitors. A flexible docking program was used to investigate the interactions between the receptor and the new ligands. We have performed molecular interaction fields (MIFs) calculations with the phospholipase model. Results confirm the important role of Lys49 for binding ligands and suggest three additional residues as well. We have proposed a theoretically nontoxic, drug-like, and potential novel BthTX-I inhibitor. These calculations have been used to guide the design of novel phospholipase inhibitors as potential lead compounds that may be optimized for future treatment of snakebite victims as well as other human diseases in which PLA(2) enzymes are involved.

Phospholipases A(2) (PLA(2)) are enzymes of high medical scientific interest due to their involvement in a large number of human inflammatory diseases. PLA(2) constitute a diverse family of enzymes which catalyses the hydrolysis of the sn-2 ester bond in glycerophospholipids and exhibit a wide range of physiological and pathological effects. The ubiquitous nature of PLA(2) highlights the important role they play in many biological processes, as cell signaling and cell growth, including the generation of proinflammatory lipid mediators such as prostaglandin and leukotrienes, regulation of lipid mediators. The activity and expression of several PLA(2) isoforms are increased in several human cancers, suggesting that these enzymes have a central role in both tumor development and progression and can be targets for anti-cancer drugs. On the other hand, some PLA(2) isolated from Viperidae venoms are capable to induce antitumoral activity. In summary PLA(2) from snake venoms can be a new class of anticancer agents and provide new molecular and biological insights of cancer development.

Snake Venom L-amino acid oxidases (LAAOs E.C. 1.4.3.2) are flavoenzymes broadly found in various snake venom compositions. LAAOs have become an attractive subject for molecular biology, biochemistry, physiology and medicine due to their actions on various cells and biological effects on platelets, apoptosis, hemorrhage and others. In this review we try to summarize some of these reports, with special emphasis on apoptosis, anti-protozoa, bactericidal and anti-viral activities.

L-Amino acid oxidases (LAAOs, EC 1.4.3.2) are flavoenzymes that catalyze the stereospecific oxidative deamination of an L-amino acid substrate to the corresponding alpha-ketoacid with hydrogen peroxide and ammonia production. The present work describes the first report on the antiviral (Dengue virus) and antiprotozoal (trypanocidal and leishmanicide) activities of a Bothrops jararaca L-amino acid oxidase (BjarLAAO-I) and identify its cDNA sequence. Antiparasite effects were inhibited by catalase, suggesting that they are mediated by H2O2 production. Cells infected with DENV-3 virus previously treated with BjarLAAO-I, showed a decrease in viral titer (13-83-fold) when compared with cells infected with untreated viruses. Untreated and treated promastigotes (T. cruzi and L. amazonensis) were observed by transmission electron microscopy with different degrees of damage. Its complete cDNA sequence, with 1452 bp, encoded an open reading frame of 484 amino acid residues with a theoretical molecular weight and pI of 54,771.8 and 5.7, respectively. The cDNA-deduced amino acid sequence of BjarLAAO shows high identity to LAAOs from other snake venoms. Further investigations will be focused on the related molecular and functional correlation of these enzymes. Such a study should provide valuable information for the therapeutic development of new generations of microbicidal drugs.