Phylogenetic Analyses Based on Small Subunit rRNA and Glycosomal Glyceraldehyde-3-Phosphate Dehydrogenase Genes and Ultrastructural Characterization of Two Snake Trypanosomes: Trypanosoma serpentis n. sp from Pseudoboa nigra and Trypanosoma cascavelli from Crotalus durissus terrificus

We sequenced the small subunit (SSU) rRNA and glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) genes of two trypanosomes isolated from the Brazilian snakes Pseudoboa nigra and Crotalus durissus terrificus. Trypanosomes were cultured and their morphometrical and ultrastructural features were characterized by light microscopy and scanning and transmission electron microscopy. Phylogenetic trees inferred using independent or combined SSU rRNA and gGAPDH data sets always clustered the snake trypanosomes together in a clade closest to lizard trypanosomes, forming a strongly supported monophyletic assemblage (i.e. lizard-snake clade). The positioning in the phylogenetic trees and the barcoding based on the variable V7-V8 region of the SSU rRNA, which showed high sequence divergences, allowed us to classify the isolates from distinct snake species as separate species. The isolate from P. nigra is described as a new species, Trypanosoma serpentis n. sp., whereas the isolate from C. d. terrificus is redescribed here as Trypanosoma cascavelli.

Phylogeny of snake trypanosomes inferred by SSU rDNA sequences, their possible transmission by phlebotomines, and taxonomic appraisal by molecular, cross-infection and morphological analysis

Blood examination by microhaematocrit and haemoculture of 459 snakes belonging to 37 species revealed 24% trypanosome prevalence in species of Viperidae (Crotalus durissus and Bothrops jararaca) and Colubridae (Pseudoboa nigra). Trypanosome cultures from C. durissus and P. nigra were behaviourally and morphologically indistinguishable. In addition, the growth and morphological features of a trypanosome from the sand fly Viannaniyia tuberculata were similar to those of snake isolates. Cross-infection experiments revealed a lack of host restriction, as snakes of 3 species were infected with the trypanosome from C. durissus. Phylogeny based on ribosomal sequences revealed that snake trypanosomes clustered together with the sand fly trypanosome, forming a new phylogenetic lineage within Trypanosoma closest to a clade of lizard trypanosomes transmitted by sand flies dagger. The clade of trypanosomes from snakes and lizards suggests an association between the evolutionary histories of these trypanosomes and their squamate hosts. Moreover, data strongly indicated that these trypanosomes are transmitted by sand flies. The flaws of the current taxonomy of snake trypanosomes are discussed, and the need for molecular parameters to be adopted is emphasized. To our knowledge, this is the first molecular phylogenetic study of snake trypanosomes.

Jararhagin, a snake venom metalloprotease-disintegrin, activates the Rac1 GTPase and stimulates neurite outgrowth in neuroblastoma cells

It has been shown previously that the snake venom metalloprotease-disintegrin jararhagin stimulates cell migration and cytoskeletal rearrangement, independently of its effects on cellular adhesion but possibly associated with the activation of small GTP-binding proteins from the Rho family [Costa, E.P., Santos, M.F., 2004. Toxicon 44(8), 861-870.] Here we show that jararhagin stimulates spreading, actin dynamics and neurite outgrowth in neuroblastoma cells, and that this effect is accompanied by the translocation of the Rac1 small GTPase to the membrane fraction, suggesting its activation. Stimulation of neurite outgrowth was observed within minutes and was dependent on the proteolytic activity of the toxin. These results suggest that jararhagin may stimulate neuronal differentiation, being potential tool for neuronal regeneration studies. (C) 2008 Elsevier Ltd. All rights reserved.

The three-dimensional structure of bothropasin, the main hemorrhagic factor from Bothrops jararaca venom: Insights for a new classification of snake venom metalloprotease subgroups

Bothropasin is a 48 kDa hemorrhagic PIII snake venom metalloprotease (SVMP) isolated from Bothrops jararaca, containing disintegrin/cysteine-rich adhesive domains. Here we present the crystal structure of bothropasin complexed with the inhibitor POL647. The catalytic domain consists of a scaffold of two subdomains organized similarly to those described for other SVMPs, including the zinc and calcium-binding sites. The free cysteine residue Cys(189) is located within a hydrophobic core and it is not available for disulfide bonding or other interactions. There is no identifiable secondary structure for the disintegrin domain, but instead it is composed mostly of loops stabilized by seven disulfide bonds and by two calcium ions. The ECD region is in a loop and is structurally related to the RGD region of RGD disintegrins, which are derived from I`ll SVMPs. The ECD motif is stabilized by the Cys(117)_Cys(310) disulfide bond (between the disintegrin and cysteine-rich domains) and by one calcium ion. The side chain of Glu(276) of the ECD motif is exposed to solvent and free to make interactions. In bothropasin, the HVR (hyper-variable region) described for other Pill SVMPs in the cysteine-rich domain, presents a well-conserved sequence with respect to several other Pill members from different species. We propose that this subset be referred to as PIII-HCR (highly conserved region) SVMPs. The differences in the disintegrin-like, cysteine-rich or disintegrin-like cysteine-rich domains may be involved in selecting target binding, which in turn could generate substrate diversity or specificity for the catalytic domain. (C) 2008 Elsevier Ltd. All rights reserved.

Argininosuccinate Synthetase Is a Functional Target for a Snake Venom Anti-hypertensive Peptide ROLE IN ARGININE AND NITRIC OXIDE PRODUCTION

Bj-BPP-10c is a bioactive proline-rich decapeptide, part of the C-type natriuretic peptide precursor, expressed in the brain and in the venom gland of Bothrops jararaca. We recently showed that Bj-BPP-10c displays a strong, sustained anti-hypertensive effect in spontaneous hypertensive rats (SHR), without causing any effect in normotensive rats, by a pharmacological effect independent of angiotensin-converting enzyme inhibition. Therefore, we hypothesized that another mechanism should be involved in the peptide activity. Here we used affinity chromatography to search for kidney cytosolic proteins with affinity for Bj-BPP-10c and demonstrate that argininosuccinate synthetase (AsS) is the major protein binding to the peptide. More importantly, this interaction activates the catalytic activity of AsS in a dose-dependent manner. AsS is recognized as an important player of the citrulline-NO cycle that represents a potential limiting step in NO synthesis. Accordingly, the functional interaction of Bj-BPP-10c and AsS was evidenced by the following effects promoted by the peptide: (i) increase of NO metabolite production in human umbilical vein endothelial cell culture and of arginine in human embryonic kidney cells and (ii) increase of arginine plasma concentration in SHR. Moreover, alpha-methyl-DL-aspartic acid, a specific AsS inhibitor, significantly reduced the anti-hypertensive activity of Bj-BPP-10c in SHR. Taken together, these results suggest that AsS plays a role in the anti-hypertensive action of Bj-BPP-10c. Therefore, we propose the activation of AsS as a new mechanism for the anti-hypertensive effect of Bj-BPP-10c in SHR and AsS as a novel target for the therapy of hypertension-related diseases.

A novel physiological property of snake bradykinin-potentiating peptides-Reversion of MK-801 inhibition of nicotinic acetylcholine receptors

The first naturally occurring angiotensin-converting enzyme (ACE) inhibitors described are pyroglutamyl proline-rich oligopeptides, found in the venom of the viper Bothrops jararaca, and named as bradykinin-potentiating peptides (BPPs). Biochemical and pharmacological properties of these peptides were essential for the development of Captopril, the first active site-directed inhibitor of ACE, currently used for the treatment of human hypertension. However, a number of data have suggested that the pharmacological activity of BPPs could not only be explained by their inhibitory action on enzymatic activity of somatic ACE. In fact, we showed recently that the strong and long-lasting anti-hypertensive effect of BPP-10c [

Effects of morin on snake venom phospholipase A(2) (PLA(2))

Flavonoids are potent anti-inflammatory compounds isolated from several plant extracts, and have been used experimentally against inflammatory processes. In this work, a PLA(2) isolated from the Crotalus durissus cascavella venom and rat paw oedema were used as a model to. study the effect of flavonoids on PLA(2). We observed that a treatment of PLA(2) with morin induces several modifications in the aromatic amino acids, with accompanying changes in its amino acid composition. In addition, results from circular dichroism spectroscopy and UV scanning revealed important structural modifications. Concomitantly, a considerable decrease in the enzymatic and antibacterial activities was observed, even though anti-inflammatory and neurotoxic activities were not affected. These apparent controversial results may be an indication that PLA(2) possess a second pharmacological site which does not affect or depend on the enzymatic activity. (c) 2005 Elsevier Ltd. All rights reserved.

Quercetin as an inhibitor of snake venom secretory phospholipase A2

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

BIOCHEMICAL VARIABILITY BETWEEN VENOMS FROM DIFFERENT HONEYBEE (APIS-MELLIFERA) RACES

1. The comparison of molecular exclusion cromatography profiles of venoms from sting apparatuses of Apis mellifera ligustica, Apis mellifera adansonii and Africanized honey-bees in Sephadex G-100 revealed both qualitative and quantitative differences.2. The venoms from A.m. ligustica and A.m. adansonii presented, respectively, three and two peaks characteristic of each sub-species, while Africanized honey-bee was characterized by the absence of eight peaks common to the former.3. The polypeptides with M(r) in the range from 100,000 to 7500 da correspond respectively to 62.0%, 66.6% and 68.7% of total proteins from the venon of A.m. ligustica, A.m. adansonii and Africanized honey-bees, while the peptidic fraction with M(r) range from 4100 to 2000 da corresponds to 11.4%, 32.4% and 10.2% of venom protein, respectively.

Cell cycle arrest evidence, parasiticidal and bactericidal properties induced by L-amino acid oxidase from Bothrops atrox snake venom

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

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

The isolation and biochemical/enzymatic characterization of an L-amino acid oxidase, Balt-LAAO-I, from Bothrops alternates snake venom, is described. Balt-LAAO-I is an acidic glycoprotein, pI similar to 5.37, homodimeric, M-r similar to 123, 000, whose Nterminal sequence is ADVRNPLE EFRETDYEVL. It displays a high specificity toward hydrophobic and basic amino acids, while deglycosylation does not alter its enzymatic activity. Bait-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. Bait-LAAO-I is a multifunctional enzyme with promising relevant biotechnological and medical applications. (C) 2004 Elsevier Ltd. All rights reserved.

Treatment of venous ulcers with surgical adhesives derived from snake venom

A Brazilian dictionary states that "ulcer", from the Latin word ulcuserise, is a superficial lesion on skin or mucous membrane characterized by an inflammatory process and loss of tissue. Ulcers are a relatively frequent problem that may be worsen by infection or long-time duration. In these cases, they can represent a serious public health issue, since the increased case number contributes to the burden on the public health service. In recent decades, wound treatment has advanced in technological and scientific aspects regarding both development of new products and techniques. Ongoing research in this area is providing new products for wound treatment. Among newly tested substances that have presented successful results is fibrin glue, a snake venom derivative that promotes reduction of infection and edema as well as bleeding control and pain decrease. Thus, the present study aimed to evaluate fibrin glue effects on ulcers of venous origin in 24 patients that were divided into two groups. The first one (G1) consisted of 11 patients treated without the adhesive while the second group (G2) was composed of 13 persons who were under therapy with the adhesive. The results of this study revealed that patients treated with fibrin glue evolved to a satisfactory healing when compared to the other group. Therefore, fibrin glue is an appropriate alternative for treating leg ulcers with several advantages: easy application, less pain, early hospital discharge, not transmiting diseases, lower cost than the adhesive currently available in Brazil and, finally, it is a Brazilian technology.

A NEW FIBRIN SEALANT FROM Crotalus durissus terrificus VENOM: APPLICATIONS IN MEDICINE

Fibrin sealant, a widely available tissue adhesive, has been used since 1940 in a variety of clinical applications. Commercially available fibrin sealant products are synthesized from bovine thrombin and human fibrinogen, which may transmit infectious diseases, and recipients may also develop antibodies against bovine thrombin. Bearing these disadvantages in mind, a new fibrin sealant was developed in 1989 by a group of researchers from the Center for the Study of Venoms and Venomous Animals, in São Paulo State, Brazil. The main purpose was to produce an adhesive fibrin without using human blood, to avoid transmitting infectious diseases. The components of this novel sealant were extracted from large animals and a serine proteinase extracted from Crotalus durissus terrificus snake venom. The applicability of this sealant was tested in animals and humans with beneficial results. The new fibrin sealant can be a useful tool clinically due to its flexibility and diversity of applications. This sealant is a biological and biodegradable product that ( 1) does not produce adverse reactions, ( 1) contains no human blood, ( 3) has a good adhesive capacity, ( 4) gives no transmission of infectious diseases, and ( 5) may be used as an adjuvant in conventional suture procedures. The effectiveness of this new fibrin sealant is reviewed and its development and employment are described.

Triple bothropic envenomation in horses caused by a single snake

In the present study, snakebites caused by a single Bothrops pauloensis simultaneously affecting three male adult horses are described. Whereas the first two affected horses were six years old, the third was 16 years old; they weighed respectively 555, 550 and 500 kg. All horses presented swollen muzzles. The first animal received an initial antivenom dose of 19 vials and adjuvant medication, it was also subjected to tracheostomy due to the progression of acute respiratory failure. The second and third horses respectively received 15 vials of antivenom each, in addition to adjuvant medication. Due to continuous changes in blood coagulation observed during hospitalization, the first and the second horses received five more vials of antivenom, respectively, in the second and third days of treatment. The first animal was discharged on 15(th) day and the other on the 8(th) day of hospitalization. Several factors including the main actions of Bothrops venom (coagulation, proteolytic and vasculotoxic activities), the importance of early diagnosis based on clinical history and clinical examination suggesting the diagnosis of Bothrops snakebite, the adequate doses of antivenom, and finally the immediate tracheostomy are herein discussed. The tracheostomy, required to save the life of the first horse, should only occur after the administration of antivenom and control measures for changes in blood coagulation.

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.