Snake assemblage in a disturbed grassland environment in Rio Grande do Sul State, southern Brazil: Population fluctuations of Liophis poecilogyrus and Pseudablabes agassizii

A mark-recapture study of a snake assemblage using pitfall traps with drift fences was carried out in a disturbed grassland environment (e.g. cattle breeding and cultivations), located in the Pampa Biome, in the central region of the Rio Grande do Sul State, southern Brazil. From February 2001 to January 2004 we caught 272 snakes belonging to 20 species from the following families: Elapidae (5%), Viperidae (10%), and Colubridae (85%). The assemblage had a unimodal seasonal pattern of activity, and the highest number of captures occurred between September and May. There was a positive and significant correlation between the number of captures and monthly minimum and maximum average temperatures. Recruitment was observed from January to April. During the study, the area was affected by human activities, which altered the community structure: Pseudablabes agassizii was negatively affected by habitat devastation while Liophis poecilogyrus took advantage of this. Our results reinforced the impression that Pseudablabes agassizii is a habitat specialist species. We extend the understanding of the susceptibility of this species to environmental destruction in open natural environments of South America, and propose its use as a potential bio-indicator of the Pampa biome. We also discuss the importance of conservation strategies for snakes in grasslands of southern Brazil. © Finnish Zoological and Botanical Publishing Board 2007.

Use of fibrin glue derived from snake venom in the repair of deep corneal ulcers - Experimental study in dogs (Canis familiaris, Linnaeus, 1758)

Fibrin glue has been researched as an alternative method for tissue synthesis and is known for its capability to promote hemostasis at the application site, good approximation of wound edges and fast healing. The current study consisted in the application of fibrin glue derived from snake venom as treatment for experimental corneal ulcers. Twenty-one dogs had their corneas experimentally prepared through lamellar keratectomy (of standardized diameter and depth). Animals were divided into seven groups of three animals each. Six experimental groups were periodically evaluated and collection was carried out on the 1st, 3rd, 7th, 15 th, 30th and 60th post-operative days, whereas one control group was evaluated throughout the experiment. Analyses consisted in the clinical evolution and in the histopathological study of all operated on eyes. Results indicated that fibrin glue was efficient in repairing keratectomy wounds in dogs and contributed to an earlier healing phenomenon, avoiding edema formation and keeping corneal clearness. The use of fibrin glue derived from snake venom showed to be easy to apply, feasible with animal models and of low cost, avoiding the lesion progress and allowing fast and appropriate corneal healing.

Crystallographic portrayal of different conformational states of a Lys49 phospholipase A2 homologue: Insights into structural determinants for myotoxicity and dimeric configuration

Catalytically inactive phospholipase A2 (PLA2) homologues play key roles in the pathogenesis induced by snake envenomation, causing extensive tissue damage via a mechanism still unknown. Although, the amino acid residues directly involved in catalysis are conserved, the substitution of Asp49 by Arg/Lys/Gln or Ser prevents the binding of the essential calcium ion and hence these proteins are incapable of hydrolyzing phospholipids. In this work, the crystal structure of a Lys49-PLA2 homologue from Bothrops brazili (MTX-II) was solved in two conformational states: (a) native, with Lys49 singly coordinated by the backbone oxygen atom of Val31 and (b) complexed with tetraethylene glycol (TTEG). Interestingly, the TTEG molecule was observed in two different coordination cages depending on the orientation of the nominal calcium-binding loop and of the residue Lys49. These structural observations indicate a direct role for the residue Lys49 in the functioning of a catalytically inactive PLA2 homologue suggesting a contribution of the active site-like region in the expression of pharmacological effects such as myotoxicity and edema formation. Despite the several crystal structures of Lys49-PLA2 homologues already determined, their biological assembly remains controversial with two possible conformations. The extended dimer with the hydrophobic channel exposed to the solvent and the compact dimer in which the active site-like region is occluded by the dimeric interface. In the MTX-II crystal packing analysis was found only the extended dimer as a possible stable quaternary arrangement. © 2012 Elsevier B.V.

Bothropstoxin-I reduces evoked acetylcholine release from rat motor nerve terminals: Radiochemical and real-time video-microscopy studies

Understanding the biological activity profile of the snake venom components is fundamental for improving the treatment of snakebite envenomings and may also contribute for the development of new potential therapeutic agents. In this work, we tested the effects of BthTX-I, a Lys49 PLA2 homologue from the Bothrops jararacussu snake venom. While this toxin induces conspicuous myonecrosis by a catalytically independent mechanism, a series of in vitro studies support the hypothesis that BthTX-I might also exert a neuromuscular blocking activity due to its ability to alter the integrity of muscle cell membranes. To gain insight into the mechanisms of this inhibitory neuromuscular effect, for the first time, the influence of BthTX-I on nerve-evoked ACh release was directly quantified by radiochemical and real-time video-microscopy methods. Our results show that the neuromuscular blockade produced by in vitro exposure to BthTX-I (1 μM) results from the summation of both pre- and postsynaptic effects. Modifications affecting the presynaptic apparatus were revealed by the significant reduction of nerve-evoked [3H]-ACh release; real-time measurements of transmitter exocytosis using the FM4-64 fluorescent dye fully supported radiochemical data. The postsynaptic effect of BthTX-I was characterized by typical histological alterations in the architecture of skeletal muscle fibers, increase in the outflow of the intracellular lactate dehydrogenase enzyme and progressive depolarization of the muscle resting membrane potential. In conclusion, these findings suggest that the neuromuscular blockade produced by BthTX-I results from transient depolarization of skeletal muscle fibers, consequent to its general membrane-destabilizing effect, and subsequent decrease of evoked ACh release from motor nerve terminals. © 2012 Elsevier Ltd.

The reproductive cycle of the male sleep snake Sibynomorphus mikanii (Schlegel, 1837) from southeastern brazil

This study describes the male reproductive cycle of Sibynomorphus mikanii from southeastern Brazil considering macroscopic and microscopic variables. Spermatogenesis occurs during spring-summer (September-December) and spermiogenesis or maturation occurs in summer (December-February). The length and width of the kidney, the tubular diameter, and the epithelium height of the sexual segment of the kidney (SSK) are larger in summer-autumn (December-May). Histochemical reaction of the SSK [periodic acid-Schiff (PAS) and bromophenol blue (BB)] shows stronger results during summer-autumn, indicating an increase in the secretory activity of the granules. Testicular regression is observed in autumn and early winter (March-June) when a peak in the width of the ductus deferens occurs. The distal ductus deferens as well as the ampulla ductus deferentis exhibit secretory activities with positive reaction for PAS and BB. These results suggest that this secretion may nourish the spermatozoa while they are being stored in the ductus deferens. The increase in the Leydig cell nuclear diameter in association with SSK hypertrophy and the presence of sperm in the female indicate that the mating season occurs in autumn when testes begin to decrease their activity. The peak activity of Leydig cells and SSK exhibits an associated pattern with the mating season. However, spermatogenesis is dissociated of the copulation characterizing a complex reproductive cycle. At the individual level, S. mikanii males present a continuous cyclical reproductive pattern in the testes and kidneys (SSK), whereas at the populational level the reproductive pattern may be classified as seasonal semisynchronous. © 2012 Wiley Periodicals, Inc.

A new snake of the genus Helicops Wagler, 1830 (Dipsadidae, Xenodontinae) from Brazil

We describe a new species of Helicops from the southern Amazon Basin in the state of Mato Grosso, Brazil. It differs from all congeners by having dorsal scales in 21/21/19 rows in males and 23/21/19 rows in females, subcaudal keels, a banded dorsal color pattern, and 14-19 ventral blotches. Besides presenting information on the lepidosis and morphometric variation, we also describe the hemipenis and discuss the known distribution of the new species. © 2013 by The Herpetologists' League, Inc.

Structural and Phylogenetic Studies with MjTX-I Reveal a Multi-Oligomeric Toxin - a Novel Feature in Lys49-PLA2s Protein Class

The mortality caused by snakebites is more damaging than many tropical diseases, such as dengue haemorrhagic fever, cholera, leishmaniasis, schistosomiasis and Chagas disease. For this reason, snakebite envenoming adversely affects health services of tropical and subtropical countries and is recognized as a neglected disease by the World Health Organization. One of the main components of snake venoms is the Lys49-phospholipases A2, which is catalytically inactive but possesses other toxic and pharmacological activities. Preliminary studies with MjTX-I from Bothrops moojeni snake venom revealed intriguing new structural and functional characteristics compared to other bothropic Lys49-PLA2s. We present in this article a comprehensive study with MjTX-I using several techniques, including crystallography, small angle X-ray scattering, analytical size-exclusion chromatography, dynamic light scattering, myographic studies, bioinformatics and molecular phylogenetic analyses.Based in all these experiments we demonstrated that MjTX-I is probably a unique Lys49-PLA2, which may adopt different oligomeric forms depending on the physical-chemical environment. Furthermore, we showed that its myotoxic activity is dramatically low compared to other Lys49-PLA2s, probably due to the novel oligomeric conformations and important mutations in the C-terminal region of the protein. The phylogenetic analysis also showed that this toxin is clearly distinct from other bothropic Lys49-PLA2s, in conformity with the peculiar oligomeric characteristics of MjTX-I and possible emergence of new functionalities inresponse to environmental changes and adaptation to new preys. © 2013 Salvador et al.

Motor Recovery and Synaptic Preservation after Ventral Root Avulsion and Repair with a Fibrin Sealant Derived from Snake Venom

Background:Ventral root avulsion is an experimental model of proximal axonal injury at the central/peripheral nervous system interface that results in paralysis and poor clinical outcome after restorative surgery. Root reimplantation may decrease neuronal degeneration in such cases. We describe the use of a snake venom-derived fibrin sealant during surgical reconnection of avulsed roots at the spinal cord surface. The present work investigates the effects of this fibrin sealant on functional recovery, neuronal survival, synaptic plasticity, and glial reaction in the spinal motoneuron microenvironment after ventral root reimplantation.Methodology/Principal Findings:Female Lewis rats (7 weeks old) were subjected to VRA and root replantation. The animals were divided into two groups: 1) avulsion only and 2) replanted roots with fibrin sealant derived from snake venom. Post-surgical motor performance was evaluated using the CatWalk system twice a week for 12 weeks. The rats were sacrificed 12 weeks after surgery, and their lumbar intumescences were processed for motoneuron counting and immunohistochemistry (GFAP, Iba-1 and synaptophysin antisera). Array based qRT-PCR was used to evaluate gene regulation of several neurotrophic factors and receptors as well as inflammatory related molecules. The results indicated that the root reimplantation with fibrin sealant enhanced motor recovery, preserved the synaptic covering of the motoneurons and improved neuronal survival. The replanted group did not show significant changes in microglial response compared to VRA-only. However, the astroglial reaction was significantly reduced in this group.Conclusions/Significance:In conclusion, the present data suggest that the repair of avulsed roots with snake venom fibrin glue at the exact point of detachment results in neuroprotection and preservation of the synaptic network at the microenvironment of the lesioned motoneurons. Also such procedure reduced the astroglial reaction and increased mRNA levels to neurotrophins and anti-inflammatory cytokines that may in turn, contribute to improving recovery of motor function. © 2013 Barbizan et al.

Biochemical, functional, structural and phylogenetic studies on Intercro, a new isoform phospholipase A2 from Crotalus durissus terrificus snake venom

Crotoxin is a neurotoxin from Crotalus durissus terrificus venom that shows immunomodulatory, anti-inflammatory, antimicrobial, antitumor and analgesic activities. Structurally, this toxin is a heterodimeric complex composed by a toxic basic PLA2 (Crotoxin B or CB) non-covalently linked to an atoxic non-enzymatic and acidic component (Crotapotin, Crotoxin A or CA). Several CA and CB isoforms have been isolated and characterized, showing that the crotoxin venom fraction is, in fact, a mixture of different molecules derived from the combination of distinct subunit isoforms. Intercro (IC) is a protein from the same snake venom which presents high similarity in primary structure to CB, indicating that it could be an another isoform of this toxin. In this work, we compare IC to the crotoxin complex (CA/CB) and/or CB in order to understand its functional aspects. The experiments with IC revealed that it is a new toxin with different biological activities from CB, keeping its catalytic activity but presenting low myotoxicity and absence of neurotoxic activity. The results also indicated that IC is structurally similar to CB isoforms, but probably it is not able to form a neurotoxic active complex with crotoxin A as observed for CB. Moreover, structural and phylogenetic data suggest that IC is a new toxin with possible toxic effects not related to the typical CB neurotoxin. © 2013.

Purificação e caracterização de proteínas de venenos de serpentes que interferem na cascata de coagulação sanguínea

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

Avaliação da resposta humoral e da capacidade de neutralização do soro de camundongos Swiss inoculados com venenos nativo e irradiado com cobalto-60 de serpentes Crotalus durissus terrificus, Bothrops jararaca, Bothrops jararacussu e Bothrops moojeni

Pós-graduação em Doenças Tropicais - FMB

Estudos estruturais com fosfolipases A2 e fosfolipases A2 homólogas isoladas no veneno de Bothrops moojeni

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)