Biological and structural characterization of a new PLA(2) from the Crotalus durissus collilineatus venom

In the present article we report on the biological characterization and amino acid sequence of a new basic Phospholipases A(2) (PLA(2)) isolated from the Crotalus durissus collilineatus venom (Cdcolli F6), which showed the presence of 122 amino acid residues with a pI value of 8.3, molecular mass of 14 kDa and revealed an amino acid sequence identity of 80% with crotalic PLA(2)s such as Mojave B, Cdt F15, and CROATOX. This homology, however, dropped to 50% if compared to other sources of PLA(2)s such as from the Bothrops snake venom. Also, this PLA(2) induced myonecrosis, although this effect was lower than that of BthTx-I or whole crotoxin and it was able to induce a strong blockage effect on the chick biventer neuromuscular preparation, independently of the presence of the acid subunid (crotapotin). The neurotoxic effect was strongly reduced by pre-incubation with heparin or with anhydrous acetic acid and rho-BPB showed a similar reduction. The rho-BPB did not reduce significantly the myotoxic activity induced by the PLA(2), but the anhydrous acetic acid treatment and the pre-incu-bation of PLA(2) with heparin reduced significantly its effects. This protein showed a strong antimicrobial activity against Xanthomonas axonopodis passiflorae (Gram-negative), which was drastically reduced by incubation of this PLA(2) with rho-BPB, but this effect was marginally reduced after treatment with anhydrous acetic acid. Our findings here allow to speculate that basic amino acid residues on the C-terminal and molecular regions near catalytic site regions such as Calcium binding loop or rho-wing region may be involved in the binding of this PLA(2) to the molecular receptor to induce the neurotoxic effect. The bactericidal effect, however, was completely dependent on the enzymatic activity of this protein.

Purification and renal effects of phospholipase A(2) isolated from Bothrops insularis venom

Bothrops insularis venom contains a variety of substances presumably responsible for several pharmacological effects. We investigated the biochemical and biological effects of phospholipase A(2) protein isolated from B. insularis venom and the chromatographic profile showed 7 main fractions and the main phospholipase A(2) (PLA(2)) enzymatic activity was detected in fractions IV and V. Fraction IV was submitted to a new chromatographic procedure on ion exchange chromatography, which allowed the elution of 5 main fractions designated as lV-1 to IV-5, from which lV-4 constituted the main fraction. The molecular homogeneity of this fraction was characterized by high-performance liquid chromatography (HPLC) and demonstrated by mass spectrometry (MS), which showed a molecular mass of 13984.20 Da; its N-terminal sequence presented a high amino acid identity (up to 95%) with the PLA(2) of Bothrops jararaca and Bothrops asper. Phospholipase A(2) isolated from B. insularis (Bi PLA(2)) venom (10 mu g/mL) was also studied as to its effect on the renal function of isolated perfused kidneys of Wistar rats (n = 6). Bi PLA(2) increased perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF) and glomerular filtration rate (GFR). Sodium (%TNa+) and chloride tubular reabsorption (%TCl-) decreased at 120 min, without alteration in potassium transport. In conclusion, PLA(2) isolated from B. insularis venom promoted renal alterations in the isolated perfused rat kidney. (c) 2007 Elsevier Ltd. All rights reserved.

Quercetin as an inhibitor of snake venom secretory phospholipase A2

As polyphenolic compounds isolated from plants extracts, flavonoids have been applied to various pharmaceutical uses in recent decades due to their anti-inflammatory, cancer preventive, and cardiovascular protective activities. In this study, we evaluated the effects of the flavonoid quercetin on Crotalus durissus terrificus secretory phospholipase A2 (sPLA2), an important protein involved in the release of arachidonic acid from phospholipid membranes. The protein was chemically modified by treatment with quercetin, which resulted in modifications in the secondary structure as evidenced through circular dichroism. In addition, quercetin was able to inhibit the enzymatic activity and some pharmacological activities of sPLA2, including its antibacterial activity, its ability to induce platelet aggregation, and its myotoxicity by approximately 40%, but was not able to reduce the inflammatory and neurotoxic activities of sPLA2. These results suggest the existence of two pharmacological sites in the protein, one that is correlated with the enzymatic site and another that is distinct from it. We also performed molecular docking to better understand the possible interactions between quercetin and sPLA2. Our docking data showed the existence of hydrogen-bonded, polar interactions and hydrophobic interactions, suggesting that other flavonoids with similar structures could bind to sPLA2. Further research is warranted to investigate the potential use of flavonoids as sPLA2 inhibitors. (C) 2010 Elsevier B.V. All rights reserved.

The Intrahippocampal Infusion Of Crotamine From Crotalus Durissus Terrificus Venom Enhances Memory Persistence In Rats.

Previous research has shown that crotamine, a toxin isolated from the venom of Crotalus durissus terrificus, induces the release of acetylcholine and dopamine in the central nervous system of rats. Particularly, these neurotransmitters are important modulators of memory processes. Therefore, in this study we investigated the effects of crotamine infusion on persistence of memory in rats. We verified that the intrahippocampal infusion of crotamine (1 μg/μl; 1 μl/side) improved the persistence of object recognition and aversive memory. By other side, the intrahippocampal infusion of the toxin did not alter locomotor and exploratory activities, anxiety or pain threshold. These results demonstrate a future prospect of using crotamine as potential pharmacological tool to treat diseases involving memory impairment, although it is still necessary more researches to better elucidate the crotamine effects on hippocampus and memory.

Compartment Syndrome After South American Rattlesnake (crotalus Durissus Terrificus) Envenomation.

In order to report the outcome of a patient who developed compartment syndrome after South American rattlesnake (Crotalus durissus terrificus) envenomation, confirmed by subfascial pressure measurement and magnetic resonance imaging (MRI). A 63-year-old male was admitted 1 h after being bitten on the right elbow by a large snake, which was not brought for identification. Physical and laboratory features upon admission revealed two fang marks, local tense swelling, paresthesia, intense local pain, hypertension, coagulopathy, and CK = 1530 U/L (RV < 170 U/L). The case was initially treated with bothropic antivenom (80 mL, intravenously), with no improvement. Evolution within 13-14 h post-bite revealed generalized myalgia, muscle weakness, palpebral ptosis, and severe rhabdomyolysis (CK = 126,160 U/L) compatible with envenoming by C. d. terrificus. The patient was then treated with crotalic antivenom (200 mL, intravenously), fluid replacement, and urine alkalinization. Twenty-four-hour post-bite MRI showed marked muscular edema in the anterior compartment of the right forearm, with a high subfascial pressure (40 mmHg) being detected 1 h later. ELISA of a blood sample obtained upon admission, before antivenom infusion, revealed a high serum concentration of C. d. terrificus venom. No fasciotomy was performed and the patient was discharged seven days later without sequelae. Snakebite by C. d. terrificus with subfascial venom injection may lead to increased intracompartmental pressure.

The Neuromuscular Activity Of Bothriopsis Bilineata Smaragdina (forest Viper) Venom And Its Toxin Bbil-tx (asp49 Phospholipase A2) On Isolated Mouse Nerve-muscle Preparations.

The presynaptic action of Bothriopsis bilineata smaragdina (forest viper) venom and Bbil-TX, an Asp49 PLA2 from this venom, was examined in detail in mouse phrenic nerve-muscle (PND) preparations in vitro and in a neuroblastoma cell line (SK-N-SH) in order to gain a better insight into the mechanism of action of the venom and associated Asp49 PLA2. In low Ca(2+) solution, venom (3μg/ml) caused a quadriphasic response in PND twitch height whilst at 10μg/ml the venom additionally induced an abrupt and marked initial contracture followed by neuromuscular facilitation, rhythmic oscillations of nerve-evoked twitches, alterations in baseline and progressive blockade. The venom slowed the relaxation phase of muscle twitches. In low Ca(2+), Bbil-TX [210nM (3μg/ml)] caused a progressive increase in PND twitch amplitude but no change in the decay time constant. Venom (10μg/ml) and Bbil-TX (210nM) caused minor changes in the compound action potential (CAP) amplitude recorded from sciatic nerve preparations, with no significant effect on rise time and latency; tetrodotoxin (3.1nM) blocked the CAP at the end of the experiments. In mouse triangularis sterni nerve-muscle (TSn-m) preparations, venom (10μg/ml) and Bbil-TX (210nM) significantly reduced the perineural waveform associated with the outward K(+) current while the amplitude of the inward Na(+) current was not significantly affected. Bbil-TX (210nM) caused a progressive increase in the quantal content of TSn-m preparations maintained in low Ca(2+) solution. Venom (3μg/ml) and toxin (210nM) increased the calcium fluorescence in SK-N-SH neuroblastoma cells loaded with Fluo3 AM and maintained in low or normal Ca(2+) solution. In normal Ca(2+), the increase in fluorescence amplitude was accompanied by irregular and frequent calcium transients. In TSn-m preparations loaded with Fluo4 AM, venom (10μg/ml) caused an immediate increase in intracellular Ca(2+) followed by oscillations in fluorescence and muscle contracture; Bbil-TX did not change the calcium fluorescence in TSn-m preparations. Immunohistochemical analysis of toxin-treated PND preparations revealed labeling of junctional ACh receptors but a loss of the presynaptic proteins synaptophysin and SNAP25. Together, these data confirm the presynaptic action of Bbil-TX and show that it involves modulation of K(+) channel activity and presynaptic protein expression.

Répteis da Estação Ecológica Serra Geral do Tocantins, Brasil Central

Os Cerrados sul-americanos abrigam alta diversidade de répteis, incluindo elevado número de endemismos. No entanto, o conhecimento desta diversidade é ainda incompleto frente à acelerada transformação das paisagens naturais no Brasil central. Constituem, portanto, uma das regiões prioritárias para estudo e conservação da biodiversidade mundial. Estudos intensivos sobre a fauna de répteis do Cerrado são necessários e urgentes para melhor compreensão dos processos que levaram à sua origem e distribuição e para subsidiar ações de conservação. Por meio de métodos padronizados, amostramos duas regiões ainda inexploradas da Estação Ecológica Serra Geral do Tocantins, situada na região do Jalapão. Registramos 45 espécies de répteis para a EESGT e entorno, o que representa uma riqueza alta e comparável à de outras regiões bem amostradas do Cerrado. Curvas de acumulação e estimadores indicam que a riqueza local de lagartos e anfisbenídeos aproxima-se da riqueza real enquanto a de serpentes é subestimada. A distribuição não-aleatória das espécies na paisagem concorda com evidências anteriores sugerindo utilização diferencial dos hábitats pelos répteis. Reunindo os resultados do presente estudo com os de levantamentos prévios realizados na região, registramos 88 espécies de répteis para o Jalapão sendo oito registros novos que incluem Bachia oxyrhina uma espécie recém descrita da região. As espécies da área apresentam três padrões gerais de distribuição: (1) espécies endêmicas do Cerrado, (2) espécies compartilhadas com domínios da diagonal de formações abertas sul-americanas, e (3) espécies de ampla ocorrência, compartilhadas também com ecossistemas florestais. Prevalecem espécies de ampla distribuição, porém é grande o número de espécies típicas do Cerrado, incluindo cinco possivelmente endêmicas do Jalapão, e há contribuição importante da fauna da Caatinga. A distribuição dos répteis em escala local e regional demonstra a necessidade de considerar a heterogeneidade paisagística para o planejamento de diretrizes visando à conservação em regiões do Cerrado. Por sua grande extensão, posição biogeográfica e complexidade de relevo e tipos de hábitat, a EESGT tem papel fundamental para a preservação e conhecimento da diversidade de répteis do Cerrado.

Herpetofauna do Parque Estadual Carlos Botelho, São Paulo, Brasil

O presente trabalho consiste em um inventário da herpetofauna do Parque Estadual Carlos Botelho (PECB), localizado na região da Serra de Paranapiacaba, Estado de São Paulo. Os dados foram obtidos por meio de coletas em seis áreas dentro do PECB durante um período de 76 dias distribuídos em um ano, e também por meio de consulta a coleções científicas para obtenção de dados secundários. São apresentados resultados sobre a biologia e ocorrência das espécies no PECB e no Brasil, além de fotografias das diferentes espécies que compõem a herpetofauna do PECB. A herpetofauna do PECB pode ser considerada uma das mais diversificadas de São Paulo, com 65 espécies de anfíbios confirmadas e 59 espécies de répteis registrados neste trabalho. Das 65 espécies de anfíbios, 84% (55 spp.) são endêmicas das formações florestais da Mata Atlântica. Devido às características do relevo do PECB, foram encontrados diferentes padrões altitudinais para os anfíbios: 46% das espécies foram registradas apenas em altitudes acima de 500 m, enquanto que 9% são exclusivas das regiões abaixo de 400 m e 45% ocorrem em todas as áreas amostradas do Parque. Das 59 espécies de répteis do PECB, foram registradas 10 espécies de lagartos, 48 de serpentes e um quelônio. Dentre as serpentes coletadas no PECB, a jararaca Bothrops jararaca foi a mais frequente, com 26,9% (N = 14) do total registrado. Espécies de difícil amostragem, como Echinanthera cephalostriata (13,5%; N = 7) e Taeniophallus affinis (7,7%; N = 4), também foram numerosas no PECB. Dentre os lagartos, Enyalius iheringii foi a espécie mais abundante, com 50% (N = 16) de registros. Uma análise de agrupamento entre 25 taxocenoses de anfíbios brasileiras, incluindo o PECB, resultou na formação de quatro agrupamentos principais. A anurofauna do PECB é mais relacionada com as taxocenoses do Parque Estadual Jacupiranga (0,68) e do Parque Estadual Intervales (0,66). Estes Parques se encontram geograficamente próximos e constituem um dos maiores fragmentos preservados de Mata Atlântica no Brasil. Este trabalho é o primeiro a apresentar a lista de répteis do PECB, alem de complementar o conhecimento em relação a fauna de anfíbios do PECB.

Ultra-sonografia do fígado, aparelho renal e reprodutivo da jibóia (Boa constrictor)

Atualmente os animais silvestres têm despertado o interesse particular na criação domestica. Na medicina de animais selvagens, os exames ultra-sonográficos podem ser considerados como ferramenta para diagnosticar e prevenir doenças. Deste modo, realizou-se um estudo em 20 jibóias (Boa constrictor), a fim de caracterizar a morfologia e aparência ultra-sonográfica das estruturas presentes da cavidade celomática desses animais. Ultra-sonograficamente, o fígado apresentou-se variando de hipoecóica a levemente hiperecogênica, com margens ecogênicas e ecotextura homogênea em toda sua extensão. Os rins mostraram formato elipsóide, com cápsula fina, regular e hiperecóica. Os folículos ovarianos apresentaram formato ovóide, margens finas, regulares e discretamente hiperecóicas. As estruturas do sistema reprodutor do macho não foram evidenciadas com precisão, devido a sua ecogenicidade similar em relação às estruturas adjacentes e pela presença do "corpo gorduroso" localizado nessa região. A ultra-sonografia da cavidade celomática em jibóias demonstrou ser uma técnica rápida e de fácil acesso, permitindo identificar a morfologia, sintopia e aparência ultra-sonográfica de estruturas como o fígado, rins e de folículos vitelogênicos nas fêmeas.

Quantum Hall Effect near the Charge Neutrality Point in a Two-Dimensional Electron-Hole System

We study the transport properties of HgTe-based quantum wells containing simultaneously electrons and holes in a magnetic field B. At the charge neutrality point (CNP) with nearly equal electron and hole densities, the resistance is found to increase very strongly with B while the Hall resistivity turns to zero. This behavior results in a wide plateau in the Hall conductivity sigma(xy) approximate to 0 and in a minimum of diagonal conductivity sigma(xx) at nu = nu(p) - nu(n) = 0, where nu(n) and nu(p) are the electron and hole Landau level filling factors. We suggest that the transport at the CNP point is determined by electron-hole ""snake states'' propagating along the nu = 0 lines. Our observations are qualitatively similar to the quantum Hall effect in graphene as well as to the transport in a random magnetic field with a zero mean value.

Biochemistry of the Envenomation Response-a Generator Theme for Interdisciplinary Integration

The understanding of complex physiological processes requires information from many different areas of knowledge. To meet this interdisciplinary scenario, the ability of integrating and articulating information is demanded. The difficulty of such approach arises because, more often than not, information is fragmented through under graduation education in Health Sciences. Shifting from a fragmentary and deep view of many topics to joining them horizontally in a global view is not a trivial task for teachers to implement. To attain that objective we proposed a course herein described Biochemistry of the envenomation response aimed at integrating previous contents of Health Sciences courses, following international recommendations of interdisciplinary model. The contents were organized by modules with increasing topic complexity. The full understanding of the envenoming pathophysiology of each module would be attained by the integration of knowledge from different disciplines. Active-learning strategy was employed focusing concept map drawing. Evaluation was obtained by a 30-item Likert-type survey answered by ninety students; 84% of the students considered that the number of relations that they were able to establish as seen by concept maps increased throughout the course. Similarly, 98% considered that both the theme and the strategy adopted in the course contributed to develop an interdisciplinary view.

Peptidase activities in the semen from the ductus deferens and uterus of the neotropical rattlesnake Crotalus durissus terrificus

To understand the role of peptidases in seminal physiology of Crotalus durissus terrificus, intra- and inter-seasonal activity levels of acid (APA), basic (APB), puromycin-sensitive (APN-PS) and puromycin-insensitive neutral (APN-PI), cystyl (CAP), dipeptidyl-IV (DPPIV), type-1 pyroglutamyl (PAP-I) and prolyl-imino (PIP) aminopeptidases as well as prolyl endopeptidase (POP) were evaluated in soluble (SF) and/or membrane-bound (MF) fractions of semen collected from the ductus deferens of the male reproductive tract and from the posterior portion of the uterus. Seminal APB, PIP and POP were detected in SF, while other peptidases were detected in SF and MF. Only the convoluted posterior uterus in winter and autumn had semen. Relative to other examined peptidases, in general, APN-PI, APN-PS and APB activities were predominant in the semen from the uterus and throughout the year in the semen from the ductus deferens, suggesting their great relevance in the seminal physiology of C. d. terrificus. The levels of peptidase activities in the ductus deferens semen varied seasonally and were different from those of semen in the uterus, suggesting that their modulatory actions on susceptible peptides are integrated to the male reproductive cycle events and spermatozoa viability of this snake.

Peptidase activities in Crotalus durissus terrificus semen

To understand the role of peptidases in seminal physiology of Crotalus durissus terrificus, activity levels of representative enzymes in semen and their sensitivities to inhibitors, cofactors, and peptide hormones were evaluated. The existence of seminal fractions and the association of peptidases with these fractions were also characterized for the first time in snakes. The prominent inhibitors of aminopeptidases (APs) were amastatin for acid, basic, and neutral; bestatin for basic; and diprotin A for dipeptidyl-IV. Cystyl and prolylimino AN were similarly susceptible to the majority of these inhibitors. The basic and neutral were characterized as metallo-peptidases, acid AP was activated by MnCl(2), and cystyl, prolyl-imino, and type I pyroglutamyl were characterized as sulphydryl-dependent APs. Angiotensin II, vasotocin, bradykinin, fertilization-promoting peptide, and TRH altered the majority of these peptidase activities; these peptides are possible substrates and/or modulators of these peptidases. Peptidase activities were found in all seminal fractions: seminal plasma (SP), prostasome-like (PR) structures, and soluble (S-) and membrane-bound fractions (MFs) of spermatozoa. The levels of activity of each peptidase varied among different seminal fractions. In SP, the higher activities were puromycin-insensitive neutral and basic APs. in PR, the higher activity was puromycin-insensitive neutral AP. In spermatozoa, the higher activity in subcellular SF was puromycin-sensitive neutral, while in MF both puromycin-sensitive and -insensitive neutral AN were equally higher than the other examined peptidases. Data suggested that these peptidases, mainly basic and neutral, have a high relevance in regulating seminal functions of C. d. terrificus.