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Cardiovascular effects induced by snake venoms, in spite of having a crucial role in the outcome of the envenomation, have been less studied than other toxic activities displayed by these venoms. In this study we evaluated acute cardiovascular responses to Bothrops leucurus venom - Bl-V - both in vivo, in anesthetized rats, and in vitro, in isolated rat mesenteric resistance arteries. Bl-V (10–100 μg protein/kg) caused dose-dependent hypotension, followed by gradual recovery (2–20 min) to basal levels, and induced dose-dependent (1–20 μg/mL) vasodilation in pre-contracted arteries, what was more pronounced when the endothelium remained intact. These effects were partially counteracted by pre-treatment with indomethacin (cyclooxygenase inhibitor). Prior incubation of Bl-V with commercial pentavalent Bothrops antivenom also attenuated the cardiovascular effects induced by the venom, in spite of it not being among the venoms used for the development of the bothropic antivenom. Through an approach based on two chromatographic steps and mass spectrometry (MALDI-ToF and MALDI-ISD), a component with acute cardiovascular effects was isolated and identified as the basic phospholipase blD-PLA2, previously purified from the venom of B. leucurus. Taken together, our results show that, at low doses, the venom of B. leucurus induces transient, acute hypotension in anesthetized rats following systemic vasodilation in a dose-dependent way. In addition, we provide clear evidence of the involvement of the enzymatic activity of blD-PLA2 in this cardiovascular response, acting via the production of vasodilating prostanoids.

Spider venoms induce different physio-pharmacological effects by binding with high affinity on molecular targets, therefore being of biotechnological interest. Some of these toxins, acting on different types of ion channels, have been identified in the venom of spiders of the genusBiochemical and functional comparison of the venoms were carried out by SDS-PAGE, HPLC, mass spectrometry, enzymatic activities and electrophysiological assays (whole-cell patch clamp).The employed approach revealed that all three venoms had an overall similarity in their components, with only minor differences. The presence of a high number of similar proteins was evident, particularly toxins in the mass range of ~6.0 kDa. Hyaluronidase and proteolytic activities were detected in all venoms, in addition to isoforms of the toxins Tx1 and Tx2-6. All Tx1 isoforms blocked Nav1.6 ion currents, with slight differences.Our findings showed that Pn-V, Pe-V and Pp-V are highly similar concerning protein composition and enzymatic activities, containing isoforms of the same toxins sharing high sequence homology, with minor modifications. However, these structural and functional variations are very important for venom diversity. In addition, our findings will contribute to the comprehension of the molecular diversity of the venoms of the other species from

Os venenos animais são constituídos por complexas misturas de toxinas de origem principalmente protéica, com ação enzimática ou não e que são responsáveis pelos principais efeitos dos envenenamentos tais como; miotoxicidade, hemorragia, distúrbios na coagulação sanguínea e edema. Muitas plantas têm sido usadas pelo homem para o tratamento de acidentes ofídicos, mas geralmente seus usos têm sido atribuídos à superstição, e poucos são os conhecimentos científicos relacionados às características apresentadas por estas plantas. O objetivo deste trabalho foi verificar a inibição dos principais efeitos dos venenos das serpentes Bothrops jararacussu, Bothrops neuwiedi pauloensis e da abelha Apis mellifera pelo extrato vegetal de Casearia sylvestris (Flacourtiaceae), uma típica planta do Cerrado Brasileiro. Inicialmente, foi preparado um extrato aquoso a partir das folhas e do caule desta planta. Para cada ensaio de inibição, o extrato foi incubado com o respectivo veneno por uma hora antes do teste. Foram realizados ensaios para as atividades fosfolipásica A2, coagulante, fibrinogenolítica, hemorrágica miotóxica, edematogênica e toxicidade. Para a atividade fosfolipásica A2, os resultados mostram uma alta porcentagem de inibição para todos os venenos, onde testes estatísticos revelaram a eficácia do extrato vegetal tanto para o caule quanto para as folhas. Na atividade coagulante, os resultados foram estatisticamente testados pelo teste T e mostraram que apenas B jararacussu incubado com Casearia sylvestris folhas, apresentou diferenças significativas (inibição de 38%), enquanto para as outras amostras, não houve inibição significativa. Quando testamos a inibição da atividade fibrinogenolítica, encontramos que o extrato (folhas ou caule) ofereceu pouca ou nenhuma proteção para a cadeia a do fibrinogênio. A neutralização da hemorragia foi determinada por injeções intradérmicas no dorso de camundongos, assim, a atividade hemorrágica apresentou grande inibição para o extrato do caule e das folhas, sugerindo a eficiência deste extrato para esta atividade. Para a atividade miotóxica, os bons resultados obtidos também sugerem a presença de componentes capazes de interferir no efeito tóxico dos venenos, pois os cortes histológicos mostram fibras em perfeito estado para as amostras incubadas com o extrato quando comparadas à aquelas que continham apenas veneno. Na atividade edematogênica, verificou-se que o edema formado pelo veneno de Bothrops jararacussu não foi inibido significativamente pelo extrato vegetal das folhas de Casearia sylvestris, mas em relação ao controle foi ligeiramente menor. Já para o teste de toxicidade o extrato vegetal de Casearia sylvestris provocou um aumento na sobrevida dos animais que receberam injeções intraperitoniais de 2DL50 (Dose letal 50%) dos venenos de Bothrops jararacussu e Bothrops neuwiedi pauloensis incubados com o extrato do caule ou das folhas. Embora todos os animais morrerem ao longo do experimento, verificou-se que o tempo médio de sobrevida foi prolongado. Para todas as atividades, foram realizados controles negativos, utilizando somente os extratos vegetais e podemos concluir que estes extratos não induzem a nenhuma atividade. De acordo com os resultados acima, verificamos o potencial inibitório do extrato de Casearia sylvestris (Flacourtiaceae). As altas porcentagens de inibição para as atividades hemorrágica, fosfolipásica A2 e miotóxica, indicam a presença de componentes que poderão apresentar grande valor terapêutico para tratamento de envenenamentos animais e talvez até para outras desordens clínicas. Snake venoms are constituted by complex mixtures of proteases, hemorrhagic factors and phospholipases that are responsible for the main effects of the envenomations such as: myotoxicity, hemorrhage, disturbs of blood coagulation and edema. Many plants have been used in popular medicine for the treatment of snakebite, but its use has generally been attributed to the superstition and there are few scientific knowledge related to the characteristics presented by these plants. The purpose of this study was to verify the inhibition of the Bothrops jararacussu, Bothrops neuwiedi pauloensis and Apis mellifera venoms main effects by Casearia sylvestris (Flacourtiaceae) extract, a typical plant from Brazilian Savannah. Initially, an acjueous extract was prepared from the leaves and the stem of this plant and this extract was incubated with the crude venom sample by one hour before the test. We realized some activities as Phospholipase A2, blood coagulation, fibrinogenolytic, hemorrhagic, myotoxic, edema and toxicity. For Phospholipase A2 activity the results indicate a high percentage of inhibition for all the venoms, where the statistical analysis revealed that these extract inhibited significantly and that the both extracts (stem and leaves) are equally efficient. In the coagulation activity the results were statistically analyzed by the test T and showed significant differences only to B.jararacussu venom incubated with Casearia sylvestris leaves (inhibition of 38/o), while for the other samples, no significant neutralization was recorded. When we tested the inhibition of the fibrinogenolytic activity, we found that the extract (leaves or stem) offered little or no protection for the fibrinogen ct chain. The inactivation of the hemorrhagic activity was determined by intradermic injections in mice. The hemorrhagic activity presented great inhibition for the stem and leaves extract, suggesting the efficiency of this extract for this activity. For myotoxic activity, the histological observations show fibers in perfect State for the samples incubated with the extract when compared to the those that contained only venom and they indicate the presence of the components able to neutralize the toxic effects. In the edema activity was verified that the edema was not inhibited by the extract of the leaves of Casearia sylvestris, but the edema produced by the sample incubated with the extract was lightly smaller than that provoked by the venom in the extract absence. Finally, for toxicity test, the Casearia sylvestris extract provoked an increase in the lifetime of the animais that received intraperitonials injections of 2DL50 (lethal dose) of Bothrops jararacussu and Bothrops neuwiedi pauloensis venoms when these were incubated with the stem or leaves extract. Despite all the animais died along the experiment, the lifetime average was prolonged and it showed significant results for Bothrops jararacussu venom. For all the activities, negative Controls were accomplished, using only the extracts and we can conclude that these extracts don't induce to any activity. In agreement with the results above, we verified that Casearia sylvestris (Flacourtiaceae) extract can neutralize animais venoms. The high inhibition percentages for the hemorrhagic, phospholipase A2 and myotoxic activities, indicate the presence of components that could present great therapeutic value for treatment of animal envenomations and perhaps to for another clinicai disorders. Dissertação (Mestrado)

Agents that modulate the activity of high-voltage gated calcium channels (HVCCs) exhibit experimentally and clinically significant effect by relieving visceral pain. Among these agents, the toxins Phα1β and ω-conotoxin MVIIA effectively reduce chronic pain in rodent models. The molecular mechanisms underlying the chronic pain associated with acute pancreatitis (AP) are poorly understood. Hypercalcemia is a risk factor; the role of cytosolic calcium is considered to be a modulator of pancreatitis. Blockade of Ca2+ signals may be useful as a prophylactic treatment of pancreatitis. We explored the pathophysiological roles of three peptide toxins: Phα1β and its recombinant form CTK 01512-2-blockers of TRPA1 receptor and HVCCs and ω-conotoxin MVIIA, a specific blocker of N-type calcium channels in cerulein-induced AP. Cerulein injection elicits AP in rats, evidenced by an increase in hyperalgesic pain, inflammatory infiltration, amylase and lipase secretion, and reactive oxygen species, TNF-α, and p65 NF-κB levels. These effects of cerulein-induced AP were abolished by Phα1β and its recombinant form CTK 01512-2, whereas ω-conotoxin MVIIA had no effect on the induced increase in pancreatic enzyme secretion. Our results demonstrate that Phα1β and CTK 01512-2 toxins-antagonists of HVCCs and TRPA1 receptor presented an effective response profile, in the control of nociception and inflammatory process in the AP model in rats, without causing changes in spontaneous locomotion of the rats.

The biological activities observed upon envenomation by Scorpaena plumieri could be linked to both the venom and the skin mucus. Through a proteomic/functional approach we analyzed protein composition and biological activities of the venom and skin mucus. We identified 885 proteins: 722 in the Venomous Apparatus extracts (Sp-VAe) and 391 in the Skin Mucus extract (Sp-SMe), with 494 found exclusively in Sp-VAe, being named S. plumieri Venom Proteins (Sp-VP), while 228 were found in both extracts. The majority of the many proteins identified were not directly related to the biological activities reported here. Nevertheless, some were classified as toxins/potentially interesting molecules: lectins, proteases and protease inhibitors were detected in both extracts, while the pore-forming toxin and hyaluronidase were associated with Sp-VP. Proteolytic and anti-microbial activities were linked to both extracts, while the main toxic activities – cardiovascular, inflammatory, hemolytic and nociceptive – were elicited only by Sp-VAe. Our study provided a clear picture on the composition of the skin mucus and the venom. We also show that the classic effects observed upon envenomation are produced by molecules from the venomous gland. Our results add to the growing catalogue of scorpaeniform fish venoms and their skin mucus proteins. Significance In this study a large number of proteins – including classical and non-classical toxins – were identified in the venomous apparatus and the skin mucus extracts of the Scorpaena plumieri fish through shotgun proteomic approach. It was shown that the toxic effects observed upon envenomation are elicited by molecules originated from the venomous gland. These results add to the growing catalogue of scorpaeniform fish venoms and their skin mucus proteins – so scarcely explored when compared to the venoms and bioactive components of terrestrial animals. Data are available via ProteomeXchange with identifier PXD009983

PhTx2 is the most toxic fraction from the venom of the spider Phoneutria nigriventer, being responsible to sodium entry into cortical synaptosomes, increasing the release of neurotransmitters, such as l -glutamate (L-Glu) and; acetylcholine. In this study, we investigated the action of a toxin purified from; PhTx2 fraction, called PnTx2-6 or δ-CNTX-Pn2a, on L-Glu release from rat; brain cortex synaptosomes, as well as its ability to induce blood-brain barrier permeability. PnTx2-6 increased L-Glu release from rat cortical brain synaptosomes in a time- and dose-dependent manner (EC50 = ∼20 nM; Tm = 16min), as measured by a fluorimetric method. The increase of L-Glu by PnTx2-6 was inhibited by tetrodotoxin. And partially inhibited by EGTA. Calcium channel blockers ω-conotoxin MVIIC (P/Q-types) and ω-conotoxin GVIA (N-type), were able to reduce the PnTx2-6-induced release of L-Glu, while nifedipine (L-type) did not show any inhibition. These findings suggest that thew release of L-Glu by PnTx2-6 is due its primary action on sodium channels, well-known to be target of this toxin. PnTx2-6 is able to potentiate penile erection and this effect may be related with the release of l -glutamate from the CNS, besides a local effect on corpus carvenosum, as previously shown by our group. If L-Glu release and penile erection potentiation are indeed correlated, then this toxin should be able to cross the blood brain barrier (BBB). Results by immunoblotting assays indicated a change in the expression of proteins associated with the paracellular and transcellular transport at the blood-brain barrier, suggesting a BBB dysfunction mediated by PnTx2-6. Therefore, PnTx2-6 may induce the release l -glutamate in the central nervous system, when injected peripherally.

Aims Extensive evidence supports a role for voltage-gated calcium channels (VGCC) and TRPV1 receptors in pain transmission and modulation. We investigated the profile of analgesic interaction between Phα1β toxin (a VGCC blocker) and SB366791 (selective TRPV1 antagonist) in a model of acute pain induced by capsaicin. Changes in body temperature induced by combination regimens were also evaluated. Main methods Isobolographic approach with a fixed dose-ratio of combined drugs was used to determine whether antinociceptive interaction of Phα1β and SB366791 are subadditive, additive or synergic. Body temperature was obtained by thermal infrared imaging. Key findings Phα1β and SB366791 interact in a synergistic manner to cause antinociception. We found an interaction index (α) of 0.07 for Phα1β and SB366791 when these drugs were injected together intraplantarly, which indicates that in vivo interaction between these drugs is greater than additive interaction. Synergism also occurred when intraplantar SB366791 was administered simultaneously with intrathecal Phα1β (interaction index α = 0.06) suggesting a 15 fold rise in potency on the analgesic effect of these drugs when they are added together. It was observed no significant alterations in body temperature of animals treated with this combination regimen. Significance Our data reveal that Phα1β toxin potentiates in 15 fold the antinociceptive action of the TRPV1 blocker SB366791. Therefore, lower doses of these drugs are required to achieve antinociceptive effects when these agents are given in combination.

The native Phα1β – a Voltage-Gated Calcium Channel (VGCC) blocker – and its Recombinant Version – were both tested in rodent pain models with an intraplantar injections of capsaicin or formalin, a chronic constriction injury, and melanoma cancer related pain. The formalin nociceptive behaviour in the neurogenic phase was not affected by the toxin pre-treatments, while in the inflammatory phase, Phα1β and the Recombinant form caused a significant reduction. The nociception that was triggered by capsaicin, an agonist of the TRPV1 vanilloid receptor, was totally blocked by 100 pmol/site, i.t. of Phα1β or the recombinant version. For the neuropathic pain that was induced by a chronic constriction injury of the sciatic nerve, Phα1β and its Recombinant reduced the allodynia that was induced by the CCI procedure in the rats and the hypersensitivity lasted for 4 h. Fourteen days after the inoculation of the B16-F10 melanoma cells in the mice, a marked hyperalgesia was induced in the melanoma cancer pain model. Phα1β and the Recombinant form reduced the hyperalgesia with a full reversion at 100 pmol/site i.t. The inhibitory effects of the nociception that was induced by native Phα1β and the Recombinant in the studied pain models were not statistically different and they developed with no side effects.

Current chemical therapies for Chagas Disease (CD) lack ability to clear Trypanosoma cruzi (Tc) parasites and cause severe side effects, making search for new strategies extremely necessary. We evaluated the action of Tityus serrulatus venom (TsV) components during Tc infection. TsV treatment increased nitric oxide and pro-inflammatory cytokine production by Tc-infected macrophages (MO), decreased intracellular parasite replication and trypomastigotes release, also triggering ERK1/2, JNK1/2 and p38 activation. Ts7 demonstrated the highest anti-Tc activity, inducing high levels of TNF and IL-6 in infected MO. TsV/Ts7 presented synergistic effect on p38 activation when incubated with Tc antigen. KPP-treatment of MO also decreased trypomastigotes releasing, partially due to p38 activation. TsV/Ts7-pre-incubation of Tc demonstrated a direct effect on parasite decreasing MO-trypomastigotes releasing. In vivo KPP-treatment of Tc-infected mice resulted in decreased parasitemia. Summarizing, this study opens perspectives for new bioactive molecules as CD-therapeutic treatment, demonstrating the TsV/Ts7/KPP-trypanocidal and immunomodulatory activity during Tc infection.

The venom of the Brazilian armed spider Phoneutria nigriventer is a rich source of biologically active peptides that have potential as analgesic drugs. In this study, we investigated the analgesic and adverse effects of peptide 3-5 (Tx3-5), purified from P. nigriventer venom, in several mouse models of pain. Tx3-5 was administered by intrathecal injection to mice selected as models of postoperative (plantar incision), neuropathic (partial sciatic nerve ligation) and cancer-related pain (inoculation with melanoma cells) in animals that were either sensitive or tolerant to morphine. Intrathecal administration of Tx3-5 (3-300 fmol/site) in mice could either prevent or reverse postoperative nociception, with a 50 % inhibitory dose (ID50) of 16.6 (3.2-87.2) fmol/site and a maximum inhibition of 87 ± 10 % at a dose of 30 fmol/site. Its effect was prevented by the selective activator of L-type calcium channel Bay-K8644 (10 μg/site). Tx3-5 (30 fmol/site) also produced a partial antinociceptive effect in a neuropathic pain model (inhibition of 67 ± 10 %). Additionally, treatment with Tx3-5 (30 fmol/site) nearly abolished cancer-related nociception with similar efficacy in both morphine-sensitive and morphine-tolerant mice (96 ± 7 and 100 % inhibition, respectively). Notably, Tx3-5 did not produce visible adverse effects at doses that produced antinociception and presented a TD50 of 1125 (893-1418) fmol/site. Finally, Tx3-5 did not alter the normal mechanical or thermal sensitivity of the animals or cause immunogenicity. Our results suggest that Tx3-5 is a strong drug candidate for the treatment of painful conditions.

Capsaicin, an agonist of TRPV1, evokes intracellular [Ca2+] transients and glutamate release from perfused trigeminal ganglion. The spider toxin PnTx3-5, native or recombinant is more potent than the selective TRPV1 blocker SB-366791 with IC50 of 47 ± 0.18 nM, 45 ± 1.18 nM and 390 ± 5.1 nM in the same experimental conditions. PnTx3-5 is thus more potent than the selective TRPV1 blocker SB-366791. PnTx3-5 (40 nM) and SB-366791 (3 μM) also inhibited the capsaicin-induced increase in intracellular Ca2+ in HEK293 cells transfected with TRPV1 by 75 ± 16% and 84 ± 3.2%, respectively. In HEK293 cells transfected with TRPA1, cinnamaldehyde (30 μM) generated an increase in intracellular Ca2+ that was blocked by the TRPA1 antagonist HC-030031 (10 μM, 89% inhibition), but not by PnTx3-5 (40 nM), indicating selectivity of the toxin for TRPV1. In whole-cell patch-clamp experiments on HEK293 cells transfected with TRPV1, capsaicin (10 μM) generated inward currents that were blocked by SB-366791 and by both native and recombinant PnTx3-5 by 47 ± 1.4%; 54 ± 7.8% and 56 ± 9.0%, respectively. Intradermal injection of capsaicin into the rat left vibrissa induced nociceptive behavior that was blocked by pre-injection with either SB-366791 (3 nmol/site i.d., 83.3 ± 7.2% inhibition) or PnTx3-5 (100 fmol/site, 89 ± 8.4% inhibition). We conclude that both native and recombinant PnTx3-5 are potent TRPV1 receptor antagonists with antinociceptive action on pain behavior evoked by capsaicin.

Several snake species possess endogenous phospholipase A2 inhibitors (sbPLIs) in their blood plasma, the primary role of which is protection against an eventual presence of toxic phospholipase A2 (PLA2) from their venom glands in the circulation. These inhibitors have an oligomeric structure of, at least, three subunits and have been categorized into three classes (α, β and γ) based on their structural features. SbγPLIs have been further subdivided into two subclasses according to their hetero or homomeric nature, respectively. Despite the considerable number of sbγPLIs described, their structures and mechanisms of action are still not fully understood. In the present study, we focused on the native structure of CNF, a homomeric sbγPLI from Crotalus durissus terrificus, the South American rattlesnake. Based on the results of different biochemical and biophysical experiments, we concluded that, while the native inhibitor occurs as a mixture of oligomers, tetrameric arrangement appears to be the predominant quaternary structure. The inhibitory activity of CNF is most likely associated with this oligomeric conformation. In addition, we suggest that the CNF tetramer has a spherical shape and that tyrosinyl residues could play an important role in the oligomerization. The carbohydrate moiety, which is present in most sbγPLIs, is not essential for the inhibitory activity, oligomerization or complex formation of the CNF with the target PLA2. A minor component, comprising no more than 16% of the sample, was identified in the CNF preparations. The amino-terminal sequence of that component is similar to the B subunits of the heteromeric sbγPLIs; however, the role played by such molecule in the functionality of the CNF, if any, remains to be determined.

In the present work, we describe the isolation and partial structural and biochemical characterization of the first phospholipase A 2 inhibitor (γPLI) from Crotalus durissus collilineatus ( Cdc ) snake serum. Initially, the Cdc serum was subjected to a Q-Sepharose ion exchange column, producing six peaks at 280 nm absorbance (Q1–Q6). Subsequently, Q4 fraction was submitted to affinity chromatography with immobilized PLA 2 BnSP-7, a step that resulted in two fractions (NHS-1 and NHS-2). The latter contained the inhibitor, denominated γCdcPLI. The molecular mass of γCdcPLI, determined by Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF), was 22,340 Da. Partial sequences obtained by Edman degradation and by mass spectrometry (MALDI-TOF/TOF), showed similarity, as expected, to other related inhibitors. Circular dichroism (CD) analysis showed the presence of approximately 22% alpha helices and 29% beta sheets in the protein secondary structure. Additionally, CD studies also indicated no significant changes in the secondary structure of γCdcPLI when it is complexed to BpPLA 2 -TXI. On the other hand, dynamic light scattering (DLS) assays showed a temperature-dependent oligomerization behavior for this inhibitor. Biochemical analyses showed γCdcPLI was able to inhibit the enzymatic, cytotoxic and myotoxic activities of PLA 2 s. Structural and functional studies performed on this inhibitor may elucidate the action mechanisms of PLA 2 inhibitors. In addition, we hope this study may contribute to investigating the potential use of these inhibitors for the treatment of snakebite or inflammatory diseases in which PLA 2 s may be involved.

In this work, we describe the molecular cloning and pharmacological properties of an acidic phospholipase A(2) (PLA(2)) isolated from Bothrops pauloensis snake venom. This enzyme, denominated BpPLA(2)-TXI, was purified by four chromatographic steps and represents 2.4% of the total snake venom protein content. BpPLA(2)-TXI is a monomeric protein with a molecular mass of 13.6 kDa, as demonstrated by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) analysis and its theoretical isoelectric point was 4.98. BpPLA(2)-TXI was catalytically active and showed some pharmacological effects such as inhibition of platelet aggregation induced by collagen or ADP and also induced edema and myotoxicity. BpPLA(2)-TXI displayed low cytotoxicity on TG-180 (CCRF S 180 II) and Ovarian Carcinoma (OVCAR-3), whereas no cytotoxicity was found in regard to MEF (Mouse Embryonic Fibroblast) and Sarcoma 180 (TIB-66). The N-terminal sequence of forty-eight amino acid residues was determined by Edman degradation. In addition, the complete primary structure of 122 amino acids was deduced by cDNA from the total RNA of the venom gland using specific primers, and it was significantly similar to other acidic D49 PLA(2)s. The phylogenetic analyses showed that BpPLA(2)-TXI forms a group with other acidic D49 PLA(2)s from the gender Bothrops, which are characterized by a catalytic activity associated with anti-platelet effects.

The use of natural substances for the treatment of diseases or injuries is an ancient practice of many cultures. According to folklore, natural aphrodisiacs may help to raise libido and increase desire. The supposed aphrodisiacs mainly include a plethora of preparations of plants, among other substances. However, the real boundary between myth and reality has not been established yet in most cases and such boundaries must be drawn by scientific methods. A growing interest of the scientific community has been focused on animal venoms, especially those from arthropods, i.e. spiders and scorpions, which cause priapism, a prolonged and painful erection. This review highlights the studies that have been performed with venoms and toxins from arthropods known to cause priapism, among other toxic symptoms, pointing out some pharmacological approaches for better understanding this effect. To date, the venom of some spiders, mainly Phoneutria nigriventer , and scorpions, such as the yellow South American scorpion Tityus serrulatus , among others, have been known to cause priapism. Since erectile dysfunction (ED) is a growing health problem in the world, more common in patients with vascular diseases as diabetes and hypertension, the use of animal venoms and toxins as pharmacological tools could not only shed light to the mechanisms involved in erectile function, but also represent a possible model for new drugs to treat ED. Unfortunately, attempts to correlate the structure of those priapism-related toxins were unfruitful. Such difficulties lie firstly on the poor data concerning purified priapism-related toxins, instead of whole venoms and/or semi-purified fractions, and secondly, on the scarce available primary sequences and structural data, mainly from spider toxins. It has been shown that all these toxins modify the sodium (Na + ) channel activity, mostly slowing down its inactivation current. Improving the knowledge on the tertiary structure of these toxins could provide a key in the search of a new drug for ED treatment.

In the present work, we report the isolation and partial biochemical characterization of BpLec, a C-type lectin purified from Bothrops pauloensis venom by one chromatographic step on an affinity agarose column immobilized with d-galactose. This protein was homogeneous by SDS-PAGE under reducing and nonreducing conditions, and was shown to be a 33.6 kDa homodimer by MALDI TOF analysis. BpLec presented an isoeletric point of 5.36. Its partial sequence of 132 amino acids for each subunit, determined by Edman degradation, revealed high identity (between 86% and 95%) when aligned with sequences of other related proteins. BpLec was capable of agglutinating native dog and cat erythrocytes and this activity was inhibited by β-galactosides and EDTA. Its hemagglutinating activity was abolished at high temperatures and stable in any pH range. BpLec was effective in inhibiting Gram-positive but not Gram-negative bacteria. In addition, BpLec agglutinated promastigote forms of Leishmania (Leishmania) amazonensis.

Voltage-sensitive calcium channels (VSCCs) underlie cell excitability and are involved in the mechanisms that generate and maintain neuropathic and inflammatory pain. We evaluated in rats the effects of two VSCC blockers, ω-conotoxin MVIIA and Phα1β, in models of inflammatory and neuropathic pain induced with complete Freund's adjuvant (CFA) and chronic constrictive injury (CCI), respectively. We also evaluated the effects of the toxins on capsaicin-induced Ca(2+) influx in dorsal root ganglion (DRG) neurons obtained from rats exposed to both models of pain. A single intrathecal injection of Phα1β reversibly inhibits CFA and CCI-induced mechanical hyperalgesia longer than a single injection of ω-conotoxin MVIIA. Phα1β and MVIIA also inhibited capsaicin-induced Ca(2+) influx in DRG neurons. The inhibitory effect of Phα1β on capsaicin-induced calcium transients in DRG neurons was greater in the CFA model of pain, while the inhibitory effect of ω-conotoxin MVIIA was greater in the CCI model. The management of chronic inflammatory and neuropathic pain is still a major challenge for clinicians. Phα1β, a reversible inhibitor of VSCCs with a preference for N-type Ca(2+) channels, has potential as a novel therapeutic agent for inflammatory and neuropathic pain. Clinical studies are necessary to establish the role of Phα1β in the treatment of chronic pain.

Erectile dysfunction (ED) mechanisms in diabetic patients are multifactorial and often lead to resistance to current therapy. Animal toxins have been used as pharmacological tools to study penile erection. Human accidents involving the venom of Phoneutria nigriventer spider are characterized by priapism. We hypothesize that PnTx2-6 potentiates cavernosal relaxation in diabetic mice by increasing cyclic guanosine monophosphate (cGMP). This effect is neuronal nitric oxide synthase (nNOS) dependent. Cavernosal strips were contracted with phenylephrine (10(-5) M) and relaxed by electrical field stimulation (20 V, 1-32 Hz) in the presence or absence of PnTx2-6 (10(-8) M). Cavernosal strips from nNOS- and endothelial nitric oxide synthase (eNOS)-knockout (KO) mice, besides nNOS inhibitor (10(-5) M), were used to evaluate the role of this enzyme in the potentiation effect evoked by PnTx2-6. Tissue cGMP levels were determined after stimulation with PnTx2-6 in presence or absence of N-nitro-L-arginine methyl ester (L-NAME) (10(-4) M) and ω-conotoxin GVIA (10(-6) M), an N-type calcium channel inhibitor. Results showed that PnTx2-6 enhanced cavernosal relaxation in diabetic mice (65%) and eNOS KO mice, but not in nNOS KO mice. The toxin effect in the cavernosal relaxation was abolished by nNOS inhibitor. cGMP levels are increased by PnTx2-6, however, L-NAME abolished this enhancement as well as ω-conotoxin GVIA. We conclude that PnTx2-6 facilitates penile relaxation in diabetic mice through a mechanism dependent on nNOS, probably via increasing nitric oxide/cGMP production.

MALDI-TOF-TOF and de novo sequencing were employed to assess the Tityus serrulatus venom peptide diversity. Previous works has shown the cornucopia of molecular masses, ranging from 800 to 3000Da, present in the venom from this and other scorpions species. This work reports the identification/sequencing of several of these peptides. The majority of the peptides found were fragments of larger venom toxins. For instance, 28 peptides could be identified as fragments from Pape proteins, 10 peptides corresponded to N-terminal fragments of the TsK beta (scorpine-like) toxin and fragments of potassium channel toxins (other than the k-beta) were sequenced as well. N-terminal fragments from the T. serrulatus hypotensins-I and II and a novel hypotensin-like peptide could also be found. This work also reports the sequencing of novel peptides without sequence similarities to other known molecules.

The objective of this study was to verify the influence of the growth hormone (GH) and halothane (Hal) genotypes, breed and sex on carcass and meat quality. Ninety-six pigs (Pietrain, Large White and Landrace) were dissected. For each breed, 16 were noncastrated males and 16 were females. The Pietrain animals presented the greatest lean meat percentage, the heaviest ham and shoulder and the lightest belly weight and fat thickness. Landrace presented the heaviest belly. Females presented the greatest drip loss; L*, b* and fat thickness; the heaviest ham and loin; and the lightest shoulder and tenderloin. Hal–NN and Nn presented greater loin weight than nn animals. Nn animals presented smaller belly weight than homozygotes. The nn animals presented greater drip loss than NN and Nn, and greater lean meat than NN. Interestingly, GH–D2D2 animals also showed higher drip loss than those with D1D1 genotype. This investigation presents relevant associations between important economic traits and specific GH and Hal genotypes. PRACTICAL APPLICATIONS The identification of new molecular markers related to pig production has been increasing in importance, especially when these markers are related to carcass and meat quality traits, which are difficult to predict. When a marker is intensively used in a selection program, it reaches fixation, so it is important to identify new markers to replace it. This work shows, for the first time, the association of a GH polymorphism with an important meat quality trait and reinforces the possibility of its use in programs of marker-assisted selection.

We designed a peptide, PnPP-19, comprising the potential active core of the Phoneutria nigriventer native toxin PnTx2-6. We investigated its role on erectile function, and its toxicity and immunogenicity.Erectile function was evaluated by the intracavernous pressure-to-mean arterial pressure ratio during electrical field stimulation on rat pelvic ganglia. Cavernous strips were contracted with phenylephrine and relaxation was induced by electrical field stimulation with or without PnPP-19 (10(-8) M). Activity on sodium channels was evaluated by electrophysiological screening of transfected channels on Xenopus oocytes and dorsal root ganglion cells. Antibodies were detected by indirect enzyme-linked immunosorbent assay in mice previously treated with the peptide. Histopathological studies were performed with mouse organs treated with different doses of PnPP-19.PnPP-19 was able to potentiate erection at 4 and 8 Hz in vivo and ex vivo. It showed no toxicity and low immunogenicity in mice, and did not affect sodium channels or rat hearts. PnPP-19 increased cyclic guanosine monophosphate levels at 8 Hz. This effect was inhibited by L-NAME (10(-4) M). Erectile function was partially inhibited by 7-nitroindazole (10(-5) M), a selective inhibitor of neuronal nitric oxide synthase.PnPP-19 potentiates erection in vivo and ex vivo via the nitric oxide/cyclic guanosine monophosphate pathway. It does not affect sodium channels or rat hearts and shows no toxicity and low immunogenicity. These findings make it a promising candidate as a novel drug in the therapy of erectile dysfunction.

Toxins and venoms produced by living organisms have exhibited a variety of biological activities against microorganisms. In this study, we tested seven snake venoms from the family Viperidae for antibacterial activity and the activities of reversal of antibiotic resistance and inhibition of biofilm formation against 22 clinical isolates of Staphylococcus aureus. Bothrops moojeni venom exhibited anti staphylococcal activity with the lowest mean value of minimum inhibitory concentration (MIC). Moreover, reversal of antibiotic resistance was observed for combinations of B. moojeni venom (½ x MIC) and norfloxacin or ampicillin (both ½ x MIC) for 86.4% and 50% of the isolates, respectively. B. moojeni venom alone at ½ MIC inhibited 90% of biofilm formation, whereas in combination with ciprofloxacin, both at ½ MIC, a reduction on the NorA efflux pump activity was observed. The detection of in vitro mutants colonies of S. aureus resistant to B. moojeni venom was low and they did not survive. A phospholipase A2 was purified from the venom of B. moojeni and displayed anti-staphylococcal activity when tested alone or in combination with ciprofloxacin. The results presented here will contribute to the search for new antimicrobial agents against resistant S. aureus.

In the present work, we demonstrate some biochemical and functional properties of a new PI snake venom metalloproteinase (SVMP) isolated from Bothrops pauloensis snake venom (BpMP-II), in addition we evaluated its capacity to inhibit endothelial cell adhesion and in vitro angiogenesis. BpMP-II was purified after a combination of three chromatography steps and showed molecular mass of 23,000 Da determined by MALDI-TOF, an isoelectric point of 6.1 and the sequence of some fragments obtained by MS/MS (MALDI TOF\TOF) presented high structural similarity with other PI-SVMPs. BpMP-II showed proteolytic activity against azocasein, was able to degrade bovine fibrinogen and was inhibited by EDTA, 1.10 phenantroline and β-mercaptoethanol. BpMP-II did not induce local hemorrhage in the dorsal region of mice even at high doses and did not affect plasma creatine kinase (CK) levels when administered intramuscularly into the gastrocnemius muscle of mice. Moreover, this metalloproteinase decreased tEnd cells viability at concentrations higher than 20 μg/mL. With sub-toxic doses this metalloproteinase affected tEnd cell adhesion and was also able to inhibit in vitro angiogenesis. BpMP-II showed very important functional properties suggesting considerable therapeutic potential for this class of protein.

Aqueous extract from Casearia sylvestris leaves, a typical plant from Brazilian open pastures, was able to neutralize the hemorrhagic activity caused by Bothrops asper, Bothrops jararacussu, Bothrops moojeni, Bothrops neuwiedi and Bothrops pirajai venoms. It also neutralized two hemorrhagic metalloproteinases from Bothrops asper venom. Proteolytic activity on casein induced by bothropic venoms and by isolated proteases, including Bn2 metalloproteinase from B. neuwiedi venom, was also inhibited by the C. sylvestris extract in different levels. The α-fibrinogen chain was partially protected against degradation caused by B. jararacussu venom, when this venom was incubated with C. sylvestris extract. We also observed that this extract partially increased the time of plasma coagulation caused by B. jararacussu, B. moojeni and B. neuwiedi venoms. C. sylvestris extract did not induce proteolysis in any substrate assayed.

The crude aqueous extract from the leaves of Casearia sylvestris, a plant found in Brazilian open pastures, was assayed for its ability to inhibit phospholipase A2 (PLA2) activity and some biological activities of bee and several snake venoms, and of a number of isolated PLA2s. The extract induced partial inhibition of the PLA2 activity of venoms containing class I, II and III PLA2s. When tested against the purified toxins, it showed the highest efficacy against class II PLA2s from viperid venoms, being relatively ineffective against the class I PLA2 pseudexin. In addition, C. sylvestris extract significantly inhibited the myotoxic activity of four Bothrops crude venoms and nine purified myotoxic PLA2s, including Lys-49 and Asp-49 variants. The extract was able to inhibit the anticoagulant activity of several isolated PLA2s, with the exception of pseudexin. Moreover, it partially reduced the edema-inducing activity of B. moojeni and B. jararacussu venoms, as well as of myotoxins MjTX-II and BthTX-I. The extract also prolonged the survival time of mice injected with lethal doses of several snake venoms and neutralized the lethal effect induced by several purified PLA2 myotoxins. It is concluded that C. sylvestris constitutes a rich source of PLA2 inhibitors.

A basic serine protease which is active on casein and fibrinogen was purified from Bothrops moojeni venom using a single step chromatography on a CM-Sepharose fast flow column. The enzyme, MOO3, was not hemorrhagic and presented only a trace of blood-clotting activity. Synthetic chromogenic substrates (azoacasein and azoalbumin) where not hydrolyzed by MOO3. Using polyacrylamide gel electrophoresis at pH 4.3, MOO3 showed as a single protein band. Using sodium dodecyl sulfate-polyacrylamide electrophoresis, MOO3 behaved as a single-chain protein with an approximate mol. weight of 27,000, both in the presence and absence of beta-mercaptoethanol. Its pI was 7.8 by electrofocusing. The enzyme did not contain neutral carbohydrates and its N-terminal amino acid was alanine. The amino acid composition showed 249 residues/mole, a high content of hydrophilic amino acids and 14 half-cystine residues, which should account for 7 disulfide bonds. The protease cleaved the A-alpha chain faster than the B-beta of bovine fibrinogen and showed no effect on the delta-chain. Specific esterolytic activity of MOO3 on alpha-N-tosyl-l-arginine methyl ester was 29.64 mumol min-1 x mg-1. MOO3 represented 1.42% (w/w) of the initial desiccated venom. Its proteolytic activity was inhibited by beta-mercaptoethanol, leupeptin, phenylmethylsulphonyl fluoride and ethylenediamine tetraacetate.

The antibothropic complex (ABC) from opossum (species Didelphis albiventris) serum was purified by chromatography on DEAE-Sephacel. It showed an acidic character and two polypeptide chains of ca. 45 kDa and 48 kDa, respectively. Lyophilized opossum serum or the ABC (100 micrograms), as well as ethylenediamine tetraacetate (0.25 mumoles) were able to completely neutralise the hemorrhagic effect of 50 micrograms of the desiccated venoms of Bothrops moojeni, Bothrops pirajai and Bothrops jararacussu. The myotoxic (100 micrograms venom in mice) and edematogenic (90 micrograms venom in rats) activities of Bothrops moojeni and Bothrops jararacussu venoms, as well as of the major myonecrotic protein (myotoxin-I) isolated from Bothrops moojeni venom, were also totally inhibited by the ABC (200 micrograms and 270 micrograms, respectively). The lyophilized opossum serum (30 micrograms) and the ABC (30 micrograms) reduced to 50% the phospholipase A2 activity of Bothrops moojeni venom (10 micrograms). The clotting activity of Bothrops alternatus and Bothrops moojeni (20 micrograms) on bovine plasma was also significantly inhibited by the ABC (60 micrograms).

We report the comparative proteomic characterization of the venoms of Bothrops atrox, B. barnetti and B. pictus. The venoms were subjected to RP-HPLC and the resulting fractions analyzed by SDS-PAGE. The proteins were cut from the gels, digested with trypsin and identified via peptide mass fingerprint and manual sequencing of selected peptides by MALDI-TOF/TOF mass spectrometry. Around 20-25 proteins were identified belonging to only 6-7 protein families. Metalloproteinases of the classes P-I and P-III were the most abundant proteins in all venoms (58-74% based on peak area A214 nm), followed by phospholipases-A(2) (6.4-14%), disintegrins (3.2-9%) and serine proteinases (7-11%), and some of these proteins occurred in several isoforms. In contrast cysteine-rich secretory proteins and L-amino acid oxidases appeared only as single isoforms and were found only in B. atrox and B. barnetti. C-type lectins were also detected in all venoms but at low levels (~ 5%). Furthermore, the venoms contain variable numbers of peptides (

Snake Venom Metalloproteinases (SVMPs) are the most abundant components present in Viperidae venom. They are important in the induction of systemic alterations and local tissue damage after envenomation. In the present study, a metalloproteinase named BpMPI was isolated from Bothropoides pauloensis snake venom and its biochemical and enzymatic characteristics were determined. BpMPI was purified in two chromatography steps on ion exchange CM-Sepharose Fast flow and Sephacryl S-300. This protease was homogeneous on SDS-PAGE and showed a single chain polypeptide of 20kDa under non reducing conditions. The partial amino acid sequence of the enzyme showed high similarity with other SVMPs enzymes from snake venoms. BpMPI showed proteolytic activity upon azocasein and bovine fibrinogen and was inhibited by EDTA, 1,10 phenanthroline and β-mercaptoethanol. Moreover, this enzyme showed stability at neutral and alkaline pH and it was inactivated at high temperatures. BpMPI was able to hydrolyze glandular and tissue kallikrein substrates, but was unable to act upon factor Xa and plasmin substrates. The enzyme did not induce local hemorrhage in the dorsal region of mice even at high doses. Taken together, our data showed that BpMP-I is in fact a fibrinogenolytic metalloproteinase and a non hemorrhagic enzyme.

The systematic investigation of the peptidic composition of the skin secretion of Phasmahyla jandaia, a phyllomedusine anuran endemic to the southern region of the Espinhaco range in Brazil, is herein reported. By means of de novo interpretation of tandem mass spectrometric data, Edman N-terminal sequencing and similarity searches, 57 peptides - including phylloseptins, dermaseptins stricto sensu, dermatoxins, hyposins, tryptophyllins, caerulein-related, bradykinin-related, bradykinin potentiating, tyrosine-rich, and opioid peptides - were sequenced. Moreover, five peptide families without significant similarity to other known molecules were verified. Differently from most Phyllomedusinae genera, the molecular diversity in the skin of representatives of Phasmahyla remained unprospected until now. Therefore, besides disclosing novel natural variants of number of bioactive peptides, the present study contributes to the understanding of the evolution of biochemical characters of the phyllomedusines.

We report the isolation and structure–function relationship of a 23 kDa metalloproteinase named atroxlysin-I from the venom of the Peruvian Bothrops atrox (Jergon). Atroxlysin is a P-I metalloproteinase and contains 204 residues. Its proteolytic activity towards dimethylcasein is enhanced by Ca +2 but inhibited by EDTA, dithiothreitol, excessive Zn +2 and α2-macroglobulin. Unlike other structurally homologous P-I metalloproteinases, atroxlysin-I causes hemorrhages. To examine its hemorrhagic activity mechanistically, we studied its function in vitro and in vivo . It cleaved the Ala 14 –Leu 15 and Tyr 16 –Leu 17 bonds in oxidized insulin B-chain and specifically hydrolyzed the α-chains of fibrin(ogen) in a dose- and time-dependent manner. Atroxlysin-I cleaved plasma fibronectin and other extracellular matrix proteins (collagens I and IV) and the triple-helical fragment CB3 of collagen IV, but did not degrade laminin-111. Complementarily, the laminin and collagen binding integrins α 7 β 1 and α 1 β 1 were cleaved by atroxlysin. Even without catalytic activity atroxlysin-I inhibited collagen- and ADP-triggered platelet aggregation.

A proteinase, named BmooMPalpha-I, from the venom of Bothrops moojeni, was purified by DEAE-Sephacel, Sephadex G-75 and heparin-agarose column chromatography. The enzyme was purified to homogeneity as judged by its migration profile in SDS-PAGE stained with coomassie blue, and showed a molecular mass of about 24.5 kDa. Its complete cDNA was obtained by RT-PCR and the 615 bp codified for a mature protein of 205 amino acid residues. The multiple alignment of its deduced amino acid sequence and those of other snake venom metalloproteinases showed a high structural similarly, mainly among class P-IB proteases. The enzyme cleaves the Aalpha-chain of fibrinogen first, followed by the Bbeta-chain, and shows no effects on the gamma-chain. On fibrin, the enzyme hydrolyzed only the beta-chain, leaving the gamma-dimer apparently untouched. It was devoid of phospholipase A(2), hemorrhagic and thrombin-like activities. Like many venom enzymes, it is stable at pH values between 4 and 10 and stable at 70 degrees C for 15 min. The inhibitory effects of EDTA on the fibrinogenolytic activity suggest that BmooMPalpha-I is a metalloproteinase and inhibition by beta-mercaptoethanol revealed the important role of the disulfide bonds in the stabilization of the native structure. Aprotinin and benzamidine, specific serine proteinase inhibitors, had no effect on BmooMPalpha-I activity. Since the BmooMPalpha-I enzyme was found to cause defibrinogenation when administered i.p. on mice, it is expected that it may be of medical interest as a therapeutic agent in the treatment and prevention of arterial thrombosis.

Spectrophotometric profiles representing the unfolding induced by guanidine on Bothrops moojeni myotoxins-I (MjTX-I) and II (MjTX-II), Bothrops jararacussu bothropstoxin-I (BthTX-I) and Bothrops pirajai piratoxin-I (PrTX-I) were obtained and compared with those obtained with bovine ribonuclease A (RNAse) and trypsin. The molar (e1M) and percent (e1%) extinction coefficients were determined for the four myotoxins as well as for RNAse and trypsin as reference parameters. These coefficients were then used throughout this work. The changes in free energy (ΔGD H20) corresponding to zero guanidine concentration and the guanidine concentrations (Δ1 / 2) able to convert 50% of the molecules from the native to the unfolded state were determined. The values of ΔGD H20 ranged from 4.42 (BthTX-I) to 8.02 (MjTX-I) kcal / mole, compared with 6.47 and 6.88 kcal / mole for trypsin and RNAse, respectively. The values for ΔGD H20 and Δ1 / 2 showed that BthTX-I is the least stable among the four myotoxins assayed, with a Δ1 / 2 close to that of RNAse, while MjTX-II is conformationally the most stable. Monitoring of the unfolding of RNAse and PrTX-I by a 0 to 6 M urea gradient PAGE revealed transitions from the native (N) to the unfolded (U) state with ΔGN-U of 0.22 and 0.41 kcal / mole, respectively. Sigmoidal curves showed welldefined two-stage transitions for both proteins. Abbreviation Used: PLA2, phospholipase A2; BthTX-I, Bothrops jararacussu bothropstoxin I, PrTX-I, Bothrops pirajai piratoxin I, MjTX-I and -II, Bothrops moojeni myotoxins I and II; GuHCl, guanidine hydrochloride; ATEE, N-acetyl-l-tyrosine ethyl ester; ATrEE, N-acetyl-l-tryptophan ethyl ester, RNAse, bovine ribonuclease A, RC-RNAse, reduced and carboxymethylated RNAse.