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Enzymatic inhibition by natural compounds may represent a valuable adjuvant in snakebite serum therapy. The objective in this work was to evaluate possible in vitro interactions between vanillic acid and enzymes from Bothrops spp. and Crotalus durissus terrificus venoms, and also suggest a theory as how they interact based on molecular docking. Vanillic acid inhibited the phospholipase activity induced by Bothrops alternatus (∼25% inhibition); the caseinolytic activity induced by Bothrops atrox (∼30%), Bothrops jararacussu (∼44%), and C. d. terrificus (∼33%); the fibrinogenolysis induced by B. jararacussu, B. atrox, and C. d. terrificus (100%); the serine protease activity induced by Bothrops moojeni (∼45%) and Bothrops jararaca (∼66%); the hemolytic activity induced by B. moojeni (∼26%); the thrombolysis activity induced by B. atrox (∼30%) and B. jararacussu (∼20%); and the thrombotic activity induced by C. d. terrificus (∼8%). The compound was also capable of delaying the coagulation time in citrated plasma by 60, 35, and 75 Sec, when incubated with B. moojeni, B. atrox, and B. jararaca, respectively. The results obtained expand the possibilities for future pharmaceutical use of vanillic acid, considering the high homology degree among human and snake venom phospholipases A2 and proteases (involved in chronic inflammatory diseases). Also, this compound can be used as adjuvant to improve currently available treatments for ophidism victims.

Efficient drug delivery systems are currently one of the greatest challenges in pharmacokinetics, and the transposition of the gap between in vitro candidate molecule and in vivo test drug is, sometimes, poles apart. In this sense, the cell-penetrating peptides (CPP) may be the bridge uniting these worlds. Here, we describe a technique to rapidly identify unlabeled CPPs after incubation with liposomes, based on commercial desalting (size exclusion) columns and liquid chromatography-MS/MS, for peptide de novo sequencing. Using this approach, we found it possible to identify one new CPP - interestingly, a classical bradykinin-potentiating peptide - in the peptide-rich low molecular mass fraction of the Bothrops jararaca venom, which was also able to penetrate live cell membranes, as confirmed by classical approaches employing fluorescence-labeled analogues of this CPP. Moreover, both the labeled and unlabeled CPPs caused no metabolic, cell-cycle or morphologic alterations, proving to be unmistakably cargo deliverers and not drugs themselves. In sum, we have developed and validated a method for screening label-free peptides for CPP activity, regardless of their biological origin, which could lead to the identification of new and more efficient drug delivery systems. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Background The reptilian immune system is represented by innate, humoral, and cell-mediated mechanisms, involving different types of blood leukocytes. The development of optimized methods for the advanced study of origin and function of reptilian blood leukocytes is needed. Objectives The purpose of the study was to optimize leukocyte density gradient isolation protocols from snake peripheral blood samples, and characterize recovered cells by flow cytometry based on size and internal complexity for a qualitative and semi-quantitative assessment of leukocyte populations in one boa (Boa constrictor), and 2 viper species (Bothrops jararaca, Crotalus durissus). Methods Blood samples from 30 snakes (10 from each species, 5 males and 5 females) were collected in tubes with sodium heparin. Fresh blood was centrifuged with either ficoll-paque PLUS or percoll density gradients for leukocyte isolation. Flow cytometric leukocyte gates were defined based on size (forward scatter [FSC]) and internal complexity (side scatter [SSC]). Relative leukocyte differential counts after sorting the cells in these gates in one snake for each species were compared to conventional light microscopic differential counts on unsorted isolated leukocytes. Results There was no statistical difference in the relative leukocyte populations, including heterophils, azurophils, and small and large lymphocytes between samples isolated by ficoll or percoll. Four leukocyte gates were identified based on their location in FSC/SSC cytograms. The relative leukocyte differential counts after sorting in single animals showed some agreement with the light microscopy differential count on unsorted cells. Conclusions Based on FSC and SSC, 4 distinct leukocyte populations were found in ficoll or percoll density gradient isolated leukocytes from peripheral blood from boa and viper species. Further optimization of the technique should allow the performance of functional assays.

Variation in the snake venom proteome is well documented and it is a ubiquitous phenomenon at all taxonomical levels. However, variation in the snake venom peptidome is so far not described. In this work we used mass spectrometry [liquid chromatography/mass spectrometry (LC/MS) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOFMS)] to explore sex-based differences among the venom peptides of eighteen sibling specimens of Bothrops jararaca of a single litter born and raised in the laboratory. MALDI-TOFMS analyses showed individual variability among the bradykinin-potentiating peptides (BPPs), and, interestingly, four new peptides were detected only in female venoms and identified by de novo sequencing as cleaved BPPs lacking the C-terminal Q-I-P-P sequence. Similar results were obtained with venom from wild-caught adult non-sibling specimens of B. jararaca and in this case we were able to identify the gender of the specimen by analyzing the MALDI-TOF profile of the peptide fraction and finding the cleaved peptides only in female venoms. Synthetic replicates of the cleaved BPPs were less potent than the full-length BPP-10c in potentiating the bradykinin hypotensive effect, suggesting that the C-terminus is critical for the interaction of the BPPs with their mammalian molecular targets. This work represents a comprehensive mass spectrometric analysis of the peptide fraction of B. jararaca venom and shows for the first time sex-based differences in the snake venom peptidome of sibling and non-sibling snakes and suggests that the BPPs may follow distinct processing pathways in female and male individuals. Copyright © 2007 John Wiley & Sons, Ltd.

The Brazilian Atlantic Forest is one of the world's major biodiversity hotspots and is threatened by a severe habitat loss. Yet little is known about the processes that originated its remarkable richness of endemic species. Here we present results of a large-scale survey of the genetic variation at the mitochondrial cytochrome b gene of the pitviper, jararaca lancehead (Bothrops jararaca), and two closely related insular species (Bothrops insularis and Bothrops alcatraz), endemic of this region. Phylogenetic and network analyses revealed the existence of two well-supported clades, exhibiting a southern and a northern distribution. The divergence time of these two phylogroups was estimated at 3.8 million years ago, in the Pliocene, a period of intense climatic changes and frequent fragmentation of the tropical rainforest. Our data also suggest that the two groups underwent a large size expansion between 50,000 and 100,000 years ago. However, the southern group showed a more marked signal of population size fluctuation than the northern group, corroborating evidences that southern forests may have suffered a more pronounced reduction in area in the late Pleistocene. The insular species B. alcatraz and B. insularis presented very low diversity, each one sharing haplotypes with mainland individuals placed in different subclades. Despite their marked morphological and behavioural uniqueness, these two insular species seem to have originated very recently and most likely from distinct costal B. jararaca populations, possibly associated with late Pleistocene or Holocene sea level fluctuations.

Envenomation by the snake Bothrops jararaca is typically associated with hemostatic abnormalities including pro- and anticoagulant disturbances. Glycyrrhizin (GL) is a plant-derived thrombin inhibitor that also exhibits in vivo antithrombotic properties. Here, we evaluated the ability of GL to counteract the hemostatic abnormalities promoted by B. jararaca venom. GL inhibited the human fibrinogen clotting (IC50=∼1.0 mg ml−1; 1.2 mM), H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroanilide dihydrochloride hydrolysis (IC50=∼0.4 mg ml−1; 0.47 mM) and platelet aggregation (IC50=∼0.28 mg ml−1; 0.33 mM) induced by B. jararaca venom, in vitro. The in vivo effect of GL was tested in rats using a model of venous thrombosis in which intravenous (i.v.) administration of B. jararaca venom (100 μg kg−1) produced in all animals a thrombus with a mean weight of 10.6±1.7 mg. Prior administration of GL (180 mg kg−1) or antibothropic serum (27 μl kg−1) inhibited thrombus formation by 86 and 67%, respectively. Remarkably, co-administration of ineffective doses of GL and antibothropic serum markedly decreased thrombus weight, suggesting a synergistic effect. Co-administration of GL with antibothropic serum abolished venom-induced bleeding. Ex vivo clotting times showed that rat plasma was non-clotting after i.v. administration of B. jararaca venom. Treatment with GL, antibothropic serum or both before venom administration efficiently prevented this abnormality. Altogether, we demonstrate here that GL prevents both in vitro and in vivo venom-induced changes in hemostasis, suggesting a potential antiophidic activity. Keywords: Glycyrrhizin, B. jararaca venom, venous thrombosis, antibothropic serum Introduction In Brazil, snakebite accidents represent an important public health problem, with the genus Bothrops (including the species B. jararaca, B. moojeni, B. erythromelas and B. atrox) being responsible for more than 90% of the registered cases (Cardoso, 1990). A number of proteins from bothropic venoms interfere with the hemostatic system and have been characterized in detail (Markland, 1998; Castro et al., 2004) as procoagulant, anticoagulant or fibrinolytic factors (Marsh, 1994). In addition, several components may alter platelet function displaying either pro- or antiaggregating properties (Markland, 1998). The signs and symptoms presented by patients include local (pain, swelling, ecchymosis and necrosis) and systemic (blood incoagulability, hemorrhage) manifestations (Kamiguti et al., 1986; Maruyama et al., 1990). Envenomation by these snakes generally results in strong coagulopathy with persistent bleeding owing to fibrinogen degradation as well as consumption of blood coagulation factors (Maruyama et al., 1990; Kamiguti et al., 1991). On the other hand, massive blood clotting activation may cause thrombosis in small vessels (Thomas et al., 1995). Therefore, severe cases of envenomotion may lead to permanent tissue loss, disability or amputation. The effective therapeutic treatment for ophidian accidents nowadays is serotherapy (Heard et al., 1999). Nevertheless, alternative medication has been proposed. The use of heparin was first proposed in the late 40s (Ahuja et al., 1946). Although it is a well-known anticoagulant, heparin does not neutralize the thrombin-like activity of bothropic venoms and does not prevent the defibrinogenating syndrome induced by these venoms (Nahas et al., 1975). In fact, structural differences between thrombin and venom-derived thrombin-like enzymes impair the inhibitory action of heparin–antithrombin complex as well as other coagulation inhibitors such as hirudin (Castro et al., 2004). A number of studies have also reported the use of plants (or their extracts) known in popular medicine for treatment of snakebite (Mors et al., 1989; 2000; Mors, 1991; Martz, 1992; Houghton & Osibogun, 1993; Houghton & Skari, 1994). Among these studies, some focus on the inhibition of snake venom hemostatic effects. Extracts from plants widely used in India, Sri Lanka and West Africa showed significant dose-related prolongation of Echis carinatus venom-induced clot formation in human plasma (Onuaguluchi & Okeke, 1989). It has also been shown that the extract of the Brazilian plant Marsypianthes chamaedrys inhibits fibrinogen clotting induced by several Brazilian snake venoms, indicating that it affects thrombin-like enzymes (Castro et al., 2003). Glycyrrhizin (GL) is a natural triterpenoid saponin extracted from the root of a Legumisosae, Glycyrrhiza glabra (licorice), with a molecular mass of 840 Da. This compound is known for its anti-inflammatory activity (Fogden & Neuberger, 2003) and has been also characterized as a thrombin inhibitor (Francischetti et al., 1997). Thrombin, which plays a central role in the hemostatic system, displays very high specificity upon a small range of substrates. This is particularly attributed to the presence of two positively charged regions that are located at a distance from its catalytic site, named ‘anion-binding exosites', which play major roles in the recognition of substrates and inhibitors (Stubbs & Bode, 1995). In fact, it was demonstrated that GL binds to thrombin exosite 1 and blocks the enzyme action upon fibrinogen and platelets (Francischetti et al., 1997), displaying a similar mechanism to that described for the C-terminal hirudin-based non-apeptide (Krstenansky & Mao, 1987). Further studies demonstrated that GL has in vivo antithrombotic properties (Mendes-Silva et al., 2003). Based on the known coagulopathy state generated by the envenomation, we attempted to evaluate the ability of GL to counteract this condition caused by B. jararaca venom. For this purpose, we injected crude venom intravenously in rats and measured thrombus formation, ex vivo clotting times and bleeding effect. GL was effective both in vitro and in vivo. Also, GL showed a synergistic effect when combined with antibothropic serum. Our data indicate that in vitro experiments combined with the animal model herein described would be useful to evaluate compounds with potential ability to reduce the venom-induced hemostatic abnormalities.

In order to study the role of neutrophils in the acute local pathological alterations induced by Bothrops jararaca snake venom, and in the process of skeletal muscle regeneration that follows, an experimental model was developed in mice pretreated with either an anti-mouse granulocyte rat monoclonal immunoglobulin G, which induces a profound neutropenia, or an isotype-matched control antibody. B. jararaca venom induced prominent haemorrhage and oedema, but only a moderate myonecrosis. No significant differences were observed in the extent of local haemorrhage, oedema and myonecrosis between neutropenic and control mice, suggesting that neutrophils do not play a determinant role in the acute pathological alterations induced by B. jararaca venom in this experimental model. Moreover, no differences were observed in skeletal muscle regeneration between these two experimental groups. In both the cases, limited areas of myonecrosis were associated with a drastic damage to the microvasculature and a scarce inflammatory infiltrate, with the consequent lack of removal of necrotic debris during the first week, resulting in a poor regenerative response at this time interval. Subsequently, a similar regenerative process occurred in both groups, and by 30 days, necrotic areas were substituted by groups of small regenerating muscle fibres. It is suggested that the drastic effect exerted by B. jararaca venom in the microvasculature precludes an effective access of inflammatory cells to necrotic areas, thereby compromising an effective removal of necrotic debris; this explains the poor regenerative response observed during the first week and the fact that there were no differences between neutropenic and control mice. As neutropenia in this model lasted only 7 days, the successful regenerative process observed at 30 days is associated with revascularization of necrotic regions and with a successful removal by phagocytes of necrotic debris in both groups.

Between June 1997 and May 1998, 47 pit vipers (Bothrops jararaca) (Group A) were euthanased when they were brought to the Instituto Butantan by farmers, and examined postmortem; during the same period, 91 snakes of the same species (group B) were examined after they had died in an outdoor serpentarium. The majority of the parasites encountered were nematodes; lungworms, Rhabdias vellardi, and the intestinal hookworm Kalicephalus inermis were the most common. Some of the snakes in group A were heavily infested, but their lesions were mild, whereas in group B the parasites were generally accompanied by severe lesions. The parasites with a direct life cycle were more common than those with obligatory intermediate hosts, and the snakes were more commonly infected during the hotter and more humid seasons.

The bradykinin-potentiating peptides from Bothrops jararaca venom are the most potent natural inhibitors of the angiotensin-converting enzyme. The biochemical and biological features of these peptides were crucial to demonstrate the pivotal role of the angiotensin-converting enzyme in blood pressure regulation. In the present study, seven bradykinin-potentiating peptides were identified within the C-type natriuretic peptide precursor cloned from snake brain. The bradykinin-potentiating peptides deduced from the B. jararaca brain precursor are strong in vitro inhibitors of the angiotensin-converting enzyme (nanomolar range), and also potentiate the bradykinin effects in ex vivo and in vivo experiments. Two of these peptides are novel bradykinin-potentiating peptides, one of which displays high specificity toward the N-domain active site of the somatic angiotensin-converting enzyme. In situ hybridization studies revealed the presence of the bradykinin-potentiating peptides precursor mRNAs in distinct regions of the B. jararaca brain, such as the ventromedial hypothalamus, the paraventricular nuclei, the paraventricular organ, and the subcommissural organ. The biochemical and pharmacological properties of the brain bradykinin-potentiating peptides, their presence within the neuroendocrine regulator C-type natriuretic peptide precursor, and their expression in regions of the snake brain correlated to neuroendocrine functions, strongly suggest that these peptides belong to a novel class of endogenous vasoactive peptides.

Bothrops snake venoms are known to induce local tissue damage such as hemorrhage and myonecrosis. The opossum Didelphis marsupialis is resistant to these snake venoms and has natural venom inhibitors in its plasma. The aim of this work was to clone and study the chemical, physicochemical and biological properties of DM64, an antimyotoxic protein from opossum serum. DM64 is an acidic protein showing 15% glycosylation and with a molecular mass of 63 659 Da when analysed by MALDI-TOF MS. It was cloned and the amino acid sequence was found to be homologous to DM43, a metalloproteinase inhibitor from D. marsupialis serum, and to human alpha1B-glycoprotein, indicating the presence of five immunoglobulin-like domains. DM64 neutralized both the in vivo myotoxicity and the in vitro cytotoxicity of myotoxins I (mt-I/Asp49) and II (mt-II/Lys49) from Bothrops asper venom. The inhibitor formed noncovalent complexes with both toxins, but did not inhibit the PLA2 activity of mt-I. Accordingly, DM64 did not neutralize the anticoagulant effect of mt-I nor its intracerebroventricular lethality, effects that depend on its enzymatic activity, and which demonstrate the dissociation between the catalytic and toxic activities of this Asp49 myotoxic PLA2. Furthermore, despite its similarity with metalloproteinase inhibitors, DM64 presented no antihemorrhagic activity against Bothrops jararaca or Bothrops asper crude venoms, and did not inhibit the fibrinogenolytic activity of jararhagin or bothrolysin. This is the first report of a myotoxin inhibitor with an immunoglobulin-like structure isolated and characterized from animal blood.

Hofstadler-Deiques, C. 2002. The development of the pit organ of Bothrops jararaca and Crotalus durissus terrificus (Serpentes, Viperidae): support for the monophyly of the subfamily Crotalinae. — Acta Zoologica (Stockholm) 83: 175-182 The development of the pit organ and the course of the lateral branch of the ethmoidal nerve of the two South American crotalines Bothrops jararaca and Crotalus durissus terrificus are described in detail using serially sectioned material from fetal stages. Serial sections of the Asian pit vipers Agkistrodon halys halys and Trimeresurus albolabris were included in this work. The viperines, as the sister-group of the Crotalinae, and the Colubridae, Elapidae and Boidae as their outgroups were used for comparisons. New synapomorphies of the advanced subfamily Crotalinae were found: namely, absence of the foramen epiphaniale and an extracapsular course of the lateral branch of ethmoidal nerve. These results support the monophyly of the crotalines.

Habituation to captivity is difficult for some species. Understanding the motivational elements involved in predation may ease this habituation. Seventy-one Brazilian jararaca snakes (Bothrops jararaca [Wied, 1824], Viperidae, Crotalinae) recently captured and never fed in captivity were tested for predatory behavior on rodents. Lighting was adapted to allow predatory sessions to occur during the first hours of the night when these animals are more active. Up to three prey subjects were presented in a session. In the first experiment, the preference for prey size and color was assessed using albino and dark-colored rodents. In a second experiment, a group of snakes was submitted to 12 sessions during a period of almost 2 years. The strike strategy was classified in one of two categories: envenomation (E) or seizing (S). Envenomation involved a bite delivered by the snake with prompt retrieval of the head; holding the rodent in the snake’s jaws since the first strike, without retrieving the fangs and holding the prey during venom action, characterized S strike. Trailing and swallowing the dead prey always followed E strike. Results suggest that snakes fed more often on larger subjects. The color of the prey was not a relevant factor. E strike was predominant in the first predatory event in captivity. After habituation, S strike was predominant. Snakes may have a poor perception of the prey objects in captivity and adopt a strike strategy that assures the control of the prey. Also, the use of small prey subjects to ease feeding during adaptation to captivity may be less effective. Zoo Biol 20:399–406, 2001. © 2001 Wiley-Liss, Inc.

Bothrops jararaca snake venom extract has been analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrospray mass spectrometry (ESI-MS) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) in order to obtain a rapid and accurate characterization. By the SDS-PAGE and MALDI-MS techniques about fourteen components are evidenced; with the latter technique, faster analysis and a more accurate molecular weight determination have been achieved. By ESI-MS the data obtained strongly depend on the operating conditions employed. © 1997 John Wiley & Sons, Ltd.

1. Bothrops jararaca venom (BJV) caused a fall in the carotid artery blood pressure of the anaesthetized snake. This effect was tachyphylactic and was potentiated by captopril, a kininase II inhibitor; it was partially antagonized by promethazine plus cimetidine and was not affected by atropine. 2. Similar hypotensive effects were obtained by administration of trypsin or a partially purified BJV kininogenase to the snake. 3. Incubation of Bothrops jararaca plasma (BJP) with trypsin released a substance (or substances) that produced hypotension in the snake but not in the rat; this hypotensive effect was also potentiated by captopril. 4. The trypsinised plasma contracted Bothrops jararaca isolated uterus, a pharmacological preparation weakly sensitive to bradykinin. Trypsinised plasma was inactive on pigeon oviduct and rat uterus and displayed a weak action on the guinea-pig ileum. Similar effects were observed with incubates of a fraction of BJP, containing globulins, with a partially purified BJV kininogenase. 5. Like mammalian kinins, the substance(s) was(were) dialysable, thermostable in acid but not in alkaline pH, and inactivated by chymotrypsin but not by trypsin. Its(their) inactivation by BJP or BJP kininase II was inhibited by captopril. 6. These findings strongly suggest that, besides releasing histamine, BJV or trypsin release a kininlike substance (or substances) from the snake plasma. 7. Since BJV and other kininogenases active on mammalian plasma were shown to be unable to release kinins from BJP, in experiments conducted on pharmacological preparations suitable for the assay of mammalian kinins, these data also suggest that the snake Bothrops jararaca, like birds, may have developed its own kallikrein-kinin system.

Morphological and histochemical studies of the cell types in the cephalic glands of Bothrops jararaca have been performed. It is concluded: 1) mucous cells are found in the salivary labial, accessory glands; mucous-serous cells are found in the salivary labial, accessory and Harderian glands; serous-mucous cells are found only in the venom gland; 2) neutral mucosubstances and protein were found in the salivary labial, venom, accessory and Harderian glands; 3) hyaluronic acid was detected in the Harderian gland; 4) of the to sulfated acid mucosubstances, only chondroitin sulfate B was detected in the salivary labial and accessory glands; 5) sialic acid was detected in the salivary labial, accessory and Harderian glands.

Summary 1 The isolated lungs of guinea-pigs, rats and dogs, perfused with Krebs bicarbonate solution via the pulmonary artery, inactivated 95, 98 and 99% respectively of the bradykinin infused through them. 2 This inactivation process was not inhibited by 2-mercaptoethanol (1 mm) or EDTA (500 μm). 3 The inactivation was inhibited by 2:3-dimercaptopropanol (100 μm), N-ethylmaleimide (100 μm), and several of the bradykinin potentiating peptides from Bothrops jararaca, in both natural and synthetic forms. 4 The effect of these compounds, 2-mercaptoethanol, EDTA, 2:3-dimercaptopropanol, N-ethylmaleimide and Bothrops peptides on the pulmonary bradykininase activity is the same as their effect on pulmonary angiotensin I converting enzyme.

Bradykinin, resulting from the action of snake venom (Bothrops jararaca) on bovine plasma, has been isolated in an essentially pure form. It contains the five amino acids arginine, phenylalanine, proline, glycine and serine in the ratio of 2:2:2:1:1, corresponding to the amino acid composition found for bradykinin stemming from the action of trypsin on corresponding plasma proteins. Degradation studies give the following partial sequence: H·Arg-(Pro2 Gly, Ser, Phe)-Phe-Arg·OH.

The synthetic analogues of three naturally occurring peptides from the venom of Bothrops jararaca have been tested as inhibitors of angiotensin converting enzyme activity in two systems. They have widely differing absolute potencies but the relative potencies are similar in the two systems. The high inhibitory potency of the nonapeptide is compatible with a non-competitive type of inhibition.