Action Of Venom Components Research Articles

Platelet aggregation inhibitors from Bothrops alternatus snake venom

Snake venoms are a complex mixture of proteins and peptides that can activate/inhibit platelet aggregation. Bothrops alternatus venom include three main families: metalloproteinases (SVMPs), serinoproteinases (SVSPs) and phospholipases A2 (PLA2s), among other minor components. In this work, we used inhibitor cocktails (containing Na2-EDTA, PMSF and/or pBPB) to investigate the effect of these three families and of baltergin (a PIII SVMP) on platelet aggregation by a turbidmetric method using a microplate reader. Cocktails 1 (active SVMPs) and 2 (active PLA2s) significantly reduced aggregation induced by ristocetin and collagen and by collagen and thrombin, respectively. Cocktail 3 (active SVSPs) showed a mild activation of aggregation, indicating the content of thrombin-like enzymes (TLEs) in this venom is low. Cocktail 4 (active minor components) displayed inhibitory effect with all agonists assayed (ristocetin, ADP, collagen and thrombin) but at higher IC50 values. Baltergin exhibited inhibitory effect when the catalytic domain was active for ristocetin-stimulated platelet aggregation and showed a non-enzymatic mechanism of inhibition when collagen was used as agonist. It was not able to disaggregate platelet thrombus. We conclude that B. alternatus venom is a source of natural inhibitors of platelet aggregation due to the action of SVMPs and PLA2s. Other minor components such as C-type lectins likely contribute to the antiplatelet effect. The interest in knowing the action of venom components on platelet function lies both in the understanding of the pathophysiology of snake bite envenomation and in their biotechnological application.

Development of High-Throughput Fluorescent-Based Screens to Accelerate Discovery of P2X Inhibitors from Animal Venoms.

Animal venoms can play an important role in drug discovery, as they are a rich source of evolutionarily tuned compounds that target a variety of ion channels and receptors. To date, there are six FDA-approved drugs derived from animal venoms, with recent work using high-throughput platforms providing a variety of new therapeutic candidates. However, high-throughput methods for screening animal venoms against purinoceptors, one of the oldest signaling receptor families, have not been reported. Here, we describe a variety of quantitative fluorescent-based high-throughput screening (HTS) cell-based assays for screening animal venoms against ligand-gated P2X receptors. A diverse selection of 180 venoms from arachnids, centipedes, hymenopterans, and cone snails were screened, analyzed, and validated, both analytically and pharmacologically. Using this approach, we performed screens against human P2X3, P2X4, and P2X7 using three different fluorescent-based dyes on stable cell lines and isolated the active venom components. Our HTS assays are performed in 96-well format and allow simultaneous screening of multiple venoms on multiple targets, improving testing characteristics while minimizing costs, specimen material, and testing time. Moreover, utilizing our assays and applying them to the other natural product libraries, rather than venoms, might yield other novel natural products that modulate P2X activity.

Stenotrophomonas-Like Bacteria Are Widespread Symbionts in Cone Snail Venom Ducts.

Cone snails are biomedically important sources of peptide drugs, but it is not known whether snail-associated bacteria affect venom chemistry. To begin to answer this question, we performed 16S rRNA gene amplicon sequencing of eight cone snail species, comparing their microbiomes with each other and with those from a variety of other marine invertebrates. We show that the cone snail microbiome is distinct from those in other marine invertebrates and conserved in specimens from around the world, including the Philippines, Guam, California, and Florida. We found that all venom ducts examined contain diverse 16S rRNA gene sequences bearing closest similarity to Stenotrophomonas bacteria. These sequences represent specific symbionts that live in the lumen of the venom duct, where bioactive venom peptides are synthesized.IMPORTANCE In animals, symbiotic bacteria contribute critically to metabolism. Cone snails are renowned for the production of venoms that are used as medicines and as probes for biological study. In principle, symbiotic bacterial metabolism could either degrade or synthesize active venom components, and previous publications show that bacteria do indeed contribute small molecules to some venoms. Therefore, understanding symbiosis in cone snails will contribute to further drug discovery efforts. Here, we describe an unexpected, specific symbiosis between bacteria and cone snails from around the world.

Proteomic View of the Venom from the Fire Ant Solenopsis invicta Buren

Fire ants are well-known by their aggressive stinging behavior, causing many stinging incidents of medical importance. The limited availability of fire ant venom for scientific and clinical uses has restricted, up to now, the knowledge about the biochemistry, immunology, and pharmacology of these venoms. For this study, S. invicta venom was obtained commercially and used for proteomic characterization. For this purpose, the combination of gel-based and gel-free proteomic strategies was used to assign the proteomic profile of the venom from the fire ant S. invicta. This experimental approach permitted the identification of 46 proteins, which were organized into four different groups according to their potential role in fire ant venom: true venom components, housekeeping proteins, body muscle proteins, and proteins involved in chemical communication. The active venom components that may not present toxic roles were classified into three subgroups according to their potential functions: self-venom protection, colony asepsis, and chemical communication. Meanwhile, the proteins classified as true toxins, based on their functions after being injected into the victims' bodies by the fire ants, were classified in five other subgroups: proteins influencing the homeostasis of the victims, neurotoxins, proteins that promote venom diffusion, proteins that cause tissue damage/inflammation, and allergens.

Protobothrops mangshanensis bite: first clinical report of envenoming and its treatment

This case report presents envenoming by the Chinese pit viper Protobothrops mangshanensis (formerly Zhaoermia) and its treatment. A 38 year old snake breeder suffered two-fang bites to elbow by a Chinese pit viper Protobothrops mangshanensis resulting in local edema of the affected arm. No other signs of envenoming appeared. On the 5(th) day following the bite a hematoma developed on the other arm which had been mechanically injured 14 days before. Laboratory testing revealed severe coagulopathy with hypofibrinogenemia and immeasurably prolonged coagulation times. As substitution therapy with fibrinogen and fresh frozen plasma was unsuccessful and specific antivenom is not produced, antivenin against some other Asian pit vipers GREEN PIT VIPER ANTIVENIN, Thai Red Cross, Thailand was applied. Three doses of antivenom reversed the course of the hemocoagulation disorder. The case confirms the persistence of active venom components affecting coagulation, difficulty in ameliorating the hemocoagulatin disorder caused by snake venom through substitution therapy and the effectiveness of delayed treatment using antivenin. It points out the potential risk of a clinically asymptomatic progress of envenoming by snake venoms containing hemocoagulation acting components, if the hemocoagulation disorder is not investigated and suitably treated. Therapy using the GREEN PIT VIPER ANTIVENIN, Thai Red Cross, Thailand in this case of envenomation by a Protobothrops mangshanensis bite proved to be applicable and the antivenom could be characterised as a paraspecific active.

Differential gene expression profiles in the venom gland/sac of Eumenes pomiformis (Hymenoptera: Eumenidae)

To search for novel transcripts encoding biologically active venom components, a subtractive cDNA library specific to the venom gland and sac (gland/sac) of a solitary hunting wasp species, Eumenes pomiformis Fabricius (1781), was constructed by suppression subtractive hybridization. A total of 541 expressed sequence tags (ESTs) were clustered and assembled into 102 contigs (31 multiple sequences and 71 singletons). In total, 37 cDNAs were found in the library via BLASTx searching and manual annotation. Eight contigs (337 ESTs) encoding short venom peptides (10 to 16 amino acids) occupied 62% of the library. The deduced amino acid sequence (78 amino acids) of a novel venom peptide transcript shared sequence similarity with trypsin inhibitors and dendrotoxin-like venom peptides known to be K + channel blockers, implying that this novel peptide may play a role in the paralysis of prey. In addition to phospholipase A2 and hyaluronidase, which are known to be the main components of wasp venoms, several transcripts encoding enzymes, including three metallopeptidases and a decarboxylase likely involved in the processing and activation of venomous proteins, peptides, amines, and neurotransmitters, were also isolated from the library. The presence of a transcript encoding a putative insulin/insulin-like peptide binding protein suggests that solitary hunting wasps use their venom to control their prey, leading to larval growth cessation. The abundance of these venom components in the venom gland/sac and in the alimentary canal was confirmed by quantitative real-time PCR. Discovery of venom gland/sac-specific transcripts should promote further studies on biologically active components in the venom of solitary hunting wasps.

The Diversity of Bioactive Proteins in Australian Snake Venoms

Australian elapid snakes are among the most venomous in the world. Their venoms contain multiple components that target blood hemostasis, neuromuscular signaling, and the cardiovascular system. We describe here a comprehensive approach to separation and identification of the venom proteins from 18 of these snake species, representing nine genera. The venom protein components were separated by two-dimensional PAGE and identified using mass spectrometry and de novo peptide sequencing. The venoms are complex mixtures showing up to 200 protein spots varying in size from <7 to over 150 kDa and in pI from 3 to >10. These include many proteins identified previously in Australian snake venoms, homologs identified in other snake species, and some novel proteins. In many cases multiple trains of spots were typically observed in the higher molecular mass range (>20 kDa) (indicative of post-translational modification). Venom proteins and their post-translational modifications were characterized using specific antibodies, phosphoprotein- and glycoprotein-specific stains, enzymatic digestion, lectin binding, and antivenom reactivity. In the lower molecular weight range, several proteins were identified, but the predominant species were phospholipase A2 and alpha-neurotoxins, both represented by different sequence variants. The higher molecular weight range contained proteases, nucleotidases, oxidases, and homologs of mammalian coagulation factors. This information together with the identification of several novel proteins (metalloproteinases, vespryns, phospholipase A2 inhibitors, protein-disulfide isomerase, 5'-nucleotidases, cysteine-rich secreted proteins, C-type lectins, and acetylcholinesterases) aids in understanding the lethal mechanisms of elapid snake venoms and represents a valuable resource for future development of novel human therapeutics.

Comparison of Active Venom Components between Eastern Brown Snakes Collected from South Australia and Queensland

The abundance and activity of the prothrombin activator (pseutarin C) within the venom of the Eastern brown snake (Pseudonaja textilis textilis) is the primary determinant of its coagulation potency. Textilinin-1, also in this venom, is a plasmin inhibitor which is thought to exert its toxic effects through the slowing of fibrinolysis. The aim of this report is to determine if there are differences in the potency of the venom from Eastern brown snakes collected from South Australia (SA) compared to those from Queensland (QLD). A concentration of 0.4 microg/ml venom protein from six QLD specimens clotted citrated plasma in an average time of 21.4+/-3.3 s compared to 68.7+/-2.4 s for the same amount of SA venom (averaged for six individuals). The more potent procoagulant activity of the QLD venom was measured between 0.4 and 94 microg/ml venom protein in plasma. The anti-plasmin activity of textilinin was also greater in the venom of the snakes collected from QLD, causing full inhibition of plasmin at approximately 1.88 microg/ml of venom protein compared to approximately 7.5 microg/ml for the SA venoms. It is concluded that geographic differentiation of the Eastern brown snakes results in significant differences venom potency.

Conus Venom Peptides: Reflections from the Biology of Clades and Species

▪ Abstract The 500 cone snail species (Conus) use complex venoms to capture prey, defend against predators and deter competitors. Most biologically active venom components are small, highly structured peptides, each encoded by a separate gene. Every Conus species has its own distinct repertoire of 100–200 venom peptides, with each peptide presumably having a physiologically relevant target in prey or potential predators/competitors. There is a remarkable interspecific divergence observed in venom peptide genes, which can be rationalized because of biotic interactions that are species specific. The peptide families/subfamilies characteristic of clades of related Conus species are potentially useful clade markers and can be used to indicate common biological mechanisms characterizing that clade. By knowing both the distribution and the physiological function of venom peptides, a type of reverse ecology becomes possible; the peptides in a Conus venom are a molecular readout of the biotic interactions of a species or clade.

Digger wasp versus cricket: Mechanisms underlying the total paralysis caused by the predator's venom

The data presented here describe neurophysiological experiments addressing the question of cellular mechanisms underlying the total paralysis of locomotor behavior in crickets occurring after being stung by females of the digger wasp species Liris niger. The Liris venom effects have been studied by both in vivo recordings from identified neurons of the well-described giant fiber pathway and in vitro recordings from cultured neurons isolated from the terminal ganglion of crickets. The total paralysis of the prey is characterized by a general block of action potential generation as well as by a block of synaptic transmission. Intracellular recordings from neurons in intact ganglia under single electrode voltage-clamp conditions, as well as whole-cell patch-clamp recordings from cultured cricket neurons consistently show that the block of action potential generation by the Liris venom is due to a block of voltage-gated sodium inward currents in neurons of the stung ganglia. Furthermore, our data provide evidence that the Liris venom also blocks calcium currents in identified neurosecretory neurons. On the other hand, outward currents are not affected by the Liris venom. The in vitro recordings suggest that the Liris venom contains active venom components, which, at least for the observed block of inward currents, do not require a metabolic modification. Because venom application does not affect the ACh-induced EPSPs in giant interneurons, the Liris venom does not seem to influence the postsynaptic ACh receptors. The possible pre- and postsynaptic sites of venom action and the functional consequences on synaptic transmission within the giant fiber system are discussed.

Antigenic cross-reactivity of venoms from medically important North American Loxosceles spider species

We characterized the antigenic cross-reactivity of two medically important North American Loxoxceles species: L. reclusa (native to southeastern US) and L. deserta (native to southwestern US). Dermonecrosis resulting from bites from these two North American spider species are indistinguishable clinically. Polyclonal IgG antivenins directed against L. reclusa and L. deserta were raised in rabbits and used to develop specific enzyme immunoassays (EIAs). Antigenic differences in the two venoms were evaluated as follows: (1) Comparison of the sensitivities and correlation coefficient ( R 2) of anti- L. reclusa (αLoxR) and anti- L. deserta antibodies (αLoxD) in the detection of varying concentrations of the two venoms; (2) separation and western blot comparison of venom components; (3) protein sequence analysis of L. desertavenom and comparison to the L. reclusa protein sequence analysis present in a US national database; and (4) in vivo evaluation of αLoxR and αLoxD antivenins in attenuating dermal lesions (rabbit model). Correlation coefficients for αLoxR ( R 2=0.99) and αLoxD ( R 2=0.99) polyclonal antibodies in the measurements of standard concentrations of venoms were virtually identical. Western blot analysis revealed multiple common bands between the two venoms. Amino acid data (amino acids 1–35, N-terminal) of the active venom components of the two venoms revealed only three non-identical amino acids. αLoxR and αLoxD antivenins were similarly effective in blocking the development of rabbit skin lesions (ANOVA p<0.05). In summary, L. reclusa and L deserta spider venoms possess several common protein bands as identified by western blot, greater than 90% amino acid sequence identity, and marked antigenic cross-reactivity.

Role of ectoparasitoid venom in the regulation of haemolymph ecdysteroid titres in a host noctuid moth

The effects on haemolymph ecdysteroid titre of a host noctuid moth in response to parasitism or injection of extracts of venom from the external parasitoid wasp, Eulophus pennicornis, were ascertained for the penultimate (fifth) instar larva of the tomato moth, Lacanobia oleracea. Unparasitized larvae showed a marked peak of ecdysteroid titre (85 ng/ml ecdysone equiv.) at approximately 48 h into the fifth stadium. This major ecdysteroid peak was eliminated, or its timing was significantly postponed (by at least 48‐60 h), when the larvae were parasitized by E. pennicornis. Parasitized larvae failed to moult and eventually died. Saline-injected larvae showed a normal, but slightly delayed (ca. 12 h) major peak of ecdysteroid titre at 64‐66 h (ca. 140 ng/ml) that followed a smaller peak (56 ng/ml) ca. 12‐14 h earlier. As with parasitism, these ecdysteroid peaks were effectively eliminated, or significantly delayed, when larvae were injected with 0.7 gland equivalents of E. pennicornis venom. Venom-injected larvae failed to moult to the sixth instar, whereas saline-injected larvae moulted and developed normally. High-performance gel filtration chromatography was used for the initial purification of active venom components. Bioassays of the size-fractionated venom gland proteins by injection into fifth instar larvae showed that the suppression of moulting effect was confined to just two fractions (out of 12) with apparent Mr between 43,000 and 158,000. Arch. Insect Biochem. Physiol. 35:169‐178, 1997. PUBLISHED 1997 WILEY-LISS, INC.

Amplification of the classical complement pathway by the venom of the Mojave rattlesnake ( Crotalus scutulatus scutulatus)

The lytic attack of human red blood cells by complement was enhanced by the venom of the Mojave rattlesnake ( Crotalus scutulatus scutulatus). Lysis, however, of sheep red blood cells by complement was not enhanced by this venom. Western diamondback rattlesnake ( Crotalus atrox) venom was anticomplementary. Enhanced lysis of human red blood cells by Mojave venom required that the red blood cells be sensitized with antibodies and that the complement system be intact. Heat inactivation of complement (56°C, 30 min) prevented lysis of red blood cells. Moreover, heating of the Mojave venom (66°C, 60 min) prevented enhanced lysis. Lytic enhancement was time, dose and temperature dependent. It is postulated that membrane active venom components enhance the efficiency of the complete complement cascade for red blood cell lysis to occur, because venom increased the lytic reaction rate.