An Appetite for Destruction: Detecting Prey-Selective Binding of α-Neurotoxins in the Venom of Afro-Asian Elapids.

Decision letter: The evolution and structure of snake venom phosphodiesterase (svPDE) highlight its importance in venom actions

Bungarus caeruleus (Indian common krait) is a venomous snake that is responsible for most of the snakebites in India. In the present study, we report the isolation and purification of neurotoxin and the biochemical changes and pathological effects induced by injection of purified neurotoxin into rats. The purpose of this study was to compare the effects of crude krait venom and the purified toxin. Both the crude venom and a sublethal dose of 60 microg/kg B. caeruleus purified toxin significantly increased the serum levels of alkaline phosphatase, alanine aminotransferase, and urea (p < 0.05). The crude venom but not the purified toxin increased the levels of lactate dehydrogenase, aspartate aminotranseferase, creatine kinase, and glucose. The kidneys showed congestion of the vessels, hemorrhage, and necrosis in venom-injected but not in toxin treated animals. The results of this study indicate that although crude krait venom has severe lethal, hemorrhagic, nephrotoxic, and proteolytic activities, the purified neurotoxin shows only moderate toxic activity, manifested as prominent local and systemic effects.

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.

In vivo analysis of effects of venom from the jellyfish Chrysaora sp. in zebrafish (Danio rerio)

The present study was designed to examine the effects of venom from the scorpion Leiurus quinquestriatus hebraeus (Lqh) on the contractility of rat aortic rings. We first examined the effect of Lqh venom on the contractile tension of isolated rat vascular aortic rings and then whether long-term exposure to the venom reduces the contractility of vascular smooth muscle by increasing the production of nitric oxide. Following the administration of 33 microg/mL of crude Lqh venom, contractile tension increased by 18.9 +/- 11.4 percent. The administration of 2.4 x 10(-7) M noradrenaline (NA) led to a 31.6 +/- 8.2 percent increase in tension (p < 0.01). The effects induced by NA and Lqh venom were similar and additive (p < 0.01). Pretreatment with the alpha-adrenergic blocker phenoxybenzamine (0.2 microM) eliminated the effect of the venom, whereas the calcium-channel blocker verapil (8.3 microM) merely attenuated the effect. Incubation of the rings with Lqh venom for 16 to 18 h, followed by NA stimulation, led to a 15 to 20 percent decrease in tension (p < 0.001). Treatment with N-omega-nitro-L-arginine methyl ester (110 microM), a constitutional nitric oxide inhibitor, restored the tension to control values. Treatment with S-methyl-isothiourea (0.1 microM), an inducible nitric oxide synthesis inhibitor, had no effect on contractile tension. The results of the present study suggest that the effect of Lqh venom on isolated aortic rings is induced via sympathetic nerve terminals. Calcium had little effect on the smooth muscle contractility of aortic rings incubated with the venom. No evidence was found to support nitric oxide synthesis after the long-term exposure of the rings to Lqh venom.

There are numerous evidences about the effects of crude snake venoms or isolated toxins on the peripheral nervous system. However, the data on their interactions with the central nervous system (CNS) are not so abundant, since the blood-brain barrier (BBB) impedes penetration of these compounds into the brain. There are several reviews describing the interactions of particular classes of snake venom polypeptides with components of the CNS; however, no general systematics of such interactions was done. This review is the first attempt to consolidate the data about the interaction of snake venom polypeptides with the CNS. Such data will be described according to three main modes of interactions: - Direct in vivo interaction of the CNS with venom polypeptides capable to penetrate BBB. - In vitro interactions of isolated components of the CNS with crude venoms or purified toxins. - Indirect effects of snake venoms or their components on functioning of the CNS under normal or pathological conditions. Although the venom components penetrating BBB are not numerous, they seem to be the most suitable candidates for the leads in drug design. The compounds from two other groups are more abundant and better studied, but the fact that the data about their ability to penetrate BBB are still absent may substantially aggravate the potentials for their medical perspectives. Nevertheless, many of these compounds are used as biochemical tools for research of the CNS in vitro. These investigations may give invaluable information for understanding the molecular basis of CNS diseases and thus lay the basis for targeted drug design. This aspect also will be outlined in the review. Keywords: Snake venom, polypeptides, neurotoxins, central nervous system, brain

We examined the effects of Pandinus imperator scorpion venom on voltage-gated potassium channels in cultured clonal rat anterior pituitary cells (GH3 cells) using the gigohm-seal voltage-clamp method in the whole-cell configuration. We found that Pandinus venom blocks the voltage-gated potassium channels of GH3 cells in a voltage-dependent and dose-dependent manner. Crude venom in concentrations of 50-500 micrograms/ml produced 50-70% block of potassium currents measured at -20 mV, compared with 25-60% block measured at +50 mV. The venom both decreased the peak potassium current and shifted the voltage dependence of potassium current activation to more positive potentials. Pandinus venom affected potassium channel kinetics by slowing channel opening, speeding deactivation slightly, and increasing inactivation rates. Potassium currents in cells exposed to Pandinus venom did not recover control amplitudes or kinetics even after 20-40 min of washing with venom-free solution. The concentration dependence of crude venom block indicates that the toxins it contains are effective in the nanomolar range of concentrations. The effects of Pandinus venom were mimicked by zinc at concentrations less than or equal to 0.2 mM. Block of potassium current by zinc was voltage dependent and resembled Pandinus venom block, except that block by zinc was rapidly reversible. Since zinc is found in crude Pandinus venom, it could be important in the interaction of the venom with the potassium channel. We conclude that Pandinus venom contains toxins that bind tightly to voltage-dependent potassium channels in GH3 cells. Because of its high affinity for voltage-gated potassium channels and its irreversibility, Pandinus venom may be useful in the isolation, mapping, and characterization of voltage-gated potassium channels.

The nervous system is a primary target for animal venoms as the impairment of its function results in the fast and efficient immobilization or death of a prey. There are numerous evidences about effects of crude snake venoms or isolated toxins on peripheral nervous system. However, the data on their interactions with the central nervous system (CNS) are not abundant, as the blood-brain barrier (BBB) impedes penetration of these compounds into brain. This updated review presents the data about interaction of snake venom polypeptides with CNS. Such data will be described according to three main modes of interactions: - Direct in vivo interaction of CNS with venom polypeptides either capable to penetrate BBB or injected into the brain. - In vitro interactions of cell or sub-cellular fractions of CNS with crude venoms or purified toxins. - Indirect effects of snake venoms or their components on functioning of CNS under different conditions. Although the venom components penetrating BBB are not numerous, they seem to be the most suitable candidates for the leads in drug design. The compounds with other modes of action are more abundant and better studied, but the lack of the data about their ability to penetrate BBB may substantially aggravate the potentials for their medical perspectives. Nevertheless, many such compounds are used for research of CNS in vitro. These investigations may give invaluable information for understanding the molecular basis of CNS diseases and thus lay the basis for targeted drug design. This aspect also will be outlined in the review.

Objective: To confirm whetherBungarus multicinctus crude venom induces the apoptosis of K562 tumor cells and to find out the components inducing apoptosis of K562 cells from the crude venom. Methods: the crude venom separated and purified by cation exchange chromatography, and the effect of venoms on K562 was studied by MTT method and flow cytometry. Results: The crude venom began to kill K562 cells at than 8×l03ng/ml (the survival rate was 82.5%) concentration and the effect was more significant in 24 h when administrating 8×l05ng/ml (the survival rate was 29.4%) crude venom. Apoptotic bodies were observed in the K562 tumor cells by fluorescent microscopy after administration of 5 µg/ml cycloheximide (CHX) or the peak VI solution at about 8×105 ng/ml. The same results were detected by the flow cytometry. A sub-Gi peak appeared after administration of CHX or the sixth peak solution. Conclusion: The authors found that the venom can kill K562 tumor cells in time- and dose-dependent manner. However, the killing effect of the venom is not apoptosis. What’s more, the peak VI solution, a component of the crude venom can induce the apoptosis of K562 tumor cells.

Fibroblast cultures were used to study the effects of crude Walterinnesia aegyptia venom and its F1–F7 protein fractions on TCA cycle enzyme activities and mitochondrial NAD-redox state. Confluent cells were incubated with 10 μg of venom proteins for 4 hours at 37°C. The activities of all studied TCA enzymes and the non-TCA mitochondrial NADP+-dependent isocitrate dehydrogenase underwent significant reductions of similar magnitude (50–60% of control activity) upon incubation of cells with the crude venom and fractions F4, F5, and F7 and 60–70% for fractions F3 and F6. In addition, the crude and fractions F3–F7 venom proteins caused a drop in mitochondrial NAD+ and NADP+ levels equivalent to around 25% of control values. Whereas the crude and fractions F4, F5, and F7 venom proteins caused similar magnitude drops in NADH and NADPH (around 55% of control levels), fractions F3 and F6 caused a more drastic drop (60–70% of control levels) of both reduced coenzymes. Results indicate that the effects of venom proteins could be directed at the mitochondrial level and/or the rates of NAD+ and NADP+ biosynthesis.

The evolution of an aquatic lifestyle from land dwelling venomous elapids is a radical ecological modification, bringing about many evolutionary changes from morphology to diet. Diet is an important ecological facet which can play a key role in regulating functional traits such as venom composition and prey-specific targeting of venom. In addition to predating upon novel prey (e.g., fish, fish eggs and invertebrates), the venoms of aquatic elapids also face the challenge of increased prey-escape potential in the aquatic environment. Thus, despite the independent radiation into an aquatic niche on four separate occasions, the venoms of aquatic elapids are evolving under convergent selection pressures. Utilising a biolayer interferometry binding assay, this study set out to elucidate whether crude venoms from representative aquatic elapids were target-specific to the orthosteric site of postsynaptic nicotinic acetylcholine receptor mimotopes of fish compared to other terrestrial prey types. Representatives of the four aquatic lineages were: aquatic coral snakes representative was Micrurus surinamensis;, sea kraits representative was Laticauda colubrina; sea snakes representatives were two Aipysurus spp. and eight Hydrophis spp; and water cobras representative was Naja annulata. No prey-specific differences in crude venom binding were observed from any species tested, except for Aipysurus laevis, which showed slight evidence of prey-potency differences. For Hydrophis caerulescens, H. peronii, H. schistosus and M. surinamensis, there was a lack of binding to the orthosteric site of any target lineage. Subsequent testing on the in vitro chick-biventer cervicis muscle preparation suggested that, while the venoms of these species bound postsynaptically, they bound to allosteric sites rather than orthosteric. Allosteric binding is potentially a weaker but faster-acting form of neurotoxicity and we hypothesise that the switch to allosteric binding is likely due to selection pressures related to prey-escape potential. This research has potentially opened up the possibility of a new functional class of toxins which have never been assessed previously while shedding light on the selection pressures shaping venom evolution.

Crude venom of Echis coloratus was separated into seven protein fractions using 7% preparative native polyacrylamide gel electrophoresis. The effect of crude venom and seven venom protein fractions (F1-F7) from Echis coloratus on key metabolic activities of fibroblast cultures was investigated. Confluent cultures were incubated with the venom proteins for 3 h at 37 degrees C. The specific activity of phosphofructokinase, was significantly lowered upon incubation with the crude venom and with fractions 2, 3, 4 and 6. Citrate synthase activity was significantly lowered by the crude venom and by fractions 2 and 3. Glycogen phosphorylase activity was significantly increased by the crude venom and by fractions 2, 3, 4 and 6 leading to a significant concurrent drop in glycogen content. Creatine kinase activity was significantly increased by the crude venom and by fractions 3, 4, 5 and 6. Cellular ATP levels rose significantly upon incubation with the crude venom and with fractions 3, 4, 5 and 6. Incubation of cell sonicates with all the venom proteins did not significantly alter the activity or content of any of the studied parameters.

Crude venom of Echis coloratus was separated into seven protein fractions using 7% preparative native polyacrylamide gel electrophoresis. The effect of crude venom and seven venom protein fractions (F1–F7) from Echis coloratus on key metabolic activities of fibroblast cultures was investigated. Confluent cultures were incubated with the venom proteins for 3 h at 37°C. The specific activity of phosphofructokinase, was significantly lowered upon incubation with the crude venom and with fractions 2, 3, 4 and 6. Citrate synthase activity was significantly lowered by the crude venom and by fractions 2 and 3. Glycogen phosphorylase activity was significantly increased by the crude venom and by fractions 2, 3, 4 and 6 leading to a significant concurrent drop in glycogen content. Creatine kinase activity was significantly increased by the crude venom and by fractions 3, 4, 5 and 6. Cellular ATP levels rose significantly upon incubation with the crude venom and with fractions 3, 4, 5 and 6. Incubation of cell sonicates with all the venom proteins did not significantly alter the activity or content of any of the studied parameters. Copyright © 2002 John Wiley & Sons, Ltd.

Snakebite envenomation is a serious medical problem in many tropical developing countries and was considered by WHO as a neglected tropical disease. Antivenom (AV), the rational and most effective treatment modality, is either unaffordable and/or unavailable in many affected countries. Moreover, each AV is specific to only one (monospecific) or a few (polyspecific) snake venoms. This demands that each country to prepare AV against its local snake venoms, which is often not feasible. Preparation of a ‘pan-specific’ AV against many snakes over a wide geographical area in some countries/regions has not been possible. If a ‘pan-specific’ AV effective against a variety of snakes from many countries could be prepared, it could be produced economically in large volume for use in many countries and save many lives. The aim of this study was to produce a pan-specific antiserum effective against major medically important elapids in Asia. The strategy was to use toxin fractions (TFs) of the venoms in place of crude venoms in order to reduce the number of antigens the horses were exposed to. This enabled inclusion of a greater variety of elapid venoms in the immunogen mix, thus exposing the horse immune system to a diverse repertoire of toxin epitopes, and gave rise to antiserum with wide paraspecificity against elapid venoms. Twelve venom samples from six medically important elapid snakes (4 Naja spp. and 2 Bungarus spp.) were collected from 12 regions/countries in Asia. Nine of these 12 venoms were ultra-filtered to remove high molecular weight, non-toxic and highly immunogenic proteins. The remaining 3 venoms were not ultra-filtered due to limited amounts available. The 9 toxin fractions (TFs) together with the 3 crude venoms were emulsified in complete Freund’s adjuvant and used to immunize 3 horses using a low dose, low volume, multisite immunization protocol. The horse antisera were assayed by ELISA and by in vivo lethality neutralization in mice. The findings were: a) The 9 TFs were shown to contain all of the venom toxins but were devoid of high MW proteins. When these TFs, together with the 3 crude venoms, were used as the immunogen, satisfactory ELISA antibody titers against homologous/heterologous venoms were obtained. b) The horse antiserum immunologically reacted with and neutralized the lethal effects of both the homologous and the 16 heterologous Asian/African elapid venoms tested. Thus, the use of TFs in place of crude venoms and the inclusion of a variety of elapid venoms in the immunogen mix resulted in antiserum with wide paraspecificity against elapid venoms from distant geographic areas. The antivenom prepared from this antiserum would be expected to be pan-specific and effective in treating envenomations by most elapids in many Asian countries. Due to economies of scale, the antivenom could be produced inexpensively and save many lives. This simple strategy and procedure could be readily adapted for the production of pan-specific antisera against elapids of other continents.

Bee venom (BV) has variety of properties, such as anti-inflammatory potential and anti-cancer activity.It has been shown that BV has cytotoxic effects on MCF-7 cells in 2D culture. Cell encapsulation is one of the three-dimensional (3D) culture methods that can better represent the physiological conditions of natural tissues and tumors in comparison to two-dimensional (2D) culture. However, the effect of BC on 3D culture has yet studied. The present study was designed to compare the cytotoxic effects of BV on MCF-7 cells in 3D and 2D cultures for the first time. To this purpose, the MCF-7 cells were encapsulated to create a 3D culture using alginate hydrogel, a natural polysaccharide. Cytotoxicity effect of BV was investigated using 3-(4,5-Dimethylthiazoyl-2-yl)-2,5-Diphenyltetrazolium bromide (MTT) reduction and confirmed with neutral red uptake assay following venom exposure. Apoptosis was evaluated with reactive nitrogen species assay, measuring Caspase-3 activity and Comet assay. Moreover, oxidative stress was evaluated by glutathione (GSH) and catalase assays. Results of caspase-3 and comet assays showed an increase of tumoral cells apoptosis following the treatment with venom in a dose dependent manner in both 2D and 3D cultures. GSH and catalase measurements showed that oxidative stress was increased in MCF-7 cells treated with BV in a dose dependent manner in both 2D and 3D cultures. The results of the present study show that the crude venom of Apis mellifera has cytotoxic effects on MCF-7 cells in a dose dependent manner. Indeed, it was shown that the effect of the venom on MCF-7 cells in 2D culture is significantly higher than that of the 3D culture.