Viridis Venom Research Articles

Distinct regulatory networks control toxin gene expression in elapid and viperid snakes

BackgroundVenom systems are ideal models to study genetic regulatory mechanisms that underpin evolutionary novelty. Snake venom glands are thought to share a common origin, but there are major distinctions between venom toxins from the medically significant snake families Elapidae and Viperidae, and toxin gene regulatory investigations in elapid snakes have been limited. Here, we used high-throughput RNA-sequencing to profile gene expression and microRNAs between active (milked) and resting (unmilked) venom glands in an elapid (Eastern Brown Snake, Pseudonaja textilis), in addition to comparative genomics, to identify cis- and trans-acting regulation of venom production in an elapid in comparison to viperids (Crotalus viridis and C. tigris).ResultsAlthough there is conservation in high-level mechanistic pathways regulating venom production (unfolded protein response, Notch signaling and cholesterol homeostasis), there are differences in the regulation of histone methylation enzymes, transcription factors, and microRNAs in venom glands from these two snake families. Histone methyltransferases and transcription factor (TF) specificity protein 1 (Sp1) were highly upregulated in the milked elapid venom gland in comparison to the viperids, whereas nuclear factor I (NFI) TFs were upregulated after viperid venom milking. Sp1 and NFI cis-regulatory elements were common to toxin gene promoter regions, but many unique elements were also present between elapid and viperid toxins. The presence of Sp1 binding sites across multiple elapid toxin gene promoter regions that have been experimentally determined to regulate expression, in addition to upregulation of Sp1 after venom milking, suggests this transcription factor is involved in elapid toxin expression. microRNA profiles were distinctive between milked and unmilked venom glands for both snake families, and microRNAs were predicted to target a diversity of toxin transcripts in the elapid P. textilis venom gland, but only snake venom metalloproteinase transcripts in the viperid C. viridis venom gland. These results suggest differences in toxin gene posttranscriptional regulation between the elapid P. textilis and viperid C. viridis.ConclusionsOur comparative transcriptomic and genomic analyses between toxin genes and isoforms in elapid and viperid snakes suggests independent toxin regulation between these two snake families, demonstrating multiple different regulatory mechanisms underpin a venomous phenotype.

Kinome scale profiling of venom effects on cancer cells reveals potential new venom activities

The search for novel and relevant cancer therapeutics is continuous and ongoing. Cancer adaptations, resulting in therapeutic treatment failures, fuel this continuous necessity for new drugs to novel targets. Recently, researchers have started to investigate the effect of venoms and venom components on different types of cancer, investigating their mechanisms of action. Receptor tyrosine kinases (RTKs) comprise a family of highly conserved and functionally important druggable targets for cancer therapy. This research exploits the novelty of complex venom mixtures to affect phosphorylation of the epidermal growth factor receptor (EGFR) and related RTK family members, dually identifying new activities and unexplored avenues for future cancer and venom research. Six whole venoms from diverse species taxa, were evaluated for their ability to illicit changes in the phosphorylated expression of a panel of 49 commonly expressed RTKs. The triple negative breast cancer cell line MDA-MB-468 was treated with optimised venom doses, pre-determined by SDS PAGE and Western blot analysis. The phosphorylated expression levels of 49 RTKs in response to the venoms were assessed with the use of Human Phospho-RTK Arrays and analysed using ImageLab 5.2.1 analysis software (BioRad). Inhibition of EGFR phosphorylation occurred with treatment of venom from Acanthoscurria geniculata (Theraphosidae), Heterometrus swammerdami (Scorpionidae), Crotalus durissus vegrandis (Crotalidae) and Naja naja (Elapidae). Western green mamba Dendroaspis viridis venom increased EGFR phosphorylation. Eph, HGFR and HER were the most affected receptor families by venoms. Whilst the importance of these changes in terms of effect on MDA-MB-468 cells’ long-term viability and functionality are still unclear, the findings present exciting opportunities for further investigation as potential drug targets in cancer and as tools to understand better how these pathways interact.

Proteomic characterization of the venom and transcriptomic analysis of the venomous gland from the Mexican centipede Scolopendra viridis.

Animal venoms of Scolopendra species have been scarcely studied, although they have been reported to contain several bioactive compounds, some of which with potential therapeutic interest. The Mexican centipede S. viridis contains a powerful venom, capable of inflicting immediate effects on their preys. This communication is focused on the identification and description of a proteomic and transcriptomic analysis of the protein components of this venom. Several amino acid sequences similar to reported enzymes are the principal components in the S. viridis venom, but also a low number of toxins were identified. This knowledge should contribute to the understanding of the pharmacological effects caused by bites of this centipede species.

Prevalence of common thrombophilia markers and risk factors in Indian patients with primary venous thrombosis.

venous thrombosis occurs as a result of interaction of genetic and acquired factors including activated protein C resistance (APC-R), fibrinogen levels, antithrombin, protein C, protein S, lupus anticoagulants and anticardiolipin antibodies. This study was aimed at determining the prevalence of these common thrombophilia markers in Asian Indians with primary venous thrombosis. this was a cross-sectional study carried out in Mumbai. samples from 78 patients with a confirmed diagnosis of venous thrombosis and 50 controls were tested. Semi-quantitative estimation (functional assays) of protein C, protein S and antithrombin was performed. Quantitative estimation of fibrinogen was done using the Clauss method. Lupus anticoagulants were screened using lupus-sensitive activated partial thromboplastin time and β2-glycoprotein-I dependent anticardiolipin antibodies were estimated by ELISA. APC-R was measured using a clotting-based method with factor V deficient plasma and Crotalus viridis venom. Statistical analysis was performed using Epi-info (version 6). the popliteal vein was the most commonly involved site. Forty-four samples (56%) gave abnormal results. The commonest were elevated fibrinogen and APC-R (17.9% each), followed by low protein S (16.6%). this study confirms the literature findings that fibrinogen level estimation and screening for APC-R are important for the work-up on venous thrombosis patients since these, singly or in combination, may lead to a primary thrombotic episode. The frequency of the other thrombophilia markers was higher among the patients than among the controls, but without statistically significant difference.

Immunological studies of rabbit antibodies against hemorrhagic fractions of Crotalus viridis viridis venom: Role of crossreacting antibodies in neutralization

1. Crude Prairie rattlesnake ( Crotalus viridis viridis) venom was fractionated by HPLC DEAE anion exchange chromatography. Both acidic and basic protein fractions were assayed for hemorrhagic activity in vivo. 2. The acidic fractions that showed the highest hemorrhagic activities were pooled and divided into two samples. One sample was treated with EDTA and heat to inactivate and denature the hemorrhagic toxins; the other sample was left in native form. Both denatured and native samples were used as immunogens for production of polyvalent antibodies in rabbits. 3. Neutralizing ability for hemorrhage of the antiserum raised against the native sample was about 50% greater than that of antiserum raised against the denatured sample. There was no correlation between ELISA reactivity and neutralizing ability of the antisera. 4. Antiserum raised against native hemorrhagic fractions showed extensive ELISA crossreactivities with the nonhemorrhagic basic proteins. The crossreacting antibodies accounted for 61% of the total ELISA reactivity and 54% of the total neutralizing ability of the antiserum. Treatment of the hemorrhagic fractions with EDTA induced a fundamental conformational change of the molecules, which was reflected in significantly increased ELISA reactivities. Antivenom raised in rabbits had high neutralizing potency compared to Wyeth antivenom (0.38 mg IgG was able to completely neutralize the hemorrhagic lesions induced by 10 μg crude C. v. viridis venom). However, when it was quantitatively compared with commercial Wyeth antivenom, no significant advantages were found.

Biochemical characterization of hemorrhagic toxin from crotalus viridis viridis (prairie rattlesnake) venom

Hemorrhage, necrosis and edema are some of the effects often observed following snake bites. This paper reports studies on the isolation and biological properties of hemorrhagic toxin from Crotalus viridis viridis (Prairie rattlesnake) venom. A hemorrhagic toxin was isolated from C. v. viridis venom by Sephadex G-50, DEAE-Sephacel and Q-Sepharose column chromatographies. The hemorrhagic toxin from C. v. viridis venom was shown to be homogenous as demonstrated by a single band on polyacrylamide gel electrophoresis and immunodiffusion. Its molecular weight was approximately 54,000 dallons, and it contained 471 amino acid residues. The toxin possessed hemorrhagic activity with a minimum hemorrhagic dose (MHD) of 0.11 μ g, and hydrolytic activity on dimethylcasein, casein, azocasein, azoalbumin, azocoll and hide powder azure. Hemorrhagic and casein hydrolytic activities were inhibited by EDTA, o-phenanthroline or dithiothreitol. The toxin contained 1 mole of zinc per mole of protein and zinc is essential for both hemorrhagic and proteolytic activities. Hemorrhagic toxin possessed hydrolytic activity on the B-chain of insulin, which cleaves His(5)-Leu(6), His(10)-Leu(11), Ala(14)-Leu(15), Tyr(16)-Leu(17) and Phe(24)-Phe(25) bonds. This toxin also hydrolyzed Aα and Bβ chains of fibrinogen. Intramuscular injections of hemorrhagic toxin caused an increase of creatine phosphokinase activity in mice serum from 50.3 mU/ml to 1133 mU/ml. A toxin isolated from C. v. viridis venom was shown to have strong hemorrhagic activity. Partial characterization is reported for this major hemorrhagic toxin in C. v. viridis venom.

Cross reactivities of monoclonal antibodies against hemorrhagic toxins of Prairie rattlesnake ( Crotalus viridis viridis) venom

Specific monoclonal antibodies (MAbs) were produced to two hemorrhagic toxins by cell fusion between spleen cells from mice immunized with a hemorrhagic HPLC fraction of Crotalus viridis viridis venom and P3X63Ag8.653 myeloma cells. The MAbs reacted with three different epitopes of high molecular weight hemorrhagic toxins of C. v. viridis venom. A species-specific epitope was detected in a 62 kDa hemorrhagic toxin by one of the MAbs. However, none of the MAbs was able to neutralize the hemorrhagic activity of the toxins. Some epitopes of the hemorrhagic toxins are shared by many related and unrelated species. High molecular weight hemorrhagic toxins ranging from 62 to 68 kDa appeared to be present in the venom of many members of the Crotalidae family. In addition to the high molecular weight toxins, several medium to low molecular weight hemorrhagic toxins were detected in the venoms of C. v. viridis, C. adamanteus, C. atrox and C. h. horridus.

Comparison of the immunogenicity and antigenic composition of ten Central American snake venoms

S. G. Anderson, J. M. Gutiérrez and C. L. Ownby. Comparison of the immunogenicity and antigenic composition of ten Central American snake venoms. Toxicon31, 1051–1059, 1993.—The immunological reactivity of five crotaline antivenoms for the venoms of ten Costa Rican snakes was determined. Venoms from Bothrops asper, B. godmani, B. lateralis, B. nasutus, B. ophryomegas, B. schlegelii, B. nummifer, B. picadoi, Crotalus durissus durissus and Lachesis muta stenophrys were separated by SDS-PAGE, transferred to cellulose nitrate membrane and reacted against five different antivenoms. Antisera used in the immunoblotting were prepared in rabbits to the crotaline venoms from Crotalus viridis viridis (prairie rattlesnake), Crotalus durissus terrificus (South American rattlesnake), Crotalus atrox (western diamondback rattlesnake), and Bothrops atrox (fer de lance). SDS-PAGE analysis of the ten venoms indicated that all venoms had components in the high-medium mol. wt ( > 15,000) and low mol. wt (< 15,000) range, but they all had at least twice as many components in the high-medium mol. wt range. The venoms of B. nummifer and B. nasutus have the greatest number of bands (24) whereas B. asper has the lowest (17). There appeared to be no difference in immunogenicity between high-medium mol. wt components and low mol. wt components; however, with the venoms of B. nasutus, B. ophryomegas, and B. schlegelii, there were few reactions between antivenoms and low mol. wt components. Half of the ten venoms tested had the highest reactivity with antivenom against B. atrox venom. Two venoms reacted most with antivenom against C. adamanteus venom; one with antivenom to C. atrox venom; one with antivenom to C. v. viridis venom and one with antivenom to C. d. terrificus venom. Antivenom to B. atrox and C. adamanteus venoms were the most reactive whereas antivenom to C. d. terrificus was the least reactive of the five antivenoms tested. The results also demonstrated considerable cross-reactivity between venoms from snakes in the Crotalus and Bothrops genera.

Comparison of the immunogenicity and antigenic composition of several venoms of snakes in the family Crotalidae

Crude venoms from the prairie rattlesnake ( Crotalus viridis viridis), the western diamondback rattlesnake ( Crotalus atrox), the eastern diamondback rattlesnake ( Crotalus adamanteus) and the timber rattlesnake ( Crotalus horridus horridus) were used to prepare monovalent antivenoms in rabbits. Each of these four monovalent antivenoms was reacted against six different venoms using the technique of immunoblotting (Western blot) to determine the relative immunogenicity of the four venoms and to compare the antigenic composition of six venoms. In addition to the four venoms listed above, venoms from the South American rattlesnake ( Crotalus durissus terrificus) and the fer-de-lance ( Bothrops atrox) were tested. SDS-PAGE showed that C. v. viridis venom contains the greatest number of components with 20, and the greatest number (7) less than 15,000 in mol. wt. C. durissus terrificus venom contains the least number of components, having 11. Immunoblotting experiments showed that the greatest reaction between venom and antivenom is not always obtained with the homologous system although the two greatest reactions obtained in this study were for two homologous reactions: that between monovalent anti- C. v. viridis venom and C. v. viridis venom, and that between monovalent anti- C. atrox venom and C. atrox venom. For antivenoms made to C. h. horridus and C. adamanteus venoms, the greatest reaction was obtained with C. atrox venom. There appeared to be no difference in immunogenicity between high-medium mol. wt ( > 15,000) components and low mol. wt ( < 15,000) components in all systems tested except for C. atrox venom where two low mol. wt components gave a stronger reaction with the antivenom than would have been predicted based on their relative content in the venom as indicated by SDS-PAGE. If the immunoblots are scanned with a densitometer, both the qualitative (number of bands) and the quantitative (density of bands) reactions between venom and antivenoms can be taken into consideration by using a Reactivity Index (number of bands × density of bands). By comparing Reactivity Indexes of the various reactions obtained, the most cross-reactive antivenom tested was the monovalent antivenom to C. v. viridis venom, followed by anti- C. adamanteus, anti- C. atrox and anti- C. h. horridus in order of decreasing reactivity. The Reactivity Index can also be used to estimate the reactivity of a single antivenom with different venoms. The major limitation of this approach is the difficulty in standardizing the detection procedure using silver enhanced Protein A gold.

Venom characteristics as an indicator of hybridization between Crotalus viridis viridis and Crotalus scutulatus scutulatus in New Mexico

One hundred and thirteen venoms from 46 populations of Crotalus viridis viridis were screened by immunodiffusion for protein toxins antigenically similar to the phospholipase A2 (PLA) toxin 'Mojave toxin', using a polyclonal antibody to it's basic PLA subunit. Venom i.p. LD50 values in mice were recorded from 22 of the 46 populations. The venoms of three of 14 specimens from southwest (S.W.) New Mexico and one specimen from northern Arizona were immunologically positive by the immunodiffusion tests and produced low LD50 values (0.38-0.65 mg/kg) compared to all immunologically negative venoms (0.9-5.5 mg/kg). These four specimens were morphologically typical for C. v. viridis and their venoms were the only samples of 15 southern New Mexico specimens examined by reverse phase HPLC to exhibit peaks corresponding to the acidic and basic subunits of Mojave toxin. Alkaline polyacrylamide gel electrophoresis (PAGE) analysis of the recombined subunit peaks from the C.v. viridis venom from the S.W. New Mexico specimens showed more similarity to Mojave toxin from C.s. scutulatus venom than to similar toxins in C.v. concolor venom. The combined results of the immunodiffusion, lethal toxicity tests, HPLC profiles and PAGE analysis strongly suggest that the venoms of the three New Mexico specimens contain Mojave toxin(s), as a result of some previous hybridization with C.s. scutulatus. The northern Arizona specimen likely contains 'concolor toxin' through integration with C.v. concolor in its' genetic background.

Biochemical and physiological studies on a kallikrein-like enzyme from the venom of Crotalus viridis viridis (Prairie rattlesnake)

A kallikrein-like enzyme was isolated from Crotalus viridis viridis (Prairie rattlesnake) venom by Sephadex G-50, DEAE-Sephacel and heparin-Sepharose CL-6B column chromatography. The purified enzyme has a molecular mass of 32 kDa and an isoelectric point of 5.4. The enzyme catalyzed the hydrolysis of arginine esters, kallikrein substrates ProPheArgMCA and ZPheArgMCA. The specificity of the enzyme's substrate requirement is demonstrated by the fact that no proteolytic activity was detected against either dimethyl casein or fibrinogen. The enzyme also cleaves kininogen analogs to release bradykinin. Although the enzyme induced contraction of the isolated rat uterus directly at high concentrations, more forceful contractions resulted when the reaction mixture of the enzyme and bovine plasma was applied to the uterus. The reaction mixture of 5 · 10 −11 M of the enzyme and plasma caused contractions equal to that of 10 −9 M of bradykinin. Additionally the enzyme demonstrated capillary permeability-increasing activity and hypotensive activity on the anaesthetized rat, suggesting that the enzyme releases the dilator of the wall of capillaries from plasma. Uterine contraction, capillary permeability-increasing activity and arginine esterolytic activity were inhibited by diisopropyl fluorophosphate, indicating that the serine hydroxyl group is essential for enzymatic and biological activities. It was demonstrated that the NH 2-terminal region of the enzyme has significant similarities in sequence with kallikrein-like enzymes from other snake venoms and porcine pancreatic kallikrein.

Characterization of nicked myotoxin a and its effect on the sarcoplasmic reticulum calcium pump

Myotoxin a, a muscle-necrotizing polypeptide isolated from Crotalus viridis viridis (prairie rattlesnake) venom, was nicked at Met-28 by cyanogen bromide. Amino acid analysis indicated that the methionine content was reduced to zero from the original 1 mol. Judging from circular dichroism, the nicked myotoxin a had a conformation similar to that of original myotoxin. Raman spectra indicated that the conformations of the three disulfide bonds are not affected in nicked myotoxin a. Like the original toxin, nicked myotoxin a was myotoxic and inhibited calcium ion loading activity, although the inhibitory action was slightly lower than that of the original myotoxin a. Both modified and unmodified myotoxin a showed myonecrotic activity as determined by examining histological slides. The modified toxin also inhibited the formation of decavanadate-induced two-dimensional crystalline arrays of the sarcoplasmic reticulum Ca 2+-ATPase just as the original myotoxin a does.

Characterization of the biological and immunological properties of fractions of prairie rattlesnake ( Crotalus viridis viridis) venom

C. L. Ownby and T. R. Colberg. Characterization of the biological and immunological properties of fractions of prairie rattlesnake ( Crotalus viridis viridis) venom. Toxicon 25, 1329 – 1342, 1987. — Prairie rattlesnake ( Crotalus viridis viridis) venom was separated using liquid column chromatography. The fractions were tested for biological activity in mice and for immunological reactivity against polyvalent (Crotalidae) antivenom and a monovalent antivenom to the crude venom. Several of the basic fractions and most of the non-basic fractions had hemorrhagic activity. Six of eight basic fractions had direct myotoxic activity, two of the basic fractions produced edema 30 min after injection, and two were lethal. Polyvalent antivenom contained few antibodies to the fractions of this venom, reacting with only two of the basic fractions. Monovalent antivenom formed mulitiple precipitin bands with almost all of the fractions. These results clearly demonstrate that most of the venom components are antigenic and immunogenic. Immunodiffusion using the monovalent antivenom demonstrated that C. v. viridis venom contains many antigens common to 10 other crotaline venoms. One of the hemorrhagic components was present in five of the other venoms tested, one of the direct myotoxic components was present in three other venoms, and one of the lethal components was common to two other venoms. Another highly active hemorrhagic component was common to all of the venoms tested except that of Trimeresurus flavoviridis.

Ability of a mixture of antimyotoxin a serum and polyvalent (Crotalidae) antivenin to neutralize myonecrosis, hemorrhage and lethality induced by prairie rattlesnake ( Crotalus viridis viridis) venom

Mixtures containing polyvalent (Crotalidae) antivenin and antimyotoxin a serum were tested for their ability to neutralize the myotoxic, hemorrhagic and lethal activities of crude C. v. viridis venom when mixed with the venom prior to injection into white mice. A light microscopic method was used to measure the local myotoxic activity of the venom, i.e. myonecrosis index. The results show that the neutralizing ability of a 1:1 mixture of antisera for myonecrosis was 16 times that of antimyotoxin a serum alone and 63 times that of antivenin alone. There was no difference in neutralizing ability of the three ratios (2:1, 1:1, 1:2) of antivenin: antimyotoxin serum tested. Hemorrhage was measured by a new method in which the amount of hemoglobin in a muscle extract was measured after i.m. injection of test solution. The results show that the ability of a 1:1 mixture to neutralize hemorrhage was comparable to that of antivenin alone. There was no difference in hemorrhage neutralizing ability of the three ratios tested. In its ability to neutralize lethality, the 1:1 mixture was again comparable to antivenin. However, when the three different ratios of antisera were tested for neutralization of lethality the 2:1 and 1:1 ratios were as effective as antivenin alone, whereas the 1:2 ratio (antivenin: antimyotoxin serum) was less effective than antivenin alone. Thus the addition of antimyotoxin a serum to antivenin in equal proportions greatly improves the neutralization of the myotoxic activity of C. v. viridis venom and does not decrease the ability of antivenin to neutralize hemorrhage and lethality.

In vivo test of the ability of antiserum to myotoxin a from prairie rattlesnake ( Crotalus viridis viridis) venom to neutralize local myonecrosis induced by myotoxin a and homologous crude venom

Antiserum to myotoxin a was tested for its ability to prevent local myonecrosis induced by myotoxin a and C. v. viridis venom. Antiserum was injected i.v. either 5 min before or immediately, 15 min, 30 min, 1 hr or 3 hr after i.m. injection of toxin or venom. A light microscopic method was used to measure the effects of myotoxin a, i.e. vacuolation index, and whole venom, i.e. myonecrosis index. The results show that antimyotoxin a serum neutralizes the myotoxicity of a sublethal amount of myotoxin a if injected 5 min before or immediately after toxin, but not if injected 15 min after the toxin. Its neutralizing ability for crude C. v. viridis venom was considerably better, neutralizing a dose of 0.75 μg/g even if injection of antiserum was delayed for 30 min after venom injection. Thus, antimyotoxin a serum might be useful in treating myonecrosis resulting from prairie rattlesnake ( C. v. viridis) venom poisoning.

A new method for quantitating hemorrhage induced by rattlesnake venoms: Ability of polyvalent antivenom to neutralize hemorrhagic activity

Polyvalent (Crotalidae) antivenin was tested for its ability to neutralize the hemorrhagic activity of two crotaline venoms when mixed with them prior to injection. Hemorrhage was measured by two methods. In the first method an intradermal injection of venom produced a hemorrhagic spot which was quantitated by measuring diameters. In the second method the amount of hemoglobin in a muscle extract was measured after i.m. injection of venom. The results show that both methods are useful for quantitating hemorrhage induced by Crotalus viridis viridis and Crotalus atrox venoms. Antivenin neutralized the hemorrhagic activity of 240 μg C. v. viridis venom and 120 μg C. atrox venom per 0.05 ml. The question remains, can antivenin neutralize this amount of venom when injected independently of venom.

Detection of antibodies to myotoxin a and prairie rattlesnake ( Crotalus viridis viridis) venom in three antisera using enzyme-linked immunosorbent assay and immunodiffusion

Immunodiffusion and enzyme-linked immunosorbent assay (ELISA) were used to compare three antisera for their content of antibodies against myotoxin a and C. v. virdis venom. No antibodies were detected in Wyeth's polyvalent (Crotalidae) antivenin against myotoxin a using immunodiffusion, whereas ELISA indicated a low titer of such antibodies. However, antimyotoxin a serum and anti- C. v. viridis venom both had higher titers than antivenin when tested against myotoxin a and crude venom in the ELISA. These results correlate well with previous data which indicated that antiserum to myotoxin a was more effective than antivenin in neutralizing the myotoxicity of C. v. viridis venom. The high content of antibodies to myotoxin a in anti- C. v. viridis venom supports the hypothesis that this monovalent antiserum might be effective in neutralizing rattlesnake ( C. v. viridis) venom-induced myonecrosis.

Ability of antiserum to myotoxin a from prairie rattlesnake ( Crotalus viridis viridis) venom to neutralize local myotoxicity and lethal effects of myotoxin a and homologous crude venom

Antiserum to myotoxin f isolated from prairie rattlesnake ( Crotalus viridis viridis) venom was tested for its ability to neutralize the local myotoxic and lethal activities of myotoxin a and of crude C. v. viridis venom. Wyeth's polyvalent (Crotalidae) antivenin was also tested for its ability to neutralize the crude venom. Using a light microscopic method to quantitate myonecrosis, the effect of myotoxin a, i.e. vacuolation, could be distinguished from total myonecrosis induced by whole venom. The results indicate that anti-myotoxin a serum is more effective in neutralizing local myonecrosis, but polyvalent antivenin is more effective in neutralizing lethality when they are mixed with crude prairie rattlesnake venom prior to injection.

Local Tissue Oamaging (Hemorrhage and Myonecrosis) Toxins from Rattlcsnake and Other Pit Viper Venoms

AbstractSix hemorrhagic toxins have been isolated from Crotalus atrox (western diamondback rattlesnake) venom. All of them contain one mole of zinc per mole of protein, although the molecular weights vary from 24,000 to 68,000. All have proteolytic activity when tested on dimethyl-casein. Removal of zinc eliminates the hemorrhagic and proteolytic activities. Proteolytic specificity of hemorrhagic toxin a is highly specific and hydrolyzes the X-Leu bond. Hemorrhagic toxin b is less specific and hydrolyzes the X-Phe, X-Ser, and X-Leu bonds.There are several types of myonecrotic [muscle damaging) toxins, and of those, myotoxin a from C. viridis viridis (prairie rattlesnake) venom is the most extensively investigated. Myotoxin a consists of 42 amino acid residues with 3 disulfide bonds. There is no similarity in amino acid sequence and disulfide bond positions when compared to well-known post-synaptic neurotoxins (both Type I and II) of snake venoms. It is rather curious and interesting to note that there is ...

A critical evaluation of the use of toxins from Dendro- aspis viridis to block nicotinic responses at central and ganglionic synapses

Previous work by other investigators has shown that toxins prepared from Dendroaspis viridis venom block cholinergic transmission at the neuromuscular junction, as well as nicotinic transmission in frog spinal cord and in snail neurons. This suggested that these ligands may be useful for studying nicotinic receptors in the central nervous system. Thus, Dendroaspis viridis venom was fractionated into its toxin components. Only one of the fractions possessed activity as assessed by: (1) inhibition of alpha-bungarotoxin (alpha-BGT) binding at the neuromuscular junction (25% at 50 microgram toxin/ml) or (2) inhibition of the ventral root--dorsal root potential (VR--DRP), a nicotinic response in frog spinal cord. However, in the spinal cord preparation, in addition ot this blockade of the nicotinic pathway, convulsant activity and an increase in the amplitude of other root potentials was observed. Binding experiments using [125I]dendrotoxin demonstrated that the labeled compound bound to central nervous tissue such as brain or spinal cord; this was not displaced by nicotine (10(-4) M) or D-tubocurarine (10 (-4) M), a nicotinic antagonist, indicating either non-specific binding or binding to a non-nicotinic receptor. These results thus suggest that toxins from Dendroaspis viridis venom are not suitable ligands for central nicotinic receptors. In addition, as experiments also demonstrated that the dendrotoxins did not block cholinergic transmission in frog sympathetic ganglia, it contraindicates their use at ganglionic nicotinic receptors.