Crotalus Viridis Research Articles

Evidence for isomerization in myotoxin a from the prairie rattlesnake ( Crotalus viridis viridis)

Myotoxin a, from the venom of the prairie rattlesnake, Crotalus viridis viridis, exists as a temperature-dependent equilibrium of two interconverting forms. Reverse-phase high-performance liquid chromatography (RP-HPLC) shows that the two forms interconvert slowly enough at 25 °C to be seen as two separate peaks with a molar ratio of c. 1:4. Each peak can be isolated and individually injected to give the same two peaks in the same ratio of areas. The two peaks merge during chromatography at elevated temperatures, indicating an increase in the rate of interconversion. At low temperature, c. 5 °C, the individual peaks can be isolated and maintained for several days without reaching equilibrium. Mass analysis by matrix-assisted laser desorption ionization (MALDI) time-of-flight mass spectrometry shows that myotoxin a is present in both RP-HPLC peaks, suggesting that the two resolved forms are conformational isomers. Capillary zone electrophoresis (CZE) also shows two resolved, but interconvertible peaks over a range of pH values. Furthermore, RP-HPLC chromatograms of myotoxin a at concentrations from 0.013 mM to 0.41 mM maintain a consistent ratio of peak areas, without evidence of dimerization. Two-dimensional 1H-NMR nuclear Overhauser enhancement spectroscopy indicates the presence of a cis-proline peptide bond, consistent with an equilibrium mixture of cis-trans isomers; however, addition of peptidyl-prolyl cis-trans isomerase (PPI) does not enhance the rate of equilibration of the RP-HPLC peaks isolated at c. 5 °C.

Isolation and characterization of a PLA 2 myotoxin from prairie rattlesnake (Crotalus viridis viridis) venom

Isolation and characterization of a PLA 2 myotoxin from prairie rattlesnake (Crotalus viridis viridis) venom

Characterization of the major metalloprotease isolated from the venom of Crotalus viridis oreganus

Characterization of the major metalloprotease isolated from the venom of Crotalus viridis oreganus

Crotavirin, a potent platelet aggregation inhibitor purified from the venom of the snake Crotalus viridis

A potent platelet aggregation inhibitor in the venom of Crotalus viridis snake was purified to homogeneity by gel filtration chromatography and reverse phase high-performance liquid chromatography. This purified principle, named crotavirin, is a single-chain polypeptide with a mol. wt of 9200 as estimated by SDS-polyacrylamide gel electrophoresis. It inhibited the aggregation of human washed platelets induced by collagen, thrombin and thomboxane analogue (U46619) with a similar IC 50 (~ 1.0 μg/ml, 0.11 μM). The binding of fluorescein isothiocyanate-conjugated crotavirin to platelets was abolished in the presence of divalent cation chelator, EDTA, indicating that divalent cation is essential for crotavirin's binding. A monoclonal antibody, 7E3, raised against platelet glycoprotein IIb-IIIa complex blocked the binding of fluorescein isothiocyanate-conjugated crotavirin to platelets, whereas the other monoclonal antibody against glycoprotein IIb-IIIa, 10E5, had no inhibitory effect. In addition, crotavirin inhibited in a concentration-dependent manner the binding of fluorescein isothiocyanate-conjugated rhodostomin, a member of the disintegrin family, to platelets. Its binding to platelets was blocked by disintegrins e.g. trigramin and rhodostomin. It is concluded that crotavirin is a potent platelet aggregation inhibitor, which acts specifically on an epitope of glycoprotein IIb-IIIa, leading to the blockade of fibrinogen binding to glycoprotein IIb-IIIa and eventually the blockade of platelet aggregation.

Calsequestrin is essential for the Ca2+ release induced by myotoxin alpha in skeletal muscle sarcoplasmic reticulum.

Myotoxin alpha (MYTX), a polypeptide toxin purified from the venom of prairie rattlesnakes (Crotalus viridis viridis), induced Ca2+ release from the heavy fraction of skeletal sarcoplasmic reticulum (HSR), using a Ca2+ electrode. The effect of MYTX was nearly abolished by pretreatment with ryanodine, an alkaloid-based Ca2+ channel blocker. In the stopped-flow experiments, MYTX increased the choline+ permeability of HSR in the presence of calsequestrin (CS). Single channel recording experiments showed that in the presence of CS, the channel currents were markedly enhanced by MYTX applied to the cis side, but not to the trans side. However, in the absence of CS, MYTX failed to cause the excitatory effect in both the experiments. These results suggest that CS is essential for MYTX-induced Ca2+ release through the Ca2+ release channels in skeletal HSR.

Venom metering by juvenile prairie rattlesnakes, Crotalus v. viridis: effects of prey size and experience

Despite contradictory evidence, it is widely believed that venomous snakes carefully control, or ‘meter’, the quantities of venom expended when feeding upon rodent prey. The major purpose of this study was to clarify experimentally whether juvenile prairie rattlesnakes inject more venom into larger mice than smaller mice. The subjects ( N=7) were videotaped as they struck at small, medium and large mice in each of two separate trials. The quantity of venom expended was measured by enzyme-linked immunosorbent assay (ELISA) of whole-animal homogenate. In the first (‘naive’) trial, the snakes injected similar quantities of venom into all size classes of prey. But in the second (‘experienced’) trial, the snakes injected significantly more venom into larger prey. No other aspect of striking varied among prey sizes or changed in the two trials. Thus, venom expenditure was probably not a consequence of, or constrained by, some extrinsic aspect of striking, such as duration of fang contact. More likely, the rattlesnakes, with experience, attempted to inject (or meter) more venom into larger prey through intrinsic control of venom delivery. Both natural (snakebite) and artificial (syringe) envenomations indicated that mice injected with larger quantities of venom died more quickly. Because larger prey succumb less rapidly to venom, metering more venom into larger prey may be an adaptive strategy for immobilizing and killing large prey more quickly.

Intracapsular Asexual Proliferation of Mesocestoides sp. Tetrathyridia in the Gastrointestinal Tract and Mesenteries of the Prairie Rattlesnake (Crotalus viridis viridis)

Nonproliferous and proliferous tetrathyridia (Mesocestoides sp.) have been reported from natural and experimental infections in reptiles and rodents. Multiple tetrathyridia (1+) in individual capsules (host-derived) were observed for both types of tetrathyridia and in both types of animals. To determine whether proliferous tetrathyridia in this genus could replicate in host capsules of the prairie rattlesnake (Crotalus viridis viridis), 11 snakes were inoculated and maintained in 2 environmental chambers (25 C and 30 C). One snake at each temperature was removed at 4-wk intervals and killed. Sections of the gastrointestinal tract and adjacent mesenteries were histologically examined. The increase in the numbers of tetrathyridia per capsule was significant at both 25 C (P = 0.007) and 30 C (P < 0.001). Identification of 7 intracapsular tetrathyridia with binary scoleces served as collaborative evidence. This paper presents the first significant evidence for asexual replication of the proliferous tetrathyridia (Mesocestoides sp.) in a reptilian host.

Mass spectrometric immunoassay.

A new, general method of immunoassay is demonstrated. The approach is based on the microscale immunoaffinity capture of target antigens followed by mass-specific identification and quantitation using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Immunoaffinity capture of antigens effectively overcomes signal suppression effects typically encountered during traditional matrix-assisted laser desorption/ionization analysis of complex biological mixtures while simultaneously concentrating the analyte into a small volume. Mass spectrometric detection of antigens is unambiguous, as antigen signals are observed at characteristic mass-to-charge values in the mass spectrum, offering a high level of immunity to artifacts due to nonbiospecific retention of mixture components. However, the most important aspect of such mass-specific detection is the ability to use a single assay to screen biological systems for the presence of multiple, mass-resolved antigens. Analyte quantitation is possible by using a single antibody to capture both the antigen and an antigen variant which has been chemically modified to have a different mass. With proper calibration, the relative signal intensities of the two species in the mass spectrum can be used to determine the antigen concentration. Sample incubation and processing methods were such that a typical analysis could be performed in less than 1 h while subnanomolar sensitivities were maintained. The technique has been used for the rapid, selective, and quantitative screening of human blood for the presence of myotoxin a, and Mojave toxin form the venoms of the prairie rattlesnakes, Crotalus viridis viridis, and and the Mojave rattlesnake, Crotalus scutulatus scutulatus.

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.

Characterization of a reovirus isolate from a rattle snake, Crotalus viridis, with neurological dysfunction.

A virus isolate from the brain of a rattle snake with central nervous system (CNS) symptoms was identified as a reovirus. The isolate did not agglutinate pig erythrocytes and was not neutralized by antisera against avian reovirus S1133 and mammalian reovirus type 3. The cytopathic effect in Vero cells was characterized by formation of syncytial giant cells. Electrophoretic analysis of the genome revealed 10 segments of dsRNA. The isolate displayed characteristics distinct from avian and mammalian reoviruses.

Ca2+ release induced by myotoxin a, a radio‐labellable probe having novel Ca2+ release properties in sarcoplasmic reticulum

1. Myotoxin alpha (MYTX), a polypeptide toxin purified from the venom of prairie rattlesnakes (Crotalus viridis viridis) induced Ca2+ release from the heavy fraction (HSR) but not the light fraction of skeletal sarcoplasmic reticulum at concentrations higher than 1 microM, followed by spontaneous Ca2+ reuptake by measuring extravesicular Ca2+ concentrations using the Ca2+ electrode. 2. The rate of 45Ca2+ release from HSR vesicles was markedly accelerated by MYTX in a concentration-dependent manner in the range of concentrations between 30 nM and 10 microM, indicating the most potent Ca2+ releaser in HSR. 3. The Ca2+ dependency of MYTX-induced 45Ca2+ release has a bell-shaped profile but it was quite different from that of caffeine, an inducer of Ca(2+)-induced Ca2+ release. 4. 45Ca2+ release induced by MYTX was remarkable in the range of pCa between 8 and 3, whereas that by caffeine was prominent in the range of pCa, i.e., between 7 and 5.5. 5. MYTX-induced 45Ca2+ release consists of both early and late components. The early component caused by MYTX at low concentrations (30-300 nM) completed within 20 s, while the late component induced by it at higher concentrations (> 0.3 microM) was maintained for at least 1 min. 6. Both the components were almost completely inhibited by inhibitors of Ca2+ such as Mg2+, ruthenium red and spermine. 7. 45Ca2+ release induced by caffeine or beta,gamma-methyleneadenosine 5'-triphosphate (AMP-PCP) was completely inhibited by high concentrations of procaine. Procaine abolished the early component but not the late one, suggesting that at least the early component is mediated through Ca(2+)-induced Ca2+ release channels. 8. On the basis of these results, the character of Ca2+ release induced by MYTX was quite different from that caused by caffeine or AMP-PCP, suggesting that MYTX induces Ca2+ release having novel properties in HSR. MYTX is the first polypeptide Ca2+ inducer and has become a useful pharmacological tool for clarifying the mechanism of Ca2+ release from skeletal muscle SR.

Presence of heat-stable hemorrhagic toxins in snake venoms

Twenty-eight snake venoms (seven Agkistrodon venoms, six Bothrops venoms, 13 Crotalus venoms, one Sistrurus venom, and one Bitis venom) were examined for the presence of heat-stable (100°C, 5 min) hemorrhagic toxins. Both heated and unheated venoms were analyzed for their ptotein composition by SDS-PAGE, and tested for their hemorrhagic activity in vivo in mice and for their proteolytic activity on two different substrates. Heating all venoms led to the denaturation and loss of some proteins; however, most of the venoms retained a significant number of proteins. Seventeen venoms contained more than seven proteins after heating, whereas five venoms contained only one to three proteins. All but nine of the heated venoms had substantial hemorrhagic activity, and Agkistrodon piscivorus piscivorus venom had very high activity, almost four times that of the second most hemorrhagic venom from Crotalus viridis lutosus. Most venoms, heated or unheated, had activity on the two protease substrates. Using succinylated casein as the substrate, there was a wide range of activity, and heating drastically reduced the activity of all venoms except that of Crotalus ruber and Crotalus molossus molossus. With azocoll as substrate, all but two of the unheated venoms ( Crotalus adamanteus and Crotalus viridis concolor) had very high activity, whereas upon heating, all except five venoms lost essentially all of their activity. Heated venoms from snakes in the Agkistrodon genus (except for Agkistrodon blomhoffi blomhoffi, an Asian snake) retained activity on azocoll, and this activity tended to correlate better with hemorrhagic activity of the venom than did proteolytic activity on casein.

Calsequestrin is a major binding protein of myotoxin α and an endogenious Ca 2+ releaser in sarcoplasmic reticulum

Myotoxin α from prairie rattlesnakes induced Ca 2+ release from the heavy fraction of sarcoplasmic reticulum at submicromolar concentrations. 125I-Labeled myotoxin α ( 125I-myotoxin α) specifically bound to the heavy fraction. Fractionation of the solubilized heavy fraction with a spermine-agarose column gave purified calsequestrin as a major binding protein of 125I-myotoxin α. We have first indicated that calsequestrin is a target protein for Ca 2+-releasing action of myotoxin α. Calsequestrin probably plays a key role in physiological Ca 2+ release from sarcoplasmic reticulum.

Myotoxin a reduces the threshold for calcium-induced calcium release in skeletal muscle

M. L. Yudkowsky, J. Beech and J. E. Fletcher. Myotoxin a reduces the threshold for calcium-induced calcium release in skeletal muscle. Toxicon 32, 273–278, 1994.—Myotoxin a, isolated from the venom of the prairie rattlesnake Crotalus viridis viridis, induces necrosis in skeletal muscle. In isolated organelles, it has been reported that myotoxin a reduces Ca 2+ uptake into the sarcoplasmic reticulum. The effects of the toxin on Ca 2+ regulation were examined in heavy sarcoplasmic reticulum fractions from human and equine skeletal muscle. Ca 2+ uptake and release (the threshold of Ca 2+-induced Ca 2+ release) were examined by dual wavelength spectrophotometry. The toxin lowered the threshold of Ca 2+-induced Ca 2+ release in a dose-dependent manner (1–10 μM) and this effect was antagonized by ruthenium red, a Ca 2+ release channel blocker. Ca 2+ uptake into equine heavy sarcoplasmic reticulum was not decreased by myotoxin a (10 μM) when Ca 2+ release was blocked by ruthenium red. [ 3H]Ryanodine binding to equine heavy sarcoplasmic reticulum was converted from a relatively low affinity state to a higher affinity state by myotoxin a. These results suggest that the dominant effect of myotoxin a is to increase the Ca 2+ sensitivity for the opening of the calcium release channel (ryanodine receptor). Myotoxin a may prove to be a useful tool to probe the modulation of calcium release in sarcoplasmic reticulum fractions.

Aging in snakes: Results of long‐term captivity on rattlesnake (Crotalus viridis oreganus) predatory behavior

AbstractConservation, scientific study, rehabilitation, and public education concerning animals often require or take advantage of captive individuals. The long‐term effects of captivity may affect the success and strategies used in such programs. For reptiles in particular, little is known about the effects of long‐term captivity. We compared the predatory behavior of the same eight Northern Pacific Rattlesnakes before and after 7 years in captivity. We found few changes in predatory behavior. Most changes were in the rate of tongue flicks before the strike and during recovery of prey; these changes seemed related to accommodation of the snakes to conditions of captivity. However, most aspects of rattlesnake predatory behavior remained unchanged, including the normal ability to target the prey accurately and kill it quickly. Although the snakes had spent over half their lives in captivity and reached a relatively old age, their ability to strike, dispatch, and relocate prey normally was relatively unaffected by captivity or by aging. © 1994 Wiley‐Liss, Inc.

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.

Fibrinogenolytic proteases from the venoms of juvenile and adult northern pacific rattlesnakes ( Crotalus viridis oreganus)

1. 1. Venoms of Crotalus viridis oreganus show marked ontogenetic variation in protease activity. Adult venoms are approximately five-fold higher in protease (caseinolytic) activity. 2. 2. Of seven potential protease inhibitors, only EDTA and 1,10-phenanthroline caused a significant decrease in protease activity. Responses of juvenile and adult venoms were essentially equivalent, and attempts at recovery of protease activity of EDTA-treated venoms by the addition of Ca 2+ or Zn 2+ were unsuccessful. 3. 3. Gel filtration resolved two proteases from juvenile and subadult venoms with approximate M r of 100,000 and 78,000. Four proteases were resolved from adult venom, and M r estimates were 78,000, 61,000, 35,000 and 19,000. 4. 4. Proteases from juvenile and adult venoms showed fibrinogenolytic activity, each producing some unique degradation products. 5. 5. The occurrence of three “new” proteases in adult venom produced the ontogenetic increase in activity seen in the crude venoms.

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.

Interaction of Sarcoplasmic Reticulum Membranes with MyotoxinAIsolated fromCrotalus viridis viridisVenom Studied by Raman Spectroscopy

Myotoxin a is a muscle-degenerating toxin isolated from the venom of Crotalus viridis viridis (prairie rattlesnake). It is composed of 42 amino acid residues with four disulfide bonds. The presence of myotoxin a and related myotoxins is common in various snake venoms. It was found recently that myotoxin a is an uncoupler of calcium ion influx in sarcoplasmic reticulum (SR). Myotoxin a is known to interact with artificial membranes. Since the biological action of myotoxin a affects the calcium pump that is present in the SR membranes, it is of interest to investigate the interaction of SR membrane with myotoxin a by Raman spectroscopy. Raman spectroscopy is particularly useful in membrane study because the C-C and C-H stretching vibrational bands are particularly sensitive in phospholipid phase transition from gel to liquid crystal.

Community Trophic Structure: The Roles of Diet, Body Size, and Activity Time in Vertebrate Predators

We examined trophic structure in an assemblage of 17 vertebrate predators - two snakes (Pituophis catenifer and Crotalus viridis), six hawks (Circus cyaneus, Buteo jamaicensis, B. regalis, Aquila chrysaetos, Falco sparverius, and F. mexicanus), six owls (Tyto alba, Otus kennicottii, Bubo virginianus, Speotyto cunicularia, Asio otus, and Aegolius acadicus), one passerine (Corvus corax), and two mammals (Canis latrans and Taxidea taxus). These predators fed collectively on prey in nine taxonomic classes. Mammalian prey were numerically most important in 15 diets and contributed the most biomass to all 17 diets. Mean prey mass ranged from 2.2 to 711 g, and correlated with predator mass. No significant differences were apparent in food-niche breadth or mean prey mass between diurnal and nocturnal predators, but dietary overlap among predators with the same diel activity was significantly greater than that among asynchronously-active predators. Mean diet overlap was significantly greater among nocturnal predators than among diurnal predators. Four feeding guilds were defined by dietary similarity. A ground squirrel-eating guild contained one snake, three hawks, and a mammal. A jackrabbit-eating guild consisted of a hawk and a mammal. An arthropod/mammal-eating guild was composed of an owl and the raven. Four owls, one hawk, and one snake formed a small-rodent guild. One owl and one hawk were not members of guilds. Mass ratios of adjacent-sized predators were greatest within guilds.