Cardiotoxicity, inflammation, and immune response after rattlesnake envenomation in the horse.

BACKGROUND: Cardiac abnormalities are reported in rattlesnake-bitten horses. The prevalence and cause are unknown. OBJECTIVES: To detect cardiac damage in rattlesnake-bitten horses by measuring cardiac troponin I (cTnI) and evaluating ECG recordings for presence of arrhythmias, and explore causes of this cardiac damage by measuring venom excretion, anti-venom antibodies, and tumor necrosis factor alpha (TNFα). ANIMALS: A total of 20 adult horses with a clinical diagnosis of rattlesnake bite and 6 healthy adult horses. METHODS: In a prospective clinical study, bite site swabs, blood samples, and urine samples were collected at various time points from 20 horses with a clinical diagnosis of snake bite. Continuous ECG recordings were obtained on the 20 affected horses and 6 normal control horses using 24-hour holter monitors. Plasma samples were assayed for cTnI, serum samples were assayed for TNFα and anti-venom antibodies, and bite site swabs and urine were assayed for venom. RESULTS: Forty percent of rattlesnake-bitten horses (8/20) experienced myocardial damage (increased cTnI). Seventy percent (14/20) experienced a cardiac arrhythmia. There was a positive correlation between cTnI and TNFα (P < .02). Horses with cTnI ≥ 2 ng/mL were more likely to have antibody titers >5,000 (P < .05). No correlations were found between venom concentration and cTnI, anti-venom antibody titers, TNFα, or presence of arrhythmias. CONCLUSIONS AND CLINICAL IMPORTANCE: Cardiac abnormalities in this population of horses indicate that cardiac damage after rattlesnake bite is common. Rattlesnake-bitten horses should be monitored for signs of cardiac damage and dysfunction. Long-term follow-up should be encouraged to detect delayed cardiac dysfunction.

Transrectal ultrasound-integrated spectral optical tomography of hypoxic progression of a regressing tumor in a canine prostate.

The objective of this study was to evaluate if transrectal optical tomography implemented at three wavelength bands for spectral detection could monitor changes of the hemoglobin oxygen saturation (StO2) in addition to those of the total hemoglobin concentration ([HbT]) in lesions of a canine prostate, including an induced tumor modeling canine prostate cancer. Near-infrared (NIR) optical tomography was integrated with ultrasound (US) for transrectal imaging. Multi-spectral detection at 705_nm, 785_nm and 808_nm rendered measurements of [HbT] and StO2. Canine transmissible venereal tumor (TVT) cells were injected into the right lobe of a dog's prostate gland, which had a pre-existing cyst in the left lobe. Longitudinal assessments of the prostate were performed weekly over a 63-day duration by NIR imaging concurrent with grey-scale and Doppler US. Ultrasonography revealed a bi-lobular tumor-mass regressing from day-49 to day-63. At day-49 this tumor-mass developed a hypoxic core that became larger and more intense by day-56 and expanded further by day-63. The tumor-mass presented a strong hyper-[HbT] feature on day-56 that was inconsistent with US-visualized blood flow. Histology confirmed two necrotic TVT foci within this tumor-mass. The cyst appeared to have a large anoxic-like interior that was greater in size than its ultrasonographically delineated lesion, and a weak lesional elevation of [HbT]. On day-56, the cyst presented a strong hyper-[HbT] feature consistent with US-resolved blood flow. Histology revealed acute and chronic hemorrhage in the periphery of the cyst. The NIR imaging features of two other TVT nodules and a metastatic lymph node were evaluated retrospectively. Transrectal US-integrated spectral optical tomography seems to enable longitudinal monitoring of intra-lesional oxygenation dynamics in addition to the hemoglobin content of lesions in the canine prostate.

Effects of ACL myotoxin, a Lys49 phospholipase A(2) from Agkistrodon contortrix laticinctus snake venom, on water transport in the isolated toad urinary bladder.

ACL myotoxin (ACLMT) is a Lys49 phospholipase A(2)-like protein isolated from the venom of the snake Agkistrodon contortrix laticinctus. The aim of this work was to study the effect of ACLMT on water transport in the toad bladder. Water flow through the membrane was measured gravimetrically in bag preparations of the bladder. ACLMT (20 nM) increased the baseline water flow and partially inhibited arginine-vasopressin (AVP), 8-chlorophenylthio-cAMP (8-CPT-cAMP) and forskolin-stimulated water flow. The effect of ACLMT on baseline or AVP-stimulated water flow was prevented by lanthanum (0.1 mM) indicating that the effect of ACLMT on water transport may be mediated through an increase in intracellular calcium. The effect of ACLMT on baseline water flow was also prevented by nifedipine (0.1 mM) indicating the participation of exogenous calcium in this effect. Carbachol (0.1 mM) has been shown to enhance baseline water flow while inhibiting AVP-stimulated water flow. The effects of ACLMT and carbachol on baseline water flow and AVP-stimulated water flow were not additive, suggesting that both agents alter water transport by a similar mechanism. Indomethacin (10 microM) reduced the effect of ACLMT on forskolin-stimulated water flow, suggesting an increase in prostaglandin biosynthesis. These results suggest that the effects of ACLMT on water transport may be mediated by increasing intracellular calcium and stimulation prostaglandin biosynthesis.

Effects of Mannheimia haemolytica leukotoxin on apoptosis and oncosis of bovine neutrophils.

OBJECTIVE: To investigate the concentration-dependent effects of Mannheimia haemolytica (formerly Pasteurella haemolytica) leukotoxin (LKT) on apoptosis and oncosis in bovine neutrophils and to examine the role of calcium ions (Ca2+) in LKT-induced apoptosis. SAMPLE POPULATION: Neutrophils isolated from blood samples obtained from healthy calves. PROCEDURE: Neutrophil suspensions were exposed to lytic or sublytic dilutions of LKT and then examined by use of transmission electron microscopy (TEM) or gel electrophoresis. Contribution of extracellular Ca2+ to LKT-induced apoptosis was investigated by incubating neutrophils with LKT or control solutions in buffer containing 1 mM CaCl2 or in Ca2+-free buffer containing 1 mM ethylene glycol-bis (b-aminoethyl ether)-N,N-tetraacetic acid (EGTA) prior to diphenyl amine analysis. RESULTS: Examination by TEM revealed that bovine neutrophils exposed to lytic dilutions of LKT had changes consistent with oncosis, whereas neutrophils exposed to sublytic dilutions of LKT and staurosporin, an inducer of apoptosis, had changes consistent with apoptosis. Effects of sublytic dilutions of LKT on apoptosis were confirmed by gel electrophoresis. Replacement of extracellular Ca2+ with EGTA, a Ca2+ chelator, reduced apoptosis attributable to the calcium ionophore A23187, but it did not have significant effects on apoptosis induced by LKT or staurosporin. CONCLUSIONS AND CLINICAL RELEVANCE: The ability of LKT to cause apoptosis instead of oncosis is concentration-dependent, suggesting that both processes of cell death contribute to an ineffective host-defense response, depending on the LKT concentration in pneumonic lesions. Furthermore, although Ca2+ promotes A23187-induced apoptosis, it is apparently not an essential second messenger for LKT-induced apoptosis.

Expression, refolding, and activity of a recombinant nonhemorrhagic snake venom metalloprotease.

Snake venoms are rich sources of proteases that strongly affect the vascular system, by promoting blood coagulation, hemorrhage, and fibrinolysis. Hemorrhagic activity is mostly due to the enzymatic action of metalloproteases on capillary basement membrane components, such as collagen IV, laminin, and fibronectin. A few low-molecular-weight snake venom metalloproteases (svMP) have been described as being devoid of hemorrhagic activity, but they have strong direct-acting fibrinolytic activity that could be very helpful in thrombosis therapy. We have developed an expression system for production of a recombinant svMP from a cDNA (ACLPREF) coding for a small metalloprotease (ACLF) with three disulfide bonds from an Agkistrodon contortrix laticinctus (broad-banded copperhead) venom gland cDNA library. The mature protein-coding region was amplified by PCR and subcloned into the pET28a vector, and the resulting plasmid was used to transform BL21(DE3) Escherichia coli cells. Culture of the transformants at either 37 or 20 degrees C led to the overexpression of an insoluble and inactive 30-kDa protein after 1.0 mM IPTG induction. The expressed protein (rACLF) was recovered from inclusion bodies with 6 M buffered urea solution and purified on a nickel-Sepharose column followed by gel filtration chromatography, both under denaturing conditions. After treatment with dithiothreitol, protein refolding was performed by gradual removal of the denaturing agent by dialysis. The refolded recombinant protein was active in fibrin-agarose plates. The purified protein achieved a conformation similar to that of the native enzyme as judged by circular dichroism analysis.

The venomous hair structure, venom and life cycle of Lagoa crispata, a puss caterpillar of Oklahoma.

The presence of a unique population of Lagoa crispata, puss caterpillar, in western Oklahoma is reported. A detailed microscopic examination shows the structure of the L. crispata spines resemble the type 4 spines described by [Kawamoto, F., Kumada, N., 1984. Biology and venoms of lepidoptera. In: Tu, A.T. (Ed.), Handbook of Natural Toxins, Insect Poisons, Allergens and other invertebrate venoms, vol. 2, pp. 291-332 (ch. 9)]. The major food source of L. crispata are the leaves of oak (shin oak). The high tannin content of this food source results in spine extracts high in oak tannins. These extracts have activity but enzyme and toxin activity is lost with time. The gel filtration protein fractions are colored from brown to yellow and are inactive as enzymes or toxins. No hyaluronidase, protease or phosphohydrolase activity is detected in these protein fractions. The life cycle shows these caterpillars have 6 instars. Characterizations and annual emerging times of each instar are included.

Ultrastructural characterization of apoptosis in bovine lymphocytes exposed to Pasteurella haemolytica leukotoxin.

OBJECTIVE: To characterize ultrastructural changes of bovine lymphocytes exposed to Pasteurella haemolytica leukotoxin (LKT). SAMPLE POPULATION: Partially purified LKT from a wild type P. haemolytica A1 strain and inactive pro-LKT from an isogeneic mutant Phaemolytica strain. Isolated bovine lymphocytes were obtained from 2 healthy calves. PROCEDURE: Isolated bovine lymphocytes were incubated with various concentrations of LKT and pro-LKT for 3 hours at 37 C and examined by use of transmission electron microscopy. A cytochemical Klenow DNA fragmentation assay was used to examine lymphocytes for DNA fragmentation. RESULTS: Lymphocytes incubated with LKT at a high concentration (1.0 toxic U/ml) had ultrastructural evidence of cytoplasmic and nuclear membrane rupture and swelling or lysis of mitochondria. Low concentrations of leukotoxin (0.1 toxic U/ml) induced DNA fragmentation in 80% of lymphocytes. Ultrastructurally, these cells had nuclear membrane blebbing, cytoplasmic vaculation, chromatin condensation, nuclear fragmentation, and membrane-bound apoptotic bodies. Incubation of lymphocytes with LKT at extremely low concentrations (0.001 toxic U/ml) or with pro-LKT did not alter their ultrastructure. Inclusion of 0.5 mM ZnCl2 in the medium blocked leukotoxin-induced ultrastructural changes in bovine lymphocytes. CONCLUSIONS AND CLINICAL RELEVANCE: Low concentrations of LKT induce apoptosis and high concentrations induce oncotic cell lysis in bovine lymphocytes. The ability of low LKT concentrations to induce apoptosis in host leukocytes may allow bacteria to escape host immune surveillance and colonize the host.

Long-term regeneration of fast and slow murine skeletal muscles after induced injury by ACL myotoxin isolated from Agkistrodon contortrix laticinctus (broad-banded copperhead) venom.

The aim of the present work was to analyze the regenerated muscle types I and II fibers of the soleus and gastrocnemius muscles of mice, 8 months after damage induced by ACL myotoxin (ACLMT). Animals received 5 mg/kg of ACLMT into the subcutaneous lateral region of the right hind limb, near the Achilles tendon; contralateral muscles received saline. Longitudinal and cross sections (10 microm) of frozen muscle tissue were evaluated. Eight months after ACLMT injection, both muscle types I and II fibers of soleus and gastrocnemius muscles still showed centralized nuclei and small regenerated fibers. Compared with the left muscle, the incidence of type I fibers increased in the right muscle (21% +/- 03% versus 12% +/- 06%, P = 0.009), whereas type II fibers decreased (78% +/- 02% versus 88% +/- 06%, P = 0.01). The incidence of type IIC fibers was normal. These results confirm that ACLMT induced muscle type fiber transformation from type II to type I, through type IIC. The area analysis of types I and II fibers of the gastrocnemius revealed that injured right muscles have a higher percentage of small fibers in both types I and II fibers (0-1,500 microm2) than left muscles, which have larger normal type I and II fibers (1,500-3,500 microm2). These results indicate that ACLMT can be used as an excellent model to study the rearrangement of motor units and the transformation of muscle fiber types during regeneration.

Lysine 49 phospholipase A2 proteins.

The structures of several K49 PLA2 proteins have been determined and these differ as a group in several regions from the closely related D49 PLA2 enzymes. One outstanding difference is the presence of a high number of positively charged residues in the C-terminal region which combined with the overall high number of conserved lysine residues gives the molecule an interfacial adsorption surface which is highly positively charged compared to the opposite surface of the molecule. Although some nucleotide sequences have been reported, progress in obtaining active recombinant proteins has been slow. The K49 proteins exert several toxic activities, including myotoxicity, anticoagulation and edema formation. The most studied of these activities is myotoxicity. The myotoxicity induced by the K49 PLA2 proteins is histologically similar to that caused by the D49 PLA2 myotoxins, with some muscle fiber types possibly more sensitive than others. Whereas it is clear that the K49 PLA2 myotoxins lyse the plasma membrane of the affected muscle cell in vivo, the exact mechanism of this lysis is not known. Also, it is not known whether the toxin is internalized before, during or after the initial lysis or ever. The K49 PLA2 toxins lyse liposomes and cells in culture and in the latter, the PLA2 myotoxins exert at least two distinct mechanisms of action, neither of which is well-characterized. While the K49 PLA2 proteins are enzymatically inactive on artificial substrates, the toxins cause fatty acid production in cell cultures. Whether the fatty acid release is due to the enzymatic activity of the K49 PLA2 or stimulation of tissue lipases, is unknown. While there may be a role for fatty acid production in one mechanism of myotoxicity, a second mechanism appears to be independent of enzymatic activity. Although we are beginning to understand more about the structure of these toxins, we still know little about the precise mechanism by which they interact with the skeletal muscle cell in vivo.

Neutralization of edema, hemorrhage and myonecrosis induced by North American crotalid venoms in simulated first-aid treatments.

Venoms of the broad-banded copperhead (Agkistrodon contortrix laticinctus, ACL) and the prairie rattlesnake (Crotalus viridis viridis, CVV), like other crotalid venoms, cause severe local tissue damage such as edema, hemorrhage and myonecrosis. Antivenom therapy is not very effective in neutralizing this local tissue damage, and such observations support the need for an effective first-aid regimen aimed at minimizing local tissue reactions. Some of the local tissue damage induced by these venoms is due to phospholipase A2 myotoxins, and since para-bromophenacyl bromide (p-BPB), an inhibitor of PLA2 catalytic activity, has been shown to inhibit the myotoxic action of two PLA2 myotoxins, we hypothesized that this compound would inhibit part of the myotoxic activity of these crude venoms. For in vitro neutralization experiments, venoms were mixed with combinations of either p-BPB, antivenom or both prior to injection into the muscles of the lower hindlimb of mice. For in vivo neutralization experiments, mice were injected with venom followed by either topical DMSO containing p-BPB or intramuscular injection with saline containing p-BPB. A final set of mice received these same injections followed by i.p. infusions of antivenom to simulate experimental first-aid followed by hospital treatment. In the in vitro neutralization tests, edema was significantly reduced when both antagonists were used together, and there was a highly significant neutralization of ACL- and CVV-generated myonecrosis. In the in vivo neutralization experiments, hemorrhage was significantly reduced when injection of ACL venom was followed by topical DMSO-p-BPB, and myonecrosis was reduced when injection of ACL venom was followed by intramuscular injection of saline-p-BPB. Antivenom significantly reduced edema, hemorrhage and myonecrosis induced by CVV venom, but reduced only myonecrosis induced by ACL venom. Taken together, these results suggest a role for pBPB in the first-aid treatment of snakebite especially when followed by hospital treatment with antivenom.

Ability of wedelolactone, heparin, and para-bromophenacyl bromide to antagonize the myotoxic effects of two crotaline venoms and their PLA2 myotoxins.

We examined the ability of wedelolactone, heparin and para-bromophenacyl bromide to antagonize the myotoxic activity in mice of venoms from Crotalus viridis viridis and Agkistrodon contortrix laticinctus and two phospholipase A2 myotoxins, CVV myotoxin and ACL myotoxin, isolated from them. Myotoxicity was measured by the increase in plasma creatine kinase (CK) activity at two hours and histological changes in extensor digitorum longus muscle (EDL) at three hours after injection of the test solution. Both heparin and wedelolactone independently reduced the myotoxic effect of both crude venoms and both myotoxins, but wedelolactone was more effective. Wedelolactone plus heparin reduced the myotoxic effect of CVV myotoxin more than either antagonist alone. The PLA2 inhibitor, para-bromophenacyl bromide (pBPB), reduced the myotoxic effect of both myotoxins more than either wedelolactone or heparin. On the other hand, the myotoxic effect of polylysine was not reduced by either wedelolactone or para-bromophenacyl bromide, but it was reduced by heparin. These results indicate that wedelolactone, para-bromophenacyl bromide and heparin are antagonists of these two phospholipase A2 myotoxins, and that antagonism by the first two compounds may be due to a more specific interaction with these proteins than that by the latter.

Citrate inhibition of snake venom proteases.

Thirty snake venoms had a citrate content of 2.3 to 12.9%, dry basis, by an aconitase isocitric dehydrogenase coupled enzyme assay. This is a venom concentration range of approximately 30 to 150 mM citrate assuming 25% venom solids content. Inhibition of snake venom protease activity by the addition of exogenous citrate was obtained using azure blue hide powder and azocasein as substrates. Protease inhibitions of 7.5% for Crotalus atrox venom to 78% for Bothrops picadoi venom were observed with citrate. Complete inhibition of snake venom protease activity by citrate was not observed. Bothrops asper (Pacifico) venom showed a 41% protease inhibition by citrate with azocasein as the substrate and 46% inhibition of Bothrops asper (Alantico) venom protease with azure blue hide power as a substrate. Trypsin was not inhibited in this system. Citrate may inhibit some venom protease activity by forming a complex with the zinc of zinc-dependent enzymes. reserved.

Injury and recovery of fast and slow skeletal muscle fibers affected by ACL myotoxin isolated from Agkistrodon contortrix laticinctus (Broad-Banded copperhead) venom.

The response of different types of skeletal muscle fibers to a snake venom PLA2 myotoxin was tested in vivo by injecting ACL myotoxin (ACLMT) into mice. Both the soleus (slow-twitch) and gastrocnemius (fast-twitch) were examined at different time periods (3 h, 3 and 21 d) after the injection. All animals received 5 mg/kg myotoxin into the subcutaneous lateral region of the right hind limb, near the Achilles tendon; contralateral muscles were used as controls. Cross-sections (10 microm) of frozen muscle tissue were cut from the medial region of the muscle. Alternate serial sections were stained either with toluidine blue or for acid phosphatase, myofibrillar ATPase activity after alkali (pH 10.3) or acid preincubation (pH 4.3), succinate dehydrogenase or acetylcholinesterase. Several stages of necrosis were observed 3 h after ACLMT injection, in both superficial and deep regions of both muscles. In these same regions 3 d after injection, clusters of regenerated muscle fibers were present, and some of them presented AChE activity. Twenty-one days after ACLMT injection the muscle fibers of soleus and gastrocnemius presented only chronic signs of damage such as split fibers and centralized nuclei. Using m-ATPase reactions it was possible to determine that both muscle fiber types I and II were injured in both muscles. The number of type IIC fibers was significantly increased, and the number of type II fibers significantly decreased in the gastrocnemius 21 d after ACLMT injection, suggesting a change in muscle fiber type from type II to type I, through type IIC. The increased number of type IIC fibers and the presence of AChE activity in clusters of regenerating fibers and split fibers indicate that injury by ACLMT produces axonal remodeling and muscle fiber type change.

Analysis of a cDNA sequence encoding a novel member of the snake venom metalloproteinase, disintegrin-like, cysteine-rich (MDC) protein family from Agkistrodon contortrix laticinctus.

In this paper, we present a cDNA sequence encoding a full-length precursor form of a new member (ACLD) of the metalloproteinase-disintegrin-like protein family from the venom glands of Agkistrodon contortrix laticinctus (broad-banded copperhead) snake. Comparison of the deduced amino acid sequence of ACLD with those of other members of the metalloproteinase-disintegrin protein family from both mammalian and snake venom origin suggests that some conserved residues may be involved in processing of the disintegrin domain.

Pathogenesis of hemorrhage induced by proteinase H from eastern diamondback rattlesnake (Crotalus adamanteus) venom.

The pathogenesis of hemorrhage of a purified hemorrhagic toxin, proteinase H from Crotalus adamanteus venom, was studied. Female, white CD-1 mice were injected intramuscularly with sublethal doses of the hemorrhagic toxin and tissue samples were obtained at 10 min, 1, 3 and 24 hr following injection. Severe local hemorrhage was observed grossly within 10 min. Hemorrhage was observed in the connective tissue of skeletal muscle and within adjacent adipose tissue. Many larger vessels were congested with erythrocytes and platelets. By 3 hr inflammatory cell infiltration was observed and necrosis of some muscle cells was evident. Transmission electron microscopy showed that the capillary endothelium was ruptured, leading to hemorrhage per rhexis. Capillary basal laminae were disorganized and often wholly or partially absent.

Systemic hemorrhage induced by proteinase H from Crotalus adamanteus (eastern diamondback rattlesnake) venom.

The systemic effects of a purified hemorrhagic toxin, proteinase H, from Crotalus adamanteus venom, were studied. Female, white CD-1 mice were injected intravenously with proteinase H and tissue samples were obtained at 1, 3 and 24 hr after injection. Hemorrhage was observed grossly within 1 hr in several internal organs including the stomach and small intestine, the heart and the lungs. Surface discolorations thought to be petechial hemorrhages were observed in the kidneys. The livers of treated animals were visibly swollen and darkened and lobules were accentuated. Tissue samples were taken from the stomach, duodenum, heart, lungs, liver and kidneys and prepared for observation by light and electron microscopy. Frank hemorrhage was observed by light microscopy in the walls of the stomach and duodenum, in the myocardium and in the lungs. Pulmonary hemorrhage was severe, with involvement of nearly all of the pulmonary tissue within 3 hr. A1 doses of 5 micrograms/g, hepatic degeneration was observed by 3 hr. Renal glomeruli were noticeably swollen and the lumena of the proximal convoluted tubules indistinct. Closer examination by electron microscopy revealed that the endothelial cells comprising the fenestrated glomerular capillaries remained intact but signs of degeneration (i.e. cytoplasmic swelling and mitochondrial swelling) were observed. Proteinase H induces systemic hemorrhage in the heart, lungs, stomach and small intestine, renal glomerulonephropathy and hepatic degeneration.

Preliminary X-ray crystallographic data for a myotoxin from Agkistrodon contortrix laticinctus (broad-banded copperhead) venom.

A Type II phospholipase A2 myotoxin from Agkistrodon contortrix laticinctus was purified to homogeneity and crystallized. The protein had only myotoxic activity. X-ray diffraction quality crystals were obtained by the hanging drop vapour diffusion method from a crystallization solution containing 2.0 M ammonium sulphate. X-ray data were collected to a resolution of 2.3 A, and the crystals are fully characterized.

Melittin and phospholipase A2 from bee (Apis mellifera) venom cause necrosis of murine skeletal muscle in vivo.

Melittin and phospholipase A2 (PLA2) from bee (Apis mellifera) venom were rested for their ability to induce necrosis of skeletal muscle cells after intramuscular injection into mice. Light and electron microscopic examination of tissue indicated that both melittin (4 micrograms/g) and bee venom PLA2 (4 micrograms/g) caused necrosis of skeletal muscle cells within 30 min after i.m. injection. Early changes in the cells consisted of delta lesions, indicating a ruptured plasma membrane, and hypercontraction of myofibrils. By 24 hr the affected cells appeared as an amorphous mass of disorganized and disrupted myofibrils contained in an intact basal lamina. To ensure that the myotoxic activity of the melittin preparation was not due to contaminating. PLA2 activity, the preparation was treated with p-bromophenacyl bromide (p-BPB), a known inhibitor of PLA2 activity. The p-BPB-treated melittin was determined to have no detectable PLA2 activity using a sensitive muscle cell culture assay, and it still induced myonecrosis, although to a lesser extent and of a slower onset. Additionally, p-BPB treatment of purified bee venom PLA2 completely inhibited its myotoxic activity. These results indicate that both melittin and bee venom PLA2 are capable of inducing necrosis of skeletal muscle cells upon i.m. injection, and that the catalytic and myotoxic activities of bee venom PLA2 are inihibited by p-BPB. Also, melittin and contaminating PLA2 in the melittin fraction may be acting synergistically to induce a stronger and more rapid myotoxic effect than occurs with either alone.

Isolation, characterization and crystallization of a phospholipase A2 myotoxin from the venom of the prairie rattlesnake (Crotalus viridis viridis).

A myotoxin with phospholipase A2 (PLA2) activity was isolated from the venom of the prairie rattlesnake (Crotalus viridis viridis, CVV) by cation-exchange chromatography. The toxin contains 123 amino acids and has an estimated mol. wt of 14,000. It is basic, with a pI above 9. Comparison of the N-terminal 33 residues of this myotoxin with other PLA2 proteins from snake venoms showed that CVV myotoxin has highest homology (91%) to one isoform of the B component of crotoxin from Crotalus durissus terrificus venom, and less homology (73-75%) to mojave toxin from Crotalus scutulatus scutulatus venom and agkistrotoxin from Agkistrodon halys Pallas venom. It has the least homology (40-43%) to PLA2s from venom of two other snakes in the Crotalus genus which are neither neurotoxic nor myotoxic. CVV myotoxin induces the type of myonecrosis typical of snake venom myotoxins with the PLA2 structure, i.e. rapid disruption of the plasma membrane as indicated by the presence of delta lesions, hypercontraction and clumping of the myofilaments, and necrosis of affected skeletal muscle cells. Inhibition of the phospholipase activity of the toxin with p-bromophenacyl bromide inhibits the myotoxic activity, indicating that for some myotoxins with the PLA2 structure, the catalytic activity is important for myotoxic activity. This is the first report of the isolation of a non-neurotoxic, single-chain PLA2 myotoxin from the venom of a snake from the Crotalus genus.