George V. Odell

Isolation of a myotoxin from Bothrops asper venom: partial characterization and action on skeletal muscle.

A myotoxic phospholipase has been isolated from Bothrops asper venom by ion-exchange chromatography on CM-Sephadex followed by gel filtration on Sephadex G-75. The toxin is a basic polypeptide with an estimated molecular weight of 10,700. It has both phospholipase A and indirect hemolytic activities, but is devoid of proteolytic, direct hemolytic and hemorrhagic effects. When injected i.m. into mice the toxin induces a rapid increase in plasma creatine kinase levels and a series of degenerative events in skeletal muscle which lead to myonecrosis. The toxin induces an increase in intracellular calcium levels and is able to hydrolyze muscle phospholipids in vivo. Pretreatment with the calcium antagonist verapamil failed to prevent the myotoxic activity. It is proposed that B. asper myotoxin causes cell injury by disrupting the integrity of skeletal muscle plasma membrane and that myotoxicity is at least partially due to the phospholipase A activity of the toxin.

The amino acid sequence of a myotoxic phospholipase from the venom of Bothrops asper

A myotoxic, basic phospholipase A2 (pI greater than 9.5) with anticoagulant activity has been purified from the venom of Bothrops asper, and its amino acid sequence determined by automated Edman degradation. It is distinct from the B. asper phospholipase A2 known as myotoxin I [Lomonte, B. and Gutierrez, J. M., 1989, Toxicon 27, 725] but cross-reacts with myotoxin I rabbit antisera, suggesting that the proteins are closely related isoforms. To our knowledge, this is the first myotoxic phospholipase to be sequenced that lacks presynaptic neurotoxicity (iv LD50 approximately equal to 8 micrograms/g in mice). The protein appears to exist as a monomer, contains 122 amino acids, and fits with subgroup IIA of other sequenced phospholipase A2 molecules. Its primary sequence shows greatest identity with ammodytoxin B (67%), a phospholipase A2 presynaptic neurotoxin from Vipera ammodytes ammodytes venom. Hydropathy profiles of B. asper phospholipase and the ammodytoxins also show great similarities. In contrast, even though the amino acid sequence identities between B. asper phospholipase and the basic subunit of crotoxin remain high (64%), their hydropathy profiles differ substantially. Domains and residues that may be responsible for neurotoxicity are discussed.

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 micrograms C. v. viridis venom and 120 micrograms C. atrox venom per 0.05 ml. The question remains, can antivenin neutralize this amount of venom when injected independently of venom.

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.

Abstract 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. Wyeths 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.

Composition and properties of tarantula Dugesiella hentzi (Girard) venom

Abstract The proteins, peptides and free amino acids of tarantula, Dugesiella hentzi (Girard), venom have been separated by chromatographic techniques and partially characterized. Tarantula venom hyaluronidase has been identified as a major constituent. A protein with an approximate molecular weight of 7300 and toxic to cockroaches is a second major constituent of this venom. γ-Amino-butyric, glutamic and aspartic acids were identified as free amino acids in the venom. Gel filtration and disc gel electrophoresis indicate the presence of a peptide in tarantula venom.

Isolation and characterization of toxins from brown recluse spider venom (Loxosceles reclusa)

Two toxins have been purified to electrophoretic homogeneity from an extract of the venom apparatus of the brown recluse spider, Loxosceles reclusa. Toxin 1 is the lesioncausing agent in rabbits and is also lethal to mice and rabbits. Toxin 2 which does not appear to be toxic to mice is lethal to rabbits but does not produce lesions. The molecular weight of both toxins is approximately 34,000 as determined by sodium dodecyl sulfatepolyacrylamide-gel electrophoresis. Amino acid compositions of both toxins have been obtained. Toxicities of the toxins are not directly associated with enzymatic activities of hyaluronidase, alkaline phosphatase and 5′-ribonucleotide phosphohydrolase although these enzymes are found in the whole extract. Nevertheless, the toxic activities of toxin 1 may be related to its plasma-coagulating activity. Antisera to toxin 1, toxin 2 and the whole extract have been prepared. Antiserum to toxin 1 is effective in protection of mice against the toxin effect of the extract. None of the antisera has been effective, as tested, in reducing lesion size in rabbits.

Skeletal muscle regeneration after myonecrosis induced by crude venom and a myotoxin from the snake Bothrops asper (Fer-de-Lance).

Skeletal muscle regeneration was studied following injections of Bothrops asper venom and a myotoxin isolated from the crude venom. In toxin-injected muscle regeneration proceeded normally. By 4 days there were myotubes and small regenerating cells. The size of the cells increased by 1 and 2 weeks, and by 4 weeks regenerating cells were fully developed. The regenerated cells retained centrally located nuclei. The regenerative process in venom-injected muscle was not completely normal--by 1 and 2 weeks four main areas, based on the predominant cell type present, were observed in the tissue: (a) necrotic muscle cells; (b) regenerating muscle cells; (c) fibroblasts and collagen; (d) adipocytes. Furthermore, some nerve fibers were demyelinated. Samples obtained 4 weeks after venom injection showed an almost complete regeneration in many areas, whereas in other areas nests of small regenerating cells were surrounded by portions of adipose tissue and collagen. At four weeks regenerating cells in venom-injected muscle were significantly smaller than cells in toxin-injected and saline-injected muscles. There was a significant reduction in capillary/muscle cell ratio in areas of the muscle where hemorrhage and myonecrosis were present 30 min after injection of B. asper venom. Since B. asper venom drastically affects the microvasculature, it is proposed that impairment of regeneration after injection of crude venom is a consequence of diminished blood supply to some areas of the muscle.

Adenosine triphosphate in tarantula spider venoms and its synergistic effect with the venom toxin.

Abstract The presence of adenosine 5′-triphosphate, adenosine 5′-diphosphate and adenosine 5′-phosphate in the venoms of the tarantula spiders, Dugesiella hentzi and Aphonopelma sp. have been identified by ion exchange chromatography, thin-layer chromatography and u.v. absorption spectrometry. Adenosine 5′-triphosphate, the major nucleotide component, was present at a concentration of 28·1 μg per μl venom in D. hentzi venom, and 56·6 μg per μl venom in Aphonopelma sp. venom as assayed by the firefly luciferase method. Toxicity studies indicated that ATP had synergistic toxic effects with the major venom toxin of the D. hentzi venom. The ld 50 value of the venom toxin in mice was significantly diminished when ATP was added.

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.

Quantitation of myonecrosis induced by myotoxin a from prairie rattlesnake (Crotalus viridis viridis) venom

Using a pure myotoxin (myotoxin a) isolated from prairie rattlesnake Crotalus viridis viridis) venom, a comparison between two methods for quantitating myonecrosis in mice was made. Measurement of creatine kinase (CK) levels in the plasma showed two peaks in CK levels after myotoxin injection, whereas measurement by histological assay (vacuolation index) showed only one peak. The first peak in CK levels at 3 hr after injection did not correlate with a high vacuolation index, but did correlate with contraction of muscle induced by the toxin. However, there was good correlation between the two methods at 6, 12, 24, 48 and 72 hr after injection, at which time the muscle cells were necrotic. In the case of the first CK peak this method might be measuring contraction of the muscle, but the second peak was probably measuring altered permeability of the sarcolemma since vacuolation, i.e. swelling of sarcoplasmic reticulum, did follow. High plasma CK levels usually preceded high vacuolation indexes, indicating that CK release due to altered sarcolemma permeability preceded vacuolation. Caution should be taken when using plasma CK levels to estimate the quantity of necrotic muscle cells, so as to insure that pathologic and not physiologic changes are being measured.