Regional variation in the presence of canebrake toxin in Crotalus horridus venom.

Reverse-phase HPLC was used to isolate the PLA complex neurotoxin "canebrake toxin" from the venom of Crotalus horridus from northern Florida. Individual venoms from 107 specimens of C. horridus throughout its range were investigated for the presence of the toxin. The distribution of canebrake toxin was limited to two separate regions, including a region of Louisiana, Arkansas and Oklahoma, and a separate region from southeastern South Carolina through eastern Georgia to northern Florida. Four distinct venom types were found and designated Venoms A (neurotoxic), B (hemorrhagic), A + B (neurotoxic and hemorrhagic) and C (lacking in both neurotoxic and hemorrhagic activities).

North-south regional variation in phospholipase A activity in the venom of Crotalus ruber.

1. Twenty-seven individual venoms from the rattlesnake species Crotalus ruber from different regions were comparatively analyzed by reverse-phase HPLC and analyzed for phospholipase A (PLA) content using a polarographic assay. 2. Two fractions containing PLA activity were detected by HPLC in the venoms of all the C. ruber specimens from southern Baja, Mexico, but specimens from southern California, U.S.A., were lacking corresponding fractions and were extremely low or lacking in PLA activity in their venoms. 3. The north-south regional variation in PLA content in C. ruber venom does not correlate with the north-south ranges (based on external morphology) of the subspecies C. ruber ruber and C. ruber lucasensis.

Prolonged hypofibrinogenemia and protein C activation after envenoming by Echis carinatus sochureki.

Following envenomization by Echis carinatus sochureki, a professional snake handler developed a profound coagulopathy manifested by hemorrhage from the bite site, venipuncture sites and gums; coagulation testing revealed prothrombin and partial thromboplastin times greater than 150 seconds, a fibrinogen of 0 mg%, and marked elevation of fibrin degradation products. In addition, protein C antigen levels were undetectable. The coagulopathy was treated with cryoprecipitate; two different antivenoms were also administered with uncertain benefit. Subsequently, the properties of the venom and antivenoms were studied. Venom did not directly clot fibrinogen; however, venom concentrations as low as 0.2 micrograms/ml caused significant prothrombin activation. In addition, venom activated protein C in the absence of thrombomodulin, and this activity was inhibited by hirudin. The ability of four commercial antivenoms to neutralize the venom prothrombinase and hemorrhagic activity was measured. Three of the four antivenoms partially neutralized venom-induced prothrombin activation. Extreme differences in efficacy were found among the four antivenoms in neutralizing venom hemorrhagic activity in mice. This case illustrates the difficulty in managing the complex coagulopathy that can result from exotic snake envenomization, and identifies a new coagulant property of Echis carinatus venom (protein C activation).

Regional differences in content of small basic peptide toxins in the venoms of Crotalus adamanteus and Crotalus horridus.

1. Reverse-phase HPLC and organic solvents were used to isolate small basic peptide (SBP) toxins from the venoms of Crotalus adamanteus, C. durissus terrificus, C. horridus, C. scutulatus scutulatus, C. viridis concolor, C. viridis helleri and C. viridis viridis. 2. Acid-DEP analyses indicated a high degree of toxin purity which was obtained with a single HPLC run. 3. The combined results of HPLC, immunodiffusion and electrophoresis analyses of venoms from different geographical regions indicate that the SBP toxin content in the venoms of Crotalus adamanteus, Crotalus horridus, Crotalus scutulatus and Crotalus viridis viridis may vary regionally.

Regional variation of biochemical characteristics and antigeneity in Great Basin rattlesnake (Crotalus viridis lutosus) venom.

1. Three pooled and 20 individual venom samples of Crotalus viridis lutosus from different localities in Utah and Arizona were screened and fractionated with HPLC-anion exchange. 2. Pooled venom samples and fractions were tested for hemorrhagic, collagenase, and phospholipase activities, and reactivity to a monoclonal antibody against a hemorrhagin from C. atrox venom (CA-P-8) using ELISA. 3. The 20 individual samples were organized into four groups based on their HPLC profiles. 4. ELISA results and specific hemorrhagic activity of the venom samples displayed a variation in latitidinal distribution although from the same species.

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.

Intergradation of two different venom populations of the Mojave rattlesnake (Crotalus scutulatus scutulatus) in Arizona.

Two distinct venom populations of Crotalus scutulatus scutulatus exist in Arizona. The venom of one population (venom A) contains the toxin 'Mojave toxin' and is lacking in hemorrhagic and specific proteolytic activities. The other population (venom B) does not contain Mojave toxin but does produce hemorrhagic and proteolytic activities. The venoms of 15 Crotalus scutulatus scutulatus from regions between the venom A and venom B populations in Arizona were examined for the presence of Mojave toxin by immunochemical assay, lethality by mouse i.p. LD50, proteolytic activity and hemorrhagic activity in mice. Venom protein constituents were analyzed using reverse-phase HPLC. Seven venoms contained both the Mojave toxin of venom A and the proteolytic and hemorrhagic activities of venom B. The i.p. LD50 values of the A + B venoms were 0.4-2.6 mg/kg, compared to 0.2-0.5 mg/kg for venom A individuals and 2.1-5.3 mg/kg for the venom B individuals. HPLC illustrated that the A + B venoms exhibited a combined protein profile of venom A and venom B. These data indicate that an intergrade zone exists between the two venom types which arcs around the western and southern regions of the venom B population. Within these regions, three major venom types can occur in Crotalus s. scutulatus.

Comparative enzymatic study of HPLC-fractionated Crotalus venoms.

1. Ten venoms of the genus Crotalus (Crotalus adamanteus, Crotalus atrox, Crotalus durissus durissus, Crotalus horridus horridus, Crotalus lepidus, Crotalus polystictus, Crotalus molossus molossus, Crotalus pusillus, Crotalus scutulatus scutulatus, venom B, and Crotalus viridis lutosus) were fractionated using HPLC anion and cation exchange chromatography. 2. HPLC venom fractions were tested for hemorrhagic, hemolytic, and proteolytic activities. 3. Crude Virginia opossum (Didelphis virginiana) serum neutralized the hemorrhagic activity of HPLC fractions.

Detection of myotoxin alpha-like proteins in various snake venoms.

Ninety-five venom samples from eight snake genera (Agkistrodon, Bitis, Bothrops, Calloselasma, Crotalus, Sistrurus, Naja and Vipera) including venoms of Crotalus species of different geographical origin were assayed using immunodiffusion or an ELISA for the presence of the small basic protein, myotoxin alpha, known to cause muscle necrosis. Of the eight genera investigated, only Crotalus and Sistrurus venoms contained detectable amounts of myotoxin alpha-like proteins. The venoms of 13 out of 17 rattlesnake species investigated contained proteins immunologically similar to myotoxin alpha, including 12 Crotalus species and one Sistrurus species. The highest amounts were detected in venoms of C. exsul, C. viridis oreganus and C. v. viridis. Qualitative differences in the presence or absence of myotoxin alpha-like proteins were observed in the venoms of C. cerastes, C. horridus, C. lepidus, C. mitchelli, C. scutulatus, C. viridis and S. catenatus specimens of different geographic origin. The toxin was not detected in the venoms obtained from C. adamanteus, C. atrox, C. enyo or C. vegrandis specimens. The toxin appears to be widely distributed among rattlesnake species in the new world, but may vary qualitatively by geographical region in several species and subspecies.

Venom properties of the rattlesnakes (Crotalus) inhabiting the Baja California region of Mexico.

Seven species of rattlesnakes (genus Crotalus) from Baja, Mexico, were investigated for venom yield, protein content, lethal toxicity, 'Mojave toxin' content and hemorrhagic, esterase (BAEE), phosphodiesterase and protease activities. Venom yield was lowest in C. catalinensis and C. enyo enyo and highest in C. ruber ruber. All venoms exhibited esterase and phosphodiesterase activities, except that three (of 14) specimens of C. e. enyo lacked esterase activity. Protease activity was extremely low or absent in adult C. e. enyo, adult C. mitchellii mitchellii, adult C. viridis caliginis and juvenile C. r. ruber venoms. The venom of C. m. mitchellii was the only venom lacking hemorrhagic activity. This venom also had the highest lethal toxicity (i.p. LD50 0.13 - 0.24 mg/kg) in mice, and was the only venom that exhibited a toxin antigenically related to 'Mojave toxin'.

Geographical variation in Crotalus scutulatus scutulatus (Mojave rattlesnake) venom properties.

Individual venom samples were analyzed from 12 specimens of Crotalus scutulatus scutulatus, from north of Tucson to the extreme southeastern region of Arizona. Six of the specimens, from north of Tucson, produced venom lethal toxicity (i.p. LD50) values in mice of 2.0-6.0 mg/kg. These coincided with the values previously reported for C. s. scutulatus in the Phoenix, Arizona, region and designated as type B venom (Glenn and Straight, 1978). In contrast, the venom LD50 of six individuals from extreme southeastern Arizona, including one individual near Tucson, ranged from 0.22-0.46 mg/kg. This corresponds to the values for C. s. scutulatus venom previously reported and designated as type A venom (Glenn and Straight, 1978). Specimens with type A venom have been collected in California, Nevada, Utah and regions of Arizona. In addition to differences in lethal toxicity, the type B venom consistently exhibits a different protein profile, greater proteolytic activity, greater hemorrhagic activity and contains little or none of the major lethal toxin, Mojave toxin, compared to the type A venom. No external morphological characteristic could be found differentiating the type A venom specimens from the type B venom specimens. These findings further confirm the geographical variation of C. s. scutulatus venom in Arizona.

Lipid accumulation cells derived from porcine aorta and grown under anaerobic conditions.

Fibroblast-like cells, derived from porcine aorta, were cultured under aerobic and anaerobic conditions. Light and electron microscopic examinations, lipid composition measurements, and incorporation of radioactive precursors into lipids of these cells were performed. Anaerobically grown cells accumulated oil red O stainable droplets and within 6 hr the triacylglycerol content increased to 4 times the level determined in cells grown under aerobic conditions. This ratio remained constant throughout an additional 12 hr of growth. The fatty acid composition of the triacylglycerols which accumulated under anaerobic conditions differed from the composition of fatty acids in the triacylglycerols present in the growth medium. The cellular unesterified fatty acids of the anaerobically grown cells differed only slightly in composition from the fatty acids in the growth medium, while the unesterfied fatty acids of aerobically grown cells differed to a greater extent from those of the growth medium.