Fractionation and partial characterization of toxic components of Agkistrodon p. piscivorus (eastern cottonmouth moccasin) venom

The venom of the eastern cottonmouth moccasin snake, Agkistrodon piscivorus piscivorus, has been separated into four fractions by carboxymethyl cellulose ion-exchange chromatography. These fractions were labelled C1–C4 in order of their elution. All four fractions were lethal to mice when injected i.p. The order of potency was C1≈C2 > C4 > C3. Only C1 and C2 produced hemorrhages. Substantial arginine esterase activity was present in fractions C1 and C2. Two types of phospholipase activity were observed. That observed in C3 had a specific activity of 271 units per mg of protein, as determined titrimetrically, and is the phospholipase previously reported by others. The other phospholipase activity was in C1. Fractions C3 and C4 each contained only a single major component, as revealed by SDS gel electrophoresis. Fraction C1 was further separated by DEAE cellulose ion-exchange chromatography. Each of the fractions obtained in this separation had one or more enzymatic activities associated with it. Fraction C2 was further separated by chromatography on a Bio Gel A-0.5 m molecular sieve column. Two regions (A2 and A3) of lethal and hemorrhage-producing activity were observed. This elution profile was analyzed for the location of four different enzyme activities. No separation of l-amino acid oxidase and phosphodiesterase activities was observed, while hide powder and arginine esterase hydrolyzing activities were partially resolved.     

The effect of Bitis gabonica (Gaboon viper) snake venom on external iliac and mesenteric arterial circulation in the dog

The effects of Gaboon viper (Bitis gabonica) venom on external iliac and mesenteric arterial blood flow and resistance were investigated in eight anaesthetized, close-chest dogs. Venom doses in the range 0.125 – 0.5 mg/kg produced a profound fall in external iliac and mesenteric arterial resistance, which recovered to control values after 30 min. After a third dose of venom, the mean arterial blood pressure failed to recover and the animals died after a period of severe hypotension. External iliac arterial blood flow rose concomitantly with the fall in external iliac resistance and decreased to a value significantly below control after 30 min. Paradoxically, mesenteric blood flow fell during the period of vasodilation. The results suggest that widespread vasodilation of muscle vascular beds (of which the external iliac circulation is representative) leads to shunting of blood away from the less-dilated mesenteric circulation. Venom-induced peritoneal haemorrhage caused a fall in blood volume and increase in viscosity. These undoubtedly contributed to the severe haemodynamic deterioration of the preparations after the third injection of the venom.     

Effects of X-irradiation on the antigenic character of Agkistrodon piscivorus (cottonmouth moccasin) venom

Cottonmouth moccasin venom with its necrotizing toxin inactivated 50 to 75 per cent by X-irradiation was studied to determine the alteration of its antigenic character. Immune globulin of rabbits immunized with both normal and X-irradiated venom were compared. The globulin of rabbits immunized with X-irradiated venom showed little difference in its neutralizing titer from the globulin of rabbits immunized with normal venom. A group of animals immunized with normal venom suffered 20 per cent mortality, sloughing of tissue, and severe local reactions around the area of injection. The group of rabbits immunized with X-irradiated venom suffered no mortality or sloughing of tissue. Local reactions around the site of injection were less severe.     

Purification and characterization of fibrolase isoforms from venom of individual southern copperhead (Agkistrodon contortrix contortrix) snakes

Fibrolase, a zinc metalloproteinase possessing direct-acting fibrinolytic activity, has been previously purified from southern copperhead (Agkistrodon contortrix contortix) snake venom. We recently reported that a pool of southern copperhead venom from different geographical locations possesses two isoforms of fibrolase (fib1 and fib2) [ , S. L. et al. (1994) J. Chromat. B, in press]. We now report that venom from individual southern copperhead snakes contains the two isoforms which can be separated by a three-step high performance liquid chromatography (HPLC) procedure consisting of hydrophobic interaction chromatography, hydroxylapatite chromatography and weak cation exchange chromatography. Utilizing mass spectrometry we determined that fib1 has a molecular mass of 22,879 atomic mass units (amu) compared to 22,753 amu for fib2. These results support earlier observations during amino acid sequence analysis that a truncated version of the enzyme is produced which is missing the amino-terminal amino acid (vs. the intact enzyme 50 values (concentration of enzyme required to degrade 50% of fibrin in a micro-fibrin plate assay) are 6.4 (±1.0) μM and 5.2 (±0.8) μM for fib 1 and fib2, respectively. Therefore, loss of the amino-terminal amino acid does not appear to influence enzymatic activity. We conclude that the two isoforms of fibrolase arise from variations in the molecular processing of the enzyme by the snake venom gland rather than being caused by the pooling of southern copperhead venoms from different geographical locations.     

The effect of Bitis gabonica (gaboon viper) snake venom on blood pressure,stroke volume and coronary circulation in the dog

The effects of Gaboon Viper (Bitis gabonica) venom on cardiac output, arterial blood pressure, coronary flow and total peripheral resistance were investigated in eight anaesthetized dogs. Venom doses in the range of 0·125–0·50 mg/kg produced an abrupt fall in arterial blood pressure which recovered to near normal after 30 min. Stroke volume and cardiac output both rose during the hypotensive response and then declined to below control values. Heart rate and electrocardiogram were essentially unaltered. Coronary blood flow increased after venom and total peripheral resistance decreased. These responses tended to diminish as venom dose was increased, indicating some form of tachyphylaxis. The results indicate the presence of two different components affecting the cardiovascular system: a vasodilatory component responsible for the fall in peripheral resistance and hence arterial blood pressure and a cardiotoxic component responsible for the progressive reduction in stroke volume.     

Local heat and cold application after eastern cottonmouth moccasin (Agkistrodon piscivorus) envenomation in the rat: effect on tissue injury.

We studied the effect of local application of heat or cold on the development of tissue necrosis in envenomated rats. Anesthetized animals had 6 mg/kg venom from Agkistrodon piscivorus injected subcutaneously into the lateral aspect of a hind limb. Heat or cold was applied to the site of envenomation in the experimental groups for 4-6 hr, and the injected area was examined histologically after 24 hr. Neither local treatment, with or without the presence of systemic antivenin, significantly affected the severity of tissue necrosis induced by the venom in comparison to a control group left at ambient temperature.     

Purification of two thrombin-like enzymes from the venom of Agkistrodon caliginosus (kankoku-mamushi)

Two thrombin-like enzymes were purified from the venom of Agkistrodon caliginosus. Both were homogeneous on polyacrylamide gel electrophoresis, hydrolyzed $\text{N-α-}\text{tosyl}\text{-}\text{l}\text{-}\text{arginine}$ methylester and did not show any caseinolytic activity. The molecular weights of the enzymes were estimated to be about 34,000–37,000 and 42,000–43,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel-filtration on Sephadex G-100 column. One enzyme was purified from the venom by gel-filtration on Sephadex G-100 column, CM-Sephadex C-50 and DEAE-Sephadex A-50 chromatographies, affinity chromatography on p-aminobenzamidine-ε-aminocaproic acid-Sepharose 4B and gel-filtration on Sephadex G-100 column. The second enzyme was separated from the first enzyme by the DEAE-Sephadex A-50 chromatography mentioned in the above procedure and was isolated by affinity chromatography on arginine-Sepharose 4B and gel-filtration on Sephadex G-100 column. From 4 g of the venom, 0.88 mg of the first enzyme and 1.18 mg of the second enzyme were obtained, with specific activities of 118 and 139 NIH units per mg of protein, respectively.     

Fibrinolytic and fibrinogen clotting enzymes present in the venoms of western diamondback rattlesnake, Crotalus atrox, eastern diamondback rattlesnake, Crotalus adamanteus, and southern pacific rattlesnake, Crotalus viridis helleri

Crotalus adamenteus and C. viridis helleri have both fibrinolytic and fibrinogen clotting activities, whereas C. atrox has strong fibrinolytic activity but no detectable fibrinogen clotting activity. Fractionation of crude venom on Sephadex G-100 revealed that C. atrox has two separate fibrinolytic activities with mol. wts of 60,000 and 21,500. C. adamanteus has fibrinolytic and fibrinogen clotting activities that co-elute in the mol. wt region of 35,000. C. viridis helleri contains fibrinolytic activity with mol. wt of 21,500 and fibrinogen clotting activity with a mol. wt of 92,000. Since there is clinical interest in the possible therapeutic application of fibrinolytic and defibrinogenating agents in certain medical disorders which involve fibrin deposition, it will be of value to study enzymes with fibrinolytic and thrombin-like activity from venoms of snakes indigenous to the North American continent.     

Preclinical assessment of the ability of polyvalent (Crotalinae) and anticoral (Elapidae) antivenoms produced in Costa Rica to neutralize the venoms of North American snakes.

Polyvalent (Crotalinae) and anticoral (Elapidae) antivenoms produced by Instituto Clodomiro Picado, Costa Rica, were assessed for their ability to neutralize various toxic activities of the venoms of North American snakes of the genera Crotalus, Agkistrodon and Micrurus, in assays involving preincubation of venom and antivenom. When the intraperitoneal route of injection was utilized, polyvalent (Crotalinae) antivenom was effective in the neutralization of the venoms of Crotalus atrox, Crotalus adamanteus, Crotalus viridis viridis, Crotalus horridus atricaudatus, Agkistrodon contortrix contortrix and Agkistrodon piscivorus piscivorus, whereas the venom of Crotalus scutulatus was not neutralized. When the intravenous route was used, results differed depending on the "challenge dose" of venom employed. Polyvalent antivenom neutralized all venoms when mice were challenged with 2 LD(50)s of venom. When 5 LD(50)s were used, antivenom neutralized the venoms of C. atrox, C. adamanteus, C. v. viridis and C. h. atricaudatus, being ineffective in the neutralization of C. scutulatus, A. c. contortrix and A. p. piscivorus. Polyvalent antivenom was effective in the neutralization of hemorrhagic and myotoxic activities of all venoms studied. It also neutralized coagulant activity of C. adamanteus venom, whereas most of the venoms were devoid of clotting activity on plasma in vitro. Moreover, it neutralized defibrinating activity of the only three venoms that induced this effect (i.e. C. adamanteus, A. c. contortrix and A. p. piscivorus). Anticoral (Elapidae) antivenom neutralized lethality induced by the venom of Micrurus fulvius, using either the intravenous or the intraperitoneal routes of injection. Moreover, it neutralized myotoxic effect of this venom as well. It is concluded that polyvalent antivenom neutralizes lethality and other activities of most of the crotaline venoms tested. However, since it is ineffective in neutralizing the lethal effect of C. scutulatus venom, it is suggested that a venom containing presynaptically-active neurotoxic phospholipases A(2) related to "mojave toxin" needs to be introduced in the immunizing mixture in order to increase the neutralizing scope of this product in North America. Anticoral antivenom is highly effective in the neutralization of the venom of M. fulvius.     

Envenomation by the Mojave rattlesnake (Crotalus scutulatus scutulatus) in southern Arizona,U.S.A.

Fifteen cases of envenomation by the Mojave rattlesnake (Crotalus scutulatus scutulatus) are reviewed. Systemic effects were observed in eight patients, consisting of early hypotension (3), decreased plasma fibrinogen (3) and platelets (2), elevated fibrinolytic split products (3) and eyelid ptosis (1). Local venom effects were most common and included swelling (15), ecchymosis (10), bleb formation (6) and necrosis (3). Effects upon neuromuscular transmission were neither common nor a clinical problem. Treatment consisted of i.v. crystalloid solution (15) and antivenin (12).     

Specificity of antibodies to the reconstituted crotoxin complex,from the venom of South American rattlesnake (Crotalus durissus terrificus), using enzyme-linked immunosorbent assay (ELISA) and double immunodiffusion

Some differences between the reaction of antiserum to reconstituted crotoxin complex, to ‘native’ crotoxin and to whole Crotalus durissus terrificus (South American rattlesnake) venom were detected by enzyme-linked immunosorbent assay (ELISA) and by immunodiffusion. ELISA showed the presence of antibodies to the components of the crotoxin complex, phospholipase A₂ and crotapotin. When the antiserum was tested against North American rattlesnake venoms some species gave a positive reaction (C. horridus atricaudatus and C. basiliscus), whilst the venoms of other species were virtually negative (C. durissus totonacus, C. horridus horridus, C. viridis viridis and C. atrox). Confirmation of antigenic similarities between crotoxin and Mojave toxin (from the venom of C. scutulatus scutulatus) was obtained with ELISA and some implications of these species differences in antigen-antibody reaction are discussed. No paraspecificity was observed with heterologous snake venoms from other Crotalidae, Elapidae, Hydrophiidae or Viperidae. ELISA also showed a lack of cross-reactivity of the antiserum to heterologous purified phospholipases A₂ from Enhydrina schistosa, Naja nigricollis or Apis mellifera venoms or from porcine pancreas. The antiserum reacted to the homologous phospholipase A₂ inactivated with p-bromophenacyl bromide as well as to the detoxified crotoxin complex reconstituted with this modified phospholipase A₂. This may be useful in the raising of high titres of antibody to crotoxin.     

Measurement of blood concentrations of FK506 (tacrolimus) and its metabolites in seven liver graft patients after the first dose by h.p.l.c.-MS and microparticle enzyme immunoassay (MEIA).

1. Blood and urine concentrations of the macrolide immunosuppressant FK506 and its metabolites were measured in seven orthotopic liver transplant patients after the first oral dose of FK506 (0.04 +/- 0.02 mg kg-1) used as primary immunosuppressant. A specific h.p.l.c.-MS assay was used, allowing the measurement of parent drug and eight metabolites. Results were compared with those obtained using a microparticle enzyme immunoassay (MEIA). 2. Blood drug concentrations were described by an open two compartment model with first-order absorption giving the following mean data: tmax: 1.9 (h), Cmax: 17.4 (microgram l-1), AUC: 328.1 (microgram l-1 h), t1/2,1: 0.74 (h). The terminal elimination half-life was estimated at about 26 h using the h.p.l.c.-MS assay. 3. The metabolites found in blood were demethyl-FK506 and demethyl-hydroxy-FK506, while in urine FK506 and eight of its metabolites were detected.     

Toxic essential oils: Anxiolytic,antinociceptive and antimicrobial properties of the yarrow Achillea umbellata Sibth. et Sm. (Asteraceae) volatiles

Many plant species are used for medicinal purposes without the knowledge of their possible toxic effect. The ethnopharmacologically renowned genus Achillea L. (Asteraceae) is even more troublesome in this respect since different taxa are believed to have the same beneficial properties as A. millefolium. According to the median lethal i.p. dose (LD₅₀ = 853 mg/kg, mice), the volatiles of Achillea umbellata Sibth. et Sm. are more toxic than the thujone-containing essential oils (LD₅₀ > 960 mg/kg). A GC–MS analysis of A. umbellata oil revealed the presence of a series of fragranyl esters (six new natural products). The major constituents of this oil, the rare monoterpene alcohol fragranol and fragranyl acetate, and one more ester (benzoate), as well as the oil itself, showed antianxiety, analgesic and, in some instances, paralyzing properties at 50–150 mg/kg but these are very likely sign of intoxication and not of possible beneficial effects of the plant volatiles. Testing of antimicrobial activity demonstrated that the oil possesses moderate activity against pathogenic microorganisms, but the effect of the oil differs in pro- and eukaryotic cells. According to the results obtained, fragranol may be considered as the main active principle responsible for the observed activity/toxicity.     

Caption: A tricolour overlay of the ryanodine receptor (red), caveolin‐3 (green) and sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA,blue) in a rat ventricular myocyte. See Jayasinghe et al. pp 469‐476.

Cover image: Cover caption: A tricolour overlay of the ryanodine receptor (red), caveolin‐3 (green) and sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA, blue) in a rat ventricular myocyte. See Jayasinghe et al. pp 469‐476.

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