South American Rattlesnake Crotalus Durissus Research Articles

DIAGNOSING ACUTE COMPARTMENT SYNDROME

Compartment syndrome has been defined as “a condition in which increased pressure within a limited space compromises the circulation and function of the tissues within that space”.1 It is most commonly seen after injuries to the leg2-5 and forearm6-8 but may also occur in the arm,9 thigh,10 foot,11-13 buttock,14 hand15 and abdomen.16 It typically follows traumatic injury, but may also occur after ischaemic reperfusion injuries,17 burns,18 prolonged limb compression after drug overdose19 or poor positioning during surgery.20-24 Furthermore, subclinical compartment syndromes may explain the occurrence of a variety of postoperative disabilities which have been identified after the treatment of fractures of long bones using intramedullary nails.25 Approximately 40% of all acute compartment syndromes occur after fractures of the tibial shaft26 with an incidence in the range of 1% to 10%.26-30 A further 23% of compartment syndromes are caused by soft-tissue injuries with no fracture and fractures of the forearm account for 18%.26 Acute compartment syndrome is seen more commonly in younger patients, under 35 years of age31 and therefore leads to loss of function and long-term productivity in patients who would otherwise contribute to the country’s workforce for up to 40 years.

Crotoxin acceptor protein isolated from Torpedo electric organ: binding properties to crotoxin by surface plasmon resonance

Crotoxin, a potent neurotoxin from the South American rattlesnake Crotalus durissus terrificus, is a heterodimeric phospholipase A 2 (EC 3.1.1.4), which blocks the release of acetylcholine from peripheral neurons. We previously have suggested the existence of a 48 kDa crotoxin-binding protein in the presynaptic membranes of the electric organ of Torpedo marmorata. Here, we report the purification and characterization of this protein that we called the crotoxin acceptor protein from Torpedo (CAPT). The membranes of electric organs from Torpedo were solubilized with a detergent (4% (w/v) Triton X-100) and CAPT was isolated by affinity chromatography on a crotoxin column. SDS–PAGE showed that the purified protein was homogeneous and cross-linking studies with radioiodinated crotoxin confirmed that it had retained its toxin-binding properties. The purified CAPT has similar molecular mass as crocalbin, a crotoxin-binding protein isolated from porcine brains, yet anti-crocalbin antiserum failed to recognize CAPT. Surface plasmon resonance biosensor technology was used to measure the specific interaction between crotoxin and solubilized CAPT. Using this method, it was possible to follow CAPT throughout the purification procedure. As well, an apparent dissociation constant ( K d app) of 3.4 nM was calculated for the interaction of pure CAPT and crotoxin from the dissociation rate constant ( k off=1.2×10 −2 s −1) and the association rate constant ( k on=3.5×10 6 M −1s −1).

Age-related biological activity of South American rattlesnake (Crotalus durissus terrificus) venom

An in vitro and in vivo comparative study was performed on the effects of Crotalus durissus terrificus venoms from a mother and its 15 newborns. The venoms were tested for protein content, lethality, proteolytic, myotoxic, hemorrhagic, and phospholipase A2 activity. The minimum coagulant dose in plasma and human fibrinogen, protrhombin, and Factor II activations were analyzed. The venoms were also analyzed by polyacrylamide gel electrophoresis (PAGE). This showed that despite similar total protein content, the biological effects of the venoms were different. Venom from young snakes exhibited higher enzymatic and coagulant activities and higher myotoxicity compared to the mother’s. In addition, the PLA2 content paralleled myotoxicity. However, no difference could be detected in their toxicity (LD50 0.08 mg/Kg). High incidence of blood coagulation disorders and elevated circulating myoglobin may characterize systemic envenoming by young C. d. terrificus.

Characterization of the crotalus durissus terrificus venom by atomic force microscopy

Atomic force microscopy was used to study the morphology of crude venom from the South American rattlesnake crotalus durissus terrificus. The results indicate that the crystallized crude venom has a high surface roughness with pores of diameters around 100 nm. Coiled filamentary structures of diameters of about 200 nm and lengths greater than 1000 nm, and large concave structures of diameter around 1000 nm, have also been observed dispersed over the main solid surface of the venom. Such structures may be related to aggregates of toxic enzymes or to bacteria or both. The coiled filamentary structures appear to be entangled on the pores of the venom surface in contrast to the large concave structures, which appear not to be bonded to the principal crystallized surface.

A proposed 3D structure for crotamine based on homology building, molecular simulations and circular dichroism

Crotamine, isolated from the venom of the South American rattlesnake Crotalus durissus terrificus is a strongly basic 42-aminoacid polypeptide belonging to the small basic myotoxin family. As no tridimensional structure is available for this myotoxin subfamily, despite its important pharmacological interest, we propose in this paper a theoretical 3D model for crotamine. Starting from a homology modelling procedure, followed by intensive molecular dynamics (MD) simulations in water and complementary CD experiments, the designed 3D model is the first example of a tridimensional structure in this family of small basic myotoxins. Crotamine, therefore, belongs to a newly identified structural family presenting a common fold also found in β-defensin and antopleurine-B. The proposed 3D model will be used for future calculations about crotamine aggregation and interaction with membranes.

Blood biochemical profile of the South American rattlesnake (Crotalus durissus terrificus) in captivity

Blood samples were collected from 180 healthy specimens of the South American rattlesnake, Crotalus durissus terrificus, in captivity. All animals were in good clinical condition. Normal biochemical reference values were established for the following: total proteins, albumin, globulins, uric acid, creatinine, urea, glucose, triglycerides, cholesterol, total lipids, calcium, phosphorus, potassium, sodium, chloride, magnesium, GOT (AST), GPT (ALT), and alkaline phosphatase (ALP). Samples were obtained by venipuncture of the ventral tail vein. Values were compared with published data for Boidae, Elapidae, and Viperidae.

Coagulopathy following lethal and non-lethal envenoming of humans by the South American rattlesnake (Crotalus durissus) in Brazil.

The South American tropical rattlesnake (Crotalus durissus subspp) is responsible for approximately 10% of bites from venomous snakes in Brazil. We studied 24 victims of bites by this species over 3 years, in south-eastern Brazil, particularly investigating haemostatic alterations. Thirteen patients were defined as moderately envenomed and 11 as severe. There were two deaths, which were not attributed to venom-induced haemostatic disturbances. However, envenoming by C. durissus is frequently associated with haemostatic disorders, which are probably attributable mainly to the action of the thrombin-like enzyme, with possible additional effects secondary to the powerful myotoxic activity of the venom.

Random amplified polymorphisms between two South American subspecies of rattlesnakes (Crotalus durissus collilineatus and Crotalus durissus terrificus)

The present work was made to determine the suitability of RAPD analysis for the identification of specimens of two subspecies of South American rattlesnakes (Crotalus durissus terrificus and Crotalus durissus collilineatus). The 11 arbitrary primer sequence tested amplified a total of 161 bands, of which 31% were polymorphics when compared with Crotalus specimens. The combining results from different primers allowed the identification of all the specimens under analysis. Several characteristic bands of each subspecies were identified. Dendrogram, based on RAPD markers, show three groups that corresponded to the subspecies of Crotalus, and to Bothrops jararaca specimen.

Bactericidal activity of PLA2 isoform isolated from the South American rattlesnake Crotalus durissus terrificus

Bactericidal activity of PLA2 isoform isolated from the South American rattlesnake Crotalus durissus terrificus

Crotoxin, the major toxin from the rattlesnake Crotalus durissus terrificus, inhibits 3H-choline uptake in guinea pig ileum.

We examined the effect of crotoxin, the neurotoxic complex from the venom of the South American rattlesnake Crotalus durissus terrificus, on the uptake of 3H-choline in minces of smooth muscle myenteric plexus from guinea pig ileum. In the concentration range used (0. 03-1 microM) and up to 10 min of treatment, crotoxin decreased 3H-choline uptake by 50-75% compared to control. This inhibition was time dependent and did not seem to be associated with the disruption of the neuronal membrane, because at least for the first 20 min of tissue exposure to the toxin (up to 1 microM) the levels of lactate dehydrogenase (LDH) released into the supernatant were similar to those of controls. Higher concentrations of crotoxin or more extensive incubation times with this toxin resulted in elevation of LDH activity detected in the assay supernatant. The inhibitory effect of crotoxin on 3H-choline uptake seems to be associated with its phospholipase activity since the equimolar substitution of Sr2+ for Ca2+ in the incubation medium or the modification of the toxin with p-bromophenacyl bromide substantially decreased this effect. Our results show that crotoxin inhibits 3H-choline uptake with high affinity (EC25 = 10 +/- 5 nM). We suggest that this inhibition could explain, at least in part, the blocking effect of crotoxin on neurotransmission.

Effect of heating on the toxic, immunogenic and immunosuppressive activities of Crotalus durissus terrificus venom

The venom of South American rattlesnake Crotalus durissus terrificus is very toxic but poorly immunogenic and it has an immunosuppressive ability. The heating of venom at 56, 70 or 100°C for 30 min caused a diminution in the lethal, phospholipase A 2 and myotoxic activities. SDS-PAGE analysis of the heated venom showed that the proteins of higher molecular weights were the most affected by heating whereas proteins with lower molecular weights (20,000–14,000) were the most resistant to heating. The immunosuppressive effect was studied in mice immunized with human serum albumin (HSA) 1 h after receiving either heated venom or heated crotoxin. The heating of venom at 70 or 100°C reduced its immunosuppressive effect whereas crotoxin had its suppressive effect reduced only when heated at 100°C. The heating of venom at 56, 70 or 100°C did not change its immunogenicity. These results suggest that heat treatment may be a useful technique to help in the production of antiserum to crotalid venom.

Pulmonary thrombosis caused by South American rattlesnake (Crotalus durissus terrificus) venom

Pulmonary thrombosis caused by South American rattlesnake (Crotalus durissus terrificus) venom

A novel toxin from South American rattlesnake (Crotalus durissus terrificus) venom

A novel toxin from South American rattlesnake (Crotalus durissus terrificus) venom

Neutralizing human anti crotoxin scFv isolated from a nonimmunized phage library.

Combinatorial phage display technology offers a new possibility for making human antibodies which could be used in immune therapy. We explored the use of this technology to make human scFvs specific for crotoxin, the main toxic component of the venom of the South-American rattlesnake Crotalus durissus terrificus. Crotoxin, a phospholipase A2 neurotoxin constituted by the association of two subunits, exerts its lethal action by blocking neuromuscular transmission. This is the first report of human anticrotoxin scFvs (scFv 1, scFv 6 and scFv 8) isolated from a naive library of more than 1010 scFv clones with in vivo neutralizing activity. Nevertheless, differences are observed at the level of biological and immunological effects. Only scFv 8 is able to reduce the myotoxicity induced by crotoxin and scFv 1 is capable of altering the in vitro enzymatic activity of this toxin. All three scFvs recognize a region of one subunit located at the junction with the other one. Moreover these scFvs share strong amino acid homologies at the level of either the heavy or the light chain. Taken together, our results suggest that the use of human anticrotoxin scFvs may lead to a new and less aggressive passive immune therapy against poisoning by the venom of Crotalus durissus terrificus.

Quantification of crotamine, a small basic myotoxin, in South American rattlesnake ( Crotalus durissus terrificus) venom by enzyme-linked immunosorbent assay with parallel-lines analysis

Intraspecific variation in Crotalus durissus terrificus venom composition was studied in relation to crotamine activity. Crotamine induces paralysis in extension of hind legs of mice and myonecrosis in skeletal muscle cells. To determine whether the venom of crotamine-negative rattlesnake contains a quantity of myotoxin incapable of inducing paralysis, we have developed a very sensitivity immunological assay method, an enzyme-linked immunoabsorbent assay (ELISA), capable of detecting 0.6 ng of purified crotamine. The parallel-lines analysis of ELISA data showed to be useful because it shows the reliability of the experimental conditions. A variation in the amount of myotoxin in the crotamine-positive venom was observed, but not less than 0.1 mg of crotamine per mg of venom. It was not possible to detect it in crotamine-negative venom even at high venom concentrations.

Primary culture of venom gland cells from the South American rattlesnake ( Crotalus durissus terrificus)

Primary cultures of venom gland cells from the South American rattlesnake ( Crotalus durissus terrificus) were attempted. At first, six different cell types were obtained including potentially secreting epithelial-like cells. Nonepithelial cell cultures were later invaded by fibroblast-like cells. Cultures of epithelial-like gland cells were successfully maintained, after testing different culture conditions by varying the media, incubation temperature, use of dissociating agents and adhesion substrates. The best results were achieved using plates precoated with rattlesnake skin collagen and incubation in CMRL 1415 modified for snake gland cells plus 10% fetal calf serum at 30°C. The presence of venom could be demonstrated in the supernatant of five out of six epithelial-like gland cell cultures tested by ELISA, in the very first passages. After the third passage, however, venom amounts dropped to undetectable values. A total of 23 venom gland cell lines were obtained and are kept frozen in the laboratory; among them, five epithelial-like gland cell lines with up to 12 passages, that were continuously cultured for more than 30 weeks. The methodology described here was successfully applied to C. d. terrificus kidney cells culturing, developed to be used as negative control.

Studies on the specificity of CNF, a phospholipase A 2 inhibitor isolated from the blood plasma of the South American rattlesnake ( Crotalus durissus terrificus). I. Interaction with PLA 2 from Lachesis mutamuta snake venom

A phospholipase A 2 inhibitor has been previously purified and cloned from the blood plasma of the South American rattlesnake, Crotalus durissus terrificus. This inhibitor, named CNF for Crotalus neutralizing factor, interacts with crotoxin, the main neurotoxin from C. d. terrificus venom, abolishing its phospholipase A 2 activity. Crotoxin is a heterodimer of an acidic subunit (CA) and a basic phospholipase A 2 (CB). CNF acts by forming a stable non-toxic complex with CB, replacing CA in the toxic CA–CB of crotoxin. In the present investigation, we have shown that CNF has a broader specificity. It is able to inhibit the PLA 2 activity of the whole venom from the bushmaster snake ( Lachesis muta muta), a species evolutionary related to Crotalus. Inhibition experiments have been carried out with four PLA 2 active components isolated from L. m. muta venom, one basic and three acidic ones. CNF inhibition is not restricted to the basic PLA 2, but extended to the three acidic forms as well.

Erratum to “Comparison of the biological activities in venoms from three subspecies of the South American rattlesnake ( Crotalus durissus terrificus, C. durissus cascavella and C. durissus collilineatus)” : [Comp. Biochem. Physiol. 123 C (1999) 61–73

Erratum to “Comparison of the biological activities in venoms from three subspecies of the South American rattlesnake ( Crotalus durissus terrificus, C. durissus cascavella and C. durissus collilineatus)” : [Comp. Biochem. Physiol. 123 C (1999) 61–73

Field test of a paradigm: hysteresis of heart rate in thermoregulation by a free-ranging lizard (Pogona barbata).

The discovery that changes in heart rate and blood flow allow some reptiles to heat faster than they cool has become a central paradigm in our understanding of reptilian thermoregulation. However, this hysteresis in heart rate has been demonstrated only in simplistic laboratory heating and cooling trials, leaving its functional significance in free-ranging animals unproven. To test the validity of this paradigm, we measured heart rate and body temperature (Tb) in undisturbed, free-ranging bearded dragons (Pogona barbata), the species in which this phenomenon was first described. Our field data confirmed the paradigm and we found that heart rate during heating usually exceeded heart rate during cooling at any Tb. Importantly, however, we discovered that heart rate was proportionally faster in cool lizards whose Tb was still well below the 'preferred Tb range' compared to lizards whose Tb was already close to it. Similarly, heart rate during cooling was proportionally slower the warmer the lizard and the greater its cooling potential compared to lizards whose Tb was already near minimum operative temperature. Further, we predicted that, if heart rate hysteresis has functional significance, a 'reverse hysteresis' pattern should be observable when lizards risked overheating. This was indeed the case and, during heating on those occasions when Tb reached very high levels (> 40 degrees C), heart rate was significantly lower than heart rate during the immediately following cooling phase. These results demonstrate that physiological control of thermoregulation in reptiles is more complex than has been previously recognized.

Effects of temperature on lung and blood gases in the South American rattlesnake Crotalus durissus terrificus

The effects of temperature on lung and blood gases were measured in the South American rattlesnake ( Crotalus durissus terrificus). Arterial blood and lung gas samples were obtained from chronically cannulated animals at 15, 25, and 35°C. As expected for reptiles, arterial pH fell with increased temperature (0.018 U °C −1 between 15 and 25°C and 0.011 U °C −1 between 25 and 35°C) while lung gas PCO 2 rose from 5.8 mmHg at 15°C to 13.2 mmHg at 35°C. Concurrently, lung gas PO 2 declined from 132 mmHg at 15°C to 120 mmHg at 35°C, and arterial PO 2 increased from 33 to 76 mmHg in that temperature range. Arterial haemoglobin O 2 saturation rose from 0.53 at 15°C to 0.83 at 25°C but became slightly reduced (0.77) with a further elevation of temperature to 35°C. Arterial haemoglobin concentration increased from 1.96 to 2.53 mM between 15 and 35°C, consistent with higher demands on oxygen delivery to tissues at elevated temperatures. Moreover, the substantial increase of haemoglobin O 2 saturation between 15 and 25°C conforms to the idea that reduction of the central vascular right-to-left shunt (pulmonary bypass of systemic venous return) is associated with high metabolic demands.