Isolation of a new L-amino acid oxidase from Crotalus durissus cascavella venom

A novel L-amino acid oxidase (LAO) (Casca LAO) from Crotalus durissus cascavella venom was purified to a high degree of molecular homogeneity using a combination of molecular exclusion and ion-exchange chromatography system. The purified monomer of LAO presented a molecular mass of 68 kDa and pI estimated in 5.43, which were determined by two-dimensional electrophoresis. The 71st N-terminal amino acid sequence of the LAO from Crotalus durissus cascavella presented a high amino acid sequence similarities with other LAOs from Colloselasma rhosostoma, Crotalus adamanteus, Agkistrodon h. blomhoffi, Agkistrodon h. halys and Trimeresurus stejnegeri. LAO displayed a Michaelis-Menten behavior with a kilometer of 46.7 mu M and an optimum pH for enzymatic activity of 6.5. Casca LAO induced a dose-dependent platelet aggregation, which was abolished by catalase and inhibited by indomethacin and aspirin. These results suggest that the production of H2O2 is involved in subsequent activation of inflammatory enzymes, such as thromboxane. Casca LAO also inhibited the bacterial Growth of Gram-negative (Xanthomonas axonopodis pv passiflorae) and Gram-positive (S. mutans) strains. Electron microscopy assessments of both bacterial strains suggest that the hydrogen peroxide produced by LAO induce bacterial membrane rupture and consequently loss of cytoplasmatic content. This LAO exhibited a high antileishmanic activity against the promastigote of Leishmania amazonensis in vitro, its activity was dependent on the production of hydrogen peroxide, and the 50% inhibitory concentration was estimated in 2.39 mu g/ml. (C) 2005 Elsevier Ltd. All rights reserved.

Automated NMR structure determination and disulfide bond identification of the myotoxin crotamine from Crotalus durissus terrificus

Crotamine is one of four major components of the venom of the South American rattlesnake Crotalus durissus terrificus. Similar to its counterparts in the family of the myotoxins, it induces myonecrosis of skeletal muscle cells. This paper describes a new NMR structure determination of crotamine in aqueous solution at pH 5.8 and 20 degrees C, using standard homonuclear (1)H NMR spectroscopy at 900 MHz and the automated structure calculation software ATNOS/CANDID/DYANA. The automatic NOESY spectral analysis included the identification of a most likely combination of the six cysteines into three disulfide bonds, i.e. Cys4-Cys36, Cys11-Cys30 and Cys18-Cys37; thereby a generally applicable new computational protocol is introduced to determine unknown disulfide bond connectivities in globular proteins. A previous NMR structure determination was thus confirmed and the structure refined. Crotamine contains an alpha-helix with residues 1-7 and a two-stranded anti-parallel beta-sheet with residues 9-13 and 34-38 as the only regular secondary structures. These are connected with each other and the remainder of the polypeptide chain by the three disulfide bonds, which also form part of a central hydrophobic core. A single conformation was observed, with Pro13 and Pro21 in the trans and Pro20 in the cis-form. The global fold and the cysteine-pairing pattern of crotamine are similar to the beta-defensin fold, although the two proteins have low sequence homology, and display different biological activities. (c) 2005 Elsevier Ltd. All rights reserved.

K-ATP(+) Channels-Independent Analgesic Action of Crotalus durissus cumanensis venom

The effect was investigated of the K+ channel blocker, glibenclamide, on the ability of Crotalus durissus cumanensis venom (CDCM) to promote peripheral antinociception. This was measured by formalin-induced nociception in male Swiss mice. CDCM (200 and 300 mu g/kg) produced an antinociceptive effect during phase 2 in the formalin test. The effect of CDCM (200 mu g/kg) was unaffected by the ATP-sensitive K+ channel blocker glibenclamide (2 mg/kg). These results suggest that CDCM is effective against acute pain. However, the ATP-sensitive K+ channels pathway is not contributable to the antinoeiceptive mechanism of CDCM.

Functional and structural analysis of two fibrinogen-activating enzymes isolated from the venoms of Crotalus durissus terrificus and Crotalus durissus collilineatus

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Histopathological evaluation in experimental envenomation of dogs with Crotalus durissus terrificus Venom

The present work evaluated histopathological aspects in experimental envenomation of dogs with Crotalus durissus terrificus venom. Twenty-eight mixed breed adult dogs were divided into three groups of seven animals each: Group I only venom; Group II - venom + 50ml antiophidic serum + fluid therapy; Group III venom + 50ml antiophidic serum + fluid therapy + urine alkalization. Lyophilized venom of Crotalus durissus terrificus was reconstituted in saline solution and inoculated subcutaneously at the dose of 1mg/kg body weight. Three animals of each group were subjected to euthanasia, and their muscular tissue, brain, spleen, kidneys, heart, lungs, stomach, small and large intestines, and popliteal lymph node fragments were collected for histopathological evaluation. There was myonecrosis in the inoculated limb, renal tubular degeneration, lymphoid hyperplasia of spleen, and unspecific reactive hepatitis. These results show the antigenicity and action of the venom on the immune system.

Reproductive biology of the neotropical rattlesnake Crotalus durissus from northeastern Brazil: a test of phylogenetic conservatism of reproductive patterns

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Modulation of the Pharmacological Activities of Secretory Phospholipase A2 from Crotalus durissus cascavella Induced by Naringin

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Energetic cost of predation: Aerobic metabolism during prey ingestion by juvenile rattlesnakes, Crotalus durissus

We investigated the cost of prey ingestion in the South American rattlesnake, Crotalus durissus, to see if the capacity to generate energy aerobically could be a constraint on the size of the prey that can be ingested. To accomplish this goal, we measured time and aerobic metabolism (inferred from oxygen consumption) of juvenile C. durissus ingesting prey ranging from 10 to 50% of their own body mass. Time needed for prey ingestion increased with prey size, with prey representing 10 and 20% of snake size being ingested with the same effort. Whole animal rates of oxygen consumption increased linearly with prey size, but at a slower pace for snakes ingesting prey larger than 30% of their body mass. Aerobic factorial power input necessary for prey ingestion increased with prey size, and for snakes ingesting prey representing 50% of their body mass it equaled the aerobic factorial scope for exercise. For the maximum prey size tested, the aerobic derived energy necessary for prey ingestion represented 0.02% of the total energy content of the prey. Within the prey size range we studied, the cost of ingestion did not constitute any constraint on the size of the prey that can be ingested. These constraints are set by morphological (gape size), ecological (predation risk), and, probably, by physiological parameters, as suggested by the tendency of V̇O2 during ingestion to increase at a slower pace at relative larger prey sizes.

Laboratory evaluation of young ovines inoculated with natural or 60Co-irradiated Crotalus durissus terrificus venom during hyperimmunization process

Laboratory profile of young ovines was studied in order to evaluate and compare their antiserum production from natural and Cobalt-60 irradiated Crotalus durissus terrificus (C.d.t.) venoms. The parameters analyzed included complete blood count, and urea, creatinine, aspartate aminotransferase, total proteins, albumin and globulin serum measurements. Three groups of six animals each were used. Group 1 (G1) received natural C.d.t. venom; Group 2 (G2) received irradiated C.d.t. venom; and Group 3 (G3) was used as control and did not receive venom, only adjuvants, using seven venom inoculations. During the experimental period, animals were fortnightly weighed. According to clinical and weight evaluation, sheep in post-weaning phase showed no changes in their physiological profiles but had excellent weight gain. The parameters analyzed were not statistically different (p<5%) among the groups tested. The hyperimmunization process was successfully accomplished with the production of specific antibodies against Crotalus durissus terrificus venom. Results bring a new possibility of utilizing ovines in the commercial production of anticrotalic serum, which may be used to treat human and animal envenomation. Its production cost may be reduced by subsequent use of hyperimmunized sheep for human consumption.

Frequency of rhabdiasid nematodes in wild Crotalus durissus terrificus (Serpentes, Viperidae) from Botucatu region, São Paulo State, Brazil

The aim of the present study was to evaluate the frequency of rhabdiasid nematodes in recently captured Crotalus durissus terrificus snakes from São Paulo State, Brazil. Fifty snakes (34 males and 16 females) were studied and each one was evaluated for the presence of that nematode at the moment of receipt at the Institution and after 90 days of quarantine inside individual cages. Tracheopulmonary washes were examined. Snakes that died during quarantine underwent necropsy and lung examination. Analysis of the results obtained at the two evaluation times (0 and 90 days), in addition to the data obtained during necropsies, showed that 44% (18 males and 4 females) of the C. d. terrificus snakes were naturally infected by rhabdiasid nematodes. These data demonstrate the parasitism level in natural conditions and are important for the sanitary handling of these reptiles in captivity.

Experimental envenomation with Crotalus durissus terrificus venom in dogs treated with antiophidic serum - Part I: Clinical evaluation, hematology and myelogram

The present study aimed at evaluating clinical and laboratory aspects during experimental envenomation by Crotalus durissus terrificus in dogs treated with antiophidic serum. Twenty-one dogs were divided into three groups of seven animals each. Group I received 1mg/kg venom (sc); Group II received 1mg/kg venom (sc), 50mg antiophidic serum (iv), and fluid therapy including 0.9% NaCl solution (iv); and Group III received 1mg/kg venom (sc), 50mg antiophidic serum (iv), and fluid therapy including 0.9% NaCl solution containing sodium bicarbonate diluted to the dose of 4mEq/kg. The clinical signs of ataxia, sedation, flaccid paralysis, mydriasis, eyeball paralysis, mandible ptosis, sialorrhea, vomiting and diarrhea observed in the dogs were very similar to those observed in humans. The decrease in hemoglobin, hematocrit, erythrocyte, platelet and fibrinogen levels, prolongation of clotting time, prothrombin time (PT) and activated partial thromboplastin time (APTT), as well as hypocellularity in the bone marrow characterized anemia, thrombocytopenia and blood incoagulability, as well as hypofibrinogenemia and decreased bone-marrow activity. Important bleeding was not observed. Increased numbers of leukocytes and neutrophils and decreased numbers of lymphocytes and eosinophils characterized an acute inflammatory response and stress caused by generalized pain. The employed antiophidic serum was effective and all animals survived.

Experimental envenomation with Crotalus durissus terrificus venom in dogs treated with antiophidic serum - Part II: Laboratory aspects, electrocardiogram and histopathology

The present work shows laboratory aspects, electrocardiogram and histopathology results during experimental envenomation by Crotalus durissus terrificus in dogs treated with antiophidic serum. Twenty-one dogs were divided into three groups of seven animals each. Group I received 1mg/kg venom (sc); Group II received 1mg/kg venom (sc), 50mg antiophidic serum (iv) and fluid therapy including 0.9% NaCl solution (iv); and Group III received 1mg/kg venom (sc), 50mg antiophidic serum (iv) and fluid therapy including 0.9% NaCl solution containing sodium bicarbonate diluted to the dose of 4mEq/kg. Urinalysis showed brown urine, proteinuria, occult blood and myoglobinuria. Respiratory acidosis and hypotension were also observed. At the venom inoculation site, there was discreet edema, popliteal lymph node response, musculature presenting whitish areas and necrotic myositis with myoregenerative activity. There was not evidence of electrocardiographical and biochemical alterations.