Effects of 60Co gamma radiation on crotamine

Ionizing radiation can change the molecular structure and affect the biological properties of biomolecules. This has been employed to attenuate animal toxins. Crotamine is a strongly basic polypeptide (pI 10.3) from Crotalus durissus terrificus venom composed of 42 amino acid residues. It induces skeletal muscle spasms leading to a spastic paralysis of hind limbs in mice. The objective of the present study was to carry out a biochemical study and a toxic activity assay on native and irradiated crotamine. Crotamine was purified from C.d. terrificus venom by Sephadex G-100 gel filtration followed by ion-exchange chromatography, and irradiated at 2 mg/ml in 0.15 M NaCl with 2.0 kGy gamma radiation emitted by a 60Co source. The native and irradiated toxins were evaluated in terms of structure and toxic activity (LD50). Irradiation did not change the protein concentration, the electrophoretic profile or the primary structure of the protein although differences were shown by spectroscopic techniques. Gamma radiation reduced crotamine toxicity by 48.3%, but did not eliminate it.

Intoxicação por veneno de sapo em um canino

O sapo do gênero Bufo possui nas suas glândulas paratóides uma secreção mucóide contendo toxinas como bufaginas e Bufotoxinas, que são esteróides cardiogênicos. Os cães podem atacar os sapos, entrando em contato com o veneno por meio das mucosas. Um canino, da raça Bulldog Francês, foi encaminhado ao Setor de Patologia Veterinária da Universidade Federal do Rio Grande do Sul (UFRGS) para a necropsia com histórico de provável intoxicação por veneno de sapo. Na necropsia o canino apresentava pulmões aumentados de volume, avermelhados e com edema, e rins de coloração vermelho-escura. As alterações microscópicas indicaram congestão, hemorragia e edema pulmonar. Nos rins, no baço e nos linfonodos foi observada congestão. As análises toxicológicas para os venenos de rotina foram negativas. Porém, a investigação do veneno de sapo a partir de cromatografia por camada delgada e gasosa demonstrou resultado positivo, revelando ser esta a causa da morte do canino.

EFFECT OF APIS-MELLIFERA BEE VENOM AND GAMMA-RADIATION ON BONE-MARROW CELLS OF WISTAR RATS TREATED INVIVO

To determine whether the venom of Apis mellifera can exert a radioprotective effect, by reducing the frequency of chromosome aberrations induced by radiation, five different experiments were performed on bone marrow cells of Wistar rats.Animals weighing about 100 g were injected intraperitoneally with different venom concentrations (1.0 or 0.5 mul) 1 or 24 h before, or 30 min after being submitted to 3 or 4 Gy of gamma radiation, and sacrificed 24 h after the last treatment. For each experiment in addition to the group of animals submitted to combined treatment (venom + radiation) and to their control, there was also one group treated with radiation only and another treated with venom only. A decrease in the frequency of chromosome aberrations, and fragments in particular, as well as in the number of cells with aberrations was observed in the experiments in which venom was administered 24 h before irradiation, and the effect was more marked at the higher venom concentration (1 mul/100 g weight).

Structure and biological activities of eumenine mastoparan-AF (EMP-AF), a new mast cell degranulating peptide in the venom of the solitary wasp (Anterhynchium flavomarginatum micado)

A new mast cell degranulating peptide, eumenine mastoparan-AF (EMP-AF), was isolated from the venom of the solitary wasp Anterhynchium flavomarginatum micado, the most common eumenine wasp found in Japan. The structure was analyzed by FAB-MS/MS together with Edman degradation, which was corroborated by solid-phase synthesis. The sequence of EMP-AF, Ile-Asn-Leu-Leu-Lys-Ile-Ala-Lys-Gly-Ile-lle-Lys-Ser-Leu-NH(2), was similar to that of mastoparan, a mast cell degranulating peptide from a hornet venom; tetradecapeptide with C-terminus amidated and rich in hydrophobic and basic amino acids. In fact, EMP-AF exhibited similar activity to mastoparan in stimulating degranulation from rat peritoneal mast cells and RBL-2H3 cells. It also showed significant hemolytic activity in human erythrocytes. Therefore, this is the first example that a mast cell degranulating peptide is found in the solitary wasp venom. Besides the degranulation and hemolytic activity, EMP-AF also affects on neuromuscular transmission in the lobster walking leg preparation. Three analogs EMP-AF-1 similar to 3 were snythesized and biologically tested together with EMP-AF, resulting in the importance of the C-terminal amide structure for biological activities. (C) 2000 Elsevier B.V. Ltd. All rights reserved.

Crystal structure of myotoxin-II: A myotoxic phospholipase A2 - Homologue from Bothrops moojeni venom.

The crystal structure of Myotoxin-II (MjTX-II), a Lys49 PLA(2)-homologue from Bothrops moojeni venom has been determined and refined at 2.0 Angstrom to a crystallographic residual of 19.7% (R-free = 28.1%). MjTX-II is a dimer in the crystal, with the monomers in the asymmetric unit related by a two-fold symmetry axis running through the dimer interface. The dimers of MjTX-II and the Lys49 PLA(2) from B. asper venom are similar, however the relative orientations of the monomers suggests a flexible dimer interface, which serves as a hinge between the two molecules.

Molecular determinants of binding of a wasp toxin (PMTXs) and its analogs in the Na+ channels proteins

The structural specificity of alpha-PMTX, a novel peptide toxin derived from wasp venom has been studied on the neuromuscular synapse in the walking leg of the lobster. alpha-PMTX is known to induce repetitive action potentials in the presynaptic axon due to sodium channel inactivation. We synthesized 29 analogs of alpha-PMTX by substituting one or two amino acids and compared threshold concentrations of these mutant toxins for inducing repetitive action potentials. In 13 amino acid residues of alpha-PMTX, Arg-1, Lys-3 and Lys-12 regulate the toxic activity because substitution of these basic amino acid residues with other amino acid residues greatly changed the potency. Determining the structure-activity relationships of PMTXs will help clarifying the molecular mechanism of sodium channel inactivation. (C) 2000 Elsevier B.V. Ireland Ltd. All rights reserved.

Crystallization of bothropstoxin II isolated from the venom of Bothrops jararacussu

A myotoxic phospholipase A(2), bothropstoxin II, which exhibits low hydrolytic activity, was crystallized and X-ray diffraction data were collected to a resolution of 2.2 Angstrom. Preliminary analysis reveals the presence of three molecules in the asymmetric unit. Copyright (C) 1996 Elsevier B.V. Ltd.

Structural and functional characterization of neuwiedase, a nonhemorrhagic fibrin(ogen)olytic metalloprotease from Bothrops neuwiedi snake venom

A fibrino(geno)lytic nonhemorrhagic metalloprotease (neuwiedase) was purified from Bothrops neuwiedi snake venom by a single chromatographic step procedure on a CM-Sepharose column, Neuwiedase represented 4.5% (w/w) of the crude desiccated venom, with an approximate Mr of 20,000 and pI 5.9, As regards the amino acid composition, neuwiedase showed similarities with other metalloproteases, with high proportions of Asx, Glx, Leu, and Ser, Atomic absorption spectroscopy showed that one mole of Zn2+ and one mole of Ca2+ were present per mole olf protein. The cDNA encoding neuwiedase was isolated by RT-PCR from venom gland RNA, using oligonucleotides based on the partially determined amino-acid sequences of this metalloprotease. The fall sequence contained approximately 594 bp, which codified the 198 amino acid residues with an estimated molecular weight of 22,375. Comparison of the nucleotide and amino acid sequences of neuwiedase with those of other snake venom metalloproteases showed a high level of sequential similarity, Neuwiedase has two highly conserved characteristics sequences H(142)E(143)XXH(146)XXG(140)XXH(152) and C164I165M166. The three-dimensional structure of neuwiedase was modeled based on the crystal structure of Crotalus adamanteus Adamalysin II. This model revealed that the zinc binding site region showed a I high structural similarity with other metalloproteases,, the proteolyitc specificity, using the B beta-chain of oxidized insulin as substrate, was shown to be directed to the Ala(14)-Leu(15) and Tyr(16)-Leu(17) peptide bonds which were preferentially hydrolyzed. Neuwiedase is a A alpha,B beta fibrinogenase, Its activity upon the A alpha chain of fibrinogen was detected within 15 min of incubation. The optimal temperature and pH for the degradation of both A alpha and B beta chains were 37 degrees C and 7.4-8.0, respectively. This activity was inhibited by EDTA and 1,10-phenantroline, Neuwiedase also showed proteolytic activity upon fibrin and some components of the extracellular matrix. However, it did not show TAME esterase activity and was not able to inhibit platelet aggregation. (C) 2000 Academic Press.

EFFECT OF PRETREATMENT WITH VENOM OF APIS-MELLIFERA BEES ON THE YIELD OF GAMMA-RAY INDUCED CHROMOSOME-ABERRATIONS IN HUMAN BLOOD-LYMPHOCYTES

Venom of the honey bee Apis mellifera induced a protective effect against the induction of dicentric chromosomes by gamma radiation (2.0 Gy) in human peripheral blood lymphocytes which the cultures were treated with 0.00015 mul venom/1 ml medium 6 h before irradiation. In cultures to which the venom was added immediately before irradiation with 0.25, 1.0 and 2.0 Gy, no significant differences in number of dicentric chromosomes induced was observed when compared to cultures submitted to irradiation only. The venom did not induce clastogenic effects nor did it increase the frequency of sister chromatid exchanges.

Kinetic and mechanistic characterization of the Sphingomyelinases D from Loxosceles intermedia spider venom

Envenomation by arachnids of the genus Loxosceles leads to local dermonecrosis and serious systemic toxicity mainly induced by sphingomyelinases D (SMase D). These enzymes catalyze the hydrolysis of sphingomyelin resulting in the formation of ceramide-phosphate and choline as well as the cleavage of lysophosphatidyl choline generating the lipid mediator lysophosphatidic acid. We have, previously, cloned and expressed two functional SMase D isoforms, named P1 and P2, from Loxosceles intertnedia venom and comparative protein sequence analysis revealed that they are highly homologous to SMase I from Loxosceles laeta which folds to form an (alpha/beta)(8) barrel. In order to further characterize these proteins, pH dependence kinetic experiments and chemical modification of the two active SMases D isoforms were performed. We show here that the amino acids involved in catalysis and in the metal ion binding sites are strictly conserved in the SMase D isoforms from L. intermedia. However, the kinetic studies indicate that SMase P1 hydrolyzes sphingomyelin less efficiently than P2, which can be attributed to a substitution at position 203 (Pro-Leu) and local amino acid substitutions in the hydrophobic channel that could probably play a role in the substrate recognition and binding. (c) 2005 Elsevier Ltd. All rights reserved.

Crystallization and preliminary X-ray diffraction analysis of a novel Arg49 phospholipase A(2) homologue from Zhaoermia mangshanensis venom

Zhaoermiatoxin, an Arg49 phospholipase A(2) homologue from Zhaoermia mangshanensis (formerly Trimeresurus mangshanensis, Ermia mangshanensis) venom is a novel member of the PLA(2)-homologue family that possesses an arginine residue at position 49, probably arising from a secondary Lys49 -> Arg substitution that does not alter the catalytic inactivity towards phospholipids. Like other Lys49 PLA(2) homologues, zhaoermiatoxin induces oedema and strong myonecrosis without detectable PLA(2) catalytic activity. A single crystal with maximum dimensions of 0.2 x 0.2 x 0.5 mm was used for X-ray diffraction data collection to a resolution of 2.05 angstrom using synchrotron radiation and the diffraction pattern was indexed in the hexagonal space group P6(4), with unit-cell parameters a = 72.9, b = 72.9, c = 93.9 angstrom.

ANTAGONISM OF THE MYOTOXIC EFFECTS OF BOTHROPS-JARARACUSSU VENOM AND BOTHROPSTOXIN BY POLYANIONS

The effects of heparin and other polyanions on the myotoxicity of Bothrops jararacussu venom and purified bothropstoxin (BthTX) were investigated. The release rate of creatine kinase (CK) from isolated extensor digitorum longus muscle and the plasma CK activity of mice were used to quantify the results. The myotoxic effects of B. jararacussu venom or BthTX were inhibited by preincubation of these agents with one of the following: a heterogeneous heparin preparation (designated 'heparin'), low mol. wt heparin (H-4500) or dextran sulfates (DS-8000 and DS-500,000). Non-sulfated dextran (D-40,000) and two chondroitin sulfates were ineffective. The antimyotoxic effects of the polyanions are ascribed to their forming inactive acid-base complexes with the basic myotoxins of Bothrops venoms. Gel-filtration experiments in Sephadex provided direct evidence for complex formation between heparin and BthTX. Intravenous (i.v.) administration of H-4500 or DS-8000 opposed the increase in plasma CK activity induced by a subsequent i.m. injection of venom or BthTX. In contrast, pretreatment with i.v. heparin or DS-500,000 enhanced the venom-induced increase in plasma CK activity. This effect was not observed (1) when the animals were treated with a polyvalent antivenom, which inhibits the coagulation and local stasis induced by Bothrops venoms, and (2) when BthTX, which has no thrombotic or hemorrhagic properties, was the myotoxic agent. The potentiation of the venom-induced increase in plasma CK activity by heparin and DS-500,000 is ascribed to improved washout of the CK released from damaged fibers, because of the anticoagulant properties of the drugs.