Poster

POSTER

Purification and characterization of peptide toxins from the sea anemone Bunodosoma cangicum

Zaharenko, A.J.^{I, II}; Oliveira, J.S.^{I, II}; Freitas, J.C.^{I, II}; Portaro, F.C.V.^II; Pimenta, D.C.^II; Yamane, T.^II; Konno, K.^II

^IDepto. de Fisiologia-Instituto de Biociências, USP, São Paulo, Brazil

^IICenter for Applied Toxinology, CAT/CEPID, FAPESP, Instituto Butantan, São Paulo, Brazil

^{Correspondence}

AIM: To purify and identify neurotoxic and haemolytic compounds previously reported in the venom of the sea anemone Bunodosoma cangicum.

MATERIALS AND METHODS: The venom was obtained by electrical stimulation of the whole animals in artificial sea water. It was lyophilized and gel-filtered in a Sephadex G-50 column, yielding five peaks. The haemolytic fraction was detected in mouse erythrocyte suspension and the neurotoxic fraction assayed in crab leg nerve bioassay and further purified by RP-HPLC (214nm) in a Beckman C-18 (150mm x 4.6mm) analytical column performed in linear gradient from 5% to 50% of buffer B in 25min, revealing three major peaks. These peaks were analyzed by Q-TOF and MALDI-TOF mass spectroscopy. SDS-PAGE was also carried out with the haemolytic fraction in order to determine the approximate molecular weight of the cytolysins.

RESULTS: The cytolytic compounds showed molecular masses between 10kDa and 20kDa, suggesting that they belong to the typical class of anemone cytolysins. Two of the three major peaks obtained from HPLC showed strong neurotoxic activity and molecular masses of 4156Da and 4975Da. The former's molecular mass is near to those of some known sea anemone K+ channel blockers (BgK: 4282Da and ShK: 4061Da). The latter is probably related to the long sea anemone peptides which are known to delay the inactivation of the voltage-gated Na+ channels. ATX I, II and V from Anemonia sulcata and cangitoxin from B. cangicum have molecular masses closely related to this compound.

CONCLUSIONS: We conclude that the polypeptidic mixture obtained directly from nematocyst discharge by electrical stimulation may provide us less levels of the body contaminants with great amounts of neurotoxic and haemolytic compounds. Some of these fractions may prove to be novel K+ and Na+ channel toxins. We are currently determining the primary sequence of these neurotoxic peptides.

ACKNOWLEDGEMENTS: CEBIMar- USP, FAPESP.

A novel hemolytic peptide isolated from the skin secretion of the Brazilian tree-frog Hyla biobeba (Anura: Hylidae)

Matsushita, R.H.^{I, II}; Fontes, W.^II; Sousa, M.V.^II; Schwartz, C.A.^I; Schwartz, E.F.^I; Sebben, A.^I; Castro, M.S.^{I, II}

^ILaboratório de Toxinologia, Departamento de Ciências Fisiológicas, IB, Universidade de Brasília

^IILaboratório de Bioquímica e Química de Proteínas, Centro Brasileiro de Serviços e Pesquisas em Proteínas, Departamento de Biologia Celular, IB, Universidade de Brasília, Brasília, DF, Brasil

^{Correspondence}

Several biologically active components such as biogenic amines, alkaloids, steroids, peptides and proteins occur in amphibian skin secretions. These compounds exhibit a large array of pharmacological effects and have different functions. Many of them are probably used in self-defense against predators and may protect against microbial infections. Hemolytic activity has been described in the skin secretions of the toad Bombina variegata, the newt Triturus cristatus and the caecilian Siphonops paulensis. In this report, we describe the purification and partial characterization of a novel hemolytic peptide isolated from the skin secretions of adult specimens of the tree-frog Hyla biobeba Bokermann and Sazima, 1973. The frogs were collected in the Federal District and maintained in captivity at the University of Brasília. Secretions were obtained by mild electrical stimulation and were freeze-dried. Aliquots of dried secretion were applied to a C₈ reversed-phase column (Pharmacia Biotech, 4.6 x 250 mm). The eluted fractions were collected manually, lyophilized and tested for hemolytic activity. One hemolytic fraction was rechromatographed using a C18 reversed-phase column (Vydac 218TP54, Separations Group). The purified hemolysin was analyzed by MALDI-TOF (Reflex IV, Bruker) and sequenced by automated Edman degradation using an ABI 477A sequencer. This bioactive peptide had a molecular mass of 1.86 kDa and shared high sequence identity with other hemolytic peptides previously isolated from Hyla species by our group.

Financial support by: FUB/UnB and FINATEC.

Purification and characterization of a putative antimicrobial peptide isolated from the frog Odontophrynus cultripes skin secretion

Sena, G.^I; Fontes, W.^II; Sousa, M.V.^II; Schwartz, C.A.^I; Schwartz, E.F.^I; Sebben, A.^I; Castro, M.S.^{I, II}

^ILaboratório de Toxinologia, Departamento de Ciências Fisiológicas/IB, Universidade de Brasília

^IICentro Brasileiro de Serviços e Pesquisas em Proteínas, Departamento de Biologia Celular/IB, Universidade de Brasília, Brasília/DF, Brasil

^{Correspondence}

Amphibian cutaneous secretions have been studied since a long time ago because they carry a large range of noxious substances. For example, they contain bioactive compounds represented by biogenic amines, steroids, alkaloids, peptides and proteins. Many of these substances are used in self-defense against predators and microorganisms. In this report, we describe the purification and characterization of one putative antimicrobial peptide obtained from Odontophrynus cultripes (Leptodactylidae) cutaneous secretion. Adult specimens of O. cultripes were collected in Luziânia/GO and maintained in captivity at the University of Brasília. Cutaneous secretion was obtained by mild electrical stimulation, lyophilized and stored at –20°C. The dried secretion was applied onto a C8 reversed-phase column. One eluted fraction was individually rechromatographed using a C18 reversed-phase column and resulted in the purification of one homogenous peptide. This purified fraction was submitted to mass spectrometry analysis using MALDI-TOF technique (Reflex IV, Bruker Daltonics) and to Edman degradation using an automated protein sequencer (477A-120A, Applied Biosystems). This novel peptide has 18 amino acid residues and a molecular mass of 1.800 Da. This peptide shows sequence similarities to other antimicrobial peptides from amphibian species included in Brevenin and Uperin families.

Financial support by: PIBIC/CNPq, FUB/UnB and FINATEC.

Recombinant toxins from Triatoma infestans salivary gland - molecular cloning and three dimensional structure

Feijó, G.^{I, II}; Martins, N.F.^III; Santana, J.M.^I; Lozzi, S.P.^I; Teixeira, A.R.L.^I

^ILaboratório Multidisciplinar de Pesquisa em Doença de Chagas, Universidade de Brasília

^IIUniversidade Católica de Brasília

^IIILaboratório de Bioinformática, EMBRAPA - Recursos Genéticos e Biotecnologia, Brasília, DF, Brasil

^{Correspondence}

Saliva of bloodsuckers contains various regulators of some hemostatic stages. Some saliva proteins activate the host’s fibrinolytic system (Basanova et all, 2002). The understanding of these proteins structure and functions could lead to a feasible mean to control insect-borne diseases. For this purpose we have carried on studies on biochemical, molecular purification and modeling of salivary proteins from the kissing bug Triatoma infestans. Therefore, a cDNA library was constructed in the l ZAP phage, and rabbit antibodies were used for immunescreening. Three gene sequences were selected and transferred to high expression plasmids where recombinant proteins were obtained in prokaryotic cells: Triatin, Infestilin and triatox have been obtained in sufficient amounts. Preliminary tests show hemorrhagic activity in vitro. The sequences were submitted to bioinformatics analysis and molecular modeling prediction. A similarity search performed with BLASTp showed low identity with nitrophorin and others salivary gland proteins for the three genes. Against the protein data bank the BLAST search selected the thrombin inhibitor form T. pallidipenis as a possible template for molecular modeling with 30% identity between the template and the targets. The threading structure prediction techniques for homology modeling revealed the scaffold similar to the lipocalin family. Lipocalins are remarkably diverse at the sequence and function level yet have highly conserved structures. The conserved motifs fold in highly symmetrical all-b structure dominated by a single eighth stranded antiparallel beta-strand flattened in its elliptical shape. This work present the three dimensional structure of the three proteins and discuss the structure-function relationship.

Delta - endotoxins structure database

Martins, N.F.^I; Togawa, R.C.^I; Batista, J.A.N.^II; Oliveira, O.B.N.^II; Dias, S.C.^II; Silva, S.M.B.^II; Fragoso, R.R.^II; Quezado, M.^II; Grossi de Sa, M.F.^II

^ILaboratório de Bioinformática, EMBRAPA - Recursos Genéticos e Biotecnologia, Brasília, DF, Brasil

^IILaboratório de Biologia Molecular EMBRAPA - Recursos Genéticos e Biotecnologia, Brasília, DF, Brasil

^{Correspondence}

The delta endotoxin (or cry protein) are produced in great variety with different species of Bacillus thuringiensis. Some proteins are specific against insects in the orders Lepidoptera, Diptera and Coleoptera. Once ingested by the host, the toxin are activated by gut proteases. The active protoxins bind to receptors on the brush border of the intestinal epithelium and create 'leakage channels' of a diameter of about 10-20 angstroms. This subsequently leads to insect death due to starvation and septicemia. The toxicology data provided nowadays are sufficient to demonstrate the specificity but not the mode of action of delta-endotoxins. Although all the effects and benefits are well established around the world there is still a remain question about the mechanism of action at the molecular level. Actually, the database has around 100 genes, but only 5 three dimensional structures are available in the PDB databank. Detailed structural and biochemical knowledge of BT d-endotoxins would permit the redesign of these toxins to customize host range, alter activity, improve stability etc. In this context, genetic engineering and molecular modeling together are able to add some important information about the structure and function relationship. This work presents the structural databases of 50 d-endotoxins from different families. The major goal was to build a molecular modeling databank which will allow further studies at the atomic level. All models were built using MODELLER and validated by PROCHECK and PROSA II. The structure –function relationship is discussed and the families conserve in their structures singular features that may explain the specificity of the toxins.

Crotamine 3D structure - homology building simulation and circular dichroism

Martins, N.F.^I; Siqueira, A.^II; Cartier, A.^II; Brown, D.^III; Maigret, B.^{II, III}

^ILaboratório de Bioinformática - Embrapa - Recursos Genéticos e Biotecnologia, Brasília, Brasil

^IILaboratory of Theoretical Chemistry, UMR CNRS/UHP 756, Université Henri Poincaré, France

^IIILaboratory of Organic Materials, Université Savoie, France

^IVInstituto de Química, UnB, Brasil

^{Correspondence}

Snake venoms contain proteins that are responsible for their neurotoxic, cardiotoxic, hemorrhagic and myotoxic activities. The myotoxin family comprises several subclasses involved in citolysis, myofibril clumping and hypercontraction. The general biological action of crotamine is the depolarization of cell membranes on the voltage sensitive sodium channels. The toxin also seems to alter calcium ion influx by opening the ryanocine receptor. Crotamine isolated from the venom of Crotalus durissus terrificus is a strongly basic 42-aminoacid polypeptide belonging to the small basic myotoxin family. As no three-dimensional structure is available for this myotoxin subfamily, despite the considerable amount of work, there is no clear understanding on the structure-function relationship. The common patterns for this myotoxin family provides a myotoxin signature consisting of three disulfide bridges which are important to the protein fold. FASTA and BLAST searches revealed 36% identity with b-defensin family. The 1BNB structure was used as a template to the model. The 3D coordinates of the crotamine model was obtained by homology modeling and several rounds of molecular dynamics calculations were carried out. The analysis of the RMSD map for each structure during the 1 ns MD simulation in vacuum showed that the structure was very stable along the calculations. After procheck verification the model was analyzed for its interactions. Mainly H-bonds were detected between Asp29 side chain and Lys7 and Arg33 respectively. In conclusion, crotamine appears to have a stable core organized around the loops and maintained by a b-strand. A weak helical tendency is visible in the model and which can be correlated to the CD results.

Catalytic activity of D49- versus K49-phospholipase A2: Molecular dynamics reveals a new hypothesis

Martins, N.F.^I; Cartier, A.^II; Brown, D.^III; Maigret, B.^{II, III}

^ILaboratório de Bioinformática - Embrapa - Recursos Genéticos e Biotecnologia, Brasília, Brasil

^IILaboratory of Theoretical Chemistry, UMR CNRS/UHP 756, Université Henri Poincaré, France

IIILaboratory of Organic Materials, Université Savoie, France

^IVInstituto de Química, UnB, Brasil

^{Correspondence}

Secreted PLA2s (sPLA2) are subdivided in two classes, I (pancreas, lung, spleen and cobra) and class II (crotalid and viper venoms and human non-pancreatic secretory PLA2s) which is subdivided into two classes: D49 and K49. K49 myotoxins are distinct and form a highly conserved protein family but in addition to the substitution at position 49, the K49 have other invariant residues not found in the D49 molecules. The D49 enzymes are high catalytic activity on artificial phospholipid but K49 has a few or no hydrolytic activity on artificial membranes. The modeling procedure of K49 enzyme from Bothrops godmani itself was initiated from the 1GOD and 1PIR structures. After preliminary calculations the whole system was relaxed until a stable energy minimum. Next 1 ns of Molecular Dynamics (MD) was recorded. All calculations were performed with ddgmq domain-decomposition parallel MD software. For each trajectory a conformation was recorded each 4 ps and 250 conformations were analyzed. Our calculations demonstrate that the calcium ion and the imidazole side chain are on opposite side of the conserved helix and the polarization of water by calcium is not sufficient to be translated so far from the polarization site. This means that another proposition has to be found to explain the lack of activity of K49 PLA2. Our calculations clearly reveal the role of Tyr69 in the activation of the catalytic water molecule and strongly suggest that the calcium ion is no more stabilized around the protein, moving from its starting location to the bulk. This simulation proves that calcium ions will not be anchored to another site on K49 PLA2.

Structural and functional analyses of BmjMIP, a PLA2 myotoxin inhibitor a-type protein from Bothrops moojeni snake plasma

Soares, A.M.^{I, II}; Marcussi, S.^I; Giglio, J.R.^II, Guerra-Sá, R.^II; França, S.C.^I; Ward, R.J.^III; Arantes, E.C.^IV

^IDepartamento de Biotecnologia, UNAERP

^IIDepartamento de Bioquímica e Imunologia, FMRP/USP

^IIIDepartamento de Química, FFCLRP/USP

^IVDepartamento de Física e Química, FCFRP/USP, Ribeirão Preto-SP, Brazil.

^{Correspondence}

A protein, which neutralizes the enzymatic, toxic and pharmacological activities of various basic phospholipases A₂ homologues from the venoms of B. moojeni, B. pirajai and B. jararacussu, has been isolated from Bothrops moojeni snake plasma by affinity chromatography using immobilized myotoxins on Sepharose gel. Biochemical characterization of this myotoxin inhibitor protein (BmjMIP) showed it to be an oligomeric glycoprotein with a M_r of 23,000 to 25,000 for the monomeric subunit. BmjMIP was stable in the pH range from 4.0 to 12.0, between 4°C and 80°C, even after deglycosilation. The role of the carbohydrate moiety was investigated and found not to affect the in vitro function of the inhibitor. The corresponding cDNA obtained by RT-PCR from the liver of this snake has 500 pb which encodes a mature protein of 166 amino acids which includes a 19 amino acid signal peptide. The primary structure of BmjMIP showed a high similarity with other snake phospholipase A₂ inhibitors (PLIs) in which the recognition domain for carbohydrates (CRDs) and the glycosylation site (Asn103) are conserved. Circular dichroism spectroscopy indicates that no significant alterations in the secondary structure of either the BmjMIP or the target protein occur upon interaction. The BmjMIP has a wide range of inhibitory properties against basic PLA₂s from Bothrops venoms (anti-enzymatic, anti-myotoxic, anti-edema inducing, anti-cytotoxic, anti-bactericidal and anti-lethal). However, the inhibitor had a reduced ability to neutralize the biological activities of crotoxin B, the PLA₂ homologue associated with crotapotin in Crotalus durissus terrificus snake venom.

Financial supported: FAPESP, CNPq, UNAERP.

Alkylation of histidine residues of Bothrops moojeni venom and its myotoxins I and II attenuates their toxicity without any harmfull effect on their antigenicity against other venoms and toxins

Soares, A.M.^{I, II}; Sestito, W.P.^II; Andrião-Escarso, S.H.^II; Marcussi, S.^I; Stabeli, R.G.^II; Cunha, O.A.B.^II; Vieira, C.A.^II; Giglio, J.R.^II

^IDepartamento de Biotecnologia, UNAERP

^IIDepartamento de Bioquímica e Imunologia, FMRP-USP, Ribeirão Preto-SP, Brazil

^{Correspondence}

Myotoxin-I (MjTX-I) and Myotoxin-II (MjTX-II) were purified to homogeneity from the venom of Bothrops moojeni by ion-exchange chromatography on CM-Sepharose. M_r of MjTX-I or II, estimated by SDS-PAGE, and that of MjTX-II by mass-spectrometry, were 13,500 and 13,698, respectively. After alkylation of B. moojeni crude venom (MjCV) and MjTX-II by p-bromophenacyl bromide (BPB), increases in the i.p. LD50 from 6.0 to 15.7 mg/kg and from 8.0 to 45.0 mg/kg (mice) were observed, respectively. In addition, doses of 5 LD50 of alkylated MjTX-I did not cause a single obit in mice. Also no myonecrosis was detected for the alkylated toxins, although both of them still induced edema. Antibodies to native and modified crude venom or myotoxins cross-reacted with 12 purified class II myotoxic phospholipases A2 found in snake venoms of the genus Bothrops. Myotoxic PLA2s from class I and class III were not recognized by the above antibodies. These results suggest that the overall antigenic structure is conserved among class II myotoxic PLA2s, despite differences in their corresponding amino acid sequences. Anti-MjTX-I-BPB and anti-MjTX-II-BPB rabbit serum, obtained against the modified myotoxins, were more efficient than those obtained against the native myotoxins. In neutralization experiments, pre-incubation of crude venom or isolated myotoxins with antibodies to the native or modified toxins inhibited their PLA₂ and myotoxic activities. Therefore, alkylation of His-48 by BPB strongly reduces the local tissue damage induced by B. moojeni venom or isolated myotoxins while keeping and even increasing antigenicity, thus leading to an enhanced and more efficient antiophidian serum production procedure for practical purposes.

Financial supported: FAPESP and CNPq.

Analysis of the venom of a Bothrops jararaca clutch keep in captivity

Travaglia-Cardoso, S.R.; Rocha, M.M.T.; Furtado, M.F.D.

Laboratório de Herpetologia- Instituto Butantan – São Paulo, Brazil

^{Correspondence}

A Bothrops jararaca clutch (12 males, 11 females) kept in captivity was analysed using the mass and biometry parameters. When they were 4 years old (adults) the males mass varied from 110 to 140g and the total lenght (TL) from 790 to 960mm, the females mass from 335 to 755g and the TL from 1160 to 1495mm. Venom samples were collected (first extration) individually and the following parameters were analysed: weight of liquid and dry venom, total protein content and its eletrophoretic profile (SDS-PAGE). Females produced more venom than males when liquid (t= 0,05(2)21 = 16,81, P < 0,001) or liofilized venoms (t= 0,05(2)21 = 31,03, P < 0,001) were considered. The water content of the venom varied from 72 to 81%, independently from sex or size of the animals. Regression analysis showed a significant relation between the amount of the produced venom and the TL of the females (F 0,05(1) = 13,234, P < 0,001). This was not true for males. The total protein content varied significantly among the males (662 to 1148 mg/mg of venom) and among the females (755 to 1251 mg/mg of venom). The electrophoretic profile presented greater intensity in the weights 60, 30 and 14 kDa. All the females presented 12 homogeneous bands. Sexual variation was found in the region 30 to 40 kDa: the females presented 4 clearcut bands and the males showed 4 to 8 bands superposed. The females were bigger, presented greater amount of venom and this was fairly homogeneous. The males were smaller and the venom presented greater variability in amount and in protein composition. One may infer that these variations are related to the feeding habits of the species B. jararaca.

Quality control in snake venom

Furtado, M.F.D.; Rocha, M.M.T.; Travaglia-Cardoso, S.R.

Laboratório Herpetologia – Instituto Butantan – São Paulo, Brazil

^{Correspondence}

The Laboratory of Herpetology maintains snakes in captivity to provide venom for scientific research and antivenom production. National reference venoms (Bothrops jararaca and Crotalus durissus terrificus, the latter from the state of São Paulo) are used in the titration of their respective antivenoms produced nationwide. Antigens are also prepared for the immunization of horses in antivenom production. Venom milked from healthy snakes is collected in an ice bath then centrifuged at 1300g for 30 min at 4ºC and the supernatant is frozen at -20ºC. The venoms are subsequently thawed, homogenized and aliquoted in flasks for lyophilization. Annual batches are prepared using the largest number of extractions possible in order to provide greater homogeneity of the final product for each species. In 2001 venom batches were produced for Micrurus corallinus, M. frontalis, Lachesis muta, Bothrops alternatus, B. atrox, B. jararaca, B. jararacussu, B. leucurus, B. neuwiedi, B. moojeni, Crotalus durissus ssp, C. d. terrificus, C. d. collilineatus, B. jararaca reference batch 05 and a bothropic pool. The pH of the venom varied from 6.2 to 6.4 for the genus Crotalus and from 5.3 to 5.7 for the other species. The variation in the protein content of the venom in natura (mg of protein/ml of venom) was significant (P<0.001) among the species analyzed, with M.corallinus showing the highest protein concentration (367mg/ml of venom). The protein content varied from 838.3 µg/mg of venom (B. jararacussu) to 1381.7 µg/mg of venom (M. corallinus). The LD50 varied from 0.05mg/Kg (C. durissus) to 9.88mg/Kg (L. muta). The SDS-PAGE electrophoretic profiles showed bands between 67 to 14kDa to 67kDa for Bothrops, and 12-67 kDa for L. muta, a band of 32kDa and others more intense bands below 12.2kDa for Crotalus and two weak bands between and 43kDa and 69 kDa for Micrurus, with others more intense bands below 14.4kDa. The prrocedures and preliminary tests described above have been incorporated into our routine fo each batch of venom produced.

Comparative study of venom of Philodryas olfersii and Philodryas patagoniensis (Serpentes Colubridae)

Rocha, M.M.T.^I; Acosta de Pérez, O.^II; Furtado, M.F.D.^I

^ILaboratório Herpetologia – Instituto Butantan, São Paulo, Brazil

^IIFacultad Ciências Veterinárias, Universidad Nacional Del Nordeste, Argentina

^{Correspondence}

Nowadays 65% of the existing snakes belong to Colubridae family. Some, but not all, present complex glands denominated Duvernoy’s glands, considered to be homologous to the glands of proteroglyphous and solenoglyphous snakes, which produce toxic secretion or “venom”. This group of snakes has been reported to cause human fatal accidents. The genus Philodryas has restrict distribution to South America, with 15 species, with only P. olfersii receiving attention in regard to the elucidation of its venom composition. A comparative study was performed on the pharmacology, biochemistry and pathology of venoms of P. olfersii and P. patagoniensis. The venoms induced lethal, hemorrhagic, myotoxic, no coagulant and desfibrinating effects, showing also proteolytic activity. The results showed that the P.olfersii venom has protein level of approximately 1mg/mg venom, while the P.patagoniensis venom presented 645mg/mg venom. The protein electrophoretic profile presents similar mobilities with components predominantly under 67kDa, with P.olfersii venom presenting greater number of bands. The profile of glycoproteins is very similar to that of proteins, showing that the majority of the components of these venoms are glycoproteins. The proteolytic activity is high, with values above 200U/mg in both venoms. The hemorrhagic activity ensues rapidly, with DMH of 23mg at 4 hours in P.olfersii, and 27µg at 2 hours in P.patagoniensis. P.olfersii presents DL50 of 58.85 µg/mouse and P.patagoniensis, 62.43 µg/mouse. Histologically after inoculation of 50mg/venom i.m., the hemorrhage sets up 15 min in both species. In 30min P.olfersii venom caused myonecrosis and inflammatory infiltrate, whereas in P.patagoniensis they arise in 60min. Venoms of both species presented the same activities, with variations in intensity and mainly in kinetics of action.

Isolation and enzymatic studies of a new thrombin-like enzyme BaIII-4 isolated from Peruvian Bothops atrox venom

Ponce-Soto, L.A.^I; Toyama, M.H.^{I, II}; Novello, J.C.^I; Yarlequé Chocas, A.^III; Marangoni, S.^I

^IDepartaments of Biochemistry Institute of Biology State University of Campinas (UNICAMP)

^IIDepartaments of Physiology and Biophisics Institute of Biology State University of Campinas (UNICAMP) Campinas, SP Brazil

^IIIFaculty of Biological Sciences, National University of San Marcos (UNMSM), Lima – Perú

^{Correspondence}

OBJECTIVE: A new thrombin-like serine protease was isolated from the venom of Bothrops atrox and was partially characterized.

METHODS AND RESULTS: The thrombin-like enzyme was isolated from B.atrox venom using molecular exclusion and reverse phase HPLC and mol mass of ~34 kDa in Tricine SDS-PAGE 12.5%.

The enzyme purified BaIII-4 had a high content of Asx, Glx, Gly, Ser, Ala and Pro, with 12 half Cys. The N-terminal amino acid sequence was: VIGGD ECDIN EHPFL AFMYY SPRYF CGMTL INQEW. BaIII 4 had a Km and Vmax of Km 7.1 X 10-4 M and Vmax 3.8 X 10-2 nmoles p-NA/min/mg. The enzymatic and biological activities (fibrinogenolitc action and platellet aggregation) of the enzyme were inhbited by protein aprotinin, PMSF, soybean trypsin inhibitor, TLCK and DA2 II (on anti-hemorrhagic protein from Didelphis albiventris opossum serum).

CONCLUSION: BaIII-4 showed similar Km and Vmax to those of thrombin-like enzymes from snake venoms. The inhibition by PMSF, TLCK, aprotinin and soybean trypsin inhibitor suggest a trypsin-like catalitic mechanism.

Financial Support: FAPESP

Unusual neurotoxic effect chicken biventer cervicis preparation of the PLA2 (F6) of crotoxin isolated from Crotalus durissus collilineatus venom

Ponce-Soto, L.A.^I; Toyama, M.H.^{I, II}; Rodrigues-Simioni, L.^III; Novello, J.C.^I; Marangoni, S.^I

^IDepartments of Biochemistry of Medical Sciences, State University of Campinas (UNICAMP) Campinas - BRAZIL

IIDepartments of Physiology and Biophysics /I.B of Medical Sciences, State University of Campinas (UNICAMP) Campinas - BRAZIL

^IIIDepartments of Farmacology Faculty of Medical Sciences, State University of Campinas (UNICAMP) Campinas - BRAZIL

^{Correspondence}

OBJECTIVES: To evaluate the activity unusual neurotoxic of a PLA2 (F6) of crotoxin isolated from C.d.collilineatus purified in a system of HPLC-RP, Using preparations of muscle biventer cervicis from chicken, through the technical conventional myographic (Fisiografo Gould RS 3400), in vat of 5 mL content nutritive solution of Krebs, at 37ºC. A preparations were subjected to one constant tension of 0.5 g and stimulated using a bipolar electrodes and pulses supremaximals of 0.1 Hz of frequency and 0.2 ms duration from a Stimulator Grass S48.

METHODS AND RESULTS: The PLA2 (F6) was purified by using HPLC-RP. Experiments controls were carried out besides the treated ones with two concentrations: 10 and 20 mg/mL. The addition of ACh (60 and 120 mM) or KCl (13.4 mM) before and after the addition of the PLA2 (F6) in preparations stimulated indirectly, I cause contract. It was observed that the PLA2 (F6) blocks the contractile response in dose-dependent manner (10 and 20 mg/mL) was determined for each. The 50% paralysis times was 42 ± 4, and 30 ± 1, (n=3) respective after 120 min observation. The PLA2 (F6) inhibited the twitch-tension of the contractile response irreversible in to the direct stimulus and it was able to reduce the contract induced by the ACh (10 mg/mL) 17 ± 16.66% and (20 mg/mL) 24 ± 14.52 % (n=3). The reduction of the answer to KCl (13.4mM) it was of 16 ± 8.02%.

CONCLUSION The PLA2 (F6) from C. d. collilineatus blocked the twitch-tension induced in the muscle biventer cervicis of chicken in a dose-time-dependent way, suggest that the neurotoxic action of crotoxin is strongly influenced by PLA2 (F6).

Financial Support: FAPESP

Effect of isolated PLA2 isolated from the Crotalus durissus collilineatus on the insulin secretion of rodents

Nogueira, T.C.A.^I; Ferreira, F.^I; Filiputti, E.^I; Toyama, M.H.^II; Marangoni, S.^II; Boschero, A.^I; Carneiro, E.M.^I

^IDepartment of Physiology and Biophysics, Institute of Biology, State University of Campinas

^IIDepartment of Biochemistry, Institute of Biology, State University of Campinas

^{Correspondence}

OBJECTIVES: Physiological action evaluation of isolated PLA2 on isolated B-cells of Langerhans.

MATERIAL AND METHODS: The isolated islets were incubated with toxins in presence of 2.8 mM and 16.7 mM of glucose as well as 30mM of potassium. After each protocol the insulin quantification was made to evaluate the action of toxin. At 2.8mM of glucose the isolated toxin (5.6 mg/ml) increased 4.9 folds the insulin secretion but in 16.7 mM of glucose this increasing was 1.9 fold higher than to control (p<0.05). In potassium presence only the isolated PLA2 increase 4 fold the insulin secretion (p<0.05). This toxin in 16.7 mM of glucose also significantly stimulated the calcium intake in 3.3 fold to than 16.7 mM of glucose but did not affected the glucose metabolism of isolated B-cells. In presence of Tetrodotoxin (TTX) or nifedipine did not change the increasing of insulin secretion induced by PLA2 as well as the Dexamethasone or incubation of PLA2 with heparin. Yet when the islet was incubated with heparin, the insulin secretion stimulated by PLA2 in presence of glucose 16.7 was partially inhibited.

CONCLUSION: The isolated PLA2 induce a insulin secretion independently of the B-cells stimulation (Glucose 2.8) and can be involved with intake calcium.

Characterization of structure and biological activities of novel crotamine isoform isolated from Crotalus durissus cascavella (maracamboia)

Toyama, M.H.^I; Oliveira, D.G.^I; Nogueira, T.C.A.^II; Rodrigues-Simioni, L.^III; Belian, L.O.S.^IV; Carneiro, E.M.^II; Monteiro, H.S.A.^V; Martins, A.M.C.^V; Boschero, A.C.^II ; Marangoni, S.^I

^IDepto Bioquímica, UNICAMP

^IIDepto Fisiologia e Biofísica, UNICAMP

^IIIDepto Farmacologia, UNICAMP

^IVInstituto Biológico de São Saulo, Campinas

^VDepto Fisiologia e Farmacologia, Universidade Federal do Ceará

^{Correspondence}

OBJECTIVES: In this work we analyzed four Crotalus durissus cascavella venoms provide from the northeast region of Brazil and isolated a novel crotamine-like isoform from the one rattlesnake group found in Fortaleza (Ceará State). Its primary structure, neurotoxic, insulin secretacogue, and bactericidal characterization was present.

METHODS AND RESULTS: Four Crotalus durissus cascavella rattlesnake venoms were analyzed in the molecular exclusion and reverse-phase HPLC. The analysis of the venom reveled some differences between then, but the most significant differences it was found to venom from the Fortaleza. This venom showed a high content of crotamine and the Convulxin is not the major fraction. Its novel protein showed a 90% of homology with other small basic neurotoxins as crotamine isolated from Crotalus durissus terrificus. It showed pH values of 9.5 and a molecular mass of 4.4 kDa. This protein it was characterized by strong facilitation and pre-synaptic effect at doses of 1mg, 3mg and 5mg and 10 mg/ml (maximum response). Using doses of 5mg this protein induced a strong insulin secretion in nom stimulated and stimulated state. The neurotoxic as well insulin secretion was strongly inhibited by TTX. This protein showed strong bactericidal effect in the both Gram-positive as well as in the Gram-Negative bacteria.

CONCLUSION: The geographical variation is very common to the Bothrops venom but there are lithe work described to Crotalic venom. C. durissus cascavella showed a significant variation to Convulxin content, the crotamine probably has important role to rattlesnake venoms found in Ceará and Crotoxin do not exhibit variation in its content but its subunits present significant variation. This novel crotamine acts on the sodium channel of the nerve and other excitatory cell as B-cell isolated from the Langerhans islet from rodents. Probably the bactericidal effect found to this crotamine is involving the sodium channel.

Prothrombin activator type C. (Lopap) and bristles extract of Lonomia obliqua actions on human coagulation and fibrinolytic factors

Reis, C.V.^I; Fritzen, M.^I; Flores, M.P.A.^I; Juliano, L.^II; Romero-Ramos, C.R.^III; Azevedo, I.L.M.J.^III; Ho, P.L.^III, Chudzinski-Tavassi, A.M.^I

^ILaboratório de Bioquímica e Biofísica, Instituto Butantan, São Paulo, SP

^IIDepartamento de Biofísica, UNIFESP, São Paulo

^IIICentro de Biotecnologia, I. Butantan, São Paulo

^{Correspondence}

Lonomia obliqua venom produces a severe consumption coagulopathy that can lead to a hemorrhagic syndrome. Here we describe some activities of the Lonomia obliqua bristles’ crude extract (LOBCE) and LOPAP on blood coagulation and fibrinolytic factors. LOBCE is able to clot the plasma, but not purified fibrinogen and shows prothrombin (LOPAP) and Factor X activators. In addition neither FXIII inhibition nor degradation (SDS-PAGE) is observed. Fibrinolytic activity on cross-linked fibrin is not found, however a fibinogenolytic activity on a-chains of fibrinogen occurred and the generated products are not similar from those induced by plasmin. In Solid Fibrin Assay no plasminogen activation is observed. Lopap hydrolysed fluorescent peptides derived from prothrombin sequences binding sites recognized by the thrombin (Arg284-Thr285) (Km 0,3269 mM and Kcat 2,91 s^-1) or by FXa (Arg320-Ile321). Lopap hydrolyse prothrombin independently of the prothrombinase complex, but its activity is strongly increased by ions calcium and phospholipids. A cDNA library from mRNA of bristles was constructed in pGEM11zf(+) plasmid. The Lopap cDNA (600bp) was amplified from this library by PCR using degenerated primers corresponding to N-terminal sequence of protein. This fragment was sequenced and sub-cloned in E. coli expression vector and the active recombinant Lopap with 6xHis fusion at the N-terminus was obtained. Aleatory clones showed a great variety of genes for several proteins of the insects. Of these, some proteins already present similarity with other known and presents in databases, among which we can highlight a BPP, a catepsin, and a hemolysin. Other DNAs didn't present homology with any present well-known sequence in databases, and they can also be studied. Lonomia obliqua extract contains procoagulant proteins that leading to a consumption coagulopathy. A fibrinogenolytic activity occurs also, although the component responsible for this activity is not able to degrades cross-linked fibrin. Also Lopap can be classified as a type C prothrombin activator.

(Supported by FAPESP, CNPq)

Isolation and characterization of a thrombin-like enzyme form the venom of the snake Bothrops insularis (jararaca Ilhoa)

Wermelinger, L.S.; Oliveira-Carvalho, A.L.; Castro, H.C.; Zingali, R.B.

Departamento de Bioquímica Médica, ICB UFRJ

^{Correspondence}

OBJECTIVES:B. insularis is a peculiar arboreal Brazilian snake found exclusively on Queimada Grande Island (São Paulo) that feeds only on birds. This venom presents a prominent fibrinoclotting activity (Selistre and Giglio, 1987). Our laboratory showed that ecotin, a trypsin/chymotrypsin inhibitor from E. coli, is able to interfere with this activity. Our main objective was to isolate and characterize the clotting factor from this venom that is affected by ecotin.

METHODS AND RESULTS:B. insularis crude venom was gel filtrated on Sephacryl S200HR column. The fraction that had fibrinoclotting activity affected by ecotin were further purified using an ecotin-agarose column. The eluted material (0.01 N HCl, 0.5 M NaCl, pH=2.0) showed a single band of activity in gelatin gel and also presented fibrinoclotting activity. Ecotin was able to enhance the fibrinoclotting activity by 50% with an EC50= 91 microM.

CONCLUSION AND PERSPECTIVES: Here in, we describe the purification a thrombin–like enzyme from the venom of a B. insularis, interestingly this protein has its activity enhanced by ecotin. The sequence of this protein will be determined, the characterization and the mechanism of interaction with ecotin are now in progress.

Financial support: CNPq, FAPERJ.

Neuronal nitric oxide synthase, PKG and ATP-sensitive K+ channels are involved in the antinociception induced by Crotalus durissus terrificus venom (CdtV)

Picolo, G.^I; Cassola, A.C.^II; Cury, Y.^I

^ILaboratório de Fisiopatologia, Instituto Butantan

^IIInstituto de Ciências Biomédicas, USP, Brasil

^{Correspondence}

The CdtV exerts peripheral antinociceptive effect in the carragenin (CG) and PGE2- induced hyperalgesia, mediated by d- (Eur. J. Pharmacol. 391:55-62, 2000) and k and d- peripheral opioid receptors, respectively, and by L-arginine-NO-cGMP pathway stimulation. Here we investigated the isoform of nitric oxide synthase (NOS) responsible for NO production and the participation of PKG and K+ channels in this effect. The rat paw pressure test, using CG or PGE2 as nociceptive stimulus, was used to evaluate pain threshold. The test was applied before and 3 h after the i.pl. injection of CG (200 µg/paw) or PGE2 (100 ng/paw). The antinociception induced by CdtV (200 µg/kg, p.o.) was blocked by 7-NI (50 µg/paw), but was not altered by L-NIL (50 µg/paw), neuronal and inducible NOS inhibitors, respectively. Rp-cGMP triethylamine (1.5 or 3 µg/paw), a PKG inhibitor, was able to partially reverse venom effect. TEA (640 µg/paw) or 4- AP (100 µg/paw) and charybdotoxin (2 µg/paw) or apamin (10 µg/paw), blockers of voltage-dependent and Ca⁺²-sensitive K+ channels, respectively, did not modify the antinociception. Glybenclamide (80 µg/paw), a blocker of ATP-sensitive K+ channels, blocked the venom effect. These data suggest that: a) neuronal nitric oxide cause analgesia, independently of the type of opioid receptor stimulated (k or d), b) the effect of CdtV involves the generation of neuronal NO, the stimulation of PKG and the opening of ATP-sensitive K+ channels.

Financial support: FAPESP, CNPq.

Interspecific variations of Loxosceles spiders venoms

de-Oliveira-Lima, K.C.; Gonçalves-de-Andrade, R.M.; Magnoli, F.C.; Ferreira-Junior, J.M.C.; Tambourgi, D.V.

Laboratório de Imunoquímica, Instituto Butantan, Brazil

^{Correspondence}

Loxosceles gaucho, Loxosceles intermedia and Loxosceles laeta are the main spiders responsible for Brazilian aracneism. They provoke a local necrotic lesion and rarely cause systemic effects. Recently, we demonstrated that the variation in the genetic background of the victim and intraspecies differences in the composition of the venoms are important factors determining the severity of the loxoscelism. The aim of this study was to evaluate other factors that could contribute to gravity of the loxoscelism, as inter-species variations of the venoms. Comparative analysis of L. gaucho, L. laeta and L. intermedia venoms by SDS-PAGE revealed a distinct pattern of bands, ranging from 10 to 200 kDa. To assess the ability of Loxosceles venoms to induce C-dependent haemolysis, human erythrocytes were treated with different amounts of the three venoms species and incubated with NHS. Results showed that venoms were able to render erythrocytes susceptible to lysis, however, a more potent haemolytic inducer’s effect was determined for L. gaucho venoms. Dermonecrotic activity was tested in adult rabbits by intradermal injection of the Loxosceles venoms. Typical loxoscelic lesions were observed, although L. gaucho venoms induced a larger loxoscelic lesion. L. laeta and L. intermedia venoms produced a stronger oedema reaction than L. gaucho venom. Beside the differences observed in the haemolytic and dermonecrotic assays we also investigated possible differences in the sphingomyelinase activity by testing the sphingomyelin cleaving ability of Loxosceles venoms. The results showed a similar sphingomyelinase activity among the species. In conclusion, Loxosceles venoms species exhibit differences in their toxic effects and these may contribute to the variability in the accidents severity caused by Loxosceles spiders.

Supported by FAPESP and The Wellcome Trust

Toxic effects of a recombinant Bothropstoxin-I on mice neuromuscular junction

Oliveira, M.^I; Dal Pai-Silva, M^II; Spencer, P.J^III; Gallacci, M.^I

^IDepartamentos de Farmacologia, IB, UNESP, Botucatu, SP

^IIDepartamentos de Morfologia, IB, UNESP, Botucatu, SP

^IIILaboratório de Radiobiologia IPEN, SP

^{Correspondence}

INTRODUCTION: The aim of this work was to characterize the toxic effects of a recombinant bothropstoxin-I (rBthTX-I), expressed in E. coli, on mice neuromuscular junction. The neuromuscular and the myotoxic effects of rBthTX-I were comparatively evaluated to the native BthTX-I, isolated from Bothrops jararacussu venom (n-BthTX-I). It was also evaluated the ability of heparin to neutralize both the neuromuscular and the myotoxic effects of that toxins.

METHODS: Twitches were directly and indirectly evoked on sciatic nerve-extensor digitorum longus (EDL) muscle preparation by supramaximal strength pulses and recorded by a polygraph. Myotoxicity was evaluated on phrenic-diaphragm preparation by optical and electronic microscopic analysis. Both toxins were tested at 1 mM.

RESULTS: nBthTX-I and rBthTX-I abolished indirect in 60 and 15 minutes, respectively. Direct contractions were reduced in 70% and 90% by nBthTX-I and rBthTX-I, respectively, within 120 minutes. Both nBthTX-I and rBthTX-I induced significant muscle damage (edema, round fibers, and areas of fibers devoid of myofibrils). Pre-incubation of toxins with heparin (27.5 mg/ml) prevented the blockade of both direct and indirect contractions, as well as, the muscle damage.

DISCUSSION: This study suggests that the pharmacological profile of rBthTX-I is similar to that of nBthTX-I. In view of this, rBthTX-I could be an useful tool for future site-directed mutagenesis studies intending to clarify the structure-function relationship of BthTX-I.

Supported by: CAPES.

Characterization of secreted products from C. diphtheriae, B. pertussis and C. tetani (DPT) bacteria in growth media - preliminary data

Perpétuo, E.A.^I; Juliano, L.^II; Fratelli, F.^III; Prado, S.M.A.^III; Lebrun, I.^I

^ILaboratory of Biochemistry and Biophysics, Butantan Institute

^IIDepartment of Biophysics, UNIFESP

^IIISection of Anaerobic Vaccines, Butantan Institute, SP-Brasil

^{Correspondence}

OBJECTIVES: To characterize secreted proteins from Corynebacterium diphtheriae, Bordetella pertussis and Clostridium tetani bacteria, to observe the production process steps, to identify possible production markers and new products. For these purposes, different fluorescent substrates were used to determine new enzymatic activities present in production batch.

METHODS AND RESULTS: 1-Purification of DPT toxins by gel filtration chromatography 2-Characterization of DPT toxins by electrophoresis and enzymatic activities, using fluorescent substrates. Four fractions were obtained by chromatography of tetanus toxin and the main active fraction was assayed on different fluorescent substrates, all based on synaptobrevin sequence (a natural substrate cleaved by tetanus toxin). The results showed that the tetanus toxin can hydrolyze substrates with 4 to 10 amino acid residues at different rates. The toxins from. C. diphtheriae and B. pertussis were also fractionated and assayed on different fluorescent substrates. Both toxins (D and P) presented enzymatic activities. The best substrate for active acellular pertussis toxin was Abz LYLVEGQ EDDnp and for diphtheria toxin was Abz LYTPKAQ EDDnp. Electrophoresis of concentrated and purified toxins also were carried out.

CONCLUSIONS: The fluorescent substrates represent a good tool to identify and characterize new enzymatic activities which could help to follow the production process (Perpetuo et al , Biotec. and Appl. Biochem., in press). In addition, these activities will be better characterized to search new enzymes which could be used for other purposes than antigen for vaccine production.

Financial support: FAPESP

Molecular cloning of a novel family of neurotoxins active on calcium channels from the venom of the spider Phoneutria nigriventer

Cardoso, F.C.; Gomez, M.V.; Kalapothakis, E.

Departamento de Farmacologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG

^{Correspondence}

INTRODUCTION: The spider Phoneutria nigriventer is frequently involved in cases of human envenomation in Central and South of Brazil. It is known to contain in its venom several neurotoxic polypeptides. One of the fractions purified from this venom, designed PhTx3, has been shown to induce a progressive flaccid paralysis in mice when injected intracerebro-ventricularly. These observations are a strong evidence for the action of this fraction on ionic calcium channels. Tx3-6, a polypeptide found in this fraction, produced paralysis only in the hind limbs and gradual decrease in movement and aggression in mice for a period of 24h.

OBJECTIVES: Through this evidences, the aim of this work is to clone and characterize the gene coding for the neurotoxin Tx3-6 and also isoforms of this neurotoxins

METHODS AND RESULTS: Our group has constructed a cDNA library using mRNA isolated from the venom gland of Phoneutria nigriventer. With the aim to isolate the cDNA coding for Tx3-6, we use different approaches of Molecular Biology (PCR, oligonucleotide and cDNA hybridization, etc.). As a result of the screening for Tx3-6, we found an interesting family of polypeptides coding for a new family of neurotoxins. All this toxins reveals the same structure, contain a signal peptide and an intervening propeptide preceding the mature toxin. Besides, these sequences showed us an interesting conserved domain probably involved with the specificity of this toxins by its target, the ionic calcium channels.

CONCLUSIONS: The neurotoxin Tx3-6 belongs to a family of interesting neurotoxins that act on ionic calcium channels. Its sequences showed the steps required to form the mature toxins and potential conserved domains probably involved in the specificity and activity of these neurotoxins.

Physical chemistry and functional aspects of native and chemically modified crotamine

Mancin, A. C.^{I, II}; Cecchini, A. L.^{I, II}; Arantes, E. C.^II; Giglio, J. R.^I

^IDepartamento de Bioquímica e Imunologia, FMRP, USP

^IIDepartamento de Física e Química, FCFRP, USP

^{Correspondence}

Crotalus durissus terrificus snake venom induces serious neurological effects when injected in humans or experimental animals. Unlike several venoms from others species, it doesn’t induce ache or significant tissue destruction in the inoculation area. Victims usually feels a sensation of analgesia around the bite region, which becomes dormant few minutes after the accident and keeps like that for weeks or months.

Crotamine was first isolated by Gonçalves and Vieira (1950). Injection of crotamine in guinea pigs, mice, rabbits and rats caused paralysis of the hind legs, front legs contracture and breathing difficulties. This toxin has been explored in our laboratory as a molecular model for the design of analgesic drugs since it shows a morphine-like activity when assayed by the hot plate test and acetic acid induced contortion tests.

Chemical modifications of crotamine were able to attenuate or even abolish the hind legs paralysis without significantly decreasing the analgesic activity. Acethylation of the nine Lys residues, which abolished its basic character (PAGE), or reduction and caboxymethylation of the three disulfide bonds, thus destroying its native conformation (Circular Dichroism) were two of such modifications.

These data suggest an important function of the S-S bonds to keep the native conformation. Indeed, spectra of crotamine in the near and far UV, when the S-S bonds are reduced and carboxymethylated, indicate that removing these bonds leads to the loss of secondary and tertiary structures.

Regarding native crotamine, the analgesic effect was shown to be time-dose dependent via opioid receptors, but 50 fold more active than morphine on a pondered basis.

Antivenom effect of a new synthetic coumestan analog of wedelolactone

Vianna-da-Silva, N.M.^I; Moraes, R.A.M.; da Silva, A.J.M.^II; Costa, P.R.R.^II; Melo, P.A.^I

^IDepartamento de Farmacologia Básica e Clínica ICB

IINúcleo de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro

^{Correspondence}

We studied the anti-snake venom activity of a synthetic coumestan named PCALC36 by using in vitro assay and isolated muscle preparations. We evaluated the anti-hemorrhagic and the antimyotoxic effect of this substance in vivo on pre-incubation and pre-treatment protocols against some crotalid crude venoms as well as some isolated toxins. This substance at 0,1-10 micromolar reduced by 95% the rate of CK release induced by B. jararacussu (25 mcg/ml) in vitro from the mouse Extensor digitorum longus muscle (EDL). In vivo, the preincubation with PCALC36 (0,03-5 mcg/g b.w.) neutralized from 4 to 92% the myotoxic effect of B. jararacussu venom (1 micg/b.w.). Also, this coumestan at 3 mcg/g neutralized the venoms (1 mcg/g b.w.) of Agkistrodon contortrix laticinctus and Crotalus viridis viridis by 50% and 70%, respectively, and reduced the myotoxicity of Bothropstoxin I or II (2.5 mcg/g) by 35% and 75% respectively. PCALC36 (0.01-6 mcg/g b.w.) when pre-incubated with B. jararaca venom (1 mcg/g b.w.) abolished its hemorrhagic effect. Pre-treatments with PCALC36 (0,3-20 mcg/g b.w.) reduced about 45 and 75% the myotoxic and hemorrhagic effects of B. jararacussu and B. jararaca venoms. Above 90% of phospholipase A2 and proteolytic activities of B. jararacussu and B. jararaca venoms (10 mcg/ml) were inhibited by PCALC36 (3-400 micromolar). These data indicate that the coumestan PCALC36 is able to neutralize the main effects of the these studied viperid venoms, protecting the muscle cells from the sarcolemmal damage and preventing the hemorrhagic lesions.

Support: PRONEX (No. 41.96.0888.00), FAPERJ, FUJB-UFRJ, CNPq.

Production of a ribosome-inactivating protein from callus culture of Abrus pulchellus. Preliminary studies

Silva, A.L.C.^I; Horta, A.C.G.^II; Moreira, R.A.^II; Beltramini, L. M.^I; Araújo, A.P.U.^I

^IGrupo de Biofísica Molecular e Espectroscopia – IFSC/USP

^IILaboratório de Lectinas e Glicoconjugados- Depto. de Bioquímica e Biologia Molecular/UFC

^{Correspondence}

Plant ribosome-inactivating proteins (RIPs) are RNA–N-glycosidases that cleave a specific ribosomal RNA site preventing the normal protein synthesis. Pulchelin is a Type 2 RIPs, like ricin and abrin, consisting on a lectin specific galactose-binding subunit, which recognizes sites on the surface of a target-cell, and one ribosome-inactivating N-glicosidase subunit. These proteins form a class of highly toxic molecules which specifically kill cell by their N-glycosidase enzymatic activity under the 60S ribosomal subunit. Although their potency to kill cells is remarkable, the RIPs have been explored in the chemical construction of drugs to be used as “biological missile” in the clinical treatment of human diseases. With the aim for investigate the production of pulchelin in callus culture, cotyledon segments of Abrus pulchellus immature seeds were inoculated in basal medium MS supplemented with different concentrations of auxin (2,4-D), citokinin (kinetin and BA) and sucrose in order to determine the best callus induction. The explants were maintained in a dark growth room at 28 + 2°C. The presence of the mRNA from Pulchelin was observed in callus by RT-PCR technique. The calli obtained after 35 days period were freeze dried, macerated and submitted to protein extraction with buffers of different pH values (2.6, 4.0, 6.0, 7.6 and 10.0) and the proteins concentration in the extracts was determined by Bradford method. The pH 7.6 and 10 buffers were the most efficient to extract the largest amount of protein. The calli crude extract (pH 7.6) showed hemagglutinating activity against rabbit blood cells and showed a high toxicity to mice when administered intraperitoneally. The crude extract was also submitted to affinity chromatography on a Sepharose-4B column. The retained protein peak released by 0.1M D-galactose showed to be composed by two main bands in polyacrylamide gel electrophoresis, in denaturating conditions, with a similar pattern to results obtained with seeds.

(Supported by CNPq)

Biological and immunochemical characterization of Loxosceles adelaida spider venom (Araneae, Sicariidae) from caves of Petar - Ribeira Valley - São Paulo

Pretel, F.D.; Gonçalves-de-Andrade, R.M.; Tambourgi, D.V.

Laboratório de Imunoquímica, Instituto Butantan, São Paulo, SP, Brazil

^{Correspondence}

Loxosceles is the most venomous spider in Brazil, and envenomation causes dermonecrosis and complement-dependent haemolysis.

The aim of this study was to characterize some of the biological and immunochemical properties of the venoms from Loxosceles spiders captured in the caves of “Parque Estadual Turístico do Alto Ribeira”, (PETAR). The spiders that have already been captured in these caves were identified as Loxosceles adelaida. Despite of the fact that accidents have never been reported, this species could have toxic components in its venom and thus cause envenomation in human beings. As Loxosceles adelaida belongs to the gaucho’s group, Loxosceles gaucho venom was also tested in order to compare their activities. SDS-PAGE analysis of L. adelaida and L. gaucho venoms showed differences in the number and size of the protein bands. Moreover, lectin blot assays also showed distinct patterns of glycosilated components in their venoms. L. adelaida and L. gaucho venoms were fully able to induce complement dependent haemolytic and dermonecrotic reactions. Rabbit serum against L. gaucho venom was able to recognize several components of L. adelaida venom. The present results show that L. adelaida venom can induce all the toxic effects reported for the Loxosceles venom of medical importance and reinforce the necessity of better understanding the molecular mechanisms of Loxosceles venoms for the development of an efficient therapy.

Supported by FAPESP, CAPES and CAT

Loxosceles adelaida (Araneae, Sicariidae) in the touristic park of Ribeira Valley, São Paulo, Brazil

Andrade, R.M.G.^I; Pretel, F.D.^I; Galati, E.A.B.^II; Tambourgi, D.V.^I

^ILaboratório de Imunoquímica, Instituto Butantan

^IIDepartmento de Epidemiologia, Faculdade de Saude Pública, USP, São Paulo, SP, Brazil

^{Correspondence}

Studies about the effect of venoms from sinanthropic Loxosceles spiders’ species are frequently reported, however, analysis from those living in natural environment has not been performed. Loxosceles are present in several different habitats, including the carstic environment, being in Brazil the most common troglophile arachnid. Frequently, they are found in the walls of the entrance of the cave as well as in the shade zone where rocks and a bit of light are present. These characteristics of the milieu favor the reduction of the humidity and so, the presence of the troglophile fauna. Caves are attracting the attention of a large number of tourists. The "Parque Estadual Turístico do Alto Ribeira", Ribeira Valley, SP, represents an important touristic and scientific research area due to the association between the tropical forest and the caves system. To characterize the venoms of wild species of Loxosceles and compare them to those of sinathropic species, we are conducting explorations into the interior of the caves from the Speleological Province of the Ribeira Valley in order to capture, identify, breed and analyze the venom of these spiders. The first exploitation was performed in the caves: “Morro Preto” and its neighborhood, “Alambari de Baixo” and “Laboratório I”. Loxosceles spiders captured until now, in these areas, were identified by morphological analysis as Loxosceles adelaida. This species is being currently kept and bred in the “Laboratório de Imunoquímica, Instituto Butantan” for venom, taxonomic and ecological studies.

Supported for FAPESP, CAPES and CAT

A study of scorpion accidents in the Federal District, Brazil, from 1991 to 2000

Yoshizawa, M.A.C.^I; Caldas, E.D.^II

^IDiretoria, Vigilância Ambiental, SAIN, Estrada do Contorno do Bosque, Lote 4

^IIUniversidade de Brasília, Campus Universitário, Brasília, DF, Brazil

^{Correspondence}

The urbanization of the Federal District has increased the number of accidents caused by scorpions in recent years. From 1991 to 2000, the Division of Environmental Vigilance received 4,526 requests for community visitis related to the presence of scorpions and there were 1,346 notifications of accidents. In this study, 977 of these notifications were investigated. The accidents occurred predominantly (93.5%) in the urban zone, mostly during the rainy season (59.4%). The North Lake and South Lake regions accounted for the highest incidences, with 34.2 and 33.9 accidents per 100,000 inhabitants, respectively. Tityus serrulatus, Tityus fasciolatus and Bothriurus araguayae were the species identified in the accidents. The number of male and female victims involved in these accidents was similar. Most of the accidents (45.4%) involved individuals between 20 and 40 years, with stings mainly on the hands (34.5%) and feet (24.4%). In the urban zone, approximately 67% of the people stung received medical care within 3 h after the accident, whereas in the rural zone, this time was greater than 3 h in 51.7% of the cases. Most of the cases (96.1%) were classified as low severity and no severe accident was reported. Analgesics represented the treatment most used in individuals of all ages and accounted for 62.5% of the 550 prescriptions. Antihistamines and antiinflammatory drugs, frequently prescribed for the treatment of low severity accidents, are not recommended by the Brazilian health authorities. The correlation between the number of accidents and the community visits requested in this study provides an important basis for the implementation of control and educational programs in areas of potential risk in order to reduce the occurrence of these accidents in the Federal District.

Analyses of venom variability in wolf spider Lycosa erythrognatha

Santos, D.M.^I; Mafra, R.A.^II; Pimenta, A.M.C.^I; Alvares, E.S.S.^III; de Maria, M.^III; Cruz, J.S.^II; Bemquerer, M.P.^IV; de Lima, M.E.^I

^ILaboratório de Venenos e Toxinas Animais, Depto. de Zoologia. Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brasil

^IILaboratório de Membranas Excitáveis, Depto. de Zoologia. Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brasil

^IIILaboratório de Aracnologia, Depto. de Zoologia. Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brasil

^IVLaboratório de Físico-química de proteínas, Depto. de Bioquímica e Imunologia

^{Correspondence}

Wolf spiders from Lycosa genus, are very common in urban areas in Southeastern region of Brazil. Their venoms are poorly studied and knowing their active components can be a step forward to prospect new drugs candidates, antibiotics, biological insecticides or even molecular tools that can be used for biochemical studies involving receptor structure and function, ionic channels, proteolitics enzymes, and other functional classes. In this work, we initiate the biochemical characterization of the L. erythrognatha venom by using reverse phase high performance liquid chromatography (RP-HPLC). Differences in some components concentrations related to gender, age and time extraction were found to be significant. Different profiles for male and female venoms were also observed in MALDI-TOF mass spectrometry analyses. Electrophysiological studies, using GH3 cells, carried out with soluble fraction of L. erythrognatha adult female venoms, demonstrated a shift to more negative potentials in sodium channels activation, similar to that one verified for scorpions b-type toxins. This is the first time that this effect is described for Lycosa venoms.

Financial support: CAPES, CNPq, FAPEMIG and PRPq-UFMG.

Partial chemical characterization of the Musa sp SAP (Musacea) and its interaction with snake venom

Borges, M.H.^I; Grossi, A.C.^I; Raslan, D.S.^II; Piló-Veloso, D.^II; Alves, D.L.F.^III; de Lima, M.E.^I

^ILab. Venenos e Toxinas Animais – Depto. Bioquímica e Imunologia, ICB-UFMG

^IIDepto. Química ICEx-UFMG

^IIIDepto. Farmacologia ICB-UFMG

^{Correspondence}

Snake venoms are constituted by complex mix of toxins (enzymes or not) responsible for the main symptoms induced by venoms including hemorrhage, edema, myotoxicity, neurotoxicity and changes in blood coagulation. Many plants are used to treat snakebite and identification of effective substances to neutralize snake venoms is very important. The claim of this study was the partial chemical analysis of the Musa sp sap (MS), a plant from tropical areas and to verify their anti-snake venom action. Phytochemical and spectroscopic (IR and NMR) analysis of the MS showed the presence of sugar, saponins and tannins in high concentration. Thin layer chromatography using cellulose as support was performed with the MS and spots were developed in a presence of ninhidrin. For inhibition assays, venom solutions were mixed with MS at room temperature immediately before the test. MS inhibited 100% PLA2 activity of Bothrops jararacussu and Crotalus durissus terrificus venoms at 1:1 ratio (venom: MS, w/w). The myotoxic activities of B. jararacussu (50mg) and B. neuwiedi (50mg) were neutralized when the MS was mixed with the venoms in a 1:5 ratio (venom:MS, w/w) prior the injection. MS neutralized about 88% of the hemorrhagic activity induced by B. jararacussu venom. We also observed that MS partially increased the time of plasma coagulation caused by B. jararacussu and C. d. terrificus venoms. Our results show that Musa sp sap was able to neutralize several activities of snake venoms such as PLA2, myotoxicity, and plasma coagulation. These results suggest that compounds isolated from this extract are able to interact with animal venoms and could be useful tools for the elucidation of the action mechanisms of toxins.

FINANCIAL SUPPORT: CAPES, CNPq and FAPEMIG.

Status epilepticus induced by marinobufagin, a bufadienolide isolated from Bufo paracnemis lutz (1925) parotid glands secretions

Nogueira, R.M.D.^I; Costa, D.C.^I; Patrocínio, M.C.A.^I; Camarão, G.C.^I; Uchoa, D.E.A.^II; Silveira, E.R.^II; Scorza, F.A.^III; Santos, N.F.^III; Cavalheiro, E.A.^III; Carvalho, K.M.^IV

^IDepartamento de Fisiologia e Farmacologia, UFC, CE

^IIQuímica Orgânica e Inorgânica, UFC, CE

^IIINeurologia Experimental, UNIFESP, SP

^IVCiências Fisiológicas, UECE, CE

^{Correspondence}

OBJECTIVES: The main focus of this research was the isolation of a substance with a strong convulsant action from Bufo paracnemis Lutz (1925) parotid glands secretions, determination of its chemical structure and its behavioral and electrographic effects on central nervous system.

METHODS AND RESULTS: This substance was purified in large amounts using a reverse-phase high-performance liquid chromatography, with a C-18 preparative column. Its chemical structure was elucidated using high resolution Nuclear Magnetic Resonance analysis, allowing its identification as a steroid of the bufadienolide type, already known in the literature as Marinobufagin (14b-15b-epoxy-3b,5b-dihidroxy-20,22-bufadienolide). Our results based on behavioral and electrographic analysis showed central effects induced by systemic administration of Marinobufagin in rats and mice. The animals presented severe neurotoxic effects, tonic-clonic seizures and death. Marinobufagin DL50 was 10.5 ± l.5 mg/kg for mice and 25.0 ± 2.0 mg/kg for rats, both through intraperitoneal injection. This strong convulsant activity is dose-dependent and 5.0 mg/kg doses for mice and 20.0 mg/kg for rats, both intraperitoneal (IP), already showed behavioral changes that evolved to generalized tonic-clonic seizures. Marinobufagin induced seizures that ended up in status epilepticus that lasted more than an hour. Seizures decreased in almost 80% of the animals treated with Diazepan (10.0 and 12.5 mg/kg, IP) even though some of these animals continued to show seizures in the electrographic recordings. Seizures induced by Marinobufagin were not affected by Phenobarbital. Phenitoin was able to block generalized tonic-clonic seizures in all animals of the group.

CONCLUSION: Our results suggest that Marinobufagin could represent a pharmacological tool for the development of an epilepsy experimental model, since it had fulfilled the following requirements: i) Marinobufagin induced epileptiform activity in electrographic recordings, ii) seizures were blocked by anticonvulsant drugs such as Diazepan and Phenitoin, an essential requirement for the evaluation of the effects of new drugs to be used in epilepsy treatment.

Jararhagin stimulates epithelial cell migration, promotes actin polymerization and the recruitment of specific integrins to focal contacts

Costa, E.P; Santos, M.F.

Department of Histology and Embriology- Institute of Biomedical Sciences/USP

^{Correspondence}

Jararhagin(JG), a snake venom metalloprotease, induces the influx of leukocytes in vivo in an air pouch model, without being chemoctatic (Costa et al., 2002). In this study we analyse the direct effects of JG on epithelial cell adhesion and migration, F-actin arrangement, laminin and fibronectin distribution, as well as the expression of some laminin and fibronectin-binding integrins.

METHODS AND RESULTS: IEC-6 cells were grown to confluence, when 1/3 of the monolayer was removed in order to stimulate spontaneous migration. Migrating cells were treated with several doses of JG, and maximum stimulation was obtained with 1,25 mg/ml (Matrigel) or 5 mg/ml (treated plates). Cell adhesion was also studied in the presence of JG during 20 min. An inhibition of 45% and 40% was observed with the doses of 1,25 and 5 mg/ml JG, rescpectively. The distribution of some integrins (a2 and av subunits, a6b1), fibronectin and laminin was studied using immunofluorescence with comercial antibodies (Chemicon) and confocal analysis (Nikon). F-actin was stained with rodhamine-phalloidin (Molecular Probes). JG stimulated actin polymerization in stress fibers, filopodia and membrane ruffles after 15 min. The pericellular fibronectin matrix was lost in migrating cells, while laminin distribution was less affected. The toxin also stimulated the recruitment of a6b1 and av integrin subunit to focal contacts. The distribution of the a2 subunit, also present in focal contacts, was not altered.

CONCLUSION: Jararhagin stimulates the migration of epithelial cells in vitro through a mechanism the involves both quantitative and qualitative changes in cellular adhesion to the substrate and the formation of actin-rich cellular processes.

Financial support: FAPESP.

Influence of temperature on the neuromuscular blockade caused by the venoms of B. neuwiedi diporus, B. neuwiedi goyasensis and B. neuwiedi paranaensis in chick biventer cervicis preparations

Abreu, V.A.^I; Leite, G.B.^I; Furtado, M.F.^II; Simioni, L.R.^I

^IDepartamento de Farmacologia, Faculdade de Ciências Médicas, Universidade Estadual de Campinas (UNICAMP) Campinas, SP, Brasil

^IILaboratório de Herpetologia, Instituto Butantan, São Paulo, SP, Brasil

^{Correspondence}

OBJECTIVES: The influence of temperature on the neuromuscular activity of the venoms of three subspecies of the pitviper Bothrops neuwiedi was studied using chick biventer cervicis muscle preparations.

METHODOLOGY: Venoms were collected from specimens (diporus, goyasensis and paranaensis) by manual extraction and lyophilized. Male HY-LINE W36 chicks 4-8 days old were used. After anesthesia with chloral hydrate (300 mg/kg, i.p.) the muscles were removed and mounted in a 5 ml organ bath (Ginsborg and Warriner, 1960) containing Krebs solution (mM): NaCl 118.7, KCl 4.7, CaCl2 1.88, KH2PO4 1.17, MgSO4 1.17, NaHCO3 25.0, and glucose 11.65, pH 7.5, aerated with 95% O2 and 5% CO2. The experiments were done at 22°C or 37°C. Stimuli were given via a bipolar platinum ring electrode (0.1 Hz, 0.2 ms, 8 V, GRASS S48 stimulator) and the contractions were recorded on a Gould RS 3400 physiograph. Contractures to exogenous acetylcholine (ACh, 60 µM and 120 µM for 60 s) and KCl (20 mM for 180 s) were obtained to test for myotoxic and neurotoxic activities. After stabilization for 20 min, ACh, KCl and venoms were added to the bath. ANOVA/MANOVA for repeated measures was used to examine the significance.

RESULTS:B. n. diporus venom (50 µg/ml) was more active in inducing neuromuscular blockade at 22ºC than the other venoms (62 ± 3.2% blockade after 120 min, n=3), B. n. goyasensis venom was more potent at 37ºC (58 ± 5% blockade after 120 min, n=3), whereas the B. n. paranaensis venom blockade (50 µg/ml, n=3) was not significantly affected by temperature. Changes in temperature did not affect the neuromuscular activity at high concentrations (100-200 µg/ml).

CONCLUSION: Only the activity of B. n. diporus venom was significantly altered by temperature, suggesting that the neurotoxic action was temperature-dependent at avian neuromuscular junctions.

Financial support: FAEP/UNICAMP.

A proteomic approach to prospect new structural families of bioactive peptides in Tityus serrulatus scorpion venom

Pimenta, A.M.C.^{I, IV}; Mansuelle, P.^I; Stocklin; R.^II; Favreau, P.^II; Diniz, C.R.^III; de Lima, M.E.^IV; Bougis, P.E.^I; Martin-Eauclaire, M.F.^I

^ILaboratoire de Biochimie - Ingénierie des Protéines, UMR 6560, Marseille, France

^IIAtheris Laboratoires, Geneve, Switzerland, Centro de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias, Belo Horizonte, Brazil

^IIILaboratório de Venenos e Toxinas Animais, Dept. de Bioquímica e Imunologia, ICB, UFMG, Belo Horizonte, Brasil

^{Correspondence}

Scorpion venoms are a rich mixture of peptides that act in many fronts from ionic channels in excitable tissues to cardiovascular and respiratory systems. Due their lethality, long toxins that alters the closing and opening kinetics of Na+ channels, called respectively a- and b-toxins, were firstly described and are well known. Those toxins are peptides ranging from 6000 to 7000 Da, constrained by four disulfide bonds and more representative in a quantitative sense. Smaller peptides, ranging from 3500 to 4500 Da, folded by three or four disulfide bonds, are known as K+-channels toxins and have a higher variability in terms of primary structure. To date, less than 15 sequences were completely established for both Na+- and K+- channel toxins in Tityus serrulatus venom. Besides, different peptide families that could respond to a variety of biological activities other than well known neurotoxic effects, have been neglected. Here, we describe novel biologically active peptides belonging to different structural families, discovered by a structurally driven approach. By using different liquid chromatography/mass spectrometry coupled platforms in a proteomic approach, we were able to screen for new molecules in T. serrulatus venom. At least 380 molecular masses were observed only in toxic fractions of this venom. On plate enzymatic digestions followed by MALDI-TOF mass spectrometry analysis, were also used to establish structural features of some peptides.

Support: CAPES/COFECUB, CNRS, INSERM, CNPq and FAPEMIG.

Endothelial cell activation mediated by berythractivase

Chudzinski-Tavassi, A.M.; Negrotto, S.; D’Atri, L.P.; Bezerra da Silva, M.; Pozner, R.G.; Lazzari, M.A.; Schattner, M.

Department of Thrombosis and Hemostasis, Hematological Research Institute, National Academy of Medicine, National Research Council (CONICET), Buenos Aires, Argentina

^{Correspondence}

The venom of Bothrops erythromelas, a species which occurs in northeastern Brazil, has a much higher procoagulant activity than other Bothrops species. Berythractivase is a 78 kDa metalloproteinase purified from B. erythromelas venom and its primary structure has been deduced from cDNA. In vitro, berythractivase is a prothrombin activator. Since Bothrops snake venoms induce local inflammatory lesions and disseminated intravascular coagulation (DIC), we have evaluated the effect of erythractivase on human umbilical vein endothelial cells (HUVEC). Morphological alterations were observed in HUVEC after 1 h of incubation with berythractivase (5 mg/ml). Flow cytometry showed that berythractivase (Be) increased the expression of ICAM-1 (Control 12±1 vs Be 21±3 arbitrary fluorescence units (AFU), p<0.05, n=6) and E-selectin (Control 7±1 vs Be 25±1 AFU, p<0.05, n=4), without modifying the levels of VCAM-1 (Control 4±1 vs Be 4±0.5 AUF, n=4). Berythractivase increased the release of von Willebrand Factor (vWF) by 153% (p<0.05, n=4) and up-regulated prostacyclin (Control 1.3±0.2 vs Be 2.8±0.7 ng/ml, n=5, p<0.05) and IL-8 (Control 1.0±0.2 vs Be 3.1±0.1 ng/ml, n=6) levels (all measured by ELISA). vWF secretion was blocked by pretreating berythractivase with bothropic antiserum or EDTA, indicating a direct action of the proteinase. The prothrombotic and proinflammatory endothelial cell responses induced by berythractivase may be involved in the local lesions and systemic effects observed in humans envenomed by Bothrops snakes.

Ion channel formation by the marine sponge Geodia corticostylifera extract in bilayer membranes

Rangel, M.^I; Procopio, J.^II; Konno, K.^III; Freitas, J.C.^I

^IDepartamento de Fisiologia, Instituto de Biociências e CEBIMar, USP, São Paulo

^IIDepartamento de Fisiologia, Instituto de Ciências Biomédicas, USP, São Paulo

^IIICentro de Estudo de Insetos Sociais, Instituto de Biociências, UNESP, Rio Claro

^{Correspondence}

OBJECTIVES: In the present work we studied ion channel formation into bilayer membranes by fractions of the extract of G. corticostylifera.

METHODS: The methanol-water extract prepared from G. corticostylifera specimens collected in São Paulo State coast, Brazil, was fractionated in an acetonitrile-water gradient in a Sep Pak Vac C18 cartridge, resulting in 9 fractions, each three were solubilized in 20, 50 and 90% CH3CN, respectively. The haemolytic fraction (the second one with 50% CH3CN solubility) was tested, in concentrations of 1-4mg/ml, in azolecitin membranes formed in a circular hole of a polypropylene wall coupled to an acrylic chamber. Electrical measurements were made with a patch-clamp amplifier, set into voltage-clamp. Data were converted to digital form through an A/D interface and acquired by a computer program.

RESULTS: Macroscopic and unitary current-voltage relations of the channels were non-linear, showing pronounced rectification, favouring current passage from the CIS (were the fraction was added) to the TRANS side. The channels were shown to be cation-selective when Na+ was tested against Cl-, and more selective to Na+ against K+. Channel conductance was 18pS (-100mV pulse, 145mM NaCl + 10mM CaCl2 solution). Channel openings apparently were modulated by Ca++.

CONCLUSIONS: The channels formed by the haemolytic fraction may present an assymetry related to the side of incorporation into the membrane, as suggested by the current-voltage relations. Further experiments with pure ionophore isolated from the sponge will be performed and a model for the channel will be proposed.

Granted by FAPESP.

Construction of a recombinant baculovirus with a novel insecticide toxin LiTx3 of spider Loxosceles intermedia

Silvestre, F.G.^I; Castro, C.S.^I; Olortegui, C.C.^I; Gomez, M.V.^I; Ribeiro, B.M.^II; Kalapothakis, E.^I

^ILaboratory of Molecular Biology of the Toxins, Farmacology Department, ICB, UFMG, Belo Horizonte, MG

^IILaboratory of Eletronic Microscopic, Celular Biology Department, ICC, UnB, Brasília, DF

^{Correspondence}

Baculoviruses are used as vectors of heterologous gene expression by the introduction of a desired gene in its genome in the place of a nonessential one under control of a strong promoter. Aiming at the recombinant construction of one baculovirus, for the production of a bioinsecticide of high efficiency and specificity for the lizard of the plague of the maize, we have worked in the identification and toxin tests of the Loxosceles intermedia spider with insecticidal activity for insects of agricultural importance. Our group, from the construction of a cDNA library made with mRNA purified from the venom gland of Loxosceles, isolated the LiTx3. The molecular screening of the clone coding LiTx3 was carried out using degenerate probes, and the automatic sequencing of positive clones, allowed the characterization of LiTx3. This toxin presented a hydrophobic signal peptide, a propeptide signal rich in residues of glutamate with arginine as the last residue. The clone also revealed the sequence of the mature toxin and the termination codon beyond the polyadenylation signal. The analysis of the amino acid sequence using the basic local alignment search tool (BLAST) did not show any similarity to described toxins, but the number and the distribution of cisteine is well conserved, following the standard of other toxins. This toxin was subcloned in the vector pSyn XIVVI+ X3 and cotransfection using the linearized DNA (vSynVI- gal) of the AcMNPV virus, directed for the mutated promoter of the polyhedrin. The infection was carried out using the cells Tn5B and the success of the construction was verified by PCR. The expression of LiTx3 in this system was verified by RT-PCR and slot-blot with antibody anti-venom Loxosceles. Preliminary tests with Spodoptera frugiperda had demonstrated insecticidal effectiveness with reduction in the time of life of this plague.

Supported by: CAPES, CNPq, PADCT and Fapemig

Localization of crotamine gene of Crotalus durissus terrificus by fluorescent in situ hybridization (fish)

Oguiura, N.^I; Svartman, M.^II; Batistic, R.F.^I; Almeida, T.M.B.^IV; Rádis-Baptista, G.^III; Yamane, T.^III; Vianna-Morgante, A.^II

^ILab. de Herpetologia do Instituto Butantan

^IIDepto. de Biologia do Instituto de Biociências da USP

^IIILab. de Toxinologia Molecular

^IVLab. De Genética do Instituto Butantan

^{Correspondence}

Crotamine belongs to a closely related group of crotamine-like proteins, sharing up to 98% of similarity, present in most rattlesnake venoms. However, its presence and amount can vary according to the subspecies or the geographic locality of a given population. The quantity of this toxin ranges from 10 to 40% of dried venom. The crotamine gene of crotamine-plus Crotalus durissus terrificus consists of approximately 1800bp: three exons separated by a long phase-1 (900bp) and a short phase-2 (140bp) introns. Exon 1 codifies the 5’-untranslated region and the first 19 amino acids of signal peptide, exon 2 codifies a total of 42 amino acids, 3 belonging to the signal peptide and 39 to the crotamine. Exon 3 codifies the last 3 amino acids of the mature toxin, the terminal lysine which is removed after post-translation processing and the 3’-untranslated region.

In this work, we mapped the crotamine gene to metaphase chromosomes of C. d. terrificus. Chromosome preparations were obtained from bone marrow. The probe, MR20 phage DNA containing the crotamine gene, was labeled with biotin by nick translation. FISH was performed as described by Viegas-Péquignot (1992) with minor modifications and immunodetection was performed with avidin conjugated with FITC.

The crotamine gene mapped to the telomeric region of the long arm of chromosome 2 in C. d. terrificus crotamine-plus karyotype. The intensity of the signal differed between the two homologues in all cells analyzed, what could be due to different number of copies of the crotamine gene. The variable amounts of crotamine found in the venom of crotamine-plus C. d. terrificus could reflect differences in gene copy numbers.

Financial support: FAPESP 99/02675-6

Putative antimicrobial peptides isolated from the skin secretion of the frog Leptodactylus ocellatus

Nascimento, A.C.^I; Fontes, W.^II; Sousa, M.V.^II; Schwartz, C.A.^I; Schwartz, E.F.^I; Sebben, A.^I; Castro, M.S.^{I, II}

^ILaboratório de Toxinologia, Departamento de Ciências Fisiológicas/IB, Universidade de Brasília

^IILaboratório de Bioquímica e Química de Proteínas - Centro Brasileiro de Serviços e Pesquisas em Proteínas, Departamento de Biologia Celular/IB, Universidade de Brasília, Brasília/DF, Brasil

^{Correspondence}

The exudate of frog skin contains an array of gene-encoded peptides with diverse biological functions. The main functions attributed to this repertoire of peptides are preventing microbial infection, assisting wound healing and helping to avoid predation. In the present report we describe the purification and characterization of two hemolytic peptides obtained from Leptodactylus ocellatus cutaneous secretion. Adult specimens of Leptodactylus ocellatus were collected in Distrito Federal region and maintained in captivity at the University of Brasília. Skin secretion was obtained by mild electrical stimulation and lyophilized. The dried secretion (5.0 mg) was dissolved in 0.1% (v/v) TFA/water and loaded onto a C8 reversed-phase column (Sephasil Protein, 4.6 x 250 mm, Pharmacia Biotech).. Elution was performed using a linear gradient of acetonitrile, at a flow rate of 0.8 mL/min. The absorbance was monitored at 216 nm. Fractions were manually collected, lyophilized and tested for hemolytic activity. Two hemolytic fractions were individually rechromatographed using a C18 reversed-phase column (Vydac 218TP54, 4.6 x 250 mm, Separations Group), yielding symmetric peaks. The purified toxins were analyzed by m-ESIMS (API 300, Sciex-PE) and submitted to automated Edman degradation using an ABI 477A sequencer, resulting in unambiguous sequences. A search using non-redundant database by BLASTP revealed homology to amphibian antimicrobial peptides included in the brevinins family.

Financial support by: FUB/UnB and FINATEC.

Tx4(5-2), a new toxin from Phoneutria nigriventer venom elicites the glutamate uptake inhibition displayed by PhTx4 toxic fraction

Oliveira, L.C.^{I, II}; Figueiredo, S.G.^V; Pimenta, A.M.C.^II; Mansuelle, P.^III; Cordeiro, M.N.^IV; Richardson, M.^IV, Diniz, C.R.^IV; Rochat, H.^III; de Lima, M.E.^II

^IDepto de Fisiologia e Farmacologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais

^IIDepto de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais

^IIILaboratoire de Biochimie - Ingénierie des Protéines, Marseille, France

^IVCentro de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias

^VDepto de Ciências Fisiológicas, Centro Biomédico, Universidade Federal do Espírito Santo

^{Correspondence}

Several pools of neurotoxic peptides obtained from fractionated Phoneutria nigriventer venom induce different toxicological effects. One of them, PhTx4, is highly toxic toward insects and displays only a slight toxicity when injected in mice. Also, this fraction contains a peptide class that is able to inhibit glutamate uptake in preparations of mammalian central nervous systems. In this work a new toxin Tx4(5-2) was isolated from the PhTx4 fraction by reverse phase and anion exchange steps using high performance liquid chromatography (HPLC). Some biological features and the complete amino acid sequence were established. This toxin leads to immediate excitatory effects when injected in house flies and cockroaches) by intrathoracical injections. Intracerebroventricular injections of 0.03 mg of Tx4(5-2) in mice resulted in no apparent signs of intoxication. Pharmacological characterization carried out in rat brain synaptosomes by using [³H]-L-glutamate, showed that the whole PhTx4 fraction as well as Tx4(5-2), Tx4(6-1) and Tx4(5-5) pure toxins were able inhibit the glutamate uptake in the micromolar concentration range. Tx4(5-2) inhibits the glutamate uptake in a dose dependent manner, with an IC50 of approximately 1mM. Edman sequencing of Tx4(5-2) revealed a 48 residues long polypeptide, containing 10 cysteines and cross-linked by 5 disulfide bridges. Molecular mass measured by MALDI-TOF mass spectrometry was 5203.99 Da, which is very close from calculated MM (5199.99). Tx4(5-2) is highly homologous to the Tx4(6-1) and Tx4(5-5) toxins previously described from the same fraction.

Hematological, hemostatic and biochemical disturbances induced by Crotalus durissus terrificus envenomation in dogs

Sousa-e-Silva, M.C.C.^I; Tomy, S.C.^I; Tavares, F.L.^I; Navajas, L.C.^II; Larsson, M.H.A.^II; Sano-Martins, I.S.^I

^ILaboratório de Fisiopatologia, I. Butantan

^IIClínica Médica, Fac. Med. Vet. Zoot., USP, SP-Brazil

^{Correspondence}

The aim of this study was to investigate the hematological, hemostatic and biochemical alterations induced by Crotalus durissus terrificus venom (VCdt) in dogs. Three groups of animals were used: an experimental group (E) that was injected intramuscularly with VCdt (1 mg/kg) and 2 hours later was treated with Crotalus antivenom (i.v.), a control group(C) that was injected with saline (i.m.), and a control group (AV) injected with saline (i.m.) and 2 hours later was treated with Crotalus antivenom. Blood samples for biochemical, hematological and hemostatic parameters were collected at 24 hours before, 2 h after venom or saline administration, and 24 and 48 hours following serumtherapy. The levels of alkaline phosphatase and alanine aminotransferase were increased in E and AV groups at 24 and 48 hours after serumtherapy. The high levels of the enzymes creatine kinase, aspartate aminotransferase and myoglobin demonstrate that the animals developed rhabdomyolysis. These alterations were not normalized 48 h after the administration of antivenom. No alterations were observed for other analyzed biochemical parameters. No evidence of erythron disturbances was observed. However, 2 hours after envenomation, a persistent neutrophilic leukocytosis was observed, which lasted even after serumtherapy. The animals of E and AV groups presented eosinopenia at 24 hours after serumtherapy. After 2 hours of envenomation the animals presented altered coagulation time, prothrombin time and activated partial thromboplastin time, fibrinogen and alfa2-antiplasmin consumption, increased levels of fibrinogen/fibrin degradation products (FDP) and platelet hypoaggregation. The increased levels of FDP and the decreased alfa2-antiplasmin levels indicate secondary activation of the fibrinolytic system. Our data suggest that the biochemical and hemostatic disturbances induced by Cdt venom in dogs are related to its myotoxic and thrombin-like activities.

Hematological alterations induced experimentally in rabbits by the venom of the spider brown Loxosceles gaucho

Tavares, F.L.^I; Barbaro, K.C.^II; Sousa-e-Silva, M.C.C.^I; Santoro, M.L.^I; Sano-Martins, I.S.^I

^ILaboratório de Fisiopatologia do Instituto Butantan

^IILaboratório de Imunopatologia do Instituto Butantan, SP-Brazil

^{Correspondence}

Human accidents by Loxosceles sp, brown spider, cause dermonecrosis and occasionally intravascular hemolysis, disseminated intravascular coagulation (DIC), renal failure and death. The aim of this work was to characterize in rabbits the hematologic responses induced by the administration (i.d.) of Loxosceles gaucho venom and the evolution of systemic reaction. At 3, 24, 48, 72 and 120 hours after the i.d injection of crude venom (10µg/kg), animals were anesthetized and the blood collected from the carotid artery after a surgical procedure. The results showed an initial leukopenia (3 and 24 hours), at 72 hours an intense polymorphonuclear leukocytosis. A significant thrombocytopenia was observed at 3 and 24 hours whereas thrombocytosis occurred in 120 hours. Erythrocytes count, packed cell volume, and hemoglobin levels were decreased at 72 and 120 hours. Haptoglobin and fibrinogen were increased, but hyperproteinemia was not observed, wich might be compensated by hemodilution, explaining the decreased of the hematological parameters. In addition, coagulation factors V, VII, VIII, IX, X and XI were increased at 120 hours. Bilirrubins and plasma hemoglobin, aspartate aminotransferase, alanine aminotransferase, glutamil gamatransferase, albumin, blood urea nitrogen and creatinine were within normal range. Our present results showed that the rabbits develops no systemic reaction of loxoscelism, as observed in humans cases, since intravascular hemolysis and DIC were not verified. However, the thrombocytopenia and leukopenia in the initial time intervals are probably related to the establishment of the lesion, confirming that rabbits are a good model for the study of cutaneous necrosis of loxoscelism.

Support: FAPESP.

Relationship between venom antigenaemia and severity of Bothrops jararaca envenoming in Brazil

França, F.O.S.^{I, IV}; Barbaro, K.C.^II; Fan, H.W.^I, Cardoso, J.L.C.^I; Sano-Martins, I.S.^III; Tomy, S.C.^III; Lopes, M.H.^IV; Warrell, D.A.^V; Theakston, R.D.G.^VI; Butantan Institute Antivenom Study Group^{I, III}

^IHospital Vital Brazil, Instituto Butantan, São Paulo, Brazil

^IILab. Immunopathology, Instituto Butantan, São Paulo, Brazil

^IIILab. Pathophysiology, Instituto Butantan, São Paulo, Brazil

^IVDepartment of Infectious and Parasitic Diseases, Faculty of Medicine, University of São Paulo, Brazil

^VCentre for Tropical Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK

^VILiverpool School of Tropical Medicine, Liverpool, UK

^{Correspondence}

A association between the clinical severity of Bothrops jararaca envenoming on admission and serum venom and plasma fibrinogen levels in 137 human snake bite victims admitted to Hospital Vital Brazil, Instituto Butantan, São Paulo less than 48 hours after the bite is reported. Other variables such as age, sex, site of the bite, use of tourniquet and the time between the accident and antivenom therapy, bleeding, the whole blood clotting test (WBTC20) on admission showed no association with either severity or serum venom. Venom antigenaemia was measured using enzyme immunoassay (EIA) before antivenom administration. The mean serum venom level in patients with mild envenoming was significantly lower than in the group with moderate envenoming (p = 0.0007). Patients with levels of plasma fibrinogen > 1.5 g/L had a lower mean admission serum venom level than patients with plasma fibrinogen levels < 1.5 g (p = 0.025). Also, patients admitted with a tourniquet in place had significantly higher fibrinogen levels than those who had had no tourniquet applied (p = 0.002). The multiple logistic regression model showed that the independent risk factors for severity of Bothrops accidents were bites which occurred at different sites other than leg and forearm, serum venom levels above or equal to 400 ng/ml and the use of a tourniquet. In conclusion, a rapid test to quantify serum venom levels prior to antivenom therapy may contribute to an improvement in the initial evaluation of severity in Bothrops accidents.

Rhabdomyolysis in presumed viscero-cutaneous loxoscelism: report of two cases

França, F.O.S.^{I, II}; Barbaro, K.C.^III; Abdulkader, R.C.R.M.^IV

^IHospital Vital Brazil, Instituto Butantan

^IIClínica de Moléstias Infecciosas e Parasitárias, Hospital das Clínicas, Faculdade de Medicina, Universidade de São Paulo

^IIILaboratório de Imunopatologia, Instituto Butantan

^IVLaboratório de Fisiopatologia Renal, Faculdade de Medicina, Universidade de São Paulo. São Paulo, SP, Brazil

^{Correspondence}

Loxoscelism is an important form of spider envenomation in Brazil. From 1990 to 1993 Loxosceles spiders were the responsible agent for 36.6% out of 17.785 arachnid accidents reported in Brazil. In humans, Loxosceles spider bites usually cause a relatively painless dermonecrotic lesion (87 to 99% of the cases) characterized by erythema, swelling and tenderness which evolves to necrosis, ulcer and scar. In viscero-cutaneous loxoscelism (1 to 13% of the cases), besides the typical cutaneous lesion, there is systemic involvement with intravascular hemolysis, thrombocytopenia, disseminated intravascular coagulation and acute renal failure. Until now, discoloured urine in viscero-cutaneous loxoscelism has been attributed only to haemoglobinuria induced by the intravascular hemolysis caused by the venom. Here two cases (in Brazil) of viscero-cutaneous loxoscelism with rhabdomyolysis and acute renal failure are described. Both patients presented severe oedema, erythema and dermonecrosis at the bite site. Elevated creatine kinase levels were found in both cases (6,841 and 1,718 U/L) associated with severe acute renal failure (one required dialysis for 50 days). The observed rhabdomyolysis was probably induced by the intense inflammatory process caused by the venom that may have spread down into the muscular layers. Therefore, in viscero-cutaneous loxoscelism, rhabdomyolysis secondary to intense local tissue damage can occur and should be considered as a contributing factor in acute renal failure. Creatine kinase should be monitored in viscero-cutaneous loxoscelism to avoid acute renal failure and to reduce the severity of any renal damage.

Inhibition of myotoxic and phospholipase activities of crotoxin by suramin

Arruda, E.Z.; Fernandes, F.F.A.; Moraes, R.A.M.; Pinheiro, D.A.; Melo, P.A.

Departamento de Farmacologia Básica e Clínica, ICB, UFRJ, Rio de Janeiro, RJ, 21941-590, Brazil

^{Correspondence}

Suramin is an enzymatic inhibitor and an uncoupler of G-protein from receptors. We investigated the protective effect of suramin on myotoxic activity of crotoxin (CTX) in mice. The myotoxicity was evaluated in vivo by intramuscular injections (i.m.) of CTX (0.5mg/kg) prepared in physiological saline solution (PSS, 0.1ml). The potential protective effects of suramin were evaluated using two different experimental approaches. In protocol A, CTX was pre-incubated with 1.0mg suramin (15 min, 37°C, in vitro), and then injected i.m. into mice at a dose of 0.5mg/kg (in vivo). In protocol B, CTX was i.m. injected 15 min prior to suramin (1.0mg/kg, i.v.). Before and 2h after the i.m. injections, the animals were lightly anesthetized with diethyl ether and the blood was collected by orbital puncture. The plasma was separated by centrifugation and stored at 4°C for subsequent determination of creatine kinase (CK) activity. Intramuscular injection of CTX increased the CK plasma activity from the control level 71.1 ± 7.4 U/L (n=25) to 943.33 ± 95.93 u/L (n=5). In both protocols, A and B, suramin reduced the increase of CK plasma activity 70% and 60%, respectively. In the study of phospholipase activity by the turbidimetric method (Marinetti et. al., 98:554, 1964), the CTX concentration response curve was determined (1-100mcg/ml) and pre-incubation of suramin reduced phospholipase activity by almost 30%. These data indicate that suramin reduced CTX phospholipase activity in vitro and also its myotoxicity in vivo.

Financial support: CAPES, FAPERJ, CNPq, PRONEX, FUJB-UFRJ.

Individual, intra-specific and gender variations in Tityus bahiensis venom: how different are they?

Dutra, A.A.A.; Pimenta, A.M.C.; de Lima, M.E.

Laboratório de Venenos e Toxinas Animais (LVTA), Dept. de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

^{Correspondence}

The objective of our study was to analyze the soluble fraction of Tityus bahiensis scorpion venom and to search for individual and intra-specific differences, specially due to gender and extraction event. Scorpions were collected at Ouro Preto, Minas Gerais between october 2001 and march 2002 and each one received an individual mark. Scorpions were milked after an starvation period of two weeks and then fed with crickets. The venoms were analyzed by Reversed-Phase (Supelco - C18 column) High Performance Liquid Chromatography and by SDS – PAGE electrophoreses. Many qualitative and quantitative intra-specific differences were observed in both chromatographic and electrophoretic profiles. Also, venoms from females were consistently more complex than those from males and profiles from the same specimen shown that the concentration and some protein constituents had changed from one extraction to another. These results have shown how a venom from a single species can be variable. A deep knowledge of these variations may be useful not only in biotechnology, to prospect new bioactive molecules, but also in evolution studies.

Eristostatin/alkaline phosphatase fusion protein: an enzymatic marker for a_IIbb₃ integrin

Butera, D.^{I, II}; McLane, M.A.^III; Paquette-Straub, C.^III; Ducancel, F.^IV; Moura-da-Silva, A.M.^{I, II}

^ILab. Imunopatologia-Instituto Butantan, Brazil

^IIDepto. Bioquímica, IQ/USP, Brazil

^IIIUniversity of Delaware, USA

^IVCEA Saclay, France

^{Correspondence}

The a_IIbb₃ integrin is the platelet fibrinogen receptor, essential for aggregation. Its low expression is related to severe coagulation disorders such as Glanzmann disease. In addition, a_IIbb₃ is over-expressed during metastasis in tumor cells of different origins. Thus, quantitation of a_IIbb₃ integrin using selective markers as Eristostatin, a snake venom disintegrin, is an important task for evaluating these pathologies. In this work, an enzymatic marker selective for a_IIbb₃ was produced using alkaline phosphatase (APv) tagged eristostatin (Er). Fusion protein was obtained by cloning and expressing Eristostatin DNA into the pLIP6-GN vector. The Er/APv fusion protein was identified by SDS-PAGE and by western blotting using both anti-Er and anti-AP antibodies. The fusion protein showed enzymatic AP activity similar to wild APv. The potential use of the hybrid protein for a_IIbb₃ integrin assay was tested in a one-step dot blot using immobilized cells incubated with the marker and developed by AP substrate. Er/APv showed selectivity towards platelets and a_IIbb₃ integrin transfected cells and reacted with the same region as unlabeled Er, as analyzed in competition assays. The dot-blot test was then standardized with respect to platelet concentration dotted onto nitrocellulose membranes, concentration of Er/APv, time and temperature of incubation. The optimized assay uses physiological concentrations of platelets, incubation times of 30 minutes, room temperature, with minimal concentrations of 10 pM Er/APv. Our data present a novel tool, Er/APv, with potential use in diagnosis of disorders where the a_IIbb₃ integrin is involved.

Support: FAPESP 00-11732/2, FAPESP 00-13651/0 and FAPESP-CEPID/CAT.

Reactivity of neutralizing antibodies elicited by natural and recombinant TsNTxP (Tityus serrulatus non toxic protein)

Mendes, T. M.^I; Kalapothakis, E.^I; Guatimosin, S.C.^I; Maria, W. S.^II; Granier, C.^III; Chávez-Olórtegui, C.^II

^ILaboratório de Biologia Molecular de Toxinas, Departamento de Farmacologia, ICB, UFMG, BH, MG, Brasil

^IIFUNED, BH, MG, Brasil

^IIIInstitut Biotechnonogie et Pharmacologie, Université Montpellier, France

^{Correspondence}

Inoculation of scorpion venom exerts noxious effects in experimental animals and in victims of scorpion stings. This venom elicits a complex patter of clinical signs and symptoms that can lead to death. The fractionation of the venom of T. serrulatus leads to the isolation of lethal, toxic and non-toxic components. Previously we have shown that the TsNTxP a non-toxic protein purified from the Tityus venom or the recombinant TsNTxp (rec TsNTxp), can induce an increase in the level of circulating antibodies sufficient to neutralize the toxic effects of the venom. However, small differences between the antibodies generated against TsNTxp and recTsNTxp were observed. Thus, in order to identify the epitopes recognized by anti-TsNTxP native and anti-recTsNTxP antibodies, we prepared by methods of multiple peptide synthesis sets of overlapping continuous and discontinuous peptides corresponding to the amino acid sequence of TsNTxP. At the end of the synthesis, the peptides remain covalently bound to the membrane and are simultaneously assayed for antibody reactivity. The pattern of binding given by antibodies generated with TsNTxP native or recombinant using mice, rabbit and sheep, showed to be different by the identification of distinct antigenic regions of the TsNTxP sequence. In conclusion, the results suggest that the structure adopted by recTsNTxP-MBP fusion protein does not resemble completely the conformation of the native protein. However, the antibodies generated against recTsNTxp were able to neutralize the venom of Tityus serrulatus.

Supported by: FAPEMIG, CNPq, Pronex, PADCT.

Molecular phylogenetic analysis of Crotalus durissus (Viperidae:Crotalinae) subspecies. Inference from mitochondrial DNA

R. T. Yassaka^I; Rádis-Baptista, G.^I; Callefo, M.E.V.^II; Furtado, M.F.^II; Yamane, T.^I

^ILab. of Molecular Toxinology, I. Butantan

^IILab. of Herpetology, I. Butantan, Vital Brazil

^{Correspondence}

The present work aims at, using sequences of conserved genes, to assist the systematic of serpents of the Crotalus genus. For this study, were collected, in the limits of the Southeastern region of Brazil, 20 individuals of the Crotalus durissus terrificus subspecies, 3 individuals of the Crotalus durissus collineatus subspecies and 4 individuals of the Crotalus genus - whose morphologic classification was not possible. The blood was taken from the esofagic vein. Mitochondrial DNA was purified by SDS and proteinase K method. Oligonucleotide primers, specific for conserved domains of cytochrome b and subunit 4 of the NADH dehydrogenase - both of the mitochondrial DNA – were used in PCR reactions. The amplified products that correspond to the segments of cytochrome b gene (758 bp) and gene of subunit 4 of the NADH (900 bp) were cloned, propagated in E. coli cells and sequenced. At the present moment, the sequences are being analyzed using different programs, among them, the EditSeq and the SeqMan in the package of DNAStar software (Lasergene, Madison, WI). The alignment of the sequences is based on the Clustal method.

Both nucleotide and amino acid sequences are used for the cladistic analysis and for construction of phylogenetic tree. The consistence of those relationships is proved by maximum parsimony and maximum likelihood methods, based on reliable coefficients (Bootstrap, Half Jack-Knife).

The fragments of mitochondrial DNA genes can supply information to complement the morphological analysis and establish the systematic of these taxa, Crotalus durissus subspecies. Molecular data from Crotalus durissus ruruima is being added, to corroborate this analysis. Comparison of the sampling loci and the monophilyletism of the taxa, contributes to elucidate the history and the taxonomic classification of this group, emphasizing differences in this subspecies level or the polymorphic aspects.

Financial support: FAPESP, FUNDAP

Cloning and purification of a C-type lectin from Bothrops insularis (BIL) venom

Guimarães-Gomes, V.^I; Oliveira-Carvalho, A.L.^I; Junqueira-de-Azevedo, I.L.M.^II; Dutra, D.L.S.^I; Castro, H.C.^I; Ho, P.L.^II; Zingali, R.B.^I

^IDepartment of Medical Biochemistry, Federal University of Rio de Janeiro / Brazil

^IICenter of Biotechnology, Butantan Institute. São Paulo / Brazil

^{Correspondence}

Lectins are carbohydrate–binding molecules that specifically recognize diverse sugar structures and mediate a variety of biological process. These proteins require two or more sugar-binding sites in order to cross-link cells. A protein family named C-type lectins has been described in venoms from Viperidae snake and these proteins display a high homology in the primary sequence although showing completely diverse biological actives. In this work we present the characterization of a lectin from the Bothrops insularis venom (BiL). The purification of a lectin from this snake venom was performed as follows: crude venom applied onto a Thiodigalactoside (TDG) - Epoxy Sepharose column and the retained fractions were eluted with lactose and further submitted to a Gel Filtration (Superdex G-75) on FPLC system. The elution pattern showed a major peak that migrated as a single 28 KDa or 14 KDa bands in SDS - PAGE under non reducing and reducing conditions, respectively. The minimal dose necessary to hemagglutinating tripsinized cells 2% was 1,9 ng and is a calcium-dependent process. The major ligands to this lectin were lactose and galactose. Characterization of the lectin primary structure was done by cDNA cloning and confirmed by N-terminal and internal peptide sequencing. An immunological comparison using anti-BiL antibody showed that this lectin presents an antigenic identity with the lectins from B. jararaca, Naja mocambique mocambique and some plant lectins (Pisum sativum e Lens culinaris). Interestingly no immunogenic similarity was observed with the botrojaracin. BiL is a new lectin from B. insularis venom that shares high sequence similarity with C - type lectins from snake venoms.

Financial Support: CNPq, CEE, Finep, FAPERJ, FAPESP.

Influence of food frequency in the ontogeny of Loxosceles laeta (Araneae, Sicariidae)

Gonçalves-de-Andrade, R.M.; Kupper, A.N.; Marinho, P.A.; Tambourgi, D.V.

Laboratório de Imunoquímica, Instituto Butantan, São Paulo, SP, Brazil

^{Correspondence}

Loxosceles laeta is the specie of brown spider that produces the most toxic venom. Cases of human death after envenomation by spiders, in Santa Catarina state, Brazil are associated with this specie. Studies about the mechanisms of the venom action are being developed to create the basis of an efficient treatment since serum therapy does not work efficiently. To perform these studies it is necessary breeding and kept these spiders in laboratory and only adult spiders are used to extract the venom.

Loxosceles laeta spiders become adult generally after one year. In order to study factors affecting the time for spider development, we investigated the influence of feed frequency in the spider ontogeny. Ninety-six spiderlings hatched of the same egg sac were fed with larvae of Tenebrio mollitor and distributed in three groups: 32 animals received food every day, 32, three times per week and 32, one time per week. The inter-moults time was registered in weeks for each instar and group, and data were submitted to a two-tail t-Test (P<0.05). The results showed statistically significant differences between the inter-moults periods of the spider groups which were fed every day and three times per week, as compared with the spiders group that have been fed one time per week. The moult time was always bigger for spiders that were fed one time per week. These data indicates that the best food frequency for a faster spider development, without affecting their length and avoiding time work consume, is three times per week.

Supported by The Wellcome Trust.

First record of Lonomia sp (Lepidoptera, Saturnidae) in the Speleological Province of Ribeira Valley, São Paulo, Brazil

Galati, E.A.B.^I; Marassá, A.M.^II; Gonçalves-de-Andrade, R.M.^III

^IDepartamento de Epidemiologia, Faculdade de Saúde Pública, USP

^IILaboratótio de Parasitoses Sistêmicas, Instituto Adolpho Lutz

^IIILaboratório de Imunoquímica , Instituto Butantan, São Paulo, SP, Brazil

^{Correspondence}

Lonomia sp, which larvae produce toxins that cause Haemorrhagic Syndrome, is reported for the first time in Iporanga county, situated in the Speleological Province of the Ribeira Valley, São Paulo State, Brazil.

One last instar larva was collected in the yard of a residence of "Bairro Serra", a district of Iporanga county. This district is located on the banks of the Betary River, a tributary of the Ribeira River, on the road that links Iporanga and Apiaí counties, about 5 km from the Santana cave, the principal tourist attraction of the "Parque Estadual Turístico do Alto Ribeira", PETAR.

This area encloses dwelling-houses inhabited by residents and several hostels which lodge tourists that visit the caves at weekends and holidays.

The environment on which the larva was collected consist of an orchard with some remaining large primitive Atlantic forest trees which are surrounded by primary woods. The larva was collected on May 26 2002, during a nocturnal capture of phlebotomine sand flies (Diptera, Psychodidae) using Shannon traps, between 3 and 4 a.m., on the ground close to the traps.

The ecosystem shared by “Bairro Serra” resemble the ones in the south of Brazil, Santa Catarina and Rio Grande do Sul State, where the accidents with Lonomia sp caterpillar are frequently reported.

Considering the mortality rate caused by this caterpillar venom in the south of Brazil, an evaluation of the contact risk with this larvae in this ecosystem is necessary to prevent accidents involving residents and tourists.

Supported by FAPESP - process 00/06811-0.

Pharmacological modulation of hyperalgesia induced by Phoneutria nigriventer spider venom

Zanchet, E.M.; Cury,Y.

Laboratory of Pathophysiology, Butantan Institute, SP, Brazil

^{Correspondence}

AIM OF INVESTIGATION: Despite the clinical relevance of local pain observed in Phoneutria nigriventer (Pn) envenomations, no information is available concerning the algogenic activity of Pn venom. The present study was undertaken to characterize the hyperalgesic response caused by Pn venom in rats.

METHODS: Male Wistar rats were injected by intraplantar (i.pl.) route with Pn venom (0.01, 0.1, 1, 3 and 10 µg/paw) or saline. Pain threshold was assessed before and 1, 2, 4 and 24 hr after venom injection using the rat paw pressure test.

RESULTS: The venom caused hyperalgesia that peaked 4h (16, 42, 51, and 61% for each dose, respectively) after injection decreasing thereafter. The hyperalgesic response (1µg venom/paw) was blocked by pre-treatment with dexamethasone (1 mg/kg, s.c), GR 94800 (10µM/paw), capsazepine (120 µM/kg, i.v.), AP5 (40 pM/paw) and partially reduced by methisergide (5 mg/kg, i.p.), anti-IL-1 antibody (20 µg/kg, i.v.), 7-nitroindazol ( 50µg/paw), meloxican (200 µg/paw), GR82334 (10µM/paw), MK801 (100pM/paw) and CNQX (100 nM/paw). Treatment with promethazine (5 mg/kg, i.p.), Hoe-140 (0.6 mg/kg, i.v.), anti-TNF-a antibody (0.2 µg/paw), CGRP 8-37 (400 nM/kg, i.v) indomethacin (4 mg/kg, i.v.), zileuton (100 mg/kg, p.o.) or guanethidine (30 mg/kg, s.c., for 3 days) did not modify this phenomenon.

CONCLUSIONS: The present data suggest that hyperalgesia induced by Phoneutria nigriventer venom is, at least partially, mediated by interleukin-1, prostaglandins generated by COX 2, neuronal nitric oxide, by stimulation of capsaicin sensory neurons and activation peripheral NK1 and NK2 takykinin receptors and NMDA and non-NMDA receptors.

Acknowledgments: Supported by grants from FAPESP (00/06965-8), CAPES and Fundação Butantan

Partial isolation and characterization of thrombin-like BuII-3 from the venom of Bothrops alternatus

Ribeiro, D.A.^I; Toyama, M.H.^{I, II}; Ponce-Soto, L.A.^I; Marangoni, S.^I; Novello, J.C.^I

^IDepartments of Biochemistry Institute of Biology State University of Campinas (UNICAMP) Campinas, SP Brazil

^IIPhysiology and Biophisics Institute of Biology State University of Campinas (UNICAMP) Campinas, SP Brazil

^{Correspondence}

OBJECTIVE: In this work we showed the purification and partial characterization of thrombin-like isolated from the venom of Bothrops alternatus.

METHODS AND RESULTS: This clotting serine protease (BuII-3) was isolated using a combination of molecular exclusion and reverse phase HPLC, respectively.

The molecular mass homogeneity was showed by tricine SDS-PAGE that in presence or absence of DTT reveled an electrophoretical band of approximately 31 kDa.

This protein showed high catalytic activity on the DL – BAPNA (N-Benzoyl-DL-arginine p-nitroanilide) as well as on Aa-chain of bovine fibrinogen, both assayed at 37°C in presence of Ca⁺².

The N-terminal sequence of BuII-3 was IIPPVVINEHRSLVAI. Amino acid analysis showed Asx/42, Glx/29, Ser/21, Gly/18, His/8, Arg/12, Thr/10, Ala/22, Pro/17, Tyr/11, Val/17, Met/4. half Cys/5, Ile/18, Leu/21, Phe/21 and Lys/21. BuII-3 showed high content of Asx and Glx and five Cys, suggesting an acid character of this protein. This protein showed similar disulfide bridges pattern found in other thrombin-like.

CONCLUSION: In conclusion, the purification protocol described here provided an efficient recovery of this enzyme can yield sufficient amounts of enzyme for detailed studies of its biological activities. Bu-II-3 showed similar structural and fibrino(geno)litic serine protease to those of thrombin-like enzymes from snake venoms.

Financial Support: CAPES-CNPq, FAPESP

Characterization of main enzymatic properties of isolated basic PLA2 of the Bothrops jararacussu and Crotalus durissus ssp using chromogenic substrate 4-(nitro-3-octanoyloxy) benzoic acid

Bonfim, V.L.; Toyama, M.H.; Ponce-Soto, L.A.; Novello, J.C.; Marangoni, S.

Department of Biochemistry, Institute of Biology/ State University of Campinas (UNICAMP), Campinas, SP, BRAZIL

^{Correspondence}

OBJECTIVES: In this work, we showed the main enzymatic characteristics of several basic PLA2 from the Bothropic and Crotalic venoms using chromogenic substrate laminate.

METHODS AND RESULTS: In this work we performed several enzymatic studies on the Bj-IV and Bj-V, F6 C.d.cascavella and F6 C.d.collilineatus using a 96 well micro plates system (SpectraMax 340 Molecular Devices). The comparison kinetics parameters between the PLA2s were temperature, pH optima, substrate concentration, influence of ions, PLA2 activity and inhibition of PLA2 activity by crotapotins. Maximum enzyme activity temperature occurred in Bj-IV and Bj-V, F6 C.d.casca and F6 C.d.colli went around 35ºC–40ºC. The pH optima went around to the of 7.5 at 8.3. All PLA2 study here required Ca²⁺ (10mM) for full activity. However those showed enzymatic activity on minimum concentration of the 1mM Ca²⁺. The PLA2 Bj-IV and Bj-V were diminished by cations Cu²⁺ and Zn²⁺, and by Cu²⁺ and Mg²⁺ in the presence and absence of Ca²⁺, respectively. The crotapotins from rattlesnake venom significantly inhibited the enzymatic activity of PLA2s as well as other PLA2 sources including the bee and bovine pancreas

CONCLUSION: All PLA2 assayed in this experimental showed an allosteric behavior using chromogenic substrate 4-(nitro-3-octanoyloxy) benzoic acid under our experimental conditions. Both PLA2 isoforms (Bj-IV and Bj-V) from B.jararacussu, F6 from C.d.coll and F6 from C. d. casca, exhibition a lot of similarity of activity kinetic because they are Asp49 with characteristic physical-chemical similar. The inhibition found by crotapotin against several PLA2 suggest a possible usage of this protein as anti-inflammatory drug.

Isolation of a new basic PLA2 from Bothrops jararacussu. Partial biochemical and biological characterization

Bonfim, V.L.; Toyama, M.H.; Ponce-Soto, L.A.; Oliveira, D.G.; Novello, J.C.; Marangoni, S.

Department of Biochemistry, Institute of Biology/UNICAMP – BRAZIL

^{Correspondence}

OBJECTIVES: In this work, we isolated and characterized a basic PLA2 from the BthTX-II and present partial biological and biochemical characterization of this isoform.

METHODS AND RESULTS: The Bothrops jararacussu venom was fractionated on the HPLC ion-exchange chromatography (Protein Pack SP 5PW-Waters). The sample Bj-V (BthTX-II isoform) was subjected to reversed phase HPLC to further purification. The Bj-V showed allosteric enzymatic behavior with a non-micellar substrate, especially at low concentrations. Maximum catalytic activity occurs at a temperature of 35-40°C and at pH 7,5-8,3. Full PLA2 activity required Ca²⁺ but was inhibited by Cu²⁺ and Zn²⁺; Cu²⁺ and Mg²⁺ inhibited this isoform when in the presence and absence of Ca2+, respectively. The enzymatic activity of Bj-V was significantly inhibited by differently crotapotins from rattlesnakes venoms. The Bj-V inhibited 92% of the growth of Clavibacter michiganensis sp michiganensis colonies. The N-terminal sequence (DLWQWGQMILKETGKLP...) showed a high degree of homology with basic D49 PLA2 myotoxins and amino acid analysis proved a high content of hydrophobic and basic amino acids, as well as 14 half-cysteine residues.

CONCLUSION: The ion-exchange HPLC and RP-HPLC allow the purification of a new basic PLA2 isoform (Bj-V) from the BthTX-II fraction. This novel enzyme showed a allosteric behavior similar to the one found in Bj-IV (BthTX-II isoform too) but the N-terminal sequence showed slight differences between both isoforms, suggesting a minor differences in its structure. The venom from Bothrops jararacussu showed high antibacterial activity and isolated fraction inhibited 92% of the growth of Clavibacter michiganensis sp michiganensis bacteria.

Financial Support: FAPESP

Effect of ACL myotoxin on the water transport in the isolated toad urinary bladder

Leite, R. S.; Selistre-de-Araujo, H. S.; Franco, W.

Departamento de Ciências Fisiológicas UFSCar – São Carlos – S.P. – Brazil

^{Correspondence}

Phospholipase A2-like myotoxins induce necrosis of skeletal muscle cells by a not well understood mechanism. There are evidences in the literature that these myotoxins influence directly or indirectly the hydromineral transport in biological tissues. Myotoxin ACL belongs to the family of the K49PLA2, purified from Agkistrodon contortrix laticinctus venom, which increases the basal water transport and promotes a decrease of the vasopressin hydrosmotic effect in this epithelium. These effects could be mediated, in part, by an increase of cytossolic calcium. Thus, the objective of the present work was to study the role of the Ca⁺² ion in the myotoxin ACL hydrosmotic action in presence of calcium antagonists in the toad bladder in vitro. Water flow was measured gravimetrically by Bentley’s technique (J. Endocrinol. 17: 201-209, 1958) using an osmotic gradient of 4 atm in hemibladders from same animal. Control hemibladders were treated with myotoxin (10nM) and experimental hemibladders received myotoxin plus lanthanum (50mM). Water flow was 3,13±0,35ml/min (n=7) and 1,42±0,25 ml/min respectively, and the difference was significant (p<0,01) between these values. This result shows a decrease of 54% in the myotoxin hydrosmotic action. In other experiments, control group was treated myotoxin and the experimental group received myotoxin associated with nifedipine (10mM) resulting in a water flow 2,96±0,49ml/min (n=8) and 1,81±0,20ml/min respectively, showing a decrease of 38% of the myotoxin hydrosmotic effect. The results suggest that ACL myotoxin increases cytossolic calcium through the release of Ca⁺² stored in the endoplasmic reticulum and also by an increase of the influx these ions via nifedipine sensitive channels.

Financial support: CAPES

Biochemical studies on the Tx3 group of toxins, active on ion-channels, obtained from the venoms of the spiders Phoneutria nigriventer and Phoneutria spp

Ferreira-de-Oliveira, R.^{I, IV}; Richardson, M.^I; Figueiredo, S. G.^II; de Lima, M. E.^III; Calegário-Oliveira, L.^{I, III}; Bloch Jr., C.^V; Diniz, C. R.^I; Cordeiro, M. N.^I

^IFundação Ezequiel Dias - DCPD - Belo Horizonte - MG

^IIUFES - Dep. de Ciências Biológicas - Vitória - ES

^IIIUFMG - ICB - Departamento de Bioquímica e Imunologia - Belo Horizonte - MG

^IVUFMG - ICEx - Departamento de Química - Belo Horizonte - MG

^VEmbrapa - CENARGEN- Brasília - DF

^{Correspondence}

Comparative biochemical studies on venoms of spiders of the genus Phoneutria have enabled the isolation of various peptides with biological activity against insects and mammals (Ferreira-de-Oliveira et al., 2002). These have been classified in groups (PhTx1, PhTx2, PhTx3 and PhTx4) on the basis of the effects they induced in biological assays (Rezende et al., 1991). In more recent work we have begun to compare the polypeptides of the type Tx3 obtained from the venom of a presently unidentified Phoneutria spp with those obtained from Phoneutria nigriventer. The various components were purified by a combination of different steps of liquid chromatography (HPLC and FPLC) and their purity checked by PAGE (acid/urea) and mass spectrocospy. Biological assays performed with mice gave an indication of the toxicity for mammals of each component, via the production of effects such as increased respiration, anti-clockwise gyration, reduction of muscle tone, paralysis of the rear legs, flaccid paralysis and death. The similarities observed underline the need for new pharmacological investigations such as those which indicated that the Tx3 toxins from Phoneutria nigriventer act on Ca²⁺ and K+ ion channels. (Reis et al., 2000, Kushmerick et al., 1999, Guatimosim et al., 1997, Prado et al. 1996).

Financial support: CNPq and FAPEMIG.

Comparative biochemical studies on the venons from Brazilian spiders of the genus Phoneutria

Ferreira-de-Oliveira, R.^I; Richardson, M.^I; Figueiredo, S. G.^II; de Lima, M. E.^III; Calegário-Oliveira, L.^{I, III}; Diniz, C. R.^I; Cordeiro, M. N.^I

^IFundação Ezequiel Dias - Centro de Pesquisa e Desenvolvimento - Belo Horizonte - MG

^IIUniversidade Federal do Espírito Santo - Dep. de Ciências Biológicas - Vitória - ES

^IIIUniversidade Federal de Minas Gerais - ICB - Departamento de Bioquímica e Imunologia - Belo Horizonte - MG

^{Correspondence}

Studies on the spider Phoneutria nigriventer initiated by DINIZ (1963) led to the discovery of novel proteins and peptides with biological activity in mammals and insects, and this has stimulated us to carry out further comparative studies on the venoms from other species of the same genus. A study of possible geographical variation, within a single species, was also made possible by the collection of specimens of Phoneutria reidyi from three well separated localities. In addition to characterizing the groups of toxins found in each species, we have carried out a preliminary study on the proteolytic enzymes in each of the venons. The biochemical characterization was performed using various techniques such as phase reverse and ion exchange chromatography on FPLC and HPLC systems, electrophoreses (PAGE) utilizing SDS-Tricine or Urea/Propionic acid, and biological assays in insects (flies) and mammals (mice). Proteolytic enzyme activity was studied using the QuantiCleave^TM kit (Pierce Chemical Company). Amongst the many proteins obtained in pure and characterized form, several deserve mention because of their toxicity for mammals or insects, in which a number were lethal, in addition to provoking various reactions such as agitation, increasing tactile sensitivity, salivation, itching, flaccid and spastic paralysis.

Supported by CNPq, FAPEMIG and FUNED.

Frei Cristóvão de Lisboa (1583-1652): early toxinology in Brazil

Cardoso, J.L.C.^{I, III}; Domingos, M.O.^II; Haddad Jr., V.^{I, IV}

^IHospital Vital Brazil,Instituto Butantan

^IILab.Esp.Microbiologia,I. Butantan

^IIIDepto Dermatologia,UNITAU

^IVFac.Medicina-UNESP, Botucatu

^{Correspondence}

The manuscript “História dos Animais e Árvores do Maranhão – History of the Animals and Trees of Maranhão”- by the Franciscan Friar Cristóvão de Lisboa (1583-1652) is considered to be the first document about the Natural History of Portuguese America. It even precedes two other highly regarded works published in 1648: “Natural History and Medicine of the West Indies” and “Natural History of Brazil” by Guilherme Piso and Jorge Marcgrave, respectively, who both came to Brazil with the Dutch prince Maurício de Nassau, in the XVII century.

Cristovão de Lisboa arrived in Brazil in May 1624. There he went deep into the center of the North region, which nowadays is represented by the state of Maranhão, where he spent three years (1624-1627) collecting and recording information about the fauna and flora of the region. He was one of the first people in Portuguese America to describe the existence of poisonous fish such as “baiacu” (Tetrodon psitaccus) and “arraia do mar” (Dasyatis gymnura) and poisonous plants such as “mandioca brava” (Manihot esculenta), “timbó” (Indigofera suffruticosa), “timbó-açu” (Loncholarpus nicou) and “caraguatᔠ(Furcraea hexapetala). He also described the biological effects of various venoms and how they were utilized by the natives in the region. His work generated four volumes of which three were lost, probably in the Great Earthquake of Lisbon in 1755. History of the Animals and Trees of Maranhão was the only volume ever found, being discovered in 1930 in a second hand bookshop in Portugal, but published only in 1967. This manuscript has 194 pages with 259 drawings of fish, birds, plants, reptiles and mammals.

Friar Cristóvão de Lisboa returned to Portugal in 1635. He died in Angola in 1652.

Clinical aspects of human envenoming caused by Lactrodectus geometricus (Theridiidae) in S. Paulo

Cardoso, J.L.C.^{I, III}; Brescovit, A.D^IV; Haddad JR., V. ^{I, II}

^IHospital Vital Brazil, I.Butantan

^IIFac.Medicina, UNESP, Botucatu

^IIILab.Artrópodes, I.Butantan

^IVDepto Dermatologia, UNITAU

^{Correspondence}

Among the three Latrodectus sp ( black-widow spider) found in Brazil, L.geometricus C.L.Koch, 1841 is a the commonest species, widely scattered all over the country. The spiders are small, reaching up to 12mm in length. They live in urban areas, buildings their webs close to walls around buildings and houses, but hardly ever invade domiciliary places. Up to now, human accidents caused by L. geometricus have been considered mild and are scarcely documented.

Report case : PRT.HVB 93294 - A 34 year old lady was stung when getting into a car. After the sting, local pain and a local erythematous circular lesion with horripilation of hair follicles developed. A few hours after the accident the patient experienced severe pain and went to a hospital where a potent analgesic intravenous analgesic, and parenteral analgesics. Crises of acute local pain lasts despite the medicaments and in the 3th day after the accident she came to HVB with local erithematous patch and pain when the photos were taken. The spider responsible for the accident was caught and identified as female L. geometricus.

One week after bite the patient was recovered and no clinical signs or symptoms were observed at that time.

Study of antinociceptive and anti-inflammatory activities present in the Crotalus durissus collilineatus whole venoms with and without crotamine

Moreira, K.G.^I; Amora, D.N.^II; Rocha, M.G.^II; Silva, R.M.^II; Camarão, G.C.^II; Silva, N.J.^III; Cardi, B.A.^I; Nogueira, R.M.D.^II; Carvalho, K.M.^I

^ILaboratório de Neurofarmacologia da Universidade Estadual do Ceará

^IIUniversidade Federal do Ceará

^IIIUniversidade Católica de Goiás

^{Correspondence}

BACKGROUND: Experimental evidences of the antinociceptive activity of Crotalus durissus terrificus whole venom have been studied and strongly attributed to the action of crotamine. However, clinical evidences show that antinociceptive activity is also present in Crotalus durissus terrificus whole venom without crotamine. The aim of this study was to compare the antinociceptive and anti-inflammatory activities present in the Crotalus durissus collilineatus whole venoms with and without crotamine.

METHODS AND RESULTS: Free crotamine-C.d. collilineatus venom (CdcN) and crotamine positive C.d. collilineatus venom (CdcP) were used at doses of 40 and 80mg/Kg, i.p. on Swiss mice (20-25g, male). Acetic acid-induced writhing test (0,6%, 0,1ml/10g, i.p.) showed a significantly decreased stretching after both CdcN dose injection (p<0,001), while CdcP caused these effects just after 80mg/Kg. Using formalin test, we could observe a decreased time of licking the paw injected for both CdcP doses in the two phases (p<0,05). CdcN just showed decreased pattern at 80mg dose on second phase (p<0.05). Carragenin-induced paw edema showed significantly decrease for all Cdc samples (p<0.05). No significant changes were observed on heat plate or Rota-rod tests after Cdc samples injection when compared with controls.

CONCLUSIONS: These results show that Cdc venom (N or P) posses similarly peripheral analgesic activity, but CdcP is more effective as anti-inflammatory than CdcN. Taken together, they suggest that crotamine is not the only responsible for these activities, opening perspectives for the existence of other unknown substances involved.

Isolation and partial characterization of the L-amino acid oxidase from B.alternatus snake venom

Stábeli, R.G.; Oliveira, E.B.

Faculty of Medicine of Ribeirão Preto – University of São Paulo – SP – Brazil

^{Correspondence}

L-amino acid oxidase (LAO, EC 1.4.3.2), one of the major components of many snake venoms (up to 30%), catalyzes the oxidative deamination of L-amino acids producing the corresponding a-ketoacids, hydrogen peroxide and ammonia. The aim of the present work is to isolate and characterize the LAO of the B. alternatus venom. The enzyme was purified by sequential affinity chromatography on Sepharose – Zn++, Sephacryl S-200 gel filtration and isoeletric focusing. This latter step yielded three active forms of the enzyme with pIs 4.2, 4.6 and 5.2. The homogeneity of the enzyme with pI 5.2 (LAO-5.2) was demonstrated by SDS-PAGE and reverse-phase HPLC over C-4 column, thus, LAO-5.2 was used for further characterization. The enzyme seems to be a dimer based on the MW determination by SDS-PAGE (66,000) and gel filtration on S-200 column (120,000). Substrate specificity toward different amino acids was determined by measuring the rate of degradation of individual amino acids present in the incubation mixture. Tyr, Phe, Met and Leu were, by far, the most rapidly oxidized substrates while Ile and Cys-Cys were degraded at rates 10-20 times slower.

LAOs have been purified from various snake venoms and most of their biological effects are believed to be, at least in part, mediated by the hydrogen peroxide released during the enzymatic reaction. We trust that highly purified LAO preparation, such as the one derived from B.alternatus venom here described, are important tools to further our understanding of the participation of LAO in promoting apoptosis and others effects such as cytotoxicity, platelet aggregation, haemorrhage, hemolysis and edema.

A novel peptide from the venomous mollusk Conus regius

Braga, M.C.V.^I; Cassola, A.C.^II; Freitas, J.C.^I; Olivera, B.M.^III

^IIB-USP, São Paulo-SP, Brazil

^IIICB-USP

^IIIDept. Biology, University of Utah-UT, USA

^{Correspondence}

Conus is a genus of carnivorous mollusks that paralyze their prey by injecting a complex mixture of biologically active peptides that interact with receptors and ion channels. As no Brazilian species have been studied so far, and as each species has a wide variety of novel peptides with pharmacological interest, we have initiated a characterization of peptides in the Conus regius venom. This abstract will describe a primary structure of a new peptide, its neurotoxic effect and its blocking effect on K+ and Na+ voltage-dependent channels. Conus regius venom was purified by HPLC, its major component was sequenced and its mass spectrum was measured by MALDI-TOF. The sequence revealed a 43 amino acid peptide (4,688 Da) with four disulfide cross-links. The biological activity of the purified peptide was tested by intracranial injection in mice. The higher the concentration injected, the more sensitive to touch the animals became. The pattern of cysteines (C-C-CC-CC-C-C) with four disulfide cross-links and the excitatory effects characterize a new group of the conotoxins, the “I superfamily” (Shetty et al., unpublished results), in which this Conus regius peptide can be included. The activity of this peptide on K+ and Na+ voltage-dependent conductances of the somata of neurons in rat dorsal root ganglion was tested by patch-clamp experiments, in the whole-cell configuration. In high concentrations (mM range), the toxin reduced both currents, indicating that it is an unspecific blocker of K+ and Na+ voltage-dependent channels in mammal cells. Further research is in course in order to characterize the action of the Conus regius I-conotoxin.

Financial support: FAPESP.

Acknowledgments: IBAMA, Águas Claras Diving Center, CEBIMAR-USP and University of Utah.

A P-Superfamily toxin encoded from Conus regius cDNA

Braga, M.C.V.^I; Freitas, J.C.^I; Yamane, T.^II; Rádis-Baptista, G.^II

^IIB-USP ([e-mail])

^IIInstituto Butantan, São Paulo-SP, Brazil

^{Correspondence}

Each species of Conus has 50 to 200 different biologically active peptides, which are encoded in its venom duct. This variety of peptides is responsible for the efficient prey capture in this marine mollusk group. A new group of the conotoxins, the “P-superfamily”, is characterized by the presence of 6 cystein residues (-C-C-C-C-C-C-) and elicits a spasmodic activity in mice. In order to search for P-superfamily toxin precursors in the Brazilian mollusk Conus regius, collected in the Fernando de Noronha Archipelago, a cDNA library was constructed from poly(A+) RNA isolated from its venom duct. Based on the published sequence of conotoxins belonging to the P-Superfamily, a primer was designed, and a PCR homology screening initiated. The amplified cDNA was cloned and sequenced. The sequence analysis revealed a pre-propeptide with a highly conserved signal peptide sequence (96%). The non-cystein residues of the mature toxin show around 80% of divergence, while the cystein residues show 100% similarity, when compared against Tx9.1 and Gm9.1 from C. textile and C. gloriamaris, respectively. Based on signal peptide sequence similarity and the conserved position of cystein residues, it is highly probable that the present toxin belongs to the "P-superfamily" of conopeptides. In concordance with the nomenclature, it is designated Rg9.1.

Financial support: FAPESP.

Acknowledgments: IBAMA, Águas Claras Diving Center, CEBIMar-USP and Instituto Butantan.

Crystal structure of miotoxin II from Bothrops moojeni bound to a fatty acid at 1.8 Å resolution

Fontes, M.R.M.^I; Soares, A.M.^II; Giglio, J.R.^III; Arni R.K.^IV

^IDepto. de Física Biofísica, Inst. de Biociências UNESP, Botucatu

^IIDepto. de Biotecnologia-UNAERP, Ribeirão Preto

^IIIDepto. de Bioquímica, Fac. de Medicina de Ribeirão Preto USP - Ribeirão Preto

^IVDepto. de Física, IBILCE UNESP, S. J. do Rio Preto

^{Correspondence}

Phospholipases A2 are common components of bothropic venoms responsible for disruption of the cell membrane integrity via hydrolysis of its phospholipids, culminating with cell death. Theses enzymes have an Asp at position 49 (D49) which is a part of the Ca++ loop which promotes the catalytic activity on phosphatidylcholine from egg yolk. A new class of PLA2-like proteins has been described which, although devoid of PLA2 activity on phosphatidylcholine, due to a mutation D49K, is still highly myonecrotic. Their effect being Ca++ independent aren’t still completely understood.

In this work we show the X-ray structure determination, refinement process and relate structural characteristics of this toxin co-crystallized (MjTX-II) with fatty acid.

The MjTX-II was cDNA cloned and sequenced and pharmacological and toxic activities were tested. The toxin was co-crystallized with a fatty acid and data collection was made at LNLS (Campinas). The crystals belong to the space group P212121 with approximated cell constants a= 61.18 b=88.71 and c=51.09 Å. Data was collected up to 1.8 Å resolution. The structure was solved by Molecular Replacement method and the refinement using the CNS program, the final R-factor was 21.9% and free-R-factor 25.3%. It was observed a strong electronic density for the fatty acid for both monomers in the hydrophobic channel close to active site. The main contacts toxin-fatty acid are His48, Lys49 and the main chain of residues 28-30.

Platelet activation during Bothrops jararaca snake envenomation in rabbits

Santoro, M.L.^I; Barbaro, K.C.^II; Rocha, T.R.F.^III; Sano-Martins, I.S.^I

^ILaboratory of Pathophysiology, Instituto Butantan

^IILaboratory of Immunopathology, Instituto Butantan

^IIILaboratory of Thrombohemorrhagic diseases, HCFMUSP, São Paulo, Brazil

^{Correspondence}

Despite being well established that Bothrops jararaca snake venom causes blood coagulation and fibrinolysis disturbances, scant information is available about blood platelet disorders. In a previous investigation, we observed decreased numbers of dense bodies in circulating platelets of experimentally envenomed rabbits, suggesting that they underwent platelet activation. Herein we show that rabbits – whose platelets were previously labeled ex vivo with NHS-biotin – injected intravenously with B. jararaca venom (60 µg/kg) presented thrombocytopenia, hypofibrinogenemia, elevation of von Willebrand factor plasma levels, platelet hypoaggregation, decreased platelet ATP secretion, normal plasma levels of platelet factor 4, and invariable intraplatelet serotonin content. By flow cytometry, platelets displayed a significant decrease on the expression of a GPIIb-IIIa epitope recognized by P2 monoclonal antibody, but total GPIIb-IIIa expression, evaluated with specific polyclonal antibodies, was normal. Fibrinogen or fibrin degradation product (FDP) expression on platelet surface showed no significant alteration. Nonetheless, significant elevations of platelet P-selectin and of a ligand-induced binding site of GPIIIa were noted on circulating platelets. The percentages of circulating reticulated platelets, as well as platelet survival, of envenomed rabbits were not statistically different from control animals. We suggest that thrombin generated by procoagulating components of B. jararaca venom has an essential role in the pathogenesis of platelet and coagulation disorders in this experimental model. Increased expression of P-selectin in the experimental group demonstrates that platelets of envenomed rabbits are indeed activated in the circulation and that decreased fibrinogen or increased FDP levels are not the primary cause of the platelet dysfunction observed in bothropic envenomation.

Determination of primary structure and its bactericidal activity of new CB isolated from Crotalus durissus collilineatus

Oliveira, D.G.^I; Toyama, M.H.^I; Carneiro, E.M.^II; Boschero, A.C.^II; Joazeiro, P.P.^III; Beriam, L.O.S.^IV; Marangoni, S.^I

^IDepartamento de Bioquímica Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, Brasil

^IIDepartamento de Fisiologia e Biofísica Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, Brasil

^IIIDepartamento de Histologia e Embriologia Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, Brasil

^IVLaboratório de Bacteriologia Vegetal, Centro Experimental do Instituto Biológico de Campinas, São Paulo, S.P, Brasil

^{Correspondence}

OBJECTIVES: In this work we determined the amino acid sequence of new crotalic PLA2 from the C. d. collilineatus venom and its antibacterial activities on Xanthomonas axonopodis pv. passiflorae and C. m. sp michiganesnis.

METHODS AND RESULTS: The amino acid sequence of novel PLA2 was determined in the procise f (Applied Biosystem) and its amino acid sequence was: HLLQFNKMIKFETRRNAIPFYAFYGCYCGWGGRPKDATDRDRCCFVHDCCYEKLTGCNYKWDFYRYSLR

SGYFQCGKGTWCEQQICECDRVAAECLRRSLSTYRYGYMIYPKSRCRKPSETC. Cdcolli F6 shows higher antibacterial effect on the Gram-positive than Gram-negative bacterial strains. Thus, the inhibition of growth was of 95% and 85% for Claribacter ssp (Gran positive) and for Xanthomonas ssp, (Gran negative), respectively. Cdcolli F6 to produce cell membrane disruption allowing the penetration of the toxin into the cells (Xanthomonas ssp), and also induced several modifications principally in the LPS composition as suggested by electrophoresis analysis.

CONCLUSION: In conclusion, the bactericidal efficacy of PLA2(s), in general, and of Cdcolli F6, in particular, is dependent on both the presence of positive amino acid residue that allows electrostatic interaction between the Cdcolli F6 molecule and the bacterial membrane and their catalytic activity that hydrolysis the bacterial cell membrane.

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

Toyama, M.H.^I; Oliveira, D.G.^I; Santos, L.M.B.^II; Pereira, R.^III; Antunes, E.^III; Monteiro, H.S.A.^IV; Martins, A.M.C.^IV; Beriam, L.O.S.^V; Novello, J.C.^I; Marangoni, S.^I

^IDepartamento de Bioquímica, Faculdade de Ciências Médicas, UNICAMP

^IIDepartamento de Imunologia e Microbiologia, Inst. de Biologia, Faculdade de Ciências Médicas, UNICAMP

^IIIDepartamento de Farmacologia, Faculdade de Ciências Médicas, UNICAMP

^IVDepartamento de Fisiologia e Farmacologia, Universidade Federal do Ceará

^VLaboratório de Bacteriologia Vegetal, Centro Experimental do Instituto Biológico de Campinas, São Paulo, S.P., Brasil

^{Correspondence}

OBJECTIVES: We purified a novel L-amino acid oxidase (LAO) from Crotalus durissus cascavella snake venom and molecular, platelet aggregating, bactericidal and cytotoxic characterization was made.

METHOD AND RESULTS: This novel LAO was purified by two chromatographic steps: molecular mass and SP 5PW HPLC. The purified enzyme (CDCE-LAO) is present as a dimer on gel filtration, with an apparent M(r) of 60 kDa for the monomer as estimated by Tricine SDS-PAGE. CDCE-LAO showed high enzymatic specificity agains L-leucine (56U/mg) and induce platelet aggregation of whole and washed platelets using doses of 5,3 and 1mg/ ml. This novel protein inhibited around 85% and 35% of gram-positive and gram-negative bacterial growth rate, respectively and induced a significant decrease of viable B-cell number after stimulation with Con A in vitro. This novel protein shows high content of acidic amino acids and its n-terminal amino acid sequence was determined to be ADDRNPLAECFQENDYEEFLEIARNGLKATSNPKHVVIVGA...

CONCLUSION: This novel LAO represents around 7.5% of whole Crotalus durissus cascavella venom. In the three other samples of Crotalus durissus cascavella venom this LAO represent around 2.3 - 3.5% of whole venom. This novel protein showed significant bactericidal, platelet and haemagglutination activities and this suggests that this protein plays an important role in of blood homeostasis.

Vasopeptidase inhibition with seletivity for the active site at the C-domain of ACE by bradykinin potentiating peptides from Bothrops jararaca venom

Ianzer, D.A.^I; Portaro, F.C.V.^I; Paula, R.D.^II; Silva, C.A.^I; Gorrão, S.S.^I; Prezoto, B.C.^I; Nascimento, N.^III; Cotton, J.^IV; Dive, V.^IV; Santos, R.A.S.^II; Camargo, A.C.M.^I

^ICenter of Applied Toxinology (CAT/CEPID-Fapesp), Departamento de Bioquímica e Biofísica, Instituto Butantan - SP

^IIDepartamento de Fisiologia, ICB, UFMG

^IIILab. de Biologia Molecular, IPEN

^IVCEA, Départment d´Ingénierie et d´Estudes des Protéines (DIEP) Yvette Cedex, France. e-mail: [e-mail]

^{Correspondence}

Venoms produced by snakes are, in general, constituted by several proteases and peptides that act as toxins. The bradykinin-potentiating peptides (BPPs) are constituents of the serpents toxins and represent the first natural inhibitors of ACE discovered. The ACE enzyme possesses two catalytic sites, at the N-terminus and C-terminus domains. Although both active sites are able to convert angiotensin I into angiotensin II and inactivate Bradykinin, the site at the C-domain seems to be more specific. Moreover, another enzyme, the neutral endopeptidase (NEP) inactivates bradykinin and natriuretic peptides. Together, both enzymes of the endothelial cells are designated vasopeptidases. Thus, the development of combined inhibitors for the vasopeptidases (VPIs) is of importance for the treatment of cardiovascular dysfunction. In this work, we studied three activities concerning the BPPs: 1) Inhibition of the vasopeptidases ACE and NEP, 2) The bradykinin potentiating effect using isolated guinea pig ileum assays and 3) Effect of the BPPs on the arterial pressure of anaesthetized rat. The results obtained in the kinetic studies showed that the BPPs act as NEP inhibitors and some of these peptides are selective inhibitors for the C-terminus domain of ACE. About these results, BPP-Xc is one of the most interesting peptides, since it is able to block both the ACE C-domain and the NEP activities. This peptide also potentiated the bradykinin activity and showed the best result in potentiating the bradykinin hypotensive effect in vivo assays. In addition, preliminary studies in mices showed that the BPP-Xc was still intact in urine after i.p. injections. We concluded the BPP-Xc can be considered a model for the development of a potent drug for treatment of the human hypertension.

FAPESP, CNPq, FINEP

Cloning of antimicrobial-opioid peptides from the skin of Prhynohyas venulosa (Hylidae)

Conceição, K.^I; Rádis-Baptista, G.^I; K. Kubo, T.^II; Antoniazzi, M. M.; Jared, C.; Yamane, T.^I

^IMolecular Toxinology Lab, Butantan Institute, São Paulo

^IIMolecular Neurobiology Lab, AIST, Tsukuba, Dept. of Cellular Biology, Butantan Institute, São Paulo, BR

^{Correspondence}

The innate immunity is the key component of the host first defense mechanism against pathogens in organisms as diverse as plants, insects, lower vertebrates, and mammalian. More than six hundred structures with antimicrobial activity against encapsulated virus, bacteria and fungi have been compiled.

In general, a family of peptides belonging to one determined class is detected in different biological source. This results from the hypermutation of mature peptide and an extremely conserved signal peptide – an indication that a high selective pressure operates to preserve an efficient “cassette of secretion”.

To identify precursors of bioactive peptides in the frog skin of Phrynohyas venulosa – a Hylidae that inhabits the Brazilian “Cerrado”, a cDNA library was constructed. Based on conserved signal peptide sequence, oligonucleotides were synthesized and used to screen the recombinant bacteriophage clones. By this approach, we identified, so far, three new antimocrobial/opiod pre-propeptide precursors that share some sequence similarity with dermaseptin/dermorphin members, isolated by several research groups, from the skin of Phyllomedusa specimens.

The biochemical and molecular evolutionary aspects of these peptides are presented and discussed.

Support: FAPESP

Pharmacological characterization of the effects induced by Tityus serrulatus venom in the rat retractor penis muscle

Bomfim, J.H.G.G.^I; Giglio, J.R.^II; de Oliveira, A.M.^I; Godoy, M.A.F.^III; Arantes, E.C.^I

^IDepartamento de Física e Química - FCFRP-USP

^IIDepartamento de Bioquímica e Imunologia – FMRP USP – Ribeirão Preto -SP - Brasil

^IIIDepartamento de Farmacologia – FMRP USP – Ribeirão Preto -SP - Brasil

^{Correspondence}

OBJECTIVES: The aim of this study is to investigate the contractile and relaxant responses induced by Tityus serrulatus venom (TsV) in rat isolated retractor penis muscle (RPM).

METHODS AND RESULTS: RPM was isolated and mounted under 0,6 g resting tension in 5 ml organ bath containing physiological Krebs solution, maintained at 37°C and gassed with 5% carbon dioxide in oxygen. The results were recorded in a Harvard Universal oscillograph connected to a 133 Pentium. TsV (1-60 mg/ml) induced a concentration-dependent contractile response (E_máx = 66.23 ± 6.2% n=6 , pD2 = -1.01). In the presence of prazosin (1mM, 20 min) and guanethidine (30 mM, 10 min), TsV did not produce any contractile effect. The relaxation response was performed by contracting the muscle with bethanechol (100 mM), in the presence of prazosin (1mM, 20 min) and guanethidine (30 mM, 10 min), associated or not with L-NAME (1mM, 30 min). TsV (1-60 mg/ml) induced a concentration-dependent relaxant response (E_máx = 52.32 ± 7.5% n=7, pD2 = -1.08) in the absence of L-NAME. In the presence of L-NAME, TsV did not produce any relaxant response.

CONCLUSION: TsV induces a contractile response in rat RPM due the norepinephrine release, which was confirmed with simpathetic specific inhibitors (prazosin and guanethidine).The relaxant response is dependent on nitric oxide release, probably from nitrergic nerves, as shown by L-NAME assay.

Lonomia obliqua: the venom action on factor XIIIa

Batista, I.F.C.^I; Ribeiro, A.L.Q.^I; Prezoto, B.C.^II; Chudzinski-Tavassi, A.M.^I

^ILaboratório de Bioquímica e Biofísica

^IILaboratório de Farmacologia, Instituto Butantan, São Paulo, SP, Brazil

^{Correspondence}

Lonomia obliqua and Lonomia achelous caterpillars contain toxic substances that in contact to human skin, causes severe damage to the healthy, like urticant dermatitis, allergy of the respiratory system, severe hemorrhagic disturbances. The main problem observed in the patients who had contact to the Lonomia bristles is a severe consumptive coagulopathy, which generally leads to a hemorrhagic syndrome. Lonomia achelous venom contains a toxin that degrades FXIIIa either in vivo or in vitro.

OBJECTIVE: to analyze the action of Lonomia obliqua crude extract (LOCBE) in the plasma Factor XIIIa activity.

METHODS: The influence of the venom in the Factor XIIIa (FXIIIa) was analyzed in vitro by gel filtration chromatography and SDS-PAGE and in vivo, infusing LOCBE to wistar rats and following blood clot parameters, the FXIII and fibrinogen levels, before and after envenomation.

RESULTS: According to the gel filtration and SDS-PAGE performed in the in vitro experiments, there is no degradation or complex formation when the purified FXIII is incubated with the venom. Another important finding is that the FXIIIa activity is absolutely maintained after the incubation. Concerning to the in vivo experiments, the plasma of envenomed rats have the level of FXIIIa reduced to 50% and the fibrinogen is totally consumed. However, when defibrinogenated plasma is incubated with LOCBE no consuption was observed.

CONCLUSION: the results suggest that the reduction of FXIIIa observed in the rats and probably in envenomed patients is caused by the consumption of fibrinogen and not by the FXIIIa inhibition or degradation as occur in L. achelous patients.

Supported by FAPESP

Preliminary characterization of hepatotoxicty of crotalic venom

Teixeira, G.N.^I; Vilela-Goulart, M.G.^II; Iakimoff; A.^I; Bastos, W.P.^III; Mattos Filho, T. R.^II; Pacheco, C.S.^I; Lopes-Martins, R.A.^I; Cogo, J.C.^I; Ribeiro, W.^I

^IUniversidade do Vale do Paraíba, IP&D/ Serpentário do CEN, São José dos Campos

^IIUniversidade de Campinas, Faculdade Odontologia de Piracicaba

^IIIUniversidade Estadual de São Paulo, Faculdade de Odontologia de São José dos Campos, SP, Brazil

^{Correspondence}

The purpose of this study was to investigate the hepatoprotective effect of calcium channel blocker verapamil against the of Crotalus durissus terrificus (C.d.t.) venom. 48 Wistar male rats (250 ± 50g) were divided in 4 groups of 12 rats each. The groups were divided in 4 sub-groups (n=3) sacrificed at the time 1 h, 2h, 3h, and 12h, after saline, verapamil, venom and verapamil + venom administration. The samples were processed and centrifuged to posterior analyze of the enzymes: alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamil transferase (Gamma GT), acetylcholinesterase (AchE) and total protein (TP). Control group (G1): Inoculated only with saline, Venom group (G2): Inoculated with C.d.t. venom (100 mg of venom to each Kg of rat weight), Verapamil group (G3): Inoculated i.p. with verapamil (26,7 mg/Kg), Venom + Verapamil (G4): Pre-treated with verapamil (26,7 mg/Kg) before 2 hour of the venom administration. The group G2 showed elevations of enzymes derived from liver tissue when compared with all groups. Already G4 showed reduced liver tissue enzymes levels when compared with G2. The values of hepatic tissue enzymes at the time of 1 hour to G4 vs G2 were respectively: ALP : 6,67 ± 1,98 vs 12,52 ± 3,22 (U/L), ALT: 71,33 ± 3,77 vs 127,13 ± 6,06 (U/L), AST: 102,56 ± 41,36 vs 149,27 ± 5,15 (U/L), GGT: 124,64 ± 27,53 vs 295,95 ± 102,80 (U/L), AchE: 6,84 ± 1,73 vs 9,87 ± 0,85 (U/ml), TP: 1,93 ± 0,21 vs 3,51 ± 0,95 (g/DL). In conclusion, we suggest that calcium channel blockers may have a role to play in limiting hepatocellular damage, especially the increase of liver dysfunction mechanism due the cytoplasmatic and mithocondrial lesions, resulting, in the elevation of enzymes derived from liver tissue.

Biotechnological applications of insecticidal toxins from the venom of the brown spider Loxosceles intermedia

Castro, C.S.^I; Silvestre, F.G.^I; Ribeiro, B.M.^II; Mangili, O.C.^III; Chávez-Olórtegui, C.^IV; Gomez, M.V.^I; Kalapothakis, E.^I

^ILaboratório de Biologia Molecular de Toxinas, Departamento de Farmacologia, ICB, UFMG, Belo Horizonte/MG,

^IILaboratório de Microscopia Eletrônica, Departamento de Biologia Celular, ICC, UnB, Brasília/DF

^IIIDepartamento de Patologia Básica, Setor de Ciências Biológicas, UFPR, Curitiba/PR

^IVDepartamento de Bioquímica e Imunologia, ICB, UFMG, Belo Horizonte/MG

^{Correspondence}

It is estimated that, every year, 40% of the world’s agricultural production is lost due to the attack of pests. Although efficient and easily accessible to farmers, chemical insecticides can favor the development of resistant insects populations besides causing damage to the environment as well as men. Bioinsecticides represent an alternative for the reduction of agricultural losses, providing advantages concerning costs, ecology and human health. Our group, aiming the production of highly efficient bioinsecticides, has characterized toxins with possible insecticidal activity from a Loxosceles intermedia venom gland cDNA library. The practical limitations to get from the crude venom high yields of previously identified neurotoxins made necessary the use of heterologous expression systems. In this work, cDNAs from two new putative insecticidal toxins, called LiTx1 e LiTx2, were identified, characterized and cloned in the transfer vector pSynXIV VI+ of the Baculovirus expression system. This is one of the most powerful and versatile eukaryotic expression systems available. Its advantages includes functional activity of the recombinant protein, post-translational modifications and high expression levels, among others. Sequencing analysis confirmed the success of all the procedures conducted so far to obtain the recombinant Baculovirus. Bioassays with the new recombinant toxins will be a important step in discovering new tools for pest control.

Supported by: CNPq, PADCT, PIBIC, Pronex, FAPEMIG.

Analysis of the mechanisms involved in the development of cutaneus loxoscelism in vivo

Paixão-Cavalcante, D.^I; Gonçalves de Andrade, R.M.^I; Antoniazzi, M.M.^II; Magnoli, F.C.^I; Van Den Berg, C.W.^{I, III}; Tambourgi, D.V.^I

^ILaboratories of Immunochemistry Butantan Institute, São Paulo, Brazil

^IILaboratories of Cell Biology Butantan Institute, São Paulo, Brazil

^IIIDepartment of Pharmacology, Therapeutics and Toxicology, University of Wales, College of Medicine, Cardiff, UK

^{Correspondence}

Envenomation by spiders belonging to the genus Loxosceles commonly results in impressive local necrotic skin lesions and, more rarely, causes systemic effects, including profound intravascular haemolysis. The predominant clinical sign is a cutaneous reaction characterised by the appearance of necrosis around the bite, resulting in ulceration. In this work, we undertook a histopathological characterization of the dermonecrotic lesion produced experimentally with Loxosceles intermedia venom and evaluated the role of the complement system and endogenous proteases in the development of cutaneous loxoscelism. Adult rabbits were injected intradermally with 5 mg of L. intermedia venom or P1 toxin in the presence of 10 mM EDTA, 1,10 phenanthroline or PMSF. Alternatively, rabbits were depleted of complement by i.v. injection of cobra venom factor and then inoculated with venom. Twenty-four hours after venom inoculation, the rabbits were sacrificed and the skin was removed for histopathological analysis. Rabbits inoculated with L. intermedia venom showed an intense inflammatory cell migration into the dermis. In the sub-epidermal region, haemorrhage and disorganization of the collagen fibres was observed. The skin muscle layer showed an intense inflammatory cell influx and haemorrhage. Rabbits inoculated with P1 toxin showed the same histopathological profile as those injected with the venom. Complement depleted rabbits showed a large reduction in the inflammatory cell infiltration into the inoculated site. Both 1,10 phenanthroline and EDTA inhibited the haemorrhage and influx of inflammatory cells whereas PMSF was unable to revert any of the effects provoked by the venom. P1 toxin had the same ability as venom to induce dermonecrotic lesions. These results reinforce the idea that the sphingomyelinase toxin P1 is the initiator of the dermonecrotic reaction. The complement system and endogenous metalloproteinases are also involved in the development of the lesion induced by L. intermedia venom.

Supported by FAPESP and The Wellcome Trust.

Analysis of the mechanisms involved in the development of cutaneus loxoscelism in vitro

Paixão-Cavalcante, D.^I; Tambourgi, D.V.^I; Gonçalves de Andrade, R.M.^I; Magnoli, F.C.^I; Van Den Berg, C.W.^{I, II};

^ILaboratory of Immunochemistry, Butantan Institute, São Paulo, SP, Brazil

^IIDepartment of Pharmacology, Therapeutics and Toxicology, University of Wales, College of Medicine, Cardiff, UK

^{Correspondence}

Envenomation by spiders belonging to the genus Loxosceles commonly results in impressive local necrotic skin lesions with scar formation and ulcers that heal slowly. To examine whether Loxosceles intermedia venom and its sphingomyelinase (SMAse) toxin P2 affect the morphology and viability as well as the expression of surface proteins in cultures of dermal and epidermal cells, human primary keratinocytes, the epidermal carcinoma cell line A431 and human and rabbit primary fibroblasts were treated with different concentrations of venom, purified SMAse P2, phorbol myristic acid (PMA), Staphylococus aureus Smase, TNF-a or C2-ceramide. Alterations in cell morphology were assessed by phase contrast light microscopy. Cell viability was determined by incubating the cells with 10% Alamar Blue for 1 h at 37ºC. The absorbance of the supernatants was read at 560 nm and 595 nm and the cell viability was expressed as (A560-A595)_treatment/(A560-A595)_control x 100. To assess the expression of cell surface antigens, cells were treated with different concentrations of L. intermedia venom for 1 h at 37ºC. Thereafter, the cells were incubated with specific antibodies against membrane cofactor protein (MCP), CD59, decay accelerating factor (DAF), MHC class I, a2-microglobulin and epidermal growth factor receptor followed by rabbit anti-mouse Ig/FITC antibody and analysis by flow cytometry. Treatment with L. intermedia venom and purified SMAse P2 decreased cell viability and altered the morphology of all cells studied. S. aureus Smase, TNF-a, PMA and C2-ceramide did not produce such effects. Venom treatment also decreased the expression of MCP, MHC I and a2-microglobulin in all cells. L intermedia venom and P2 protein have toxic effects on the main cell types present in skin.

Supported by FAPESP and The Wellcome Trust.

Epitope mapping and synthesis of peptides derived from TSNTxP for the production of neutralizing antibodies against Tityus serrulatus scorpion venom

Alvarenga, L.; Machado, C.; Granier, C.; Chávez-Olórtegui, C.

Fundação Ezequiel Dias, Belo Horizonte, MG, Brasil and Institut de Biotechnologie et Pharmacologie, CNRS UMR 5094 Faculté de Pharmacie, Montpellier -France

^{Correspondence}

A non–toxin protein (TsNTxP) isolated from the venom of scorpion Tityus serrulatus (Ts) induces which antibodies cross-react with several toxins from the venom, in contrast to anti-toxin antibodies which are toxin specific. The use of defined synthetic epitopes could be used for the antivenom preparation and production of vaccines against scorpionism.

OBJECTIVES: Use of Spot method of multiple peptide synthesis to identify epitopes derived from toxins of the Ts venom for the preparation of neutralizing antivenoms.

MATERIALS AN METHODS: The Spot-method was used to prepare immobilized overlapping peptides covering the amino acid sequence of TsNTxP and TsIV, an a-type toxin of the Ts venom. Rabbit sera anti-TsNTxP and anti-Ts IV were produced and their binding in the membrane was detected by using a alkaline-phosphatase conjugate anti-rabbit. The reactive peptides were synthesized as soluble peptides and coupled to KLH for use as immunogens.

RESULTS: Three antigenic regions were discovered with anti-TsNTxP, one N-terminal, one central and the C-terminal part. One epitope in the C-terminal of Ts IV was identified and used for production of antibodies. Quantities of venom equivalent to 13.5 LD50 were neutralized by 1 ml of the anti-peptide serum. The anti-peptide antibodies had a high reactivity against Ts venom, moderate binding for T. bahiensis, T. cambridgei, T. stimurus and were unable to recognize Androctonus australis and Centrutoides sculpturatus venoms.

CONCLUSION: These results show that peptides derived from the sequence of scorpion toxins appear to be an alternative for the antivenom preparation against scorpionism.

Supported by: CNPq, INSERM.

Jararhagin-induced pro-inflammatory cytokines in mouse experimental model

Clissa, P.B.^I; Laing, G.L.^II; Taylor, M.J.^II; Theakston, R.D.G.^II; Mota, I.^I; Moura-da-Silva, A.M.^I

^ILaboratório de Imunopatologia, Instituto Butantan – São Paulo, Brasil

^IILiverpool School of Tropical Medicne – Liverpool, UK

^{Correspondence}

Accidents caused by Bothrops jararaca snake are characterized by haemorrhage, prominent local tissue damage, and acute inflammation, which may result in permanent sequel. Specific antivenom is not capable of neutralizing venom-induced local inflammation suggesting that venom toxins can activate endogenous mechanisms. Venom metalloproteinases are able to process pro-TNF-a and TNF-a antibodies reduced the size of metalloproteinase induced necrotic lesions in mice, suggesting the importance of cytokines on the severity of envenomation. The present study evaluates the cytokines induced by jararhagin, a metalloproteinase/disintegrin isolated from B. jararaca venom, in vitro (Murine Peritoneal Adherent Cells-MPACs) and in vivo (mouse footpad). Jararhagin induced mRNA expression for TNF-a, IL-1b and IL-6 in both MPACs and footpad systems. Soluble TNF-a (130pg/paw, after 2 hours) IL-1b (400pg/paw, after 4 hours) and IL-6 (4500pg/paw, after 4 hours) were detected in the footpad homogenates. However, in culture supernatants, jararhagin enzymatic activity degraded the cytokines and their levels were detected only after inhibition of jararhagin proteolytic activity with EDTA. The role of Jararhagin disintegrin/cystein-rich domains for the local release of cytokines was also studied using Jararhagin-C isolated from B. jararaca venom. Disintegrin/cystein rich domains play an important role in cytokine release. Levels of TNF-a were similar in mice treated with either complete jararhagin or isolated disintegrin/cystein-rich domains. A reduction of approximately 50% was observed in IL-6 and IL-1b levels after treatment with jararhagin-C in comparison with treatment using the role toxin. This study showed the presence of cytokines in a pro-inflammatory model, induced by snake venom metalloproteinases both in the cell culture and also at the site of venom injection.

Financial support: FAPESP

Complement activation by Tityus serrulatus venom and TsTX-I

Bertazzi, D.T.; Talhaferro, V.L.; Lazzarini, M.; Azzolini, A.E.C.S.; Assis-Pandochi, A.I.; Arantes, E.C.

Departamento de Física e Química, FCFRP-USP, Ribeirão Preto, SP, Brasil

^{Correspondence}

AIM: The effect of Tityus serrulatus venom and a purified toxin (TsTX-I) on the lytic activity of the classic (CP) and alternative (AP) pathways was examined in order to assess the involvement of complement in the acute inflammatory reaction seen following venom injection.

METHODS AND RESULTS: Male Wistar rats (200 g) received 500 mL of venom (150 mg/kg, i.p.) and were sacrificed 0.5, 1, 2, 4, 24 and 48 h later. Rats treated with TsTX-1 (150 mg/kg, i.p.) were sacrificed 1 and 24 h after toxin injection. A kinetic assay was used to assess the effect of venom and toxin on complement. Serum was mixed with erythrocyte antibody (CP) or rabbit cell (AP) suspensions and the kinetics of lysis was monitored by the change in absorbance at 700 nm. The time required for the absorbance to decrease to half its initial value (t1/2) was calculated from the lysis curve. Increased lytic activity associated with CP and AP occurred at all intervals. Venom produced activities that were 154-170% and 184-220% of the control values for CP and AP, respectively. TsTX-I increased the activities to 132-121% and 128-130% for CP and AP, respectively. The lytic activity of serum from rats treated with venom was abolished by zymosan, indicating that it was dependent on complement. The hematocrit was higher than in the controls from 0.5–4 h after venom administration, but recovered to normal values after 24 h.

CONCLUSIONS: The increase in the lytic activity of serum from rats treated with venom and TsTX-I may partly be the result of hemoconcentration. The elevated activity after 24 h may be a consequence of increased synthesis of complement components, as seen with other acute phase proteins in rats treated with this venom.

Hematological changes induce by Tityus serrulatus venom in rats

Cusinato, D.A.C.^I; Souza, A.M.^II; Gregório, Z.M.O.^II; Ogawa, K.C.^I; Vasconcelos, F.^I; Arantes, E.C.^I

^IDepartamento de Física e Química, FCFRP-USP, Ribeirão Preto, SP, Brazil

^IIDepartamento de Análises Clínicas Toxicológicas e Bromatológicas, FCFRP-USP, Ribeirão Preto, SP, Brazil

^{Correspondence}

OBJECTIVES: The aim of this work was to evaluate the hematological changes induced by Tityus serrulatus venom (TsV), using in vivo and in vitro assays.

METHODS AND RESULTS: Blood was collected from male Wistar rats (6 rats/group, b.w. 100 g) 30, 120 and 360 min after the intraperitoneal injection of TsV (500 mg/kg) or 0.9% NaCl (control). The following hematological parameters were determined by standard methods: red blood cell count with a Neubauer hemocytometer using Hayem diluting solution, hemoglobin concentration (Hb) by the cyanmethemoglobin method, packed red blood cell volume or hematocrit (Ht) by the microhematocrit method, and red blood cell indices (mean corpuscular volume – MCV, mean corpuscular hemoglobin – MCH, and mean corpuscular hemoglobin concentration – MCHC). The osmotic fragility (OF) of red blood cells was determined using fresh blood and various concentrations of hypotonic NaCl (0%–0.85%). The median corpuscular fragility (MCF) corresponded to the saline concentration that lysed 50% of the cells. The osmotic fragility test in vitro used blood preincubated with TsV (250 mg/mL) or 0.9% NaCl (control). TsV significantly increased the Ht (44%), Hb (13.9 g/dL) and red blood cell count (5.3x10₁₂/L) after 120 min compared to the control values (36.5%, 11.7 g/dL and 4.5x10₁₂/L, respectively). A significant decrease in the MCF (in vivo) was observed 120 min (0.40%) and 360 min (0.39%) after TsV injection relative to the control (0.43%). There were no significant changes in the other parameters assayed.

CONCLUSION: The reduction in MCF may be a consequence of the dehydration induced by TsV. The increase in Ht, Hb and red blood cell count indicated hemoconcentration. Further analyses are necessary to confirm these findings.

Lethal toxin to mice produced by E. coli isolated from chickens with swollen head syndrome

Salvadori, M.R.^I; Figueirêdo, P.^I; Furumura, M.T.^I; Chudzinski-Tavassi, A.M.^II; Baccaro, M.R.^III; Prado-Franceschi, J.^IV; Yano, T.^I

^IDepartamento de Microbiologia e Imunologia, IB, Unicamp, Campinas

^IILaboratório de Bioquímica e Biofísica, Instituto Butantan, São Paulo

^IIIDepartamento de Patologia, Faculdade de Medicina Veterinária e Zootecnia, USP, São Paulo

^IVDepartamento de Farmacologia, Faculdade de Ciências Médicas, Unicamp, Campinas

^{Correspondence}

OBJECTIVES: In this study we investigated whether E. coli isolated from chickens with Swollen Head Syndrome (SHS) present lethal activity in mice.

MATERIALS AND METHODS: The E. coli strains were cultivated in LB medium, and the culture were harvested by centrifugation, filtered through a 0.22 mm membrane filter. The pathogenicity and LD50 were determined in female BALB/c mice (20±2g, 5/groups) injected i.v. with 0.5ml of E. coli culture supernatants. The nature of the lethality-inducing molecule was identified, by heating E. coli culture supernatants at 60°C and 100°C for 30 min, and treatment with trypsin, and then testing for lethal activity. Specimens from lungs, heart, liver and kidney from mice inoculated with E. coli were collected and stained with H&E for histopathological analysis.

RESULTS ANS CONCLUSIONS: The lethal activity was heat-labile protease-sensitive and as lethal as a toxin from Bacillus cereus killing mice within 10 min. Light microscopy revealed histopathological lesions in the liver and kidney, but the principal organ affected was the lung. In conclusion, E. coli produce a novel toxin with lethal activity in mice similar to that of B. cereus strains responsible for food poisoning. Additional studies are necessary to characterize this toxin since this occurs in SHS in broiler chickens and may represent a risk to public health.

Thiol-independent activity of a cholesterol-binding enterohemolysin (EHly) produced by enteropatogenic Escherichia coli

Figueirêdo, P.M.S.; Salvadori, M.R.; Furumura, M.T.; Catani, C.F.; Yano, T.

Departamento de Microbiologia e Imunologia, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, SP, Brasil

^{Correspondence}

Enterohemolysin (EHly) produced by Escherichia coli associated with infant diarrhea, showed similar characteristics to those of thiol-activated hemolysins produced by Gram positive bacteria including inactivation by cholesterol, lytic activity towards eukaryotic cells and chemical instability. In this work, we describe the partial purification of EHly by size exclusion chromatography and show that it shares several properties with thiol-activated hemolysins (TAH). TAH have the following general characteristics, in addition to their inactivation by cholesterol: inactivation by oxidation and reactivation with reducing agents, lethality in mice and lytic activity towards eukaryotic cells through the formation of pores in the cell membrane. However, the activity of EHly was not inactivated by oxidation or by SH group blocking reagents and the hemolysin was not lethal to mice. These results suggest that E. coli EHly is not a thiol-activated hemolysin, despite its ability to bind cholesterol.

Neuromuscular action of novel toxins from Crotalus durissus terrificus venom in mouse phrenic nerve-diaphragm preparations

Hernandez-Oliveira, S.S.^I; Borja-Oliveira, C.R.^I; Toyama, M.H.^II; Marangoni, S.^II; Rodrigues-Simioni, L.^I

^IDepartamento de Farmacologia, FCM, Caixa Postal 6111,

^IIDepartamento de Bioquímica, IB, Universidade Estadual de Campinas (UNICAMP), 13083-970, Campinas, SP, Brasil

^{Correspondence}

OBJECTIVES: The venom of the South American rattlesnake, Crotalus durissus terrificus, has been studied for more than a century (V. Brazil, Rev. Med. São Paulo, 6:265, 1903). In this work, we investigated the pharmacology of some novel toxins recently identified in this venom (Toyama et al., Prot. Peptide Lett., 7:381-388, 2000).

METHODS AND RESULTS: Gel filtration of the venom on a column of Sephadex G-75 yielded four peaks, namely, convulxin (peak I), gyroxin (peak II), crotoxin (peak III) and crotamine (peak IV). In contrast, molecular exclusion HPLC (Protein Pack SW 300) under isocratic conditions gave three new components, peaks IIIa, V and VI (inter-cro), in addition to peaks (I, II, III and IV) containing the principal toxins. Indirectly stimulated (0.1 Hz, 0.2 ms) mouse phrenic nerve-diaphragm preparations suspended in Tyrode solution were incubated with fractions (20 mg/ml) for up to 120 min. Crotoxin (peak III from gel filtration on Sephadex) produced its characteristic neuromuscular blockade. Peak IV from HPLC was also crotoxin and caused facilitation followed by irreversible blockade. Peak IIIa induced a slow, progressive facilitation that lasted for 120 min. Peak V showed only slight neuromuscular action.

CONCLUSION: Molecular exclusion HPLC allows dissociation of the facilitatory and neuromuscular blocking actions of crotoxin.

Financial support: CAPES/FAEP.

Comparison of the neuromuscular activity of Crotalus durissus ruruima and Crotalus durissus terrificus venoms in mouse nerve-muscle preparations

Hernandez-Oliveira, S.S.^I; Borja-Oliveira, C.R.^I; Dal Bello, C.A.^I; Toyama, M.H.^II; Marangoni, S.^II; Rodrigues-Simioni, L.^I

^IDepartamento de Farmacologia, FCM

^IIDepartamento de Bioquímica, IB, Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brasil

^{Correspondence}

OBJECTIVES: We have compared the effects of Crotalus durissus ruruima and C. d. terrificus snake venoms on neurotransmission in mouse isolated phrenic nerve-diaphragm preparations.

METHODS AND RESULTS: Indirectly stimulated (4 x threshold, 0.1 Hz, 0.2 ms) mouse phrenic nerve-diaphragm muscle preparations suspended in Tyrode solution were incubated with venoms for up to 120 min. At 1 mg/ml, C. d. ruruima and C. d. terrificus venoms increased the twitch-tension amplitude (34.7±5.0% and 47.7±12.6%, respectively, p<0.05 vs. pre-venom amplitude) after 10 min, to reach a maximum of 137.4±21.5% and 116.0±21.4%, respectively, after 40 min (p<0.05 vs. pre-venom amplitude). This was followed by progressive, irreversible blockade (50% in 89.0±8.0 min and 93.7±11.5 min, respectively). At 10 mg/ml, C. d. ruruima and C. d. terrificus venoms produced an initial increase in twitch-tension amplitude (82.7±22.0% and 41.2±8.3%, respectively, p<0.05 vs. pre-venom amplitude) after 10 min, to reach a maximum of 166.5±28.6% and 106.3±21.5% (p<0.05 vs. pre-venom amplitude) after 30.0±5.7 min and 25.0±3.5 min, respectively. As with the lower concentration, this was followed by progressive, irreversible blockade (50% in 78.2±10.0 min and 62.5±5.3 min, respectively). At 5 mg/ml, the pharmacological effects of both venoms were not significantly different from those obtained with the other two concentrations.

CONCLUSION: These results incate that the neuromuscular action of C. d. ruruima venom in mouse phrenic nerve-diaphragm preparations is similar to that of C. d. terrificus venom.

Financial support: FAEP.

A novel crotamine function observed in the mouse embryonic stem cells

Kerkis, I.^I; Kerkis, A.^I; Rádis-Baptista, G.^II; Oliveira, E. B.^III; Yamane, T.^II

^IDept. of Genetics, Institute of Bioscience, University of São Paulo

^IIMolecular Toxinology Lab., Butantan Institute

^IIIDept. of Biochemistry, Faculty of Medicine of Ribeirão Preto, University of São Paulo

^{Correspondence}

The venom arsenal developed by poisonous animals contains toxins that disrupt the prey homeostasis – a hunting strategy evolved during million of years. Not surprisingly, the toxic repertory encompasses polypeptides with very conserved structural domains able to mimic endogenous modulators and to interrupt cell function. The toxin crotamine, a cationic peptide (4.4 kDa, pI >9.5) occurs in the venom of Crotalus durissus terrificus (South American rattlesnake).

Its 3-D structure is not yet solved, but comparison of its primary amino acid sequence, and its predicted secondary structure, with other peptides constrained also by three cysteine bridges, indicates that it could adopt a spatial conformation similar to b-defensins or knottins. Both conformation types include members of multiple functions such as ion channel modulators, antimicrobial peptides, and also cell growth factors.

Using mouse embryonic stem (ES) cells we were able to detect a novel function of crotamine peptide that is typical of cell signaling modulators. The dual function of crotamine is essentially dependent of concentration in the cell culture medium. At the concentration above 10^-4 M/ml crotamine was cytotoxic to undifferentiated ES cells. We also found that at day 3 of treatment with crotamine (10^-6 M/ml -10^-7 M/m) the differentiating ES cells produced cells of ectoderm origin (nerve cells). In addition, the Cy3-labelled crotamine was selectively included into the chromatin of proliferated ES cells. The role of crotamine in the regulation of ES cells proliferation and differentiation is discussed.

Support: FAPESP

Purification and characterization of a new peptide included in the labyrinthin family from the skin secretion of Leptodactylus labyrinthicus

Zanotta, L.C.^{I, II}; Fontes, W.^II; Sousa, M.V.^II; Schwartz, C.A.^I; Schwartz, E.F.^I, Sebben, A.^I; Castro, M.S.^{I, II}

^ILaboratório de Toxinologia, Depto. de Ciências Fisiológicas/IB

^IICBSP – Centro Brasileiro de Serviços e Pesquisas em Proteínas, Depto. de Biologia Celular/IB, Universidade de Brasília, Brasília/DF, Brasil

^{Correspondence}

It is already known that the skin secretion of anurans presents a large number of bioactive compounds that, among other activities, are involved in defending the organism against predators and microbial infections. We have previously described a new family of putative antimicrobial peptides from skin secretion of Leptodactylus labyrinthicus, called Labyrinthins. The peptides included in this family present a large number of hydrophobic residues, no cystein residues and an amidated C-terminus. Moreover, the peptides of this family have low sequence similarity to other antimicrobial peptide families. In the present report we describe the purification and characterization of a new peptide isolated from the cutaneous secretion of Leptodactylus labyrinthicus. The animals were collected in Distrito Federal region and the skin secretion was obtained by mild electrical stimulation and immediately lyophilized. Dried secretion was dissolved in 0.1% (v/v) TFA/water and applied onto a Pharmacia C8 column and elution was performed using a linear acetonitrile gradient. The fraction of interest was rechromatographed using a Vydac C18 column. Its primary structure was obtained by Edman degradation (ABI 477A sequencer) and its molecular mass was determined using a triple-quadrupole mass spectrometer (PE-Sciex API 300). The purification procedure resulted in the isolation of a highly homogenous fraction with molecular mass of 2192.60 kDa. The N-terminal sequence obtained displayed high similarity to Labyrinthins 1 and 2, recently isolated from this skin secretion by our group. Due to the observed homology, we discuss that Labyrinthins probably have antimicrobial properties.

Renal effects of crotoxin isolated from Crotalus durissus cascavella venom

Martins, A.M.C.; Toyama, M.H.; Marangoni, S.; Oliveira, D.G.; Havt, A.; Nobre, A.C.L.; Almeida, A.C.P.; Barbosa, P.S.F.; Facó, P.E.G.; Menezes, D.B.; Fonteles, M.C.; Monteiro, H.S.A.

Depto. Bioquímica, UNICAMP-SP and Federal University of Ceará-UFC

^{Correspondence}

OBJECTIVES: The most serious systemic change and primary cause of death caused by rattlesnake snake bit is acute renal failure, thus we investigate the role of the crotoxin, isolated from C. d. cascavella venom on the renal function, as well as, study its renal histological alterations.

METHODS AND RESULTS: The isolated rat kidney system was prepared following to Fonteles et al., (Am J, Physiol., 244, 235-246, 1983). After 30 minutes, the perfusion pressure (PP) was measured and samples of perfusate and urine were taken at 10 minutes intervals. Samples were analyzed for sodium, potassium, inulin and osmolality. Perfusion pressure (PP), renal vascular resistance (RVR), glomerular filtration rate (GFR), urinary flow (UF), percent of sodium tubular transport (%TNa+) and percent of potassium tubular transport (%TK+) were observed. Maximum effects were seen in the last 30 minutes of each perfusion named 120 minutes. Treated group (ctx) was compared to a control group (ct) analyzed by Student t Test (*p<0,05). Crotoxin (5 mg/ml) increased the PP (ct120 = 114.3 ± 3.2 mmHg, ctx120= 156.6 ± 5.1 mmHg*), RVR (ct120= 5.3 ± 0.05 mmHg/mL.g-1.min-1, ctx120= 7.1 ± 0.4 mmHg/mL.g-1.min-1*), GFR (ct120= 0.65 ± 0.01mL.g-1.min-1, ctx120= 1.57 ± 0.33mL.g-1.min-1*), UF (ct120= 0.15 ± 0.002 mL.g-1.min-1, ctx120= 0.42 ± 0.07mL.g-1.min-1*) and decreased %TNa+ (ct120= 81.2 ± 0.3, ctx120= 71.9 ± 2.5 *) and %TK+(ct120= 69.9 ± 0.9, ctx120= 62.8 ± 1.1 *). Proteinaceous material was observed in the renal tubules and glomerulus in all kidneys treated with crotoxin.

CONCLUSION: These results suggest that crotoxin, isolated from Crotalus durissus cascavella venom cause acute nephrotoxicity.

Finatial Support: FAPESP, CNPq

Actions of gyroxin, isolated from Crotalus durissus cascavella venom, in the isolated rat kidney

Martins, A.M.C.; Toyama, M.H.; Oliveira, D.G.; Marangoni, S.; Almeida, A.C.P.; Havt, A.; Nobre, A.C.L.; Barbosa, P.S.F.; Facó, P.E.G.; Fonteles, M.C.; Monteiro, H.S.A.

UNICAMP and Federal University of Ceará-UFC

^{Correspondence}

OBJECTIVES: Envenomation by Crotalus genus leads to coagulation disorders, myotoxicity, neurotoxicity and acute renal failure, which is the principal cause of death. Thus we investigate the actions of the gyroxin isolated from Crotalus durissus cascavella on renal function.

METHODS AND RESULTS: The isolated rat kidneys were perfused with Krebs-Henseleit solution containing 6% of bovine serum albumin in absence and in presence of toxins. The parameters studied included perfusion pressure (PP), renal vascular resistance (RVR), glomerular filtration rate (GFR), urinary flow (UF), percent of sodium tubular transport (%TNa+), percent of potassium tubular transport (%TK+) and percent of chloride tubular transport (%TCl-). Maximum effects were seen in the last 30 minutes of each perfusion named 120 minutes. Treated group (gyr) was compared to a control group (ct) analyzed by Student t Test (*p<0,05). Gyroxin (5 mg/ml) increased significantly the PP (ct120 = 114.3 ± 3.2 mmHg, gyr120 = 144.5 ± 4.5 mmHg*) and RVR (ct120 = 5.3 ± 0.05 mmHg/mL.g-1.min-1, gyr120 = 7.0 ± 0.5 mmHg/mL.g-1.min-1*) and UF in the last 30 minutes of each perfusion, but GFR (ct120 = 0.65 ± 0.01mL.g-1.min-1, gyr120 = 0.67 ± 0.04 mL.g-1.min-1) remained stable during the 120 min. of perfusion. The %TNa+ (ct120 = 81.2 ± 0.3, gyr120 = 69.9 ± 1.9*) %TK+(ct120 = 69.9 ± 0.9, gyr120 = 63.7 ± 0.9*) and %TCl- (ct120 = 78.9 ± 0.9, gyr120 = 72.0 ± 1.1*) decreased significantly after the infusion of gyroxin.

CONCLUSION: These results indicate that gyroxin participates in the toxic effect of Crotalus durissus cascavella venom on isolated rat kidney.

Supported by FAPESP, CNPq

New findings for bothropstoxin-I, a myotoxin from Bothrops jararacussu venom

Oshima-Franco, Y.; Leite, G.B.; Belo, C.A.D.; Hyslop, S.; Giglio, J.R.; Cruz-Höfling, M.A.; Rodrigues-Simioni, L.

Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), Campinas, SP, Brazil

^{Correspondence}

Bothropstoxin-I (BthTX-I) from Bothrops jararacussu venom is a Lys49 PLA2 and has myotoxic and neurotoxic activities. In this study, we investigated the mode of action of BthTX-I in mouse phrenic nerve-diaphragm preparations using myographical and electrophysiological techniques. The effects of Mn²⁺ on the neuromuscular action of BthTX-I were also examined. BthTX-I (1.4 mM) produced 50% neuromuscular blockade in 31 + 6 min whereas Mn²⁺ (0.9 mM and 1.8 mM) produced rapid blockade (50% in less than 4 min) which was spontaneously reversible at the lower concentration. Pretreating preparations with 0.9 mM Mn2+ prevented the blockade by BthTX-I. When added after BthTX-I, Mn²⁺ produced its characteristic blockade and, after washing, the twitch tension returned to pre-Mn²⁺ levels. Using d-tubocurarine (5.8 mM) showed that BthTX-I acted directly on muscle cell membranes and not on nicotinic receptors. Unlike 3,4-diaminopyridine (0.09 mM), BthTX-I (1.4 mM) did not block K+ channels, nor did it exert a specific sarcolemmal action such as seen with dantrolene (10 mM). Electrophysiological measurements revealed a novel presynaptic action for BthTX-I (0.35 mM) seen as the appearance of ‘giant’ miniature end plate potentials before any change in resting potential. Preparations preincubated with Mn²⁺ (0.9 mM, 30 min) were protected against the depolarizing action of BthTX-I (0.7 mM). These results indicate that BthTX-I acts on axolemmal and sarcolemmal membranes via Ca²⁺ channels.

N-terminal amino acid sequences of a presynaptically-active neurotoxic phospholipases from Bothrops neuwiedi pauloensis (jararaca-pintada) snake venom

Borja-Oliveira, C.R.^I; Durigon, A.M.^I; Kassab, B.H.^II; Toyama, M.H. ^II; Novello, J.C.^II; Marangoni, S.^II; Rodrigues-Simioni, L.^I

^IDepto. de Farmacologia, FCM, UNICAMP, Campinas-SP

^IIDepto. de Bioquímica, IB, UNICAMP, Campinas-SP, Brasil

^{Correspondence}

OBJECTIVES: A presynaptically-active neurotoxic phospholipase A2 (PLA2) have been isolated from Bothrops neuwiedi pauloensis snake venom. The toxin have PLA2 activity and produce complete neuromuscular blockade in chick isolated biventer cervicis preparation, without inhibiting acetylcholine-induced contractures (Borja-Oliveira et al., Revista Brasileira de Toxicologia 2001,14:113). In this work, we determined the N-terminal sequence of this PLA2, named neuwieditoxin-I, and compared it with those from other presynaptic and myotoxic PLA2 from snake venoms.

METHODS AND RESULTS:B. n. pauloensis venom was fractionated by molecular exclusion HPLC (Protein Pack 300SW column) followed by reverse phase HPLC (C-18 m-Bondapak column). Tricine SDS-PAGE in the absence or presence of dithiothreitol (DTT) showed that neuwieditoxin-I had a molecular mass of 30 kDa and 15 kDa, respectively. The N-terminal sequence DLVQFGQMILKVAGRSLPKSYGAYGCYCGWGGRGK... showed 66% homology to the Asp49 PLA2 myotoxin bothropstoxin-II and 54% homology with the presynaptic PLA2 caudoxin and taipoxin (a-subunit).

CONCLUSION: The fact that neuwieditoxin-I have Gln-4, Phe-5 and Tyr-28, amino acid residues conserved in all Asp49 PLA2 variants purified so far, and that their amino acid sequences were similar to Asp49 PLA2 strongly suggests that neuwieditoxin-I is a Asp49 PLA2.

Financial support: FAPESP

Effects of the toxic secretion of a crab on other crab species

Aggio, J.F.; Freitas, J.C.

Biosciences Institute and CEBIMar, University of São Paulo, Brazil

^{Correspondence}

The digestive secretion of the crab Mithrax hispidus may be released on some conditions and contains neurotoxins, as revealed by sucrose-gap. We investigated its effect in vivo on the cardiac activity of another crab, Hepatus pudibundus and on the feeding activity of the ghost crab, Ocypode quadrata. Animals of both species were immobilized and temporarily blinded, and platinum electrodes were glued in small holes drilled in the carapace. Electrodes were used to monitor cardiac activity (both species) and mandibular muscle activity (burst of action potentials) during eating (O. quadrata). Signals were amplified, digitized and stored in a computer for posterior analysis. Stimuli were applied to the oral region of experimental animals including the antennulae, antennae and mouth parts, which are known to possess chemorreceptors. As control stimuli we used mussel extract (50 mg/ml) and filtered sea water. Both species were clearly able to distinguish between the different stimuli. In H. pudibundus, mussel extract caused a non-beating heart to start beating and it increased the frequency of an already beating one, whereas the digestive secretion had the opposite effect (i.e., stopped a beating heart and did not start a quiescent one). Individuals of O. quadrata were fed with mussel tissue and subsequently stimulated. Sea water and mussel extract had no effect, whereas the digestive secretion caused animals to stop eating altogether or, in some cases, deorganized the rhythmic activity associated with eating. Burst durations were unaltered, but the intervals between them became irregular. Our results suggest that the digestive secretion of M. hispidus affects negatively other animals in the environment, thus being a suitable candidate for a chemical defensive system.

Geographical distribution of snakes of medical importance and snakebites in São Paulo and Paraná States

Fan, H.W.^I; Sant’anna, S.S.^I; Málaque, C.M.S.^I; Rúbio, G.B.G.^II; da Silva, E.M.^III; Leite, J.C.M.^IV; Albuquerque, M.J.^V; Franco, F.L.^I; Furtado, M.F.^I; Fernandes, W.^I; França, F.O.S.^I; Cardoso, J.L.C.^I

^IInstituto Butantan, SES/SP

^IICentro de Saúde Ambiental, SES/PR

^IIICentro de Produção e Pesquisa em Imunológicos, SES/PR

^IVMuseu de História Natural “Capão da Imbúia”, Secretaria Municipal do Meio Ambiente, Curitiba

^VSES/SP

^{Correspondence}

Distribution of snakebites and poisonous snakes are influenced by vegetation cover, modified in consequence of agricultural occupation that changed the natural landscape. The objective of this study was to analyze altogether the geographical distribution of snakebites, poisonous snakes and environmental changes. Data collection included snakebites registered in São Paulo (SP) and Paraná (PR) States between 1988 and 1997, snakes captured and maintained at Instituto Butantan (SP), Museu de História Natural (PR) and Centro de Produção e Pesquisa em Imunobiológicos (PR) herpetological collections, and secondary data from vegetation cover and percentage of agricultural occupation. Maps were obtained using a geographical information system. Bothrops jararaca showed a wide distribution in open fields, primitive and modified forest, and even in regions under anthropic influence. B. moojeni and B. neuwiedi were found predominantly in open fields. Crotalus occupied areas originally constituted by semidecidual seasoned forest and “cerrado”. Incidence of snakebites varied from 4.31 to 10.57 cases/100.000 population, decreasing through the period, both for Bothrops (88%) and Crotalus (11%). Micrurus accidents were rare. There was no correlation between municipalities where snakes occurred and nowadays vegetation cover, except for B. jararacussu in the remaining forest at Vale do Ribeira. No correlation was observed between the percentage of agricultural occupation and Bothrops snakebites, for Crotalus snakebites, a small negative correlation was observed in areas occupied by sugar cane. A decreasing tendency was observed in the number of snakebites and snakes of medical importance captured, with predominance of Bothrops. Maps of distribution of poisonous snakes and snakebites permitted a better definition of risk areas. The anthropic pressure probably had influenced the result of no correlation between areas of distribution of snakes with the vegetation cover, while some agricultural practices may had determined changes in the risk areas for snakebites.

Composition of indolealkylamines of Bufo Crucifer (Anura: Bufonidae) cutaneous secretion

Maciel, N.M.^I; Schwartz, C.A.^I; Fontes, W.^II; Sousa, M.V.^II; Castro, M.S.^{I, II}; Schwartz, E.F.^I

^ILaboratório de Toxinologia, Departamento de Ciências Fisiológicas

^IILaboratório de Bioquímica de Proteínas, Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, Brazil

^{Correspondence}

INTRODUCTION: The cutaneous secretions of amphibians contain an amazing variety of compounds such as proteins, peptides, steroids, alkaloids and biogenic amines. Indolealkylamines represent the longest known and most thoroughly investigated group of aromatic amines of the amphibian skin. The skin of Bufo provides a spectacular representation of indolealkylamines.

OBJECTIVES: To determine the composition of indolealkylamines in the skin and parotoid glands of B. crucifer.

METHODOLOGY: Specimens of B. crucifer were collected in Ubatuba, São Paulo State, Brazil. Skin extracts were obtained separately for both skin body and parotoid glands. Indolealkylamines were separated by thin-layer chromatography (TLC). Standards indolealkylamines used were serotonin (5-HT), bufotenine (BTN), dehydrobufotenine (DHB) and bufotenidine (BTD). To identify bufotionin (DHB-S) we adopted Erspamer et al. (1967). Standards were collected from TLC and analyzed by electrospray mass spectrometry to MS fragmentation.

RESULTS:B. crucifer contains BTD, DHB, DHB-S, 5-HT and BTN in the skin and DHB, DHB-S, 5-HT and traces of BTN in the parotoid glands. Spectrograms profiles of 5-HT, BTN and BTD showed resemblance to those obtained by McClean et al. (2002). The fragmentation of DHB was for the first time obtained.

CONCLUSIONS: Differences in the composition of indolealkylamines between skin and parotoid gland secretions have not been described previously. These differences indicate that parotoid glands are not only an agroupment of granular glands, suggesting differences in the activity of enzymes in two tissues analyzed. MS fragmentation was an useful tool to confirm the standards utilized to identify the indolelakylamines.

Financial support: DPP-UnB

Investigation of the blood-brain barrier breakdown mechanisms caused by Phoneutria nigriventer spider venom

Le Sueur, L.P.; Collares-Buzato, C.B.; Cruz-Höfling, M.A.

Depto. de Histologia e Embriologia– UNICAMP/Campinas, SP, Brasil

^{Correspondence}

OBJECTIVES: We have recently demonstrated that intravenous injection of Phoneutria nigriventer venom (PNV) induces blood-brain barrier (BBB) breakdown in adult rats. The increased permeability was observed in hippocampus by ultrastructural method using the extracellular tracer lanthanum nitrate. The tracer was detected in clefts between two endothelial cells and into pinocytotic vesicles, indicating that changes in BBB permeability may occur by para- and transcellular route. In this work, we investigated the effect of PNV on the expression of some proteins associated to intercellular junctional complex, responsible for the paracellular barrier properties.

METHODS AND RESULTS: The rats were injected with an i.v. single dose of 850 mg/kg PNV or saline and 5 h, 1, 5, and 9 days (n=5/each) post-injection (p.i.) they were anaesthetized and the hippocampus isolated. Biochemical analyses were done by Western Blot for occludin and b-catenin, proteins associated to the tight and adherens junctions, respectively. Both junctional proteins were localized specifically at the cell-to-cell contact region of brain vessel cells, as revealed by immunocytochemistry. In our findings no statistical differences in occludin and b-catenin proteins expression was observed when compared control and PNV-treated groups. Only a tendency to increase in occludin expression was observed at 5 h and 1 d of PNV p.i., probably indicating an enhancement of the paracellular tightness as a compensatory mechanism for the BBB breakdown occurring by transcellular route.

CONCLUSION: The BBB breakdown seen in rats injected with PNV may not occur through the paracellular transport, and the presence of the tracer in interendothelial clefts is probably due to discharge of lanthanum-filled pinocytotic vesicles at the lateral cell surface.

Financial support: FAPESP, CAPES, CNPq, FAEP.

Occurence of antimicrobial compounds in skin secretion of the toad Bufo rubescens

Cunha Filho, G.S.A.^I; Castro, M.S.^I; Sousa, M.V.^II; Fontes, W.^II; Schwartz, C.A.^I; Schwartz, E.F.^I

^ILaboratório de Toxinologia, Universidade de Brasília

^IILaboratório de Bioquímica e Química de Proteínas, Universidade de Brasília, Brasília, DF, Brasil

^{Correspondence}

INTRODUCTION: Antimicrobial peptides have been described in skin secretion of many amphibian species. These peptides act as compounds of the innate immunity in these animals. The objective of the present work was to identify and characterize antimicrobial agents from skin secretion of the toad Bufo rubescens.

METODOLOGY: Toad skin secretion was obtained by electrical stimulation and the gland secretion by manual compression. These secretions were mixed, immediately lyophilized, dissolved in 0,1% (v/v) TFA/water, and applied into a Pharmacia C8 column. The elution was performed using a double linear gradient. The fractions of interest were rechromatographed using a Vydac C18 column. The molecular weights from individual peptide fractions were obtained by using MALDI-TOF mass spectrometer. The antimicrobial activity was assayed against Escherichia coli. Hemolytic activity was assayed using human red blood cells.

RESULTS: The crude extract and two fractions from B. rubescens secretion showed inhibition against E. coli. These active fractions resulted on compounds of 1,369, 1,526, 684, 700 and 714 Da. None peptide fraction had significative hemolytic activity.

CONCLUSION: This work showed the occurrence of antimicrobial compounds of low molecular weights in the skin secretion of Bufo rubescens.

Financial Support: DPP – UnB.

Reactivity of monoclonal antibodies anti-K49 myotoxins with venoms from different snake families

Bento, l.; Tanjoni, I.; Spencer, P.J.; Fernandes, I.; Moura-da-Silva, A.M.

Laboratório de Imunopatologia, Instituto Butantan, São Paulo, BR

^{Correspondence}

Myotoxins are components widespread in venoms of snakes from different genera, responsible for muscle tissue damage adjacent to the bite. These toxins belong to two major structural groups: short chain peptides and molecules with a phospholipase A2 structure with preserved catalytic activity (D-49) or a mutated non-catalytic form (K-49). Monoclonal antibodies (MoAb) were previously raised against Bothrops jararacussu K-49 Bothropstoxin-1. In this work, we analyzed their reactivity against isolated toxin, homologous venom and venoms of 27 different species of snakes. MoAbs producing cells were expanded in vitro and subsequently injected into mice to produce the ascites. Antibody levels were assayed by ELISA. Similar titres were detected against isolated toxin or whole venom, although differing amongst the clones. When analyzed by Western blotting, MoAbs recognized a B. jararacussu venom band of approximately 28 kDa under non-reducing conditions and a 14 kDa band in reduced samples corresponding to the dimeric and monomeric forms of the myotoxin, respectively. Four antibodies reacted with reduced samples, three reacted preferentially with non-reduced samples and two were non-reactive. MoAbs were then tested by dot blot, under native conditions with venoms of 27 species of snakes belonging to VIPERIDAE, ELAPIDAE and COLUBRIDAE families. MoAbs reacted preferentially with venoms of viper snakes, sub-family CROTALINAE, preferentially with Bothrops jararacussu, Bothrops jararaca, Bothrops asper,

Crotalus atrox, Crotalus adamanteus and Agkistrodon contortrix. Neutralization ability of MoAbs and their reactivity towards K-49 or D-49 myotoxins are under evaluation. Concluding, these antibodies are apparently selective to myotoxins, being an interesting tool for screening of venoms and purification of toxins by immunoaffinity.

Financial support: FAPESP and CNPq (PIBIC fellowship for L.B.)

Structure/function study of jararhagin using monoclonal antibodies and recombinant fragments

Tanjoni, I.^I; Butera, D.^I; Gutiérrez, J.M.^II; Rucavado, A.^II; Takehara, H.A.^I; Moura-da-Silva, A.M.^I; Fernandes, I.^I

^ILaboratório de Imunopatologia, Instituto Butantan, São Paulo, Brasil

^IIInstituto Clodomiro Picado, Universidad de Costa Rica, San Jose, Costa Rica

^{Correspondence}

Jararhagin, a 52 kDa hemorrhagin found in Bothrops jararaca venom, comprises a metalloproteinase (M), an ECD-disintegrin (D) and a cysteine-rich (C) domain. The M domain, due to its catalytic activity, disrupts the extracellular matrix and is responsible for the severe hemorrhagic activity, while the D/C domains block the a2b1 integrin, inhibiting collagen-dependent platelet aggregation. To further understand the structure/function relationships, 7 murine monoclonal antibodies (MAJar1 to 7) against jararhagin were produced. The regions recognized by MAJars, were mapped by dot blot using GST-fusion fragments from the D domain (JD49, JD89, JD98), C domain (JC63, JC76, JC103, JC116) and BaP1, a metalloproteinase comprising only the M domain isolated from B. asper venom. MAJar2, 6 and 7 recognized JD89 and 98, but not JD49, demonstrating their interaction with the N-terminal portion of the D domain. MAJar1 recognized all JD fragments, including JD49, suggesting its interaction with the C-terminal portion of the D domain. MAJar4 and 5 may bind zones of interaction between the D and C domains since they failed in recognizing the fragments, although they interact with D/C domains. MAJar3 recognized both JD49 and BaP1, demonstrating a spatially close interaction between the M and the C-terminal portion of D domain. Moreover, only MAJar3 completely neutralized the hemorrhage induced by jararhagin, suggesting that this interaction includes the catalytic residues. The epitope recognized by MAJAr 3 was conserved in most hemorrhagic venoms, suggesting the importance of this structure in venom hemorrhagic activity.

Financial Support: FAPESP

Blood clot and platelet aggregation inhibitors from the saliva of Amblyomma cajennense (Acari: Ixodidae)

Simons, S.M.^I; Batista, I.F.C.^I; Faria, F.^I; Barros-Battesti, D.M.^II; Labruna, M.B.^III; Chudzinski-Tavassi, A.M.^I

^ILaboratório de Bioquímica e Biofísica, Instituto Butantan, São Paulo, SP

^IILaboratório de Parasitologia e Entomologia, Instituto Butantan, São Paulo, SP

^IIIUSP - FMVZ-VPS – São Paulo, SP

^{Correspondence}

Saliva of bloodsucker contains several substances that act on the platelet aggregation, protease control, vasoconstriction, among other physiological mechanisms.

OBJECTIVES: to identify blood clotting and platelet aggregation inhibitors, from the saliva of the Amblyomma cajennense ticks.

METHODS: Crude saliva was obtained by Tatchell method with some modifications, and it was used to: check the 2D-electrophoreseis profile, inhibition of the platelet aggregation induced by collagen, and the inhibitors purification by gel filtration and ion-exchange chromatographs. The activity of the purified inhibitor on the prothrombinase complex was also analyzed.

RESULTS:Ablyomma cajennense saliva contains proteins with pI between 3.37 to 6.64 and molecular mass 306 and 16 kDa. Concerning to the platelet aggregation induced by collagen, 35% of inhibition was observed, moreover, the results obtained in the flow cytometry experiments, performed also with the crude saliva suggested that the GP II-b is modified. The Factor Xa purified inhibitor is a 70 k Da protein that shows a IC50 0.13 nM, and is able to inhibit the FXa in the prothrombinase complex. A thrombin inhibitor in the crude saliva was also found but it was not visualized in the SDS-PAGE suggesting that it could be a peptide inhibitor.

CONCLUSION:Amblyomma cajennense saliva contains protease inhibitors, which are able to maintain the successful feeding and physiological systems of the tick.

Supported by Fapesp

Purification and structural characterization of a homodimeric peptide from skin secretion of Phyllomedusa tarsius (Amphibia: Anura)

Morales, R.A.V.^{I, II}; Prates, M.V.^I; Santos, N.C.F.^I; Bloch Jr., C.^I

^IEmbrapa–Recursos Genéticos e Biotecnologia

^IIInstituto de Ciências Biológicas-Universidade de Brasília-UnB

^{Correspondence}

The amphibian skin contains granular glands that are responsible for the production of several classes of useful biotechnological compounds, such as alkaloids, opiod peptides and antimicrobial peptide. These compounds are usually associated with amphibian chemical defense against potential predators such as animals and microorganisms. In this work, we describe the structural characterization of a new homodimeric peptide found in the skin secretion of Phyllomedusa tarsius from Amazon rain forest.

The skin extracts were obtained by mild electrical stimulation, collected in Milli-Q water, and were later frozen, and lyophilized. The water-soluble fractions from the crude extract were fractionated by RP-HPLC, using a semi-preparative C18 column in a linear acetonitrile gradient with 0.1% of trifluoracetic acid and monitored in 216nm and 280nm. The fraction of interest was manually collected and submitted to a further purification step using analytical C18 column in the same conditions. The purified fraction was reduced with DTT, alkylated with iodoacetamide and had its primary structure obtained by Edman degradation in an automated system. The purity and molecular mass was analyzed in each characterization step by mass spectrometry using a MALDI-TOF (Matrix Assisted Laser Desorption Ionization Time-of-Flight) system and a-cian-4-hydroxycinamic acid as matrix.

The analyzed compound show to be a homodimeric structure of 5509.40Da composed by two identical polypeptide chains of 24 amino acid residues with 2755.21Da each, linked by a single disulphide bond between the Cys23 of each chain. Structurally it is very similar to the heavy chain of Distintin, an efficient heterodimeric antimicrobial compound from Phyllomedusa distincta previously described by our group (Batista et. al. 2001, FEBS Letters 484:85-89)

Neutralizing activity of experimental Thalassophryne nattereri fish antivenom produced in horse

Piran-Soares, A.A.^I; Takehara, H.Á.^I; Távora, J.P.F.^II; Guidolin, R.^III; Higashi, H.G.^III; Lopes-Ferreira, M.^I

^ILaboratório de Imunopatologia – Instituto Butantan, São Paulo, Brasil

^IIFazenda São Joaquim – Instituto Butantan, São Paulo, Brasil

^IIIDivisão de Desenvolvimento Tecnológico e Produção – Instituto Butantan, São Paulo, Brasil

^{Correspondence}

Poisoning due to Thalassophryne nattereri (niquim) venom constitutes a serious health problem for fishermen, particularly in the north and northeast of Brazil. The venom causes strong local effect such as intense pain, edema and necrosis. The present therapy with anti-inflammatory and analgesic drugs is ineffective. To evaluate horse serum therapy as an alternative treatment, animals were immunized with sc injections of crude venom. A strong antibody response was observed as measured by ELISA (titer of 8,192,000). Immunoblotting assay showed that the antivenom was effective to recognize all the proteins present in the venom. Regarding its specificity, the antivenom only reacted with venoms of Thalassophryne maculosa and the genus Potamotrygon (ray). Neutralization of edema, nociception, necrosis and lethality was evaluated after pre-incubating the antivenom with venom for 30 min, at 37°C. Controls were similarly treated using normal horse serum. The total neutralization of the lethality induced by 5LD₅₀ venom (455 mg) was obtained with 400 mL antivenom. Necrosis (100 mg venom) and nociception (3 mg) were also completely neutralized with 200 and 30 mL antivenom, respectively. Edema was only partially neutralized even when large amounts of antivenom were used. These data suggest that horse antivenom therapy may be a promising approach for patients damaged by T. nattereri venom.

Supported by FAPESP (01/11032-3)

Toxic activities of Thalassophryne maculosa (sapocano) fish venom

Sosa-de-Rosales, I.^I; Piran-Soares, A.A.^II; Farsky, S.H.P.^III; Takehara, H.A.^II; Lopes-Ferreira, M.^II

^IEscola de Ciências Aplicadas do Mar, Universidade do Oriente, Venezuela

^IILaboratório de Imunopatologia, Instituto Butantan, Brasil

^IIIDep. de Análises Clínicas e Toxicológica, Faculdade de Ciências Farmacêuticas, USP, Brasil

^{Correspondence}

Thalassophryne maculosa fish is widely distributed in the coasts of the north of South America, Colombia and Venezuela. The human accidents occur by inoculation of the venom through the fish spines and are characterized by severe edema, intense pain and necrosis. Preliminary data on the biological and biochemical properties of the venom showed that when crude venom was separated by SDS-PAGE at least 7 components were visualized with the major band located around 45,000 mol. wt. Subcutaneous injections of the venom in mice induced systemic effects as difficulty of movement, increase of the heart frequency and abdominal dilation, followed by death. Intradermal injection (footpad) of the venom induced nociception and edema in a dose-dependent manner, reaching a maximum with 30 mg (190 ± 33 s, 3.0 ± 0.8 mm, respectively). Effects evoked by topical applications of venom (3 mg) on cremaster muscle were visualized through intravital microscopy. Stasis was observed concomitantly with the presence of thrombi in venules and focal transient constrictions in arterioles. Even when a dose as high as 300 mg venom was used it was not possible to detect any phospholipase A2, hemorrhagic or coagulant activities. The intraperitoneal LD50, determined by probit test using groups of six mice for each dose, was 5.42 mg/Kg. These data suggest that although the Thalassophryne maculosa venom did not present the activities tested it induced serious local and systemic effects.

Supported by FAPESP (01/11032-3).

Cloning of a serine proteinase cDNA from Bothrops moojeni (caissaca) snake venom

Vitorino, A.F.C.^I; Homsi-Brandeburgo, M.I.^I; Brites, V.L.C.^II; Bauab, F.^III; Selistre-de-Araújo, H.S.^IV

^IInstituto de Genética e Bioquímica, Universidade Federal de Uberlândia (UFU), MG

^IIInstituto de Biologia, UFU

^IIIFaculdade de Medicina de Catanduva, SP

^IVDepartamento de Ciências Fisiológicas, Universidade Federal de São Carlos, SP – Brazil

^{Correspondence}

Serine proteinases may represent about 20% of total snake venom protein composition. Acting on the victim’s blood coagulation cascade, these enzymes are not lethal, but they potentialize the toxic effects derived from envenomation. Because of these characteristics, they became important tools in the study of coagulation mechanisms and in the development of diagnostic tests, being models to synthesize therapeutic agents. In this work, we have cloned the cDNA encoding a previously isolated serine proteinase (BthT1) from Bothrops moojeni (Viperidae, Crotalinae) venom. The total RNA template of RT-PCR was extracted from the frozen venom gland of an adult female snake with TRIzoL reagent (GIBCO-BRL). Primers were designed using the Multalign software, based on the known serine proteinase N-terminal sequence and the 3’UTR region of homologue proteins. The amplification product was sequenced, cloned into the pGEMT Easy plasmid (Promega), and used to transform competent cells of E. coli DH5a strain. Restriction analysis of ampicilin-resistent transformed colonies revealed the expected dimensions for the vector (1015 bp) and insert (756 bp). The alignment of translated sequences of the RT-PCR product and the insert evidenced high homology. Blast analysis in the GenBank showed that the cloned sequence shared identity with snake venom serine proteinases like batroxobin (92%) and bothrombin (86%), conserving amino acid residues characteristics of the group. Additionally, some internal peptides of the serine proteinase cleaved with Cyanogen Bromide and analyzed by HPLC will be sequenced, in order to confirm your its correspondence to the cloned cDNA.

SUPPORT: CAPES, FAPESP.

Localization of crotamine gene of Crotalus durissus terrificus by fluorescent in situ hybridization (fish)

Oguiura, N.^I; Svartman, M.^II; Batistic, R.F.^I; Almeida, T.M.B.^IV; Rádis-Baptista, G.^III; Yamane, T.^III; Vianna-Morgante, A.^II

^ILab. de Herpetologia do Instituto Butantan

^IIDepto. de Biologia do Instituto de Biociências da USP

^IIILab. de Toxinologia Molecular do Instituto Butantan

^IVLab. De Genética do Instituto Butantan

^{Correspondence}

Crotamine belongs to a closely related group of crotamine-like proteins, sharing up to 98% of similarity, present in most rattlesnake venoms. However, its presence and amount can vary according to the subspecies or the geographic locality of a given population. The quantity of this toxin ranges from 10 to 40% of dried venom. The crotamine gene of crotamine-plus Crotalus durissus terrificus consists of approximately 1800bp: three exons separated by a long phase-1 (900bp) and a short phase-2 (140bp) introns. Exon 1 codifies the 5’-untranslated region and the first 19 amino acids of signal peptide, exon 2 codifies a total of 42 amino acids, 3 belonging to the signal peptide and 39 to the crotamine. Exon 3 codifies the last 3 amino acids of the mature toxin, the terminal lysine which is removed after post-translation processing and the 3’-untranslated region.

In this work, we mapped the crotamine gene to metaphase chromosomes of C. d. terrificus. Chromosome preparations were obtained from bone marrow. The probe, MR20 phage DNA containing the crotamine gene, was labeled with biotin by nick translation. FISH was performed as described by Viegas-Péquignot (1992) with minor modifications and immunodetection was performed with avidin conjugated with FITC.

The crotamine gene mapped to the telomeric region of the long arm of chromosome 2 in C. d. terrificus crotamine-plus karyotype. The intensity of the signal differed between the two homologues in all cells analyzed, what could be due to different number of copies of the crotamine gene. The variable amounts of crotamine found in the venom of crotamine-plus C. d. terrificus could reflect differences in gene copy numbers.

Financial support: FAPESP 99/02675-6

Comparison of the local anesthetic effects on tooth pulp stimulation of a bufodienolide isolated from Bufo paracnemis venom with those of lidocaine and bupivacaine

Freitas, S.F.; Carvalho, I.F.; Cardi, B.A.; Carvalho, M.D.F.; Carvalho, D.M.F.; Carvalho, K.M.

Laboratório de Neurofarmacologia, Universidade Estadual do Ceará, Fortaleza, CE, Brazil

^{Correspondence}

OBJECTIVES: The amphibian skin secretions contain several pharmacologically active compounds, such biogenic amines, toxins, peptides, enzymes and alkaloids, protecting then from predators and microorganisms. In this study, we compare the local anesthetic effects on tooth pulp stimulation of a bufodienolide isolated from Bufo paracnemis skin secretions with those of lidocaine and bupivacaine.

METHODS AND RESULTS: The venom was dissolved in ethanol (1:3,w,v), centrifuged at 5000g/20 min and the supernatant was submitted to HPLC using a C18 column (25x250mm)(5ml/min) eluted with acetonitrile (0-40%)(30min). The purified fraction (Bpa) with local anesthetic activity was pooled and lyophilized. High resolution NMR analysis of the purified factor by application of modern pulse sequences allowed its identification as a bufodienolide-type steroid, known in the literature as telecinobufagin. Local anesthetic activity was assayed with tooth pulp stimulation test in rabbit (n=6). Bpa, in the concentration of 0.5%, presented greater effect (p<0.05, 43±3.4Volts) than bupivacaine, in this same concentration (30±2.2Volts), and than lidocaine, in concentration of 2% (32±2.6Volts). Bpa, in the concentration of 0.5%, presented similar anesthetic time (160±12,5min) with that of bupivacaine, in this same concentration (150±11,9min), but longer than that of lidocaine, in concentration of 2% (50±4,5min).

CONCLUSION: a) Bpa (0.5%) presented greater local anesthetic effect than bupivacaine (0.5%) and than lidocaine (2%), b) Bpa (0.5%) presented similar anesthetic time with that of bupivacaine (0.5%) and longer than that of lidocaine (2%), c) although the mechanism of action of Bpa is unknown, it may be a tool to develop a new class of local anesthetics.

A new local anesthetic isolated from Bufo paracnemis venom (Amphibia, Anura)

Patrocínio, M.C.A; Cortez, J.J.C.; Almeida, M.M; Evangelista, I.L.; Oliveira, R.G.S.; Cardi, B.A.; Carvalho, K.M.

Laboratório de Neurofarmacologia, Universidade Estadual do Ceará, Fortaleza, CE, Brazil

^{Correspondence}

OBJECTIVES: The anuran skin secretions contain several pharmacologically active compounds, such as biogenic amines, toxins, peptides, enzymes and alkaloids, protecting anurans from predators and microorganisms. In this study, we demonstrated the isolation, the local anesthetic effects and the structure of a bufodienolide from Bufo paracnemis skin secretions.

METHODS AND RESULTS: The venom was dissolved in ethanol (1:3, w,v), centrifuged at 5000g/ 20 min and the supernatant was submitted to HPLC using a C18 column (25 x 250 mm) (5 ml/min) eluted with acetonitrile (0-40%)(30 min). The purified fraction (Bpa) with local anesthetic activity was pooled and lyophilized. High resolution NMR analysis of the purified factor by application of modern pulse sequences such as 1H, 1H-COSY and NOESY, carbon-detected and hydrogen-detected (inverse) hetero-nuclear correlation of directly attached carbon-hydrogen (HETCOR and HMQC, respectively), as well as the long-range equivalent sequences, COLOC and HMBC, allowed its identification as a bufodienolide-type steroid, known in the literature as telecinobufagin. Local anesthetic activity was assayed by infiltration anesthesia in guinea pigs (n=6), mouse tail flick test (n=6) and cornea test in rabbits (n=6). The anesthetic time of the drugs used were: a) infiltration anesthesia: Bpa (0.5%), 181±15 min, bupivacaine (0.5%), 110±10 min, and lidocaine (2%), 49±5 min, b) mouse tail flick test: Bpa (0.5%), 152±12 min, bupivacaine (0.5%), 52±8 min, and lidocaine (2%), 18.4±4 min, c) cornea test: Bpa (0.5%), 49.4±6 min, bupivacaine (0.5%), 46.8±5 min, and lidocaine (2%), 11.5±3min.

CONCLUSIONS: A bufodienolide isolated from Bufo paracnemis skin secretions, known as telecinobufagin, induced a strong local anesthesia, more sustained (p<0.05) than those caused by lidocaine and bupivacaine.

Detection of toxic cyanobacteria and its toxins in a water treatment plant of COPASA-Minas Gerais - Brazil

Jardim, F. A.^I; Fonseca, Y.M.F.^I; Vianna, L. N. L.^I; Azevedo, S. M. F. O.^II; Ciscotto, P. H. C.^I

^ILaboratório Metropolitano da COPASA

^IINúcleo de Pesquisas de Produtos Naturais da UFRJ

^{Correspondence}

The present work intents to present the results of the hydrobiological monitoring accomplished in the water intake of the Alfenas city. In October 1998 it was detected a bloom of Cylindrospermopsis raciborskii, followed by Microcystis viridis. Toxicity test were done by mice bioassays and analyses for hepatotoxins identification by High Performance Liquid Chromatography (HPLC). The results of bioassays indicated the presence of neurotoxins (PSP). The analyses by HPLC confirmed the microcystin presence in raw water samples. With the present study the cyanobacteria species identification and the confirmation of its toxicity were done. Besides this it was possible to evaluated the cyanobacteria toxicity and immediate measures should be taken to guarantee the water quality for public consumption.

Effects of Bothrops asper (BaV) and Bothrops jararaca (BjV) venoms on the release of prostanoids and expression of cyclooxygenases

Zamuner, S.R.^I; Wallace, J.L.^II; Olivo, R.A.^I; Teixeira, C.F.P.^I

^ILaboratório de Farmacologia, Instituto Butantan, São Paulo, Brasil

^IIDept of Pharmacology and Therapeutics, University of Calgary, Canada

^{Correspondence}

Venoms from snakes of genus Bothrops cause pronounced local effects in the victims, which are characterized by necrosis, hemorrhage and edema. Previous results of our laboratory have showed that BaV- and BjV-induced paw edema is markedly reduced by indomethacin pretreatment. In the present work, the effects of the BaV and BjV on the release of prostanoids (PGE2 and PGD2) and expression of COX-1 and COX-2 were investigated. Groups of male mice were injected i.p. with BaV or BjV (250 mg/kg) or saline. Peritoneal washes were collected and centrifuged at selected periods of time and concentrations of PGE2 and PGD2 were determined by specific enzymatic immunoassay in the supernatant. Expression of COX-1 and COX-2 was evaluated by western blot analyses in leukocytes. BaV and BjV induced a significant release of PGE2 BaV but not BjV induced the release of PGD2 Both venoms increased the expression of COX-2, BaV from 3 to 12 hours and BjV at only 12 hours. Expression of COX-1 was not affect by both venoms. These results indicate that BaV and BjV induce local production of prostanoids in the site of their injection. This effect is correlated with expression of COX-2, but not COX-1. Absence of PGD2 modulation on the release of PGE2 was observed in our experimental model. Since the effect of BaV on the expression of COX-2 was seen earlier than that of BjV, the components responsible for these effects may differ for both venoms.

Financial support: FAPESP

Aspects of kinetic and biodistribution of neurotoxin from the Phoneutria nigriventer venom

Yonamine, C.M.^I; Camillo, M.A.P.^I; Troncone, L.R.P.^II

^ILaboratório de Biologia Molecular, IPEN-CNEN/SP, São Paulo, SP, Brazil

^IILaboratório de Farmacologia, Instituto Butantan, São Paulo, SP, Brazil

^{Correspondence}

The Phoneutria nigriventer spider venom contains innumerous neurotoxic peptides which interfere with voltage dependent ionic channels function. The present study was carried out with an isolated peptide from the venom. The molecular weight was 5290 daltons and induces priapism in mice at the dose of 5 mg/animal and may conduct to death. The objective was a kinetic and biodistribution study and aimed to contribute for a better understanding of its mechanism of action. The toxin was isolated after two steps: chromatography in SephadexG50 column with ammonium formiate buffer 100mM pH3.0 and, in sequence, in mRPC-C2C18 (Pharmacia) with water/TFA 0.1% and acetonitrile (grade 25-100% in 35min). The radioiodination was done by the method of modified cloramine T (Ribela et al., 1993). Mice Balb C, males, adults, fed with water and food ad libitum were kept in a room with natural cycle light and controlled temperature. The animals were endovenouslly administered and samples were collected after 5, 15 and 30min, 1, 2, 4, 7 and 24hours. The incorporated radioactivity was measured in blood, brain, heart, lung, liver, kidney-adrenal, spleen, stomach, testicle, intestine, muscle and thyroid. The blood measures were expressed as cpm/mL and statistically analyzed with the program GraphPad Prism, nonlinear regression, exponential model. For the organs the results were expressed as cpm/g of tissue. The results indicated that (a) there was a redistribution after 1hour, (b) hepatic metabolization with release of 125-Iodine, (c) it passed to be accumulated in the thyroid and (d) the renal clearence was the main elimination route.

Chronic ulcers in lower extremities of human beings caused by stings of freshwater stingrays (Potamotrygonidae) in Brazilian rivers: epidemiologic, clinic and therapeutical aspects

Haddad Jr, V^{I, II}; Cardoso, JLC^II; Ribeiro, LMG^III; Talhari, S^IV; Magalhães, MR^V; Paula Neto, JB^VI

^IUniversidade Estadual Paulista

^IIInstituto Butantan

^IIITrês Lagoas – Mato Grosso do Sul

^IVFundação Universidade do Amazonas

^VUniversidade Católica de Goiás

^VIITPAC – Hospital de Doenças Tropicais – Tocantins

^{Correspondence}

OBJECTIVE:The authors report twenty-eight accidents caused by freshwater stingrays (family Potamotrygonidae) emphasizing the clinical aspects, in special the cutaneous necrosis that evolved to chronic ulcers in lower limbs.

METHODS AND PATIENTS: The authors observed patients in rivers of North, Midwest and Southwest regions of Brazil (Araguaia, Amazon, Paraguai and Parana rivers) and clinical and epidemiological aspects of the accidents were registered.

RESULTS: The stingray was always stepped by the victim while wading in shallow waters. The mechanism of the envenomation by the caudal spine is the same for marine stingrays.

The freshwater stingrays occur in South America rivers, swamps and lakes, especially in Amazonas, Paraná and Paraguai rivers. The venom is localized in spine(s) on the caudal appendix and can to provoke intense pain, sudoresis, vomiting, diarrhea and cardiac arrythmias. The report of deaths are dubious, but the secondary infection can be very important and occasionally may provoke the death.

CONCLUSION: The necrosis appears in 12-24 hours and the ulcers in one a two weeks from the time of accident. The initial treatment for the stings was the immersion of the point injured in hot water during 30-90 minutes, since the venom of the fishes can be neutralized at high temperatures. The treatment of the ulcers is made by intensive washing, antibiotics and occasionally cutaneous grafts on the affected point, with good results.

Reference

Haddad JR, V. Atlas de animais aquáticos perigosos do Brasil. Editora Roca, São Paulo, 2000.

An outbreak of accidents probabily caused by the cubomedusa Chiropsalmus quadrumanus (Cnidaria) in Southeast region of Brazil with high percentage of systemic findings

Haddad Jr, V^I; Silva, TCR^I; Soares, CG^I; Souza, V^I; Cardoso, JLC^II

^IFaculdade de Medicina de Botucatu – Universidade Estadual Paulista

^IIFaculdade de Medicina da Universidade de Taubaté

^{Correspondence}

OBJECTIVE: The accidents provoked by cnidarians are very common in Brazilian coast and around the world. The cubomedusa Chiropsalmus quadrumanus is associated with severe accidents in USA and Caribbean with reports of deaths in children (2).

The local injuries are the main markers of the accidents: linear oedematous and erythematous plaques on the points of contact with the tentacles, blisters and superficial necrosis. The systemic findings, caused by the venom or by allergic process are not frequent in the accidents caused by Brazilian cnidarians(1). This paper reports new cases of cubomedusae accidents in Brazil.

METHODS AND PATIENTS: The authors report ten accidents observed in bathers of the Southeast region of Brazil in January/2000. By means of net-fishing, specimens of the cubomedusa Chiropsalmus quadrumanus were collected in the two beaches where the accidents happened.

RESULTS: The systemic manifestations were noted in eight patients (80%): especially dyspnea (80%), nausea and vomiting (50%), and abundant nasal secretion (20%). In an early study with Brazilian jellyfishes, the authors reported systemic manifestations in only 5% of the victims(1).

CONCLUSION : The observation of a high percentage of systemic symptoms in these accidents should alert for the probability of important injuries and the necessity of more studies about the animals and the consequences provoked by the contact with human beings.

References

1. Haddad Jr, V. Atlas de animais aquáticos perigosos do Brasil: guia médico de identificação e tratamento.Editora Roca, São Paulo, 2000.

2. Bengston, K, Nichols, MM, Schnadig, V et al. Sudden death in a child following jellyfish envenomation by Chiropsalmus quadrumanus. JAMA 266(10):1404-1406, 1991.

Novel antimicrobial peptides from the venoms of solitary eumenid wasps

Konno, K.^{I, III}; Souza, B.M.^{I, III}; Fontana, R.^{I, III}; Hirata, I,Y.^{II, III}; Juliano, M.A.^{II, III}; Juliano, L.^{II, III}; Palma, M.S.^{I, III}

^ICEIS - Department of Biology - IBRC - UNESP, Rio Claro - SP

^IIDepartment of Biophysics, EPM - UNIFESP, São Paulo - SP

^IIICAT/CEPID - FAPESP, São Paulo - SP

^{Correspondence}

Solitary wasps are known to inject their venoms to insects or spiders and paralyze the preys to feed their larvae. Therefore, the solitary wasp venoms should contain neurotoxins acting on various nervous systems. In fact, we have isolated novel peptide neurotoxins, a- and b-pompilidotoxins (PMTXs), from pompilid wasp venoms. Besides the neurotoxins, however, we have also found antimicrobial peptides, anoplin and eumenine mastoparan-AF (EMP-AF), in a pompilid and an eumenid wasp venoms, respectively. These peptides show broad-spectrum inhibitory activity against both Gram-positive and Gram-negative bacteria, and are structurally related to mastoparan, a mast cell degranulating peptide from a social wasp venom. Therefore, our recent studies indicated that the solitary wasp venom may be a new and rich source of bioactive peptides.

A further survey of a variety of solitary wasp venoms demonstrated that antimicrobial peptides are widely distributed in eumenid wasp venoms. For example, decoralin was isolated from the venom of Oreumenes decoratus and showed inhibitory activity against Gram-positive bacteria. This peptide is consisted of eleven amino acids, and the sequence is highly homologous to anoplin. Isolation, sequence determination, and biological properties of this and other antimicrobial peptides from eumenid wasp venoms will be reported.

Supported by FAPESP

Mass spectrometric characterization of two novel biologically active peptides from the venom of social wasp Polybia paulista

Souza, B. M.; Mendes, M. A.; Marques, M. R.; Cesar, L.M.M.; Santos, L. D.; Konno, K.; Palma, M.S.

CEIS - Department of Biology / IBRC – UNESP, CAT (CEPID/FAPESP) – Institute of Immunological Investigation (Millennium Institute/CNPq) – Rio Claro/SP

^{Correspondence}

The venoms of Hymenoptera have been known to cause significant effects such as systemic reactions and allergy. Currently little is known about the structure, chemical composition and mechanisms of action of the toxins present in the venom of wasps from the neotropical regions responsible for the high number of harmful stinging accidents every year.

Two novel peptides were isolated and identified from the crude venom of social wasp Polybia paulista. Specifically, using RP-HPLC, thirteen fractions were identified. Two new peptides were identified in the thirteenth fraction, with molecular weights (as determined by ESI-MS) of 1613 Da and 1659 Da. Further purification of this fraction, allowed sequence determination by ESI-MS/MS spectra to elucidate the following sequences: Ac-S A D I/L P K/Q N D K/Q I/L N G I/L D, 1613Da, and Ac-S P D M V M K/Q G I/L K/Q I/L D I/L G I/L, 1659 Da. The sequence determined by MS/MS was compared with the sequence elucidated from MALDI-TOF-PSD spectra. This comparison indicates the loss of 42 Da from each of the two peptides, confirming acetylation of the N-terminus. Specifically, both peptides contain fourteen amino acid residues each with an acetylated serine at the N-terminus side.

The following biological activities were investigated: hemolysis, chemotaxis, mast cell degranulation and antibiotic properties. The results showed that both peptides exhibit potent biological actions, most notably, mast cell degranulation activity.

Supported by FAPESP and CNPq

Structural elucidation of two new b-carbolinetoxins from the droplets of Nephila clavipes web

Marques, M.R.^I; Salles, H.C.^I; Mendes, M.A.^I; Souza, B.M.^I; Cesar, L.M.M.^I; Konno, K.; Tormena, C. F.^II; Rittner, R.^II; Palma, M.S.^I

^IDept. Biology - CEIS / IBRC – UNESP, CAT (CEPID-FAPESP) - Rio Claro, SP, Brazil

^IIPhysical Organic Chemistry Laboratory - Chemistry Institute – UNICAMP – Campinas, Brazil

^{Correspondence}

The orb-web spiders are polyphagous animals in which the web localized compounds play a very important role in the capture of prey, specifically aqueous droplets that cover the capture threads of these webs. In our field observations, we have noted that immediately after web entrapment, prey present toxicity signs, prior to venom inoculation by the spider. This fact suggests the existence of toxins in the web of Nephila clavipes. The objectives of this research are to investigate the presence of toxins in the aqueous droplets of Nephila clavipes webs, characterize these toxins biochemically, elucidate their structures and reveal the ultra-structure of the droplets. The ultra-structure of the droplets was analyzed by transmission electron microscopy and scanning electron microscopy. The droplets present regular shape, varying among oval and elliptic shape, keeping a standard organization for whole thread extension. The sections of the droplets show that them present three deferent layers surrounding of two axial fibers. The crude extract of Nephila clavipes web was chromatographed under RP-HPLC. The chromatographic profile of the crude extract presented 6 fractions. Fraction 6 was one of the major peaks. This fraction was rechromatographed, resulting in more 5 subfractions. The subfraction 6.5 was submitted to mass spectrometry (ESI-MS and ESI-MS/MS) and H1-NMR analysis. We have found two compounds of low molecular weight: 287 Da and 315 Da. The two new compounds were denominated of (I) 1-2-guanidinoethyl-6-hydroximethyl-1,2,3,4-tetrahydro-b-carboline and (II) 1-4-guanidinobuthyl-6-hydroximethyl-1,2,3,4-tetrahydro-b-carboline. These compounds are natural analogous of the trypargine, a b-carbolinetoxin isolated from skin frog. Cesar (2001) found out three b-carbolinetoxins in the venom of the social spider Parawixia bistriata, which showed significative evidence of stimulation in regions of the diencephalon of Wistars rat. Taken to gather, these data indicate that these newly toxins are involved with the process of prey paralysis or death in the Nephila clavipes web.

Supported by FAPESP and CAPES

Investigations of membrane-peptide interactions for solitary wasp peptide toxins

Alves, D.S.^I; Fossey, M.^II; Ruggiero, J.^II; Filgueira, W.^II; Konno, K.^I; Palma, MS^I

^ICEIS/UNESP- RC

^IIDept. Physics/UNESP-SJRP

^{Correspondence}

Anoplin and EMP-AF are peptides that were isolated from the venoms of the solitary wasps, that showed antimicrobial, hemolytic and mast cell degranulation activities. Earlier studies revealed that the interactions of peptides with phospholipids and polysaccharides are very important to understand the mechanisms of these peptides. The pore formation on the bacteria membrane is related to the structure of the peptides. Studies demonstrated that the a-helical conformation induce the penetration of the peptide into the cell.

The Glycosaminoglycan Heparan Sulfate (HS) occurs on the surface of the most cells as proteoglycans side chains. Heparin can be described as a highly sulfated type of HS found in the mast cell granules. We used heparin in this study due to its known interaction with peptides and its natural occurrence on mast cells.

The complex interaction heparin, peptides and liposomes was studied by using Circular Dicroism (CD). The model of liposomes that we used was Small Unilamellar Vesicles (SUVs) and the lipids were DMPC and DMPG.

The presence of heparin seems to induce a-helical conformations both for Anoplin and EMP-AP peptides, even in aqueous solutions, suggesting a feed-back mechanism when these peptides start to induce mast cell degranulation delivering heparin from the granules. The results of the CD spectra of Anoplin and EMP-AF in presence of different compositions of phospholipids indicate a high percentage of a-helical conformation it is required to observe the interactions of anoplin and EMP-AF with the membranes. The results of the present investigation surely present new understanding about the mechanism of interaction peptide-membrane, mainly to explain the antibiotic effect of these peptides and their involvement with the inflammatory process.

Neutralizing capacity of antiserum raised in rabbits against crotoxin from Crotalus durissus cascavella venom

Beghini, D.G.^I; Panunto, P.C.^II; Hyslop, S.^II; Rodrigues-Simioni, L.^II; Toyama, M.H.^I; Saraguacy-Hernandez, O.S.^II; Novello, J.C.^I; Marangoni, S.^I

^IDepto de Bioquímica, IB, Universidade Estadual de Campinas (UNICAMP)

^IIDepto de Farmacologia, FCM, UNICAMP, Campinas, São Paulo, Brasil

^{Correspondence}

OBJECTIVES: To examine the ability of antiserum against crotoxin (anti-Crtx) and of commercial antiserum in neutralizing the neurotoxic activity of crotoxin from C. d. cascavella venom.

METHODS AND RESULTS: Anti-Crtx serum was produced by immunizing rabbits with crotoxin from C. d. cascavella venom. The presence of anti-Crtx antibodies in the serum was monitored by ELISA. The antibodies produced cross-reacted with crotoxin and its subunits crotapotin and phospholipase A2. Immunoblotting showed that the anti-Crtx serum recognized crotoxin in C. d. cascavella venom, whereas commercial serum also recognized other venom proteins. The ability of antisera to neutralize the neurotoxicity of crotoxin was assessed in mouse electrically-stimulated phrenic nerve-diaphragm preparations (PND). The time required to produce 50% neuromuscular blockade in PND was 36.6±1 min (n=3) for crotoxin (10 mg/ml) and 64±9 min (n=3) for crotoxin (10 mg/ml) preincubated with affinity purified anti-Crtx IgG (toxin:antiserum ratio of 1:10), whereas with commercial antiserum there was less than 50% blockade after 120 min.

CONCLUSION: Rabbit antiserum to crotoxin showed weak neutralization of the neurotoxicity of C. d. cascavella crotoxin compared to commercial antiserum.

Financial support: FAPESP

Crystal structure of a basic myotoxic phospholipase-A2 homologue from the Bothrops neuwiedi pauloensis venom

Magro, A.J.^I; Soares, A.M.^II; Giglio, J.R.^III; Fontes, M.R.M.^I

^IDepto. de Física Biofísica, Inst. de Biociências UNESP, Botucatu

^IIDepto. de Biotecnologia-UNAERP, Ribeirão Preto

^IIIDepto. de Bioquímica, Fac. de Medicina de Ribeirão Preto-USP - Ribeirão Preto

^{Correspondence}

In Latin America the most of the ophidian accidents are caused by snakes of the genus Bothrops. In addition to systemic alterations, these poisonings are characterized by prominent local tissue damage due to myonecrosis, hemorrhage and edema. Acute muscle damage induced by these venoms is due mainly two basic myotoxic phospholipases A2 (PLA2). Its members seem to fall in two categories: enzymatically-active PLA2 (Asp49) and enzymatically-inactive PLA2 (Lys49). Myotoxins are believed to act on the sarcoplasma membrane, thus inducing a disorganization of the phospholipds, the loss of intracellular components and an influx of Ca++ ions, however, in the case of Lys49 PLA2 is not still completely understood the myotoxic mechanism. We recently studied B. neuwiedi venoms from different regions and observed two basic myotoxins found only on B. neuwiedi pauloensis of the region of Botucatu/SP. The BnSP-7 was cDNA cloned and sequenced and several biological activities were assayed and compared with those of chemically modified toxin involving specific residues.

In this work we show the X-ray structure determination, refinement process and structural characteristics of BnSP-7.

The toxin was crystallized and data collection was made using synchrotron radiation at LNLS (Campinas). The crystals belong to the space group P3121 with cell constants a=b=58.2 and c=132.0 Å. Data was collected up to 2.3 Å resolution. The dimeric toxin structure was solved by Molecular Replacement method and is currently being refined using the CNS program. The structure has excellent geometry and electronic density map.

Renal alterations promoted by Thalassophryne nattereri venom

Facó, P.E.G.; Havt, A.; Bezerra, G.P.; Barbosa, P.S.F.; Bezerra, I.S.A.M.; Martins, A.M.C.; Nobre, A.C.L.; Fonteles, M.C.; Lopes-Ferreira, M.; Monteiro, H.S.A.

Butantan Institute and Federal University of Ceará – Brazil

^{Correspondence}

The accidents with the Brazilian venomous fish, Thalassophryne nattereri (niquim), causes severe local damages including pain and necrosis. However its renal effects were never investigated. Our goal is to identify the possible renal alterations promoted by this fish using the isolated perfused rat kidney method. Wistar rats were anesthetized with sodium pentobarbitone (50mg/mL), and their kidneys were surgically removed following Fonteles and coworkers (Am. J. Physiol. 244, 235-346, 1983) technique. We used a Krebs-Henseleit perfusion solution (KHPS) modified by serum bovine albumin (6g%). Each experiment (n=6) lasted 120 minutes. We tested the effects of three doses (0.3, 1 and 3 mg/mL) named T1, T2 and T3, respectively. The venom was always administered 30 minutes after the beginning of each perfusion. The treated groups were compared to an external control group (CG) where we perfused the kidneys with only KHPS (ANOVA with *p<0,05). Thalassophryne nattereri venom altered intensely the renal parameters especially in the last 30 minutes of each experiment (120 min.). We noticed an increase after administration of all three doses in the perfusion pressure (CG120 =111.5 ± 0.34, T1120= 147.8 ± 3.28*, T2120 = 134.2 ± 0.94*, T3120 = 130.2 ± 2.68* mmHg) and renal vascular resistance (CG120 = 4.53 ± 0.07, T1120= 6.72 ± 0.15*, T2120 = 5.13 ± 0.04*, T3120 = 5.76 ± 0.13* mmHg/mL/g/min). The sodium and potassium tubular transport were reduced only with the dose of 3mg/mL (CG120 = 81.15 ± 0.2, T3120= 73.10 ± 1.07* %), (CG120 = 72.90 ± 0.84, T3120= 67.22 ± 1.63* %), respectively. The urinary flow and glomerular filtration rate were increased after 0,3 and 1 mg/mL, but decreased after 3 mg/mL. We concluded that the Thalassophryne nattereri venom was nephrotoxic and altered intensely the vascular and glomerular renal parameters, but tubular transport was only affected by the largest dose.

Supported by FUNCAP/FAPESP

Effects caused by Bothrops moojeni venom in the isolated rat kidney method

Barbosa, P.S.F.; Havt, A.; Facó, P.E.G.; Bezerra, I.S.A.M.; Aragão, B.J.M.; Martins, A.M.C.; Nobre, A.C.L.; Soares, T.F.C.; Fonteles, M.C.; Monteiro, H.S.A.

Federal University of Ceará-UFC

^{Correspondence}

Acute renal failure is one of the most common systemic complications after snakebite. However, its pathogenesis remains obscure. In this study we evaluated the renal effects of Bothrops moojeni venom in the isolated perfused rat kidneys. Adult male Wistar rats (240 - 280 g) were anesthetized with sodium pentobarbitone (50 mg/kg, i.p.) and after careful dissection of the right kidney, the right renal artery was cannulated via mesenteric artery without interrupting the blood flow as described by Fonteles and coworkers (Am. J. Physiol. 244, 235-346, 1983). The crude venom (10 mg /mL) was added to the perfusion system 30 min after the beginning of each perfusion. Maximum effects were seen in the last 30 minutes of each perfusion named 120 minutes. The renal effects were compared with a control group (CG), perfused with only modified Krebs-Henseleit solution and statistic analyses were done by Student t Test (*p< 0,05). Bothrops moojeni venom (Bm) decreased the perfusion pressure (CG120 = 113,5 ± 0,833: Bm120 = 78,8 ± 7,1), renal vascular resistance (CG120 = 5.216 ± 0,16 mmHg/mL-1. g-1.min-1, Bm120 = 3,47 ± 0,36 mmHg/mL-1. g-1.min-1), and the percentage of sodium, potassium and chloride tubular transport. In contrast, the venom increased the urinary flow (CG120 = 0,150 ± 0,002 mL-1. g-1.min-1, Bm120 = 0,397 ± 0,039 mL-1. g-1.min-1), glomerular filtration rate (CG120 = 0,617 ± 0,022 mL-1. g-1.min-1, Bm120 = 1,512 ± 0,039 mL-1. g-1.min-1), and sodium, potassium and chloride excretion. In conclusion, Bothrops moojeni venom caused intense alterations in renal physiology, including a drop in vascular resistance associated with diuresis, natriuresis and kaliuresis. Bradykinin potentiating peptides presented in this venom possibly contributed to the decrease in vascular parameters.

Supported by FUNCAP/CAPES/CNPq

Ocurrence of a somatostatin-like peptide in the skin secretion of Hyla punctata (Anura)

Prates, M.V.^{I, II}; Sforça, M.L.^III; Figueredo, R.C.R.^III; Gordo, M.^IV; Sebben, A.^V; Spisni, A.^III; Bloch Jr, C^I

^ILab. Espectrometria de Massa, Embrapa-Recursos Genéticos e Biotecnologia, Brasília-DF

^IIDepartamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília-DF

^IIICentro de Biologia Molecular Estrutural, Laboratório Nacional de Luz Síncrotron, Campinas-SP

^IVDepartamento de Biologia, Universidade do Amazonas, Manaus-AM

^VLaboratório de Toxinologia, Departamento de Biologia Animal, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília-DF

^{Correspondence}

INTRODUCTION: Anurans produce powerful host defense compounds in their skin secretion, including bioactive peptides displaying antibiotic, antiviral, antifungal, anticancer and hormonal activities (Weneger, K. L. et al. 2001, RCM 1726-1734). Recently, we have isolated a somatostatin-like neuropeptide from the skin secretion of the Brazilian hylid H. punctata.

OBJECTIVES: The main goal of this work was the purification, synthesis and 3D structure determination of the somatostin-like peptide isolated from H. punctata.

METHODS: Purification of the natural somatostatin-like peptide by RP- HPLC, purity and mass analysis by MALDI-TOF/MS, N-terminal sequencing by automatic Edman's degradation, F-moc automatic solid-phase synthesis, Circular dichroism (CD) and 2D ¹H NMR studies in H2O.

RESULTS: The 15-residue neuropeptide Hp-somatostatin was sinthesized and its molecular weight was determined to be 1832.80 Da. CD and 2D ¹H NMR studies, carried out in water indicate the presence of a preferential 3D fold.

CONCLUSIONS: Somatostatin is a 14-residue peptide hormone found in superior vertebrates, synthesized in the hypothalamus and pancreatic islets. It plays an important role in the regulation of various hormones, such as growth hormone, insulin, glucagon and gastrin (Tran. T-A., et al. 1998, J. Med. Chem. 2679-2685). The actual function of Hp-somatostatin found in the skin secretion of H. punctata is unclear. A possible explanation might be that it is part of a defence mechanism, however, this needs to be further investigated.

Support: UnB, EMBRAPA, CNPq, FAPESP.

An edematogenic peptide isolated from Leptodactylus pentadactylus skin secretion

Cordeiro, J.S.; Cardi, B.A.; Carvalho, K.M

Laboratório de Neurofarmacologia, Universidade Estadual do Ceará, Fortaleza, Ce, Brazil

^{Correspondence}

OBJECTIVES: The anuran skin secretions contain several compounds, such as enzymes, steroids, biogenic amines, alkaloids, proteins and peptides that play an important biological hole to anuran adaptation to environment and protection from predators and microorganisms. The aim of this study was to evaluate the ability of a new peptide from Leptodactylus pentadactylus skin to produce inflammatory edema in rat hind paw and to verify the mediators involved.

METHODS AND RESULTS: The skin secretion, obtained by subcutaneous injection of 500ml adrenaline (100mg/ml), was diluted in bidestilated water (1:4, v:v), lyophilized and kept at -25°C. The edematogenic peptide (EP) was purified by HPLC using a C18 column (25x250 mm) (5 ml/min)(acetonitrile 0-40%, 30 min). Its amino acid sequence (Edman, 1950), performed by an Applied Biosystem sequencer, was determined: RVDL. Intraplantar injection of male Wistar rats with the EP (0.4, 0.8 and 2mg/kg, 0.1ml/paw) induced significant (p<0.05) paw edema with peak activity at 1 hour (increase in volume: EP 0.4 mg/kg = 1.02 ml ± 0.13 vs. 0.11 ml ± 0.03 of control group). The injection of mepiramine (10mg/kg), methysergide (1mg/kg), HOE 140 (1mg/kg) and cyproheptadine (6mg/kg), 1h before intraplantar injection of EP (0,4 mg/kg) significantly inhibit (p<0.05, mepiramine 49%, methysergide 86%, HOE 140 64%, cyproheptadine 80%) the paw edema.

CONCLUSIONS: The present study provides a direct evidence of the ability of EP to induce hind-paw edema in rats and indicates a key role for serotonin and histamine in this response. Once the edema induced by substance was also inhibited by B2 antagonist, it is possible to suppose that EP could also act directly in this receptor or could be acting by the synthesis of bradykinin.

Isolation of a new toxic protein from the Leptodactylus pentadactylus (Amphibia, Anura) skin secretion

Evangelista, J. S. M.; Honório-Júnior, J.E.R.; Falcão, N.R.; Oliveira, R.G.S.; Cardi, B.A.; Carvalho, K.M.

Laboratório de Neurofarmacologia, Universidade Estadual do Ceará, Fortaleza, Ce, Brazil

^{Correspondence}

OBJECTIVES: Amphibians have undergone profound evolutionary changes to survive to predators and microorganisms, and their skin secretions containing toxins, alkaloids, biogenic amines, proteins, peptides, seems to be a paramount importance. In this paper, we demonstrated the purification and pharmacological effects of a new toxic protein from L. pentadactylus skin secretion.

METHODS AND RESULTS: Skin secretion was obtained after injection of 500ml adrenaline (100mg/ml, s.c.) in dorsal skin. After dilution in bidestilated water (1:4, v:v) and lyophilization, it was kept at -25°C. The toxin was purified by Sephacryl S-400 chromatography and its purity was evaluated by denaturing and non-denaturing gel electrophoresis. The LD50, pharmacological and toxicological analysis were evaluated after i.v. injection on mice. On SDS-PAGE only 29 kDa form was identified. The LD50 values were 1.0±0.2 mg/Kg (crude venom) and 0.5±0.03 mg/Kg (purified toxin), the dead time was less than 1 minute. The preliminary histopathological analysis showed pulmonary congestion macroscopically, without significant microscopical changes at this place or on other organs. In vitro hemolysis (washed mouse erythrocytes), using crescent toxin concentrations (0.012-0.12mg/ml), was observed. At 0.5-1.0 mg/Kg doses rate, we observed locomotion activity decreased, sub-convulsion movements, prostration, defecation/urination increased, nasal discharge, mydriasis, and dyspnea. At low doses (< 0.5 mg/Kg), the toxin induced in vivo an acute and reversible decrease of the blood pressure and in vitro an inotropic positive effect on heart, without interfering on the frequency.

CONCLUSION: Although the mechanism of action of this toxin is unknown, these results suggest that it may be a tool to study the cardiovascular functions involved on blood pressure and heart homeostasis.

Physical and biological properties of the new biological dressing constituted of Rana catesbeiana skin

Oliveira, C. R.^I; Vieira, C.M.^I; Araújo, A.C.M.^I; Adorno, J.^V; Cunha Filho, G.A.^I; Paulino, L.^II; Azevedo, R.B.^II; Kyaw, C.M.^III; Magalhães, A.V.^IV; Schwartz, E.F.^I

^ILaboratório de Toxinologia, Universidade de Brasília

^IILaboratório de Morfologia e Morfogênese, Universidade de Brasília

^IIILaboratório de Microbiologia, Universidade de Brasília

^IVDepartamento de Patologia, Universidade de Brasília

^VHospital Regional da Asa Norte, Brasília, DF, Brasil. [e-mail]

^{Correspondence}

Introduction Wound dressings are thought to promote healing. The goal of the present study was to verify the physical and biological properties of a dressing derived from the amphibian Rana catesbeiana skin (Ranafilm) and that has been used at Hospital Regional da Asa Norte, Brasilia, DF, Brazil. Methods The resistance to water loss was evaluated by the weight variation of flasks filled with saline solution and covered with Ranafilm or other commercial dressings. The evaluation of resistance tension was made by dynamometer. To the healing test, three small burns were made in rabbits dorsum after anesthesia. After dehybridization, one wound was treated with Ranafilm, another one with silver sulfadiazine cream and the third one did not receive treatment. Biopsies of the wounds were done at different stages and were immediately fixed for histology. Ranafilm was extracted with 1% acetic acid and the obtained extract was tested against Staphylococcus aureus, Escherichia coli, Acinetobacter baumani, Proteus mirabilis, Klebsiela pneumoniae e Pseudomonas aeruginosa. The hemolytic activity was evaluated on human red blood cells. Results Ranafilm was the coverage with lowest capacity of water retention and demonstrated more resistance than the most of synthetic dressings. Clinical observation and histology revealed that wounds treated with Ranafilm showed faster recuperation than ones without treatment. Ranafilm extract was capable to inhibit the growth of all bacteria used, but did not presented hemolytic activity. Discussion Although Ranafilm showed minimal resistance to water loss, it holds several benefic properties that enhance wound healing such as the property of working as a framework to cell proliferation, and the antimicrobial activity. The last one is probably related to the skin bioactive compounds which have been extensively studied by several groups.

Support: DPP-UnB, CNPq

Occurence of antimicrobial activity in parotoid gland secretion of the toad Bufo ictericus

Olah, D.^I; Castro, M.S.^I; Fontes, W.^II; Sousa, M.V.^II; Kyaw, C.^III; Schwartz, E.F.^I

^ILaboratório de Toxinologia, Universidade de Brasília, Brasília, DF, Brasil

^IILaboratório de Bioquímica e Química de Proteínas, Universidade de Brasília, Brasília, DF, Brasil

^IIILaboratório de Microbiologia, Universidade de Brasília, Brasília, DF, Brasil

^{Correspondence}

Amphibian skin secretions contain many active compounds which present a large diversity of biological activities, among them, antimicrobial activity. The aim of the present work was to identify antimicrobial compounds from parotoid gland secretion of the toad Bufo ictericus. Toad secretion was obtained by manual compression of the parotoid gland, and immediately lyophilized. The extract was ressuspended in 0,1% TFA/water (v/v) and applied into a Pharmacia C8 column. The elution was performed using a double linear gradient: TFA 0,1% / acetonitrile + 0,1% TFA. Fractions were collected manually, lyophilized, and rechromatographed using a Vydac C18 column. The antimicrobial activity of parotoid secretion and obtained fractions was assayed against Escherichia coli. Hemolytic activity was tested using human red blood cells. Molecular masses from active fractions were obtained by MALDI-TOF spectrometry. The parotoid secretion and five fractions presented antimicrobial activity. Only one of these fractions showed hemolytic activity. This chromatography procedure resulted in the isolation of fractions with molecular mass from 600 to 800kDa. The mass spectrometry data indicated that the antimicrobial activity of parotoid secretion of B. ictericus is not related to the presence of antimicrobial peptides, but probably to the presence of steroids, such as the bufadienolides. The chemical characterization of these antimicrobial compounds are in progress.

Support: DPP-UnB, PIBIC-CNPq

A study on the experimental envenomation with the venom of Tityus serrulatus (Scorpiones; Buthidae) from State of Bahia, Brazil

Silva, T.F.^I; Diaz, D.^I; Casais e Silva, L.L.^II; Barbosa, A.^III; Lira-da-Silva, R.M.^I

^IDepartamento de Zoologia, UFBA

^IIEscola Bahiana de Medicina e Saúde Pública (EBMSP)

^IIICPGM/FIOCRUZ, Bahia

^{Correspondence}

Most of the actions of the Tityus serrulatus venom are indirect and due to derangement of ion channels that result on abnormal release of adrenergic and cholinergic neurotransmitters. In the severe human poisoning by this specie, pulmonary edema is a frequent finding and the cause of death. In Bahia, despite the clinical features observed on T. serrulatus accidents be also similar to other reports, it can be stood out the benignity of the accidents. Nevertheless, between the few severe cases (7%) only vomiting was observed and no lung edema was registered. The present work was designed to characterize the effect of Bahia’s T serrulatus venom (TSV) as for their lethal, neurotoxic activities and the ability to induce lung injury. Groups of rats were inoculated with TSV (400 mg/Kg, i.v.) and observed for 1 hour to investigate the behavior related to neurotoxic effects. After 1 hour, these animals were dead by decapitation. Pulmonary edema was assessed by determination of lung weight of experimental group in relation of control animal injected with saline solution. The clinical observation showed salivation (55,6%), agitation (50%), piloerection (50%) and tachypnea (22,2%). All lungs from rats injected with TSV showed no anatomo-pathological alteration and no significant statistical differences between experimental and control lungs weight. TSV was also not toxic since no death was observed on DL50 assay in mice. These results confirm the absence of pulmonary edema on scorpion accidents in Bahia and showed a regional variation of the venom of T. serrulatus.

Skeletal muscle damage caused by Bothrops leucurus (Serpentes; Viperidae) venom

Mise, Y.F.^I; Casais e Silva, L.L.^II; Barbosa, A.^III; Lira-da-Silva, R.M.^I

^INúcleo Regional de Ofiologia e Animais Peçonhentos da Bahia (NOAP)

^IIEscola Bahiana de Medicina e Saúde Pública (EBMSP)

^IIICPGM/FIOCRUZ, Bahia

^{Correspondence}

The Bothrops leucurus snakes known as “jararaca-do-rabo-branco” has large distribution in the State of Bahia, being responsible for the majority of registered cases at the Metropolitan Region of Salvador (98%). The aim of the study is to investigate the local tissue damage cause by B. leucurus venom assessed by quantifying the release of creatine kinase (CK) and by histological analysis. The myotoxicity was evaluated in vivo by intramuscular (50 ml) of B. leucurus crude venom (25, 50 or 100 mg/ml) over the gastrocnemius muscle of the right posterior limb. Control was injected with physiological saline solution. Blood were collected by orbital puncture after 1, 3, 6, 12 and 24 hours. The plasma was separated by centrifugation and stored at 4°C for subsequent determination of creatine kinase (CK) activity using CK-NAC Liquiform test (LABTEST Diagnóstica). For histological analysis, the muscle were removed and fixed on formol 10% and processed for examination by light microscopy. The peak of liberation of CK was detected at 6 (1758,16), 3-6 (1183,67 – 1145,44) and 3 (1718,16) hours for 25, 50 and 100 mg/ml, respectively. Morphological analysis revealed that Bleu venom affected a large number of muscle fibers as show by widespread and varying degrees of necrosis. These results confirmed the clinical observations and the myotoxicity of the B. leucurus venom that caused muscle damage and release of the CK into blood plasma.

Analysis of the salivary gland proteins from leech Haementeria depressa using bidimensional electrophoresis and MALDI-TOF-MS methodologies

Ricci-Silva M.E.^I; Stocklin R.^II; Rádis-Baptista G.^III; Yamane T.^III; Favreau P.^II; Michalet S.^III; Chudzinski-Tavassi A.M.^I

^IDepto Bioquímica, Instituto Butantan, São Paulo, Brasil

^IIAtheris Lab., Genebra, Suíça

^IIIToxinologia Molecular, Instituto Butantan, São Paulo; Brasil

^{Correspondence}

The salivary extract of the Haementeria depressa leech contains several important proteins acting on blood coagulation and on the fibrino(geno)lytic systems. The two major proteins described so far from this species include the fibrinogenolytic metalloproteinase hementerin and the factor Xa inhibitor lefaxin. Our aim is to further investigate the protein and peptide content of this extract using Proteomics and Mass Spectrometry (MS) strategies. The bidimensional electrophoresis technique showed 96 spots above 25 kDa, from 133 total spots in the extract 1, indicating the feasibility of this methodology for the identification of large proteins. Preliminary study based on MALDI-TOF-MS will be also presented here. The optimization of a suitable matrix-solvent-extract combination was followed by appropriate adjustment of the instruments’ settings on a Voyager® STR Biospectrometry (Applied Biosystem) MALDI-TOF mass spectrometer providing a good compromise between sensitivity and resolution. The sample was analysed using different matrices in linear mode with delayed extraction, leading to detection of several compounds with molecular masses of interest. The main monocharged ions of m/z detected were: 2028.37, 3369.83, 3864.95, 4687.60, 5103.36, 10805.65, 14046.26, 17165.44, 28261.10, 29261.10, 29068.21 and 42318.58. Isolation, purification and further structural investigations of these compounds are presently under investigation with the aim of discovering novel bioactive compounds.

Supported by FAPESP.

Molecular identification of multiform peptides related to proteinase inhibitor from Japanese toad (Bufo bufo formosus)

Yang, X.^I; Kobayashi, S.^I; Rádis-Baptista, G.^II; Yamane, T.^II; Kubo, T.^I

^INational Institute of Advanced Industrial Science and Technology, Neurobiology Research Institute, Molecular Neurophysiology, Tsukuba, Japan

^IIInstitute Butantan, Molecular Toxinology Laboratory,São Paulo, Brazil

^{Correspondence}

Frog and toad skins are well known to be a rich source of bioactive peptides. It is also recognized that many of the amphibian skin peptides have counterparts in the gastrointestinal tract and the nervous system of higher vertebrates. Recently, there is a growing interest in therapeutic and industrial applications of antimicrobial and others bioactive peptides from the skin secretions

“Gama ointment” made from skin secretion of Japanese toads is one of the traditional medicines to treat wound, injury and infectious skin diseases. Heart stimulation, local anesthesia and diuretic effects are also observed in extract of toad skin secretions. To identify the biologically active substances we prepared a cDNA library from dorsal skin of the toad (Bufo bufo formosus), and screened the library using primers degenerated from the core sequence “CA(N/D)”, which is conserved among some of the bioactive peptides, such as chemokine family. Among the identified cDNAs we focused on the clones coding a novel and characteristic cysteine framework. Four clones so far identified show common structural features: a signal peptide sequence followed by one to five repetitive domain(s). Each of the domains contains strictly spaced eight cysteine residues but the overall degree of sequence similarity is low. This characteristic structure is also found in whey acidic protein, elafin (elastase inhibitor), chelonianin (trypsin and subtilisin inhibitor), WDNM1 protein (involved in the metastasis), Kallmann syndrome protein, caltrin-like protein II (inhibits calcium transport). Biochemical analysis of recombinant polypeptides from the clones is under investigation.

Production and immunological characterization of a neutralizing monoclonal antibody specific to dermonecrotic protein from Loxosceles intermedia spider venom

Oliveira, J.C.R.; Bohórquez, K.; Nascimento, A.L.; Martins, M.S.; Alvarenga, L.M.; Granier, C.; Chávez-Olortegui, C.

Fundação Ezequiel Dias, Belo Horizonte, MG, Brazil. Departamento de Parasitologia, ICB, Universidade Federal de Minas gerais, Belo Horizonte, MG, Brazil. Institud de Biotechnologie et Pharmacologie, Université de Montpellier, France

^{Correspondence}

A group of related proteins of 35 kDa with sphingomyelinase activity has been described as the major toxic components and is involved in the dermonecrotic, lethal activity and induction of C-mediated haemolysis. Studies confirmed the importance of monoclonal and polyclonal antibodies for the neutralization of the dermonecrosis activity of Loxosceles venoms. We have prepared monoclonal antibodies by immunization of mice with crude venom of L. intermedia. One monoclonal antibody was very efficient to neutralize the dermonecrotic effects of L. intermedia. The antigenic specificity of this monoclonal antibody was compared by an indirect enzyme-linked immunosorbent assay and immunoblotting using crude venoms from L. intermedia, L. laeta (Brazil), L. laeta (Perú) and L. gaucho to coat the microtitration plates or nitrocellulose membranes. The antibody had reactivity with L. intermedia but was unable to recognize the other Loxosceles venoms. To determine the specific antigenic region(s) of dermonecrotic protein, we have used the Spot method of multiple peptide synthesis to prepare sets of immobilized overlapping peptides of uniform size (12 mer), covering the complete aminoacid sequence of a dermonecrotic protein. Anti-dermonecrotic monoclonal antibody binding to continuous peptides, revealed epitopes in the N-terminal part of the dermonecrotic protein. This study confirm the hypothesis that effective serum therapy against loxoscelism should contain antibodies against all medically important venom species.

Supported by FAPEMIG, CNPq and INSERM

Cloning and functional expression of TX3-2 a lethal neurotoxin from the spider Phoneutria nigriventer

Ciscotto, P.H.C.; Cordeiro, M.N.; Pereira, M.N.A.; Diniz, C.R.; Diniz, M.R.V.

From the Centro de Pesquisa e Desenvolvimento.

Fundação Ezequiel Dias, Belo Horizonte (MG), Brazil

^{Correspondence}

Tx3-2, a lethal neurotoxic polypeptide of Mr 3.5 kDa with eight cysteine residues, selectively decreases L-type currents present in GH3 cells. The amino acid sequence homology with toxins of the same venom and from venoms of other spiders which bind with refined specificity to different subtypes of calcium channels, make the Tx3-2 a potential instrument for use in this kind of study. Therefore, a cDNA encoding Tx3-2 was amplified by RT-PCR technique, cloned in pET32c(+) vector DNA, and expressed as a thioredoxin-binding fusion protein in Escherichia coli. After removal of the chaperone protein from the affinity-purified product, the recombinant protein was purified by reverse phase chromatography and shown to be neurotoxic by intracerebral injection in mice. At dose levels of 5 micrograms/mouse, induced immediate clockwise gyration and flaccid paralysis, showing the same symptoms of the native toxin.

This research was funded by FAPEMIG (Fundação de Amparo à Pesquisa do Estado de Minas Gerais).

Functional expression of TX1 from the Brazilian "armed" spider Phoneutria nigriventer, a Ca⁺⁺ channel blocker

Diniz, M.R.V.^I; Theakston, R.D.G.; de Assis, J.B.; Crampton, J.M.; Diniz C.R.; de Lima M.E.

^IFrom the Centro de Pesquisa e Desenvolvimento. Fundação Ezequiel Dias, Rua Conde Pereira Carneiro, 80. 30510-010. Belo Horizonte (MG), Brazil

^{Correspondence}

The Tx1 from the venom of the Brazilian spider Phoneutria nigriventer, a lethal neurotoxic polypeptide of Mr 8kDa with fourteen cysteine residues, selectively inhibits voltage sensitive calcium channels from GH3 cells. The cDNA gene encoding this toxin was previously isolated and identified (Diniz et al. (1993) J. Biol. Chem. 268, 15340-15342). The coding region for matured toxin was amplified, cloned and expressed as thioredoxin fusion product in cytoplasm of Escherichia coli. After affinity purification and cleavage, the recombinant TX-1 displayed the same properties as the native toxin in its chromatographical behaviour, convulsive-inducing biological activity, immunological reactivity towards the antibodies raised against the toxin and binding to receptors in rat brain synaptosomes.

This research was funded by FAPEMIG (Fundação de Amparo à Pesquisa do Estado de Minas Gerais).

Inhibition of Bothrops jararaca venom hemorrhagic activity by fraction EA2MB from Croton urucurana baillon

Esmeraldino, L.E.; Veronese, E.L.G.; Ticli, F.K.; Franco, J.J.; Cintra, A.C.O.; Sampaio, S.V.

Department of Clinical, Toxicological and Bromatological Analyses, School of Pharmaceutical Sciences of Ribeirão Preto-University of São Paulo. Ribeirão Preto, SP, Brazil

^{Correspondence}

The aim of this work was to fraction the aqueous extract of de Croton urucurana and evaluated the neutralization of B. jararaca venom (BjV) hemorrhagic activity by fractions. The fractionation of the aqueous extract (AE) was performed on Amberlite column, using ethanol (Fraction EA2) and then water (fraction EA1) as eluent. The EA2 was dissolved in methanol, cleared by centrifugation and the resulting solution (EA2M) was further submitted to partition with water and butanol, which extracted fractions EA2MA and EA2MB, respectively. Antihemorrhagic and anticoagulant activities of these fractions against BjV were assayed according to Theakston and Reid (Bull. Word Health Organ. 61: 949-956, 1983). For the antihemorrhagic assays, BjV was added to fractions (1:20, w/w) and immediately injected i.d. in 90-110g Wistar rats. The hemorrhagic halos were analyzed using the computer program “Image tool”. For the anticoagulant assay, BjV was incubated whit fractions (1:20, w/w) for 1 h and 25 mL of the incubated with fractions to 200 mL of the plasma. The clotting time was measured up to the visualization of the first sings of fibrin. Fractions EA2MA e EA2MB inhibited 91.9 and 96.6%, respectively, of the effect of 2 MHD of BjV. this fractions did not increase the clotting time of MCD of BjV. Fraction EA2MB inhibited the hemorrhagic activity of 2 MHD of BjV at a ration of 20:1 (w/w) whit no effect on clotting time.

Financial support: CNPq and FAPESP.

Kinetics of TNF-a release induced by BAP1, a class PI metalloprotease isolated from Bothrops asper snake venom

Fernandes, C.M^I; Zamuner, S.R.^I; Gutiérrez, J.M.^II; Rucavado, A.^II; Teixeira, C.F.P.^I

^ILaboratory of Pharmacology, Butantan Institute, Brazil

^IIClodomiro Picado Institute, Costa Rica

^{Correspondence}

Metalloproteases are abundant enzymes in crotalinae and viperinae venoms. They are involved in venom local and systemic toxic effects which include haemorrhage, platelet alterations and myonecrosis. BaP1 is a 24 kDa metalloprotease isolated from Bothrops asper snake venom (Central America). This toxin induces not only weak haemorrhage, but also a moderate myonecrosis and oedema. In the present study we evaluated the effects of BaP1 on isolated peritoneal elicited macrophages (Mfs) analyzing the cell viability and production of tumor necrosis factor (TNF-a). Mfs from Swiss male mice were obtained 96h after intraperitoneal (i.p.) injection of thioglycollate. These cells were incubated (5 to 90min) with BaP1 (6.25 – 25mg/mL) or RPMI(control).Cell viability was assessed by Trypan Blue exclusion test. TNF-a was determined by a standard assay using L929 cell line in co-culture with Mfs. BAP1 induced a marked release of TNF-a at 5 min of incubation with all the concentrations used. However, no release was detected at 30min. A significant release of cytokine was observed only with the highest concentration of BAP1 at 60min. Significant but low concentrations of TNF-a were released from macrophages 90min after incubation with all concentrations of the toxin. The present data demonstrate that BaP1 is not directly toxic to elicited Mfs and is able to induce the release of TNF-a by these cells. This effect appears to be related to the toxin proteolytic activity at the first period and also to induction of TNF-a synthesis at the last period of incubation.

Financial Support: FAPESP

Molecular characterization and activity on platelet aggregation of three phospholipases A₂ of Bothops atrox venom

López-Lozano, J. L.^{I, III}; Sánchez, E.F.^II; Maria, W.S.^II; Sousa, M.V.^II; Ricart, C.A.O.^II; Muniz, E.G.^I; Bührnheim, P.F.^I; Morhy, L.^III

^IInstituto de Medicina Tropical de Manaus-AM

^IIFUNED-MG

^IIICBSP/Universidade de Brasília- Brasília-DF. Brazil

^{Correspondence}

Bothrops venoms contain phospholipases A2 (PLA2) isoforms with different biological activities. Snake venom PLA2 can induce effects on human platelet aggregation. In this study, we are reporting partial molecular characterization and activity on human platelet aggregation of three PLA2 of Bothrops atrox venom from Manaus region-Brazil. Three PLA2s, named Bam-PLA2-I, Bam-PLA2-II and Bam-PLA2-III, were purified. The molecular mass (by electrospray ionization mass spectrometry), N-terminal amino acid sequence, isoelectric point, PLA2 activity and activity on human platelet aggregation, induce by ADP, were determinated to each PLA2. In table 1 and 2 are sumarized molecular characteristics of the PLA2s B. atrox venom.

Table 1.
N-terminal amino acid sequence of PLA2s Bothrops atrox venom.

Table 2.
Molecular characteristics of PLA2s Bothrops atrox venom.

Only Bam-PLA2-II showed inhibitory activity of human platelets aggregation induced by ADP (10 mM, final concentration), in a dose - and - time dependent manner (IC50% = 14,2 mg).

Conclusions. Molecular analyses of N-terminal amino acid sequence and PLA2 activity suggest that Bam-PLA2-I is a PLA2-Lys49, but Bam-PLA2-II and Bam-PLA2-III are PLA2-Asp49, and inhibitory activity of PLA2s on human platelet aggregation induced by ADP could be not associated with PLA2 activity.

Clinical manifestations and treatment of envenoming by Bothrops atrox and Bothrops brazili snakes in Manaus region, Amazonas State - Brazil

Souza, A.R.B.; Muniz, E.G.; López-Lozano, J.L.; Ferreira, L.C.L; Noronha, M.D.N.

Gerência de Animais Peçonhentos – Fundação de Medicina Tropical/Instituto de Medicina Tropical do Amazonas Manaus – Amazonas – Brasil

^{Correspondence}

Bothrops species are the main cause of snake bites in humans in the amazonian rain forest. In Manaus region, 212 human victims of B. atrox and three of B. brazili were attended at Instituto de Medicina Tropical do Amazonas (FMT/IMT-AM) from 1986 to 1999. Snakes were identified at Gerência de Animais Peçonhentos – FMT/IMT-AM. Clinical symptoms and treatment of these patients are report in this study. Pain, abscess and local edema were the most frequent local manifestations of B. atrox and B. brazili snakebites. Systemic hemorrhage with coagulation disorders were observed with B. atrox (16%) and B. brazili (two cases) snake bites. Intracranial hemorrhage was also observed with both species, but only one B. atrox snake bite patient died. In B. atrox snake bites, 48.3% were classified as light cases. In B. brazili snakebite, one case was classified as moderate and two cases as severe. Some local complications in 39,0% of the patients were observed with B. atrox bites: cellulitis and abscesses were the most frequent. One accident by B. brazili showed cellulitis at the local bite. Renal failure was the single systemic complication observed by B. atrox snakebites (10%), none severe. Bothropic antivenoms were used in the treatment of both Bothrops snake, two to six hours after the snakebite. B. atrox snake bites showed early reactions after administration of antivenom at 16% of cases. Twenty four hours after intravenous administration of antivenom, in eleven B. atrox snake bites patients was detected fibrinogen level. The symptomatology observed suggest similar clinical features of envenoming by B. atrox and B. brazili snakes in Manaus region. More studies of therapeutic efficacy of bothropic antivenoms on coagulation disorders produced by B. atrox venom are necessary.

Cross-neutralization of coagulant activities of Lachesis muta muta venom by Brazilian antivenoms

Muniz, E.G.; Noronha, M.D.N.; Mendonça, M.A.B.; López-Lozano, J.L.

Gerência de Animais Peçonhentos, Instituto de Medicina Tropical de Manaus-Amazonas State, Brazil

^{Correspondence}

In Amazonian rain forest, when Bothrops-Laquesis antivenom is not available, Bothrops antivenoms are use for treatment of Lachesis muta muta snake bites. Clinical and experimental evidences (Bard et al., 1994, Colombini et al., 2000) showed that administration of Bothrops antivenom produced by Butantan Institute was not effective to neutralize coagulation disorders produced by L. m. muta venom. This work compares the efficacy of three different antivenoms produced by three Brazilian Institutes (Butantan, Fundação Ezequiel Dias and Vital Brazil) to neutralize the coagulant and defibrinating activities of L. m. muta venom from Manaus region (Amazonas State- Brazil). Minimum coagulant doses in plasma (MCD-P) and bovine fibrinogen (MCD-F) and defibrinating dose (DF) of Bothrops atrox and L. m. muta venoms were taken as references.

CONCLUSION. Bothrops antivenoms showed very low efficacy, whereas Bothrops-Crotalus and Crotalus antivenoms were more effective to neutralize coagulation activities of L. m. muta venom. These findings suggest the presence of antibodies in Crotalus antivenoms with ability to neutralize toxins of L. m. muta venom that induce coagulation disorders.

Clinical manifestations of snakebite by the bushmaster snake Lachesis muta muta in Manaus - Amazonas - Brazil

Souza, A.R.B.; Muniz, E.G.; López-Lozano, J.L.; Ferreira, L.C.L.; Noronha, M.D.N.

Gerência de Animais Peçonhentos – Fundação de Medicina Tropical/Instituto de Medicina Tropical do Amazonas Manaus – Amazonas – Brasil

^{Correspondence}

Ten cases of Lachesis snakebites, attended in the hospital of the Fundação de Medicina Tropical/Instituto de Medicina Tropical do Amazonas (FMT/IMT-AM), Manaus – Amazonas, Brazil, between 1986 and 1996, were studied. Nine patients brought the snakes that were identified as Lachesis muta muta. One of these accidents was diagnosed by ELISA method. Clinical manifestations of the accidents were studied from the patients’ records. Pain, swelling, erythema, light hemorrhages were the principal manifestations in snakebite region. These patients no showed bullae, necrosis or systemic hemorrhages. Four patients present one or more signals of vagal stimulation: blurred vision, hyper salivation, nausea, vomiting (in four patients), abdominal pain, diarrhea, shock, bradycardia (one patient showed blood pressure: 70x00 mmHg and cardiac rate: 64). Three accidents were classified as light, because they did not show hemorrhage in snakebite region, systemic hemorrhages or neurotoxic manifestations and edema restricted in snakebite region. Three accidents were classified as middle and three as severe. The blood was incoagulable in four patients and change in blood coagulation time was detected in three patients. Only one patient presented normal coagulation time. Urea and creatinine in plasma were normal in all the patients studied. Two patients received Lachesis antivenom, four patients received Bothrops-Lachesis antivenom. One patient received 19 flasks of bothropic antivenoms and the blood was incoagulable for 15 days. There was no death in all studied patients.

Inhibition of PLA2, coagulant and hemorrhagic activities of Bothrops alternatus venom by Schizolobium parahyba (Caesalpinoideae) extract

Mendes, M.M.; Vale, L.H.F.; Gebrim, L.C.P.C.; Hamaguchi, A.; Homsi-Brandeburgo, M.I.

Instituto de Genética e Bioquímica – Universidade Federal de Uberlândia

^{Correspondence}

Snake venoms are complex mixtures of proteins which cause several biological effects Vegetal extract represent an attractive source as an alternative substitute for antiserum.We reported the effects of the crude aqueous extract of leaves (EVF) from Schizolobium parahyba to neutralize the hemorrhagic, coagulant and PLA2 activities induced by Bothrops alternatus venom.The EVF was prepared by mixing the leaves with destiled water and centrifuged at low rotation for 20 min/4°C and the supernatante was lyophilized and stored at -20°C. Hemorrhagic activity was tested by intradermal injection of 2 MHD (minimun hemorrhagic dose, 10.12 mg) in Swiss male mice (n=6, 30g), distribuited in 3 groups,.G1: only VB, G2: only EVF, G3: VB+EVF/1:20 (w/w).The coagulant activit on bovine plasma was carried out at 37ºC, by using 10 mg of venom (VB) (positive control) and 50mg of EVF(negative control).The VB+EVF was used as group test in the ratios 1:1, 1:2 and 1:5 (w/w), respectively. The PLA2 activity was determined by potenciometric titration effected on egg yolk as substrat, in presence of the sodium deoxicholic and calcium cloride using the same ratios of the coagulant activity.The EVF was able to neutralize 100% of the hemorrhagic activit in the ratio 1:20. The coagulant and PLA2 activities were inhibited in a dose-dependent curve, reaching 100% in the ratio 1:5 (w/w) for both. Our results clearly indicate that the aqueous extract of the leaves from Schizolobium parahyba contains compounds capable to neutralize proteolytic and PLA2 activities induced by bothrops venoms.

Supported by CNPq, FAPEMIG and UFU.

Isolation and characterization of a novel coleoptera-selective toxin from the venom of brazilian scorpion Tityus serrulatus

Andrez, R.M.A.; Gomes, P.C.; Almeida-Gontijo, M.; Kalapothakis, E.; Chavez-Olortegui, C. ; Richardson, M.

Fundação Ezequiel Dias Belo Horizonte, MG, Brazil. Departamento de Farmacologia Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil

^{Correspondence}

Tityus serrulatus scorpion venom is a rich source of various polypeptides with diverse physiological and pharmacological activities which generally exert their action via target specific modulation of ion channel function. According to their species selectivity sodium channel toxins have been divided into mammalian and insect toxins. depending on their binding affinities and electrophysiological properties, the mammalian toxins are sub-classified into a- and b-toxins and the insect-specific toxins are subdivided into excitatory, depressant and a-insect toxins. In this work we investigated the venom of the Brazilian yelow scorpion, T. serrulatus for the purpose of identifying potent insecticidal peptide toxins.

A coleoptera-selective toxin has been isolated from T. serrulatus whole venom by a combination of conventional gel filtration (Sephadex G50), ion-exchange chromatography (CM Sepharose) and reverse phase HPLC (Vydac C4). This toxin is selectively active on Tenebrio molitor causing flaccid paralysis and death but was non-toxic to mice. As conclusion this is the first report of a T. molitor selective peptide toxin. Identification of diverse insect selective toxins offer advantages in employing these peptides selectively for pest control.

Supported by: CNPq, INSERM, FAPEMIG.

Anti-bacterial activity of the extracts of the seaweed Galaxaura marginata (Rhodophyta, Nemaliales)

Rozas, E.; Freitas, J. C.

Dept. of Physiology of Biosciences Institute, University of São Paulo, São Paulo, SP, Brazil

^{Correspondence}

Anti-bacterial and haemolytic activities of substances extracted from Galaxaura marginata (J.V. Lamouroux) were investigated using seaweed collected at São Sebastião channel (45°25’ W, 26°49’ S), SP, Brazil. Samples, collected in each season, were homogenized in ethanol:acetic acid (5:1 v/v) and partitioned with hexane, obtaining the apolar fractions (H1 and H2) and polar fractions (H3 and H4). The polar fractions were filtered through membranes of 3000 and 1000 Da cut off. All fractions were tested for haemolytic activity in mouse erythrocytes and anti-bacterial activity in several Vibrio strains (Cp1: V. Anguillarum, CM3: V. damsela, F9: V. Anguillarum-Like) both in solid and liquid media. Apolar fractions and the polar fraction H3 induced haemolysis (EC50, IC95: 0,38, 0.36-0.39 (H1), 0.47, 0.42-0.52 (H2), 2.95, 2.69-3.25 (H3) mg/mL), but did not show anti-bacterial activity. In contrast, the polar fraction H4 of each season, in concentrations ranging from 1 to 10 mg/mL, exhibited inhibitory or stimulatory effects on the bacterial growth, depending on the concentration used, but even at the highest concentration tested, their haemolytic activity was inferior to the 35%. The “summer fraction” showed the strongest anti-bacterial activity in all three strains( 91±0,9 %, CM3, 96,9± 0,5 % CP1, 94.36±0,9 % F9) and the winter fraction the weakest activity (21,6±1,9 %, CM3, 49,4± 2.1 % CP1, 78,4 ±1,9 % F9). The latter, in the lowest four concentrations, showed stimulating activity in the growth of CP1 (55,37 % over the control). This fraction also stimulated the growth of V. damsela, an effect also shown by the fall fraction in F9. Differential filtering of H4 showed that only compounds with molecular weights between 3 and 1 KD had antibacterial activity. In conclusion, G. marginata has polar substances that regulate bacterial growth, stimulating or inhibiting it in a season-dependent manner.

Key words: Galaxaura, anti-bacterial, haemolytic, stimulatory..

Peptides and genes from Centruroides noxius scorpion that affect the function of Erg K+-channels

Zamudio, F.^I; Corona, M.^I; Pardo, L.^I; Gurrola, G.^I; Scaloni, A.^II; Possani, L.D.^I

^IDepartment of Molecular Recognition and Structural Biology, Institute of Biotechnology, Universidad Nacional Autonoma de Mexico, Av. Universidad, 2001 Apartado Postal 510-3, Cuernavaca 62210 – MEXICO

^III.A.B.B.A.M. Centro Internazionale Servizi di Spettrometia di Massa, CNR, Naples 80147, Italy

^{Correspondence}

Ergtoxin-1 (ErgTx1) is the first example of a scorpion toxin that inhibits specifically only the Erg K+-channels (Gurrola et al., FASEB J. 13:953-962, 1999). We discovered that a family of similar peptides is present in the same venomous gland of the Mexican scorpion Centruroides noxius Hoffmann. At least, seven peptides and genes were cloned from this scorpion. They all contain four disulfide bridges, having either 42 or 43 amino acid residues, whose primary structure similarities varies from 76 to 97% identity. The Kd of Ergtx1 examined in Xenopus laevis oocytes expressing the human hypothalamic Erg-channels (HERG) was shown to be in the order of 7 nM, whereas that of ErgTx2 was 1.2 uM. Oxidation of the Met35 of ErgTx1 decreased by almost 4 order of magnitudes its affinity for HERG-channels, heterologously expressed in the oocyte model. This chemical modification is indicative of the possible location of the active site of this toxin when interacting with HERG-channels.

Acknowledgements: Supported in part by grants from DGAPA-UNAM (IN216900), CONACyT (31691-N and Z-005) and Howard Hughes Medical Institute (55000574), to LDP.

Purification and partial characterization of a novel phospholipase A2 from Micrurus dumerilii carinicauda coral snake venom

Belo, C.A.D.^I; Leite, G.B.^I; Toyama, M.H.^II; Marangoni, S.^II; Fontana, M.D.^I; Hyslop, S.^I; Rodrigues-Simioni, L.^I

^IDepartamento de Farmacologia, FCM, Universidade Estadual de Campinas (UNICAMP), Campinas, SP

^IIDepartamento de Bioquímica, Instituto de Biologia, UNICAMP, Campinas, SP, Brasil

^{Correspondence}

OBJECTIVE: The aim of this work was to purify and partially characterize the PLA2 from Micrurus dumerilii carinicauda venom and to examine its neuromuscular activity.

MATERIAL AND RESULTS: Venom (10 mg samples) was fractionated by reverse-phase HPLC on a Sephasil Peptide C18 5 mm St 4.6/250 column (Pharmacia Biotech) equilibrated with 0.1% trifluoroacetic acid (TFA). Proteins were eluted (1 ml/min) with a linear gradient (0-65%) of acetonitrile (66% in 0.1% TFA). The elution profile was monitored at 214 nm and 280 nm and fractions were collected manually and lyophilized. PLA2 activity was assayed colorimetrically. Sequencing was done with an Applied Biosystems model Procise f Protein Sequencer and the phenylthiohydantoin amino acid derivatives were identified in a PTH amino acid analyzer. Fractionation resulted in 22 peaks. Peak 16 contained most PLA2 activity and consisted of a single protein (confirmed by RP-HPLC). N-terminal sequencing showed the first 36 residues to be NKIDFLNMIQCTTPGREPLVAFTNYGCYCGKGGSGT which shared high homology with PLA2 enzymes from Micropechis ikaheka venom (group IB PLA2). In mouse phenic nerve-diaphragm preparations, peak 16 (5 mg/ml) produced some facilitation (15±3%, n=3) with slight neuromuscular blockade after 120 min. In contrast, peak 9 (5 mg/ml) produced rapid blockade (100% in 40 min, n=4), similar to the venom (100% in 80 min, n=3), but contained no PLA2 activity.

CONCLUSION: The PLA2 from M. d. carinicauda venom contains Lys at residue 2 and causes slight facilitation, but has only weak neuromuscular activity compared to the principal neurotoxic peak and the whole venom.

Financial support: FAPESP

Comparison of the neuromuscular effects of venoms from juvenile and adult Crotalus durissus terrificus in chick nerve-muscle preparations

Pereira, R.S.^I; Belo, C.A.D.^I; Leite, G.B.^I; Cogo, J.C.^II; Simioni, L.R.^I

^IDepartamento de Farmacologia, FCM, UNICAMP, Campinas-SP, Brasil

^IISerpentário do CEN, UNIVAP, S. J.Campos ,SP, Brasil

^{Correspondence}

OBJECTIVES: The neuromuscular activity of venoms from juvenile and adult South American rattlesnakes(Crotalus durissus terrificus) was compared using chick isolated biventer cervicis muscle preparations.

METHODOLOGY: Venoms were collected from juvenile and adult snakes by manual extraction and desiccated. Male HY-LINE W36 chicks 4-8 days old were anesthetized with cloral hydrate (300 mg/kg, i.p.) and the muscles were removed and mounted in a 5 ml organ bath (Ginsborg and Warriner, 1960) containing Krebs solution (pH 7.5, 37°C) of the following composition (mM): NaCl 118.7, KCl 4.7, CaCl2 1.88, KH2PO4 1.17, MgSO4 1.17, NaHCO3 25.0, and glucose 11.65 aerated with 95% O2 and 5% CO2. Stimuli were delivered via a bipolar platinum ring electrode (0.1 Hz, 0.2 ms, 8 V, GRASS S48 stimulator) and the contractions were recorded on a Gould RS 3400 physiograph. Contractures to exogenous acetylcholine (ACh, 60 and 120 mM for 60 s) and KCl (13.4 mM for 130 s) were obtained to test for neurotoxic and myotoxic activities (Harvey et al., 1994). After a 20 min stabilization, ACh, KCl and venoms were added to the bath. ANOVA/MANOVA for repeated measures was used to test for significance.

RESULTS: The venoms of juvenile snakes tested at five concentrations (0.5, 1, 5, 10 and 20 mg/ml) produced 50% neuromuscular blockade in 36±7,32±4,20±3,20±1 and 19±3 min, respectively (mean±SEM, n=3 each). At these same concentrations, 50% neuromuscular blockade was observed with venoms from adult snakes after 40±3,35±4,24±2,27±3 and 24±2 min, respectively (n=3 each).None of the venoms affected the contractile responses to exogenous Ach and K+.

CONCLUSION: The venoms of juvenile and adult C. d. terrificus showed similar potencies in chick neuromuscular preparations, and none of them were myotoxic, as shown by their inability to affect contractures to KCl.

Financial support : CNPq

Isolation and partial characterization of a hemorrhagic protein from Bothrops lanceolatus venom

Stroka, A.; Donato, J. L.; Hyslop, S.; Lôbo de Araújo, A.

Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), CP 6111, Campinas, SP

^{Correspondence}

INTRODUCTION:Bothrops lanceolatus venom contains hemorrhagic metalloproteinases that can be inhibited by EDTA. In this work, we have purified one of these proteins.

METHODS AND RESULTS: Fraction FI obtained by gel filtration was chromatographed on Q-Sepharose preequilibrated with 0.05 M Tris-HCl, pH 7.5. The proteins were eluted (30 ml/h) with a linear gradient (0-1.0 M) of NaCl in this buffer. The active fraction (FI2) with caseinolytic and hemorrhagic activities was dialyzed against Tris-HCl, pH 7.5, and applied to a column of Blue-Sepharose that was then eluted as described above. In all cases, the elution profile of the proteins was monitored at 280 nm. Fractions FI2, FI2a and FI2b were analyzed by SDS-PAGE in 12% gels. Fractionation of FI on Q-Sepharose resulted in a major peak (FI2) which contained all of the caseinolytic and hemorrhagic activities. Chromatography of this peak on Blue-Sepharose resulted in two fractions (FI2a and FI2b), and both had caseinolytic and hemorrhagic activities. However, only FI2b showed a single band following SDS-PAGE. The estimated molecular mass of this protein was 53.3 kDa.

CONCLUSION:Bothrops lanceolatus venom apparently contains more than one hemorrhagic protease, as shown by two peaks obtained following affinity chromatography on Blue-Sepharose. The co-elution of caseinolytic and hemorrhagic activities and the molecular mass of the protein in fraction FI2b agreed with observations for other Bothrops venoms.

Financial support: FAPESP and FAEP.

Two-dimensional electrophoresis applied for snake venom analysis and for the evaluation of its immunogenicity

Flores, M.P.A.; Ricci-Silva, M.E.^I; Furtado, M.F.D.^II; Rádis-Baptista, G.^I; Prieto da Silva, A.R.B.^III; Yamane, T.^I

^IMolecular Toxinology Lab., Institute Butantan, São Paulo, SP

^IIHerpetology Lab., Institute Butantan, São Paulo, SP

^IIIBiotechnology Center, Institute Butantan, São Paulo, SP

^{Correspondence}

The two-dimensional electrophoresis (2-D PAGE) technique, first described by O'Farrell in 1975 (J.Biol.Chem. 250:4007), is the preferred method and still the highest resolution technique for gene product analysis. By combining conventional one-dimensional SDS-PAGE and isoelectric focusing (IEF) techniques, each with resolving power of -100 proteins, it can have a theoretical resolution of 10,000 individual spots. However, e.g., HeLa cells contain as many as 23,500 poly(A)-mRNA species, and many of them are low abundance proteins. Therefore, Proteomics faces a formidable challenge, far beyond our current technical abilities, unless the question is narrowed to a subset of proteins (vide Gavin, A., et at. Nature 415: 141-147, 2002).

Venom glands of animals contain a limited number of peptides/proteins, normally less than 50, with antigenic properties, therefore, amenable to 2-D PAGE analysis. Anti-venom sera rose against a given snake venoms should contain a great number of antibodies, some of which are important for neutralizing the venom's lethal effect. Cross-reactivity is a very common phenomenon, i.e., snake venoms that were not used in immunization for the sera production, can be neutralized by immunosera raised against different snake venoms. This leads to the assumption that venom of different species of genra may present common antigens/epitopes. It is quite clear that 2-D PAGE is the most suitable for identifying cross-reacting toxins.

Our studies on Bothrops jararaca venom by 2-D PAGE and subsequent identification of antigenic components using polyvalent anti-Bothrops sera have revealed the following; (a) pl of venom components lies between 4 and 7; (b) not all components are immunogenic and it is not correlated with lhe amount present as detected by silver staining; (c) among isoforms, those with a lower pl seem to be more antigenic.

Support: FAPESP

Serum levels of creatine kinase (CK) and local tissue lesions in the envenomation by Bothrops and Crotalus snakes: Clinical - laboratorial and magnetic resonance image (MRI) evaluation

Fonseca, M.G.^{I, II}; Mathias, M.R.C.^I; Yamashita, S.^I; Morceli, J.^I; Barraviera, B.^{I, III}

^IHospital das Clínicas-Department of Tropical Diseases and Imaging Diagnosis, Medical School of Botucatu- UNESP, São Paulo, Brazil

^IIDepartment of Biology-Health Center, FAFIBE-FANORP, State of São Paulo, Brazil

^IIICenter for the Study of Venoms and Venomous Animals-CEVAP-UNESP, State of São Paulo, Brazil

^{Correspondence}

Snake bite envenomation frequently causes alterations in the CK levels associated with local tissue and systemic lesions on skeletal muscles. The present study aimed to evaluate these alterations due to envenomation by Bothrops and Crotalus snakes as well as the evaluation of the local tissue lesions by magnetic resonance images (MRI). For specific treatment, fifteen patients bitten by Bothrops (group Bothrops) and six bitten by Crotalus (group Crotalus) were hospitalized at the Tropical Diseases Section of Hospital das Clínicas of the Medical School of Botucatu. Evaluation of biochemical parameters through the dosage of the CK levels by ultraviolet kinetic method was performed after their admission. Afterwards, they were examined by MRI to screen the damaged member. In the Bothrops group, five patients showed lesions in the subcutaneous tissue, and CK mean value was 260 mUI/l. In six patients, the lesions were in perimuscular areas, CK mean value was 198 mUI/ml. Muscular lesions were found in four patients, CK mean value was 150 mUI/ml. In the Crotalus group, the lesion in subcutaneous tissue were observed in two patients, serum mean level of CK was 1014 mUI/ml. Lesion in perimuscular areas was observed in one patient, CK value of 7620 mUI/ml. Three patients showed lesions in muscular tissue, CK mean level of 4586 mUI/ml. The standard normal value of CK on plasma was 80 mUI/ml. In the Bothrops group, the level of CK was higher in the patients with lesions in subcutaneous tissue. In the patients from the Crotalus group with muscular lesions, the level of CK was higher. The resonance magnetic images showed lesions in subcutaneous tissue, perimuscular and muscular areas in both groups. Although some pacients in both groups presented no local muscular lesions as determined by MRI, the levels of CK were increased.

Support: CAPES

Mechanism of induction of complement susceptibility of erythrocytes by spider and bacterial sphingomyelinases

Tambourgi, D. V.^I; Silva, M.S.^I; Billington, S. J.^II; Andrade, R.M.G.^I; Magnol, F.C.I.^I; Glenn Songer, J.^II; Van den Berg, C.W.^III

^ILaboratório de Imunoquímica, Instituto Butantan, Brazil

^IIDepartment of Veterinary Science and Microbiology, University of Arizona, USA

^IIIDepartment of Pharmacology, Therapeutics and Toxicology, University of Wales, College of Medicine, UK

^{Correspondence}

We have recently shown that the sphingomyelinase toxins P1 and P2 from the venom of the spider Loxosceles intermedia induces Complement (C) dependent lysis of autologous erythrocytes by induction of cleavage of cell surface glycophorins (GPs) through activation of an endogenous metalloproteinase facilitating the activation of the alternative pathway (AP) of C. Phospholipase D (PLD) from Corynebacterium pseudotuberculosis shows some degree of homology with the spider sphingomyelinases and can induce similar clinical symptoms to that observed after spider envenomation. The aim of this study was to investigate if the bacterial PLD-induced haemolysis of human E was C dependent and if cleavage of GPs occurred. We show here that haemolysis of both PLD and P1 treated human E was C dependent, but while PLD mediated haemolysis was dependent on activation of the classical pathway of C, P1 induced lysis via both the classical and alternative pathway. P1 but not PLD induced cleavage of glycophorins and no change in expression of complement regulators was induced by either of the toxins. In both cases Annexin V binding sites were exposed, suggesting that the membrane asymmetry had been disturbed causing exposure of phosphatidylserine (PS) to the cell surface. Our results suggest that C susceptibility induced by L. intermedia and C. pseudotuberculosis PLD is due to exposure of PS, and the higher potency of P1 toxin can be explained by its additional effect of cleavage of glycophorins.

Supported by the Wellcome Trust as a Collaborative Research Initiative Grant to DVT and CWB

First report of the capture of Loxosceles laeta (Araneae, Sicariidae) in the west zone of São Paulo city, SP, Brazil

Andrade, R.M.G.; Tambourgi, D.V.

Laboratório de Imunoquímica, Instituto Butantan, São Paulo, Brazil

^{Correspondence}

Loxosceles laeta is one of the brown spider species responsible for loxoscelism, the most severe form of araneism in Brazil. Loxosceles laeta is very well adapted in the anthropic environment and has the widest geographic distribution among the Loxosceles species. It is probably an endemic specie in the West area of South America, although it has been introduced in the East area, as well as in the Central and North Americas. The dispersion of Loxosceles spiders is facilitated because of their criptozoic habits. In Brazil the presence of Loxosceles laeta was registered in the states of "Paraíba" to "Rio Grande do Sul". Recently, we found several specimens of Loxosceles laeta in the West area of São Paulo City, Pirituba district, in the “Pinel Psychiatry Hospital”. This hospital has an area of 77 thousand square meters, from which 32 thousands are of forest. The spiders were captured not only in the external areas as well as in the buildings of the hospital. This is the first report of the occurrence of Loxosceles laeta in the West area of São Paulo city. The presence of L. laeta has only been, previously, registered by Bucherl in 1961, in the South area of São Paulo city, Ipiranga district. Considering that loxoscelism is an important health problem in the South region of Brazil, it is necessary to investigate the dynamic and spread of the Loxosceles laeta population in the city of São Paulo.

Supported by FAPESP, The Wellcome Trust

Detection of Escherichia coli producing LT-I enterotoxin by capture immunoassay

Menezes, C.A.^I; Lima, F.A.^I; Fernandes, I.^II; Amianti, J.^I; Koga, P.C.M.^I; Trabulsi, L.R.^I; Piazza, R.M. F.^I

^ILaboratório Especial de Microbiologia, Instituto Butantan, São Paulo - SP

^IILaboratório de Imunopatologia, Instituto Butantan, São Paulo - SP

^{Correspondence}

Enterotoxigenic Escherichia coli (ETEC) is an important pathogen responsible for traveler’s diarrhea and causes more than 700,000 childhood deaths per year due to diarrhea in third-world countries. ETEC strains cause diarrhea through the action of the enterotoxins ST and LT. These strains can express ST-I , LT-I , or both. Detection of ETEC has been long relied on detection of the enterotoxins LT and/or ST through biological methods, immunoassays and molecular diagnostic techniques. The aim of present data was standardized an immunoassay for detection of diarrheagenic E. coli producing LT-I toxin. Cell fusion with B cells from lymph nodes of immunized mice with LT-I toxins resulted in one positive and stable clone, characterized as IgG2b with binding capacity of 8,4 x 10⁴ molecules to LT-I and dissociation constant of 3 x 10^-6M. This monoclonal antibody was able to inhibit 50% the ability of LT-I to bind to GM1 receptor. We also raised rabbit´s antisera to LT-I toxin. The IgG enriched fraction of this antisera showed specificity and was able to inhibit up to 85% of the binding of LT-I to its receptor. The capture assay developed using the IgG enriched fraction of anti-LT-I antisera and IgG2b anti-LT-I monoclonal, allowed a clear distinction between E. coli LT-I - producing and non-producing. Statistical analysis showed that in capture immunoassay the variances between two groups are significantly different, and these results suggests that sensibility of capture immunoassay is higher than ELISA-GM1 and Vero cells assay.

Tc30, a novel toxin from the Amazonian scorpion T. cambridgei that blocks shaker B K + channels

Batista, C.V.F.^I; Gómez-Lagunas, F.^I; Zamudio, F.Z.^I; Lucas, S.^II; Possani, L.D.^I

^IDepartment of Molecular Recognition and Structural Biology, Institute of Biotechnology, National Autonomous University of Mexico, Av. Universidad, 2001, Cuernavaca 62210 - Mexico

^IIInstituto Butantan, Av. Vital Brazil, 1500, Sao Paulo, Brazil

^{Correspondence}

The venom of scorpions constitutes a rich source of peptides toxic to a variety of organisms. In Brazil the scorpions of the family Buthidae, genus Tityus (T.) are medically important and several species have been studied, among which are: T. serrulatus, T. bahiensis, T. stigmurus. The venom of the scorpion T. cambridgei, has been less studied (Batista et al. Toxicon 40:557-562, 2002), although it contains a variety of peptides that affect Na⁺ and K+-channel function (Batista et al FEBS Lett. 486:117-120, 2000). In this communication we described the isolation, full sequence and electrophysiological studies of a novel toxin peptide (Tc30) from the venom of this Amazonian scorpion. This peptide was isolated by several steps of high performance liquid chromatography. It was sequenced by automatic Edman degradation and MS/MS fragmentation. Pure peptide was tested against Shaker B K+ channels expressed in Sf9 cells. Tc 30 has 37 amino acid residues, with molecular mass of 3871.8 a.m.u., and contains three disulfide bridges. It is relatively potent inhibitor of Shaker B K+ channels with Kd value of 74 nM. Tc 30 has a lysine in the position 27 and a tyrosine at position 36, identical to those of Charybdotoxin. Comparison of the amino acid sequence of Tc30 with data bank data showed that it belong to the Tytus II-9 subfamily and was given the number a-KTx4.4.

Acknowledgements: Supported in part by grants from DGAPA-UNAM (IN216900), CONACyT (31691-N and Z-005) and Howard Hughes Medical Institute (55000574), to LDP.

Effect of SDS and the zwitterionic surfactant HPS on the conformation and hemolytic activity of St I and St II, two isotoxins from Stichodactyla helianthus

Lanio, M.E.^I; Alvarez, C.^I; Pazos, F.^I; Martinez, D.^I; Martínez, Y.^I; Casallanovo, F.^II; Campos, A.M.^III; Abuin, E.^III; Schreier,S.^II; Lissi, E.^III

^IUniversity of Havana, Cuba

^IIUniversity of Sao Paulo, Brazil

^IIIUniversity of Santiago, Chile

^{Correspondence}

Pore forming toxins are extremely sensitive to changes in their conformation. We have shown that the hemolytic activity of Sticholysins I and II (St I and St II) is dependent of those factors that change their conformation. There are no data regarding the effect of detergents on the structural and functional characteristics of cytolysins. In the present work, the interaction of St I and St II with sodium dodecyl sulfate (SDS) and N-hexadecyl-N-N'-dimethyl-3-ammonio-1-propane-sulfonate (HPS) was characterized. At low surfactant concentrations SDS leads to the formation of aggregates and the proteins show an increase in intrinsic fluorescence intensity and reduced quenching by acrylamide, with an almost total loss of their hemolytic activity. At higher surfactant concentrations it was observed a loss of the protein-aggregated state. This produces a decrease in fluorescence intensity, increase in quenching efficiency by acrylamide, loss of the native tertiary conformation, and increase in a-helix content. However, the toxins partially recover their hemolytic activity. The results suggest that, as a consequence of the interaction with SDS micelles, the proteins adopt an a-helix-enriched, more open structure. This structure might be able to leave the micelle and convert to the native b-structure-rich conformation in the presence of red blood cell membranes, recovering the ability to form the pore. On the other hand, the binding constants of St I and St II faced to HPS were ca.0.5 - 0,7 mM-1. The changes elicited by HPS lead to a more expanded tertiary conformation than the native structure. In spite of this, the toxins fully retain their hemolytic activities. This indicates that spectroscopic changes can be poor predictors of toxin activity.

The interaction of ST I and II, two pore-forming toxins from the sea anemone Stichodactyla helianthus with membranes

Alvarez, C.^I; Lanio, M.E.^I; Nogueira, L.V.^II; Pazos, F.^I; Martinez, D.^I; Tejuca, M.^I; Casallanovo, F.^II; Shida, C.^II; Menestrina, G.^IV; Lissi; E.^III; Schreier, S.^II

^IUniversity of Havana, Cuba

^IIUniversity of São Paulo, Brazil

^IIIUniversity of Santiago, Chile

^IVCenter for Aggregate State Physics, Italy

^{Correspondence}

Sticholysins I (St I) and II (St II) are two highly hemolytic polypeptides that form oligomeric pores of radius around 1 nm in cell and lipid membranes containing sphingomyelin. The hemolytic activity of St II is ca. 5 times higher than that of St I. Binding to vesicles of phosphatidylcholine (PC): sphingomyelin (SM) produced an increase in both St I and II fluorescence intensity being larger for St I. This indicates a greater exposure of St I Trp to a less polar environment. Neither far- nor near-UV CD spectra revealed extensive conformational changes upon St I and St II binding to PC:SM vesicles. The inclusion of small proportions of a third lipid, particularly phosphatidic acid (PA) and cardiolipin (CL) into PC:SM vesicles led to a marked increase in calcein release caused by St I and St II. Probably, the insertion of the toxin channel could imply the formation in the bilayer of a nonlamellar structure, a toroidal lipid pore. This possibility is confirmed by the fact that the formation of toxin pores strongly promotes the rate of transbilayer movement of lipid molecules. Lipid-protein interaction and the degree of penetration of both toxins into PC:SM membranes was monitored by EPR. The proteins interact with the bilayer conducing to a more immobilized lipid population. St I and St II had essentially no effect on the spectra of probes labeled at C-16. We have also examined fluorescence quenching by PC-spin labeled. The smallest effect was observed for 16-PCSL, both for St I and St II, corroborating the EPR findings and could be explained considering the existence of toroidal lipid pore.

Construction and evaluation of a membrane-lytic immunoconjugate selective for colon cancer cells

Díaz,I.^I; Figueredo, R.^II; Roque, L.^II; Pazos, F.^I; Martínez, D.^I; Iznaga-Escobar, N.^II; Pérez, R.^II; Alvarez, C.^I; Lanio, M. E.^I; Tejuca, M.^I

^ICentro de Estudios de Proteínas y Departamento de Bioquímica, Facultad de Biologia, Universidad de La Habana, Calle 25 #455 e/ J e I, Vedado, Ciudad de La Habana, Cuba

^IICentro de Inmunología Molecular, P.O. Box 16040, Ciudad de La Habana 11600, Cuba

^{Correspondence}

Sticholysin I, a potent cytolysin isolated from the sea anemone Stichodactyla helianthus, was linked to the monoclonal antibody ior C5. Sticholysin I acts by forming oligomeric hydrophilic pores in the membrane of the attacked cells, impairing cellular functions and leading to osmotic lysis. The monoclonal antibody ior C5 is a murine IgG1, which recognizes the tumor associated antigen ior C2, a glycoprotein expressed at the cell surface in more than 83% of primary colorectal carcinomas. The cytolysin and the monoclonal antibody were coupled by using the heterobifunctional cross-linking reagent Sulfosuccinimidyl 4-(N-Maleimidomethyl)-cyclohexane-1-carboxylate (SMCC). Two hybrid molecules composed by one ior C5 and one or two Sticholysin I molecules were obtained, as demonstrated by electrophoresis. The conjugates called I and II, respectively, were purified by hydrophobic interaction chromatography in a Progel-TSK Phenyl-5PW column. The purified conjugates were evaluated by a binding affinity assay against an ior C2-positive colon cancer cell line (SW948), and compared with the unconjugated ior C5. The hybrid molecules were able to recognize their specific antigen in the same way that ior C5 does. Both conjugates lost most of their hemolytic activity but their residual activity was very similar. Nevertheless, when we studied their cytotoxicity on the colon cancer cell line SW948, only the conjugate II killed efficiently the cells, indicating a specific antigen-monoclonal antibody interaction of this hybrid molecule. This suggests that this could be a useful approach for immunotoxin design.

High yield purification and characterization of serine proteinases from the venoms of Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni and Crotalus durissus terrificus

Murakami, M.T.^I; Laure, H.J.^II; Greene, L.J.^II; Arni, R.K.^I

^IDepartment of Physics, IBILCE/UNESP, São José do Rio Preto, SP

^IIFaculdade de Medicina de Ribeirão Preto, USP, Ribeirão Preto, SP

^{Correspondence}

The serine proteinases encountered in snake venoms, especially in the venoms of Viperidae e Crotalidae families, have been the focus of attention since they have many medical applications, for example, Calloselasma rodhostoma Ancrod, Bothrops atrox moojeni Batroxobin, Bothrops jararaca Bothrombin, Crotalus atrox Calobin, Crotalus adamanteus Crotalase and Trimeresurus flavoviridis Flavoxobin. These enzymes are very important because that are involved in many biological process, such as plasminogen activation, factor IIa, V, X activation in the blood coagulation cascade and platelet aggregation. The aim of this work was to develop a rapid procedure for purifying large quantities of the serine proteases from the venom of Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni and Crotalus durissus terrificus in high purity for crystallographic and biochemical studies.

The serine proteinases from the venoms were purified using affinity chromatography on benzamidine-sepharose 6B as an important step. The purity and molecular masses were estimated by SDS-PAGE. The crystallizations were carried out by the hanging-drop vapor-diffusion method using 24-well tissue-culture plates. The activities of these enzymes were tested using bovine fibrinogen as substrates. These enzymes were present as single bands in silver stained SDS-PAGE gels and had molecular masses of approximately 28kDa. The sequence of the first fifteen amino acids of the enzyme from the venom of Bothrops jararacussu was determined and compared to the cDNA sequences of venom proteases available in the data bank that demonstrated high homology. The single crystals obtained prove the purity of these enzymes.

X-ray diffraction data of the enzymes from Bothrops e Crotalus sp. have been collected to high resolution and the structure determination is in progress. We are currently trying to improve the quality of the crystals of the other enzymes by including substrates and inhibitors. This work will contribute to improve our understanding of the stereochemical requirements of these enzymes.

Financial support: FAPESP and CNPq.

Cloning and DNA sequencing of a g-phospholipase A2 inhibitor in Lachesis muta muta homologous to CNF from the blood plasma of Crotalus durissus terrificus

Barcellos, C.J.^I; Estevão-Costa, M.I.^I; Velarde, D.T.^II; Cotta, G.A.^II; Fortes-Dias, C.L.^I

^ICentro de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias (FUNED), Belo Horizonte, MG

^IIDivisão de Imunobiológicos, Fundação Ezequiel Dias (FUNED), Belo Horizonte, MG

^{Correspondence}

A phospholipase A2 inhibitor, named CNF, has been previously purified and characterized from the blood plasma of C. d. terrificus (the South American rattlesnake). In the present work we have cloned and sequenced a homologous inhibitor encoded in the liver of another Crotalidae, Lachesis muta muta (the bushmaster snake). Total RNA has been extracted from the liver of one specimen using TRIZOL (GIBCO). After RT-PCR with a commercially available kit, the cDNA was amplified by PCR using specific primers to the non-coding and carboxy-terminal regions of the CNF gene. The amplicon was cloned using a TA cloning kit and putative positive colonies were screened by PCR. Two positive clones were selected and the recombinant DNAs were purified (Wizard® miniprep, Promega) and sequenced. The DNA and deduced protein sequences obtained were compared with available sequences of g-inhibitors from other snake species using the Vector NTI®software confirming the high degree of homology among them.

Financial support: FAPEMIG

Cloning and DNA sequencing of a a-phospholipase A2 inhibitor encoded in the liver of Crotalus durissus terrificus

Estevão-Costa, M.I.^I; Velarde, D.T.^II; Maciel, R.^II; Fortes-Dias, C.L.^I

^ICentro de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias (FUNED), Belo Horizonte, MG

^IIDivisão de Imunobiológicos, Fundação Ezequiel Dias (FUNED), Belo Horizonte, MG

^{Correspondence}

Several authors have reported the presence of PLA2 inhibitors in the blood plasma of snake species. These inhibitors have been divided into three classes (alpha, beta and gamma) according to their structure (Ohkura et al., 1997). In the present work we have cloned the cDNA encoding a PLA2 inhibitor from the a-class in a Brazilian snake species. Total RNA was extracted from the liver of C. d. terrificus (the South American rattlesnake) and submitted to RT-PCR in the presence of specific oligonucleotides, the design of which were based on the published sequence of a similar inhibitor isolated the blood plasma of the Chinese mamushi Agkistrodon blomhoffii siniticus (Ohkura et al., 1997). The amplification product was cloned in TA pCR 2.1 vector and putative positive colonies were screened by PCR with appropriate primers. Recombinant clones were subjected to sequencing on an automated DNA sequencer. On the basis of the sequence data generated the complete nucleotide sequence of C. d. terrificus a-PLA2 inhibitor was determined and compared to members of the a-class of inhibitors from other snake species using the Vector NTI^® software. Structurally, the a-inhibitors display motifs homologous to the carbohydrate recognition domain (CRD) of C-type lectins.

Financial support: FAPEMIG

Studies on the immunogenicity and stability of the epsilon prototoxin of Clostridium perfringens type D detoxified by controlled iodination

Parreira, M.P.; Aurélio, R.; Campos, P.C.; Júnior, A.D.C.; Lobato, F.C.F.; Heneine, L.G.D.

Immunology Laboratory, Fundação Ezequiel Dias, Rua Conde Pereira Carneiro, 80 – Gameleira, Belo Horizonte, MG, Brasil

^{Correspondence}

A survey on the efficacy of 6 vaccines commercialized in Brazil against Clostridiosis revealed a very weak or no protection in neutralization tests performed in mice (Azeved, E.O., Lobato, F.C.F.,Abreu, V.L.V, 1998, Arq. Bras. Med. Vet., 1998, v. 50, n.3, p.239-242). Most vaccines are toxoids from concentrated culture medium where the toxin is present in low amounts. The use of purified toxoid would allow for better formulation and immunogenicity of the vaccine. The epsilon prototoxin was purified by ion-exchange chromatography on DEAE-Sepharose and a single band was observed by SDS-PAGE and silver staining. The incorporation of cold iodine (Heneine, L.G.D., and Heneine, I. 2001, Toxin Reviews, v.20, n.3/4, p. 209-228) to the prototoxin resulted in the detoxification of the activated toxin by trypsin, showing no lethality when injected intravenously in mice. Serum from sheep immunized with the iodinated toxoid had high anti-epsilon titres when tested by cELISA. The purified prototoxin shows a high level of denaturation (Worthington et al, 1973. J. Vet. Res. v.40 p.143-152), a lot kept at –20 °C was totally denatured after 6 months, when evaluated by its protein content, using the coefficient exctintion method and another lot kept at –80 °C decreased its protein content by 50%. The iodinated toxoid had increased stability when kept at –20 °C and when kept at –80°C there was no decrease protein content measured by the exctintion coefficient and by the appearance of denaturation bands on SDS-PAGE. The iodinated toxoid could be tested as a vaccine antigen.

Aknowledgements: CNPq, FAPEMIG.

A competitive ELISA for the potency assay of vaccines and antitoxin sera against the epsilon toxin of Clostridium perfringens type D

Parreira, M.P.; Aurélio, R.; Campos, P.C.; Júnior, A.D.C.; Lobato, F.C.F.; Heneine, L.G.D.

Immunology Laboratory, Fundação Ezequiel Dias, Rua Conde Pereira Carneiro, 80 – Gameleira, Belo Horizonte, MG, Brasil

^{Correspondence}

Competitive ELISA (cELISA) was standardized to detect antibodies to Clostridium perfringens Type D epsilon prototoxin as an alternative to the toxin neutralization test performed in mice. Epsilon prototoxin was purified by ion-exchange chromatography on DEAE-Sepharose column and detoxified by controlled iodination and used to coat microwell plates. Sera were tested for their ability to inhibit the standard ELISA reaction between rabbit anti-epsilon immunoglobulin and the epsilon prototoxin. The potency of the test serum was calculated from a standard curve produced with the International Standard anti-epsilon serum obtained from the National Institute for Biological Standards and Control (NIBSC). The applicabitility and repetibility of the test was measured by calculating the in vitro potency of sheep anti-epsilon toxin sera, in different days using standard curves prepared in the same day and previously elaborated. The potencies obtained were averaged and compared to the potency obtained with the neutralization test in mice. The correlation observed was equal to r=0.94. Cattle sera vaccinated with clostridial vaccines against C. perfringens type C and D, were assayed for antitoxin potency in mice and with the cELISA. The correlation was r= 0.87.The results indicate that the cELISA can reliably quantify the potency of anti-epsilon toxin sera and replace the neutralization test in mice.

Acknowledgements: FAPEMIG, CNPq.

Linear protein-protein interactions in crotoxin by spot synthesis: Identification of contact sites between its subunits

Santos, R.M.M.^I; Chávez-Olórtegui, C.^I; Granier, C.^II; Fortes-Dias, C.L.^I

^ICentro de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias (FUNED), Belo Hte., MG

^IIFaculté de Pharmacie, CNRS UMR 9921, Montpellier, France

^{Correspondence}

The venom from Crotalus durissus terrificus is composed mainly of a neurotoxic protein named crotoxin, which is known to contain the bulk of the lethal toxicity of the crude venom. Crotoxin is a heterodimer composed of a basic toxic PLA2 subunit (CB) and of an acidic, non-toxic and non-enzymatic subunit (CA). In the present work we have used the SPOT method of multiple peptide synthesis to measure the ability of CA to bind to linear segments of CB. Sets of overlapping short peptides (12 residues) spanning the primary amino acid sequences of two main isoforms of CB were immobilized onto a cellulose membrane and probed with CA previously labelled with alkaline phosphatase. Colour intensities were measured after scanning the membrane and relative reactivities were calculated with reference to a black spot taken as 100 %. Four to five linear regions of CB have been mapped that might be involved in the interaction with CA in the crotoxin complex.

Financial support: FAPEMIG and CNPq

Is the phospholipase A2 (PLA2) activity of the MT-II and MT-III isolated from Bothrops asper venom important for macrophages functions?

Zuliani, J.P.^I; Sampaio, S.C.^II; Gutierrez, J.M.^III; Teixeira, C.F.P.^I

^ILab. of Pharmacology, Butantan Institute, SP – Brazil

^IILab. of Pathophysiology, Butantan Institute, SP – Brazil

^IIIClodomiro Picado Institute, San José – Costa Rica

^{Correspondence}

INTRODUCTION AND GOALS: MT-III is a PLA2-Asp49 with high enzymatic activity and MT-II, a PLA2-Lys49, devoid of catalytic activity. However both are myotoxic. In the present work we have investigated the ability of these myotoxins to induce activation of macrophages (phagocytosis, H2O2, NO and cytokines liberation) and the participation of PLA2-activity in these effects .

METHODS AND RESULTS: Macrophages were obtained from Swiss mice peritoneal cavity 96 hours after intraperitoneal injection of thioglicolate (3%). Cell viability was measured by Trypan blue exclusion test. Phagocytosis via C3b receptor, via Fcg receptor and via mannose receptor were studied with opsonized zymosan, opsonized sheep red blood cells and Candida albicans, respectively. Release of H2O2 and NO was measured by Phenol red and Griess reaction, respectively. Myotoxins (1 mg/kg) or saline were injected in the peritoneal cavity. At selected time, peritoneal washes were colected and concentrations of IL-1, IL-6 and TNF-a were measured by ELISA and L929 cytotoxicity, respectively. Both myotoxins are cytotoxic for macrophages only at high concentrations (50-200 mg/mL). MT-II affected phagocytosis via C3b, Fcg and mannose receptors (p<0,05). On the other hand, MT-III affected only phagocytosis via mannose receptor (p<0,05). Both myotoxins induced a significant release of H2O2 and NO (p<0,05) from isolated macrophages as well as an increase in concentrations of IL-1, IL-6 and TNF-a (p<0,05) in the peritoneal washes.

CONCLUSION: These data suggest that myotoxins are able to stimulate microbicidal functions of macrophages and the release of cytokines. These do not appear to be connected with PLA2 activity. Therefore, distinct domains in the molecule of the myotoxins are important for activation of the macrophage functions.

Financial Support: FAPESP.

Delineation of linear epitopes of a hemorrhagic factor from Lachesis muta muta snake venom by multiple peptide synthesis and phage display

Castanheira, P; Bohórquez, K; Martins, M.S.; Villard, S.; Granier, C.; Sanchéz, E.F.; Chávez- Olórtegui, C.

Fundação Ezequiel Dias, and Departamento de Parasitologia, UFMG, Belo Horizonte, MG, Brazil. Institut de Biotechnologie et Pharmacologie, CNRS UMR 5094, Faculté de Pharmacie, Montpellier, France

^{Correspondence}

Local or evasive hemorrhage is a major complication resulted from envenomation by Bothrops and Lachesis snake venoms. Mutalysin-II is a 22.5 kDa single chain protein, purified from the Lachesis venom with broad substrate specificity and hemorrhagic effects. We have previously shown that polyclonal antibodies and a monoclonal antibody against Mutalysin showed cross-reactivity and neutralized the hemorrhagic effects of some Bothrops and Lachesis whole venoms. These results suggest that Mutalisin-II is a very efficient antigen, which might have potential for the preparation of efficient anti-venom antisera for passive immunotherapy or even for human vaccination.

Two different approaches, the phage display technique and Spot peptide synthesis on cellulose membranes, were used to identify sequences recognized by specific antibodies against Mutalisyn-II, and define the preferred chemical composition of functional epitopes. Our results show that the functional epitope of monoclonal and polyclonal antibodies derived from the complete amino acid sequence of mutalisyn and identified by Spot synthesis methods requires precise physico-chemical properties at a limited number of positions, and that residues flanking these key residues can influence binding affinity. However, the phage display method localizes functional epitopes not related with the amino acid sequence.

The results obtained in the present study indicate that the phage display and Spot synthesis methods should be used as the techniques for the precise localization of conformational and linear funcional epitopes, respectively.

Supported by: CNPq, INSERM.

Kallikrein-like proteinase from bushmaster snake venom

Vilela, L.F.^I; Souza, C.T.^I; Bello, C.A.^I; Magalhaes, A.^I; Almeida, A.P.^II; Richardson, M.^I; Sanchez, E.F.^I

^ICentro de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias, Belo Horizonte, MG

^IIDept. de Fisiologia e Biofísica, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil

^{Correspondence}

Objectives.The experiments in this work were focused on the biochemical characterization of a kallikrein-like proteinase (LV-Ka) from Lachesis muta muta (bushmaster) snake venom. The isolated protein is a 33 kDa monomeric glycoprotein which Mr was reduced to 28 kDa after deglycosylation with PNGase F. LV-Ka showed kallikrein-like activity, releasing bradikinin from kininogen as evidenced by guinea pig bioassay. In addition, intravenous injection of the proteinase (1 mg/g) was shown to lower blood pressure in experimental rats. In vitro, the purified enzyme was shown to have neither fibrino(geno)lytic activity nor coagulant effect. LV-Ka was active upon the kallikrein substrates S-2266 and S-2302 (specific activity= 13 and 31.5 U/mg, respectively, crude venom= 0.25 and 6.0 U/mg), but had no proteolytic effect on dimethylcasein and insulin B chain. Its enzymatic activity was inhibited by NPGB, p-aminobenzamidine and PMSF, indicating that the enzyme is a serine proteinase. Approximately 76 % of its protein sequence was determined by sequencing the various fragments derived from CNBr cleavage and digestion with endoprotease. Sequence studies on the NH2-terminal region of the protein indicates that LV-Ka shares a high degree of sequence homology with the kallikrein-like enzymes EI and EII from Crotalus atrox, with LMR-47 from L.m.rhombeata and significant homology with other serine proteinases from snake venoms and vertebrate serum enzymes. Interestingly, one of the other reactions catalyzed by the venom enzyme, the activation of plasminogen was also exhibited by LV-Ka. In conclusion, these studies indicate that LV-Ka is a kallikrein-like enzyme from snake venom origin which may also participate in the extrinsic pathway of fibrinolysis.

Financial support: CNPq and FAPEMIG.

Comparative study of coagulant thrombin-like enzymes from the venoms of Brazilian and Peruvian bushmaster (Lachesis muta muta) snakes

Magalhaes, A.^I; Richardson, M.^I; Ferreira, R.N.^I; Gontijo, S.^I; Yarleque, A.^II; Magalhaes, H.P.B.^III; Block, C.^IV; Sanchez, E.F.^I

^ICentro de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias, Brazil

^IIDept. de Ciencias Biológicas, Universidad. Nacional Mayor de San Marcos, Perú

^IIIFaculdade de Farmacia, Universidade Federal de Minas Gerais, Brazil

^IVDept. de Bioquímica, Universidade de Brasilia, Brazil

^{Correspondence}

Objectives. The aim of this work was to conduct a comparative study of two thrombin-like enzymes (TLE-B and TLE-P) purified from the venoms of Lachesis muta muta snakes captured in two different geographical localities, Manaus (Brazil) and Pucallpa (Perú). TLE-B and TLE-P showed Mr values of 40 and 41 kDa, under reducing conditions on SDS-PAGE, which were reduced to 27 kDa after treatment with PNGase F. The proteinases split off fibrinopeptide A from human fibrinogen and fibrinopeptide B more slowly. In addition, the enzymes removed a 42-residue polypeptide from the NH2-terminal end of the Bb-chain. Their specific clotting activities were equivalent to 1000 and 900 NIH thrombin units/mg on human fibrinogen and 526 and 606 NIH thrombin units/mg on bovine fibrinogen for TLE-B and TLE-P, respectively. When injected into the tail veins of mice (15 – 120 ng/g mouse), both enzymes induced temporary episodes of opisthotonos and rapid rolling around the long axis of the animals. Kinetic properties of both enzymes were determined using representative chromogenic substrates. Tryptic peptide mapping of the two native enzymes suggested a large degree of structural similarity. Purified rabbit IgG against TLE-B reacted with both enzymes forming a continuous precipitin line on immunodiffusion. Furthermore, western blot and indirect ELISA were used to compare the antigenic cross-reactivity for both enzymes as well as with the venoms of L.m.muta and Bothrops snakes. Incubation of human a2-macroglobulin with each enzyme at molar ratios of 1:1 and 1:2 enzyme:inhibitor, resulted in retarding their clotting activities by about 12 times, whereas their amidolytic activities were not affected. Similarly, inhibition of their clotting activities was obtained using high concentrations of rabbit IgG (40 mg, corresponding to molar ratio enzyme:inhibitor of 1:2). We conclude that TLE-B and TLE-P are nearly identical clotting enzymes.

Financial support: CNPq and FAPEMIG.

Crotamine and crotasin, two evolutionary related peptides from the Crotalus durissus terrificus (South American rattlesnake)

Rádis-Baptista, G.^I; Hayashí, M.A.F.^II; Grego, K.F.^III; Oliveira, E.B.^IV; Yamane, T.^I

^IMolecular Toxinology Lab., Institute Butantan, São Paulo, SP

^IIBiochemistry and Biophysics Lab., Institute Butantan, São Paulo, SP

^IIIHerpetology Lab., Institute Butantan, São Paulo, SP

^IVBiochem. Dept., Faculty of Medicine, USP, Ribeirão Preto, SP

^{Correspondence}

Crotamine (myotoxin) is a small basic peptide (MW 4400, pl > 9.5), widely distributed among venoms of the genera Crotalus from the prairies of the Western United States to the pampas of South America. However, its presence/absence varies according to the subspecies and the geographic locality of a given species (Schenberg, S., 1959. Science 129:1361, Ownby, C.L., 1998. J.Toxicol. 17:213). In order to address the evolutionary relationship between crotamine-positive and -negative variants, their genomic structures were studied, to verify the structural organization of crotamine gene and why it is not transcribed in the venom glands of crotamine-negative specimens. Genomic DNA, Crt-p1, with the size of 1.8 kb, encoding the precursor of crotamine was isolated from the liver of crotamine-positive specimen, but we were unable to find similar gene in the crotamine-negative one. Instead, a 2.5 kb size gene, designated crotasin, was isolated, paralogous to crotamine gene, presenting virtually identical overall structural organization: three exons separated by two introns, a long phase-1 and a short phase-2 introns. Crotasin gene, Cts-p2, which happens to occur in both crotamine-positive and -negative ones, when compared to Crt-p1 gene, shows a considerable mutations involving a high nucleotide substitutions (134 bases), as well as deletions (23 bases) and an insertion of 892 bases, due partly to the duplication (760 bases) of intron segments. Base substitutions occur mostly in the crotamine-coding region, leading to a hypermutated peptide (only 11 out of 42 amino acids are preserved), in contrast to a highly conserved signal peptide sequence and equally conserved upstream and downstream gene flanking regions. One striking fact is that all 6 cysteine residues are preserved maintaining, probably, the same fold. Crotasin is expressed in various tissues, and at a high concentration in pancreas. The probable function of this novel peptide is under investigation.

Support: FAPESP

Effects of hementerin, a fibrino(geno)lytic metalloprotease from leech, on human platelet aggregaton. Involvement of nitric oxide pathway

Chudzinski-Tavassi, A.M.^I; Lazzari, M.A.^II; Rosenstein, R.^III; Faria, F.^I; Keller Sarmiento, M.I.^III; Alberto, F.^II; Bermejo, E.^II

^ILaboratório de Bioquímica e Biofísica, Instituto Butantan, SP. Brasil

^IIIIHEMA, Academia Nacional de Medicina de Buenos Aires

^IIIFacultad de Medicina, Dto Biología Humana, Universidad de Buenos Aires, Argentina

^{Correspondence}

Hementerin (HT) is a fibrino(geno)lytic metalloprotease of 80 kDa, purified from salivary complexes of Haementeria depressa leech. In this report was examined the effect of Hementerin on several human platelets´s parameters.

Platelet rich plasma (PRP) was preincubated with different concentrations of HT (0 - 10mg/ml), then 1 mg/ml collagen (final concentration) was added and aggregation recorded. HT inhibited platelet aggregation being IC50 of 7.5mg/ml.

To perform a time-course PRP was incubated with buffer or HT (7.5 mg/ml) at 37ºC during 0, 2, or 15 min before activation with collagen. The inhibitory effect of HT was already evident at 2 min of preincubation, meanwhile no changes on plasmatic fibrinogen concentration were observed.

Hementerin also inhibited secondary platelet aggregation induced by agonists as: 5mM ADP, 10mM adrenaline, 0.5U/ml thrombin, 0.5 mM arachidonic acid and ATP release was completed blocked.

Hementerin did not modify membrane glicoproteins as GPIIb-IIIa, Ib, Ia, IIa, IV measured by flow cytometry.

This inhibitory pattern prompted us to hypothesize an involvement of the nitridergic pathway. The inhibitory effect of HT on platelet aggregation induced by thrombin was blocked by L-NAME and L-NNA. An increase of Ca²⁺ flux in platelets preincubated with hementerin and stimulated with thrombin was observed, besides a sensitive increase in cGMP concentration, and a conversion of citrulina in L-arginina was evident indicating NO formation in platelets.

Taken together these results we postulate the following sequence in the signaling pathway triggered by HT: 1) an increase in intracellular Ca²⁺ flux, 2) an increase in platelet iNOS activity, 3) cGMP levels augmentation, and 4) inhibition of platelet aggregation through a cGMP-dependent mechanism.

Therefore, HT, an inhibitor of platelet aggregation, acting presumably through the increase in platelet iNOS activity, could become a useful tool to understand this mechanism and its possible implications on different therapeutics to inhibit platelet aggregation.

Intraspecific variability in snake venoms

Abrão, D.O.^I; Chavez-Olórtegui, C.^II; Velarde, D.T.^I

^IDivisão de Imunobiológicos-Fundação Ezequiel Dias, Conde Pereira Carneiro 80, 03550-010, Belo Horizonte, MG, Brazil

^IICentro de Pesquisas-Fundação Ezequiel Dias, Conde Pereira Carneiro 80, 03550-010, Belo Horizonte, MG, Brazil

^{Correspondence}

OBJECTIVES: There are statements in the literature about variability in snake venoms but little data. The variabilities described involve difference occuring between wild and captive snakes, snake sizes, seasonal variation and geographical origins. The objective of this work was the systematic study of the real variation exhibited by the venoms of snakes when in captivity.

METHODS: Using Crotalus durissus terríficus snakes as a model we worked with ten adult specimens coming from different regions of Minas Gerais State. After physical identifications their inicial (wild) venoms were collected. Periodically during the following year the snakes were weighed, milked and fed, in this order. The venoms were studied in terms of appearance, weight, proteins and crotamine contents measured by biological assay, Elisa and SDS-PAGE and lethality by

LD-50 in mice.

RESULTS: 1)- One of the specimens did not feed during the observation year, but despite this showed very little loss of weight and venom productivity. 2)- Only one of the wild venoms showed difference in protein contents 3)- All the snake venoms were white except one which was yellow. 4)- After a year in captivity one snake changed its venom color to yellow. 5)- Only six specimens produced venoms containing crotamine. 6)- The LD-50 amplitude varied from 6.34 to 15.52 ug/ mouse. Some of the specimen venoms presented positive and negative variations in lethality during the course of the year.

CONCLUSIONS: The changes exhibited by snake venoms during the prolonged period in captivity are important data, The variability could be intraspecies and even intraspecimen according to the snakes environment.

Support : FAPEMIG, FUNED

Antigen components for coral antivenom preparation

Mourão, M.M.; Maria, W.S.; Velarde, D.T.

Divisão de Imunobiológicos, Fundação Ezequiel Dias-FUNED

^{Correspondence}

OBJECTIVES: By government regulation the preparation of antivenom against coral snakes has to use for equine hyperimmunization a mixture of Micrurus frontalis and M. corallinus venoms. The aim of this work was to evaluate the most adequate antigen proportion of each venom component to get a most effective antivenom.

METHODS: Five group of four equines each were selected and immunized with as follows antigens : Group 1 (G1), 100 % of M. frontalis venom, G2, 75/ 25 % M. frontalis/ M. corallinus, G3, 50/ 50 % M. frontalis/ M. corallinus, G4, 25/ 75 % M. frontalis/ M. corallinus, G5, 100 % M. corallinus. During the first cycle immunization a total 18 mg of venom was used during three weeks : 6 mg in each week using CFA in first, IFA in second and saline in third week. Following cycles of immunization were as conducted the two last weeks above. The horses were bled after ten and twelve days and rested for two weeks prior to re-immunization. The study was done for four cycles. The individual and combined samples were analysed by indirect haemolysis, Elisa and ED-50 in mice.

RESULTS: The M. corallinus venom showed a very low phospholipase activity. The G2 showed the best results in neutralizing M. frontalis and M. corallinus venoms by Elisa. G2 and G3 demonstrated the highest and similar lethality protection in mice against both M. frontalis and M. corallinus venoms.

CONCLUSIONS: For the preparations coral antivenom, antigen proportions of 75/25 or 50/50 % from M. frontalis/ M. corallinus venoms could be used.

Support : FAPEMIG, FUNED

Bothropic antivenom (BjAv) efficacy on microcirculatory effects evoked by Bothrops jararaca venom (BjV). Intravital microscopic study

Battellino, C.^I; Piazza, R.^II; Silva, A.M.M.^III; Cury, Y.^IV; Farsky, S.H.P.^{I, V}

^ILaboratórios de Imunoquímica, Instituto Butantan

^IILaboratórios de Microbiologia, Instituto Butantan

^IIILaboratórios de Imunopatologia, Instituto Butantan

^IVLaboratórios de Fisiopatologia, Instituto Butantan

^VDep. De Análises Clínicas e Toxicológicas, Faculdade de Ciências Farmacêuticas, USP. São Paulo, Brasil

^{Correspondence}

INTRODUCTION: I.v. administration of BjAv neutralises systemic effects but does not completely reverse the local reaction evoked by BjV. The mechanism involved in this inefficacy has not been already known. The aims of this work were: 1) to investigate the efficacy of BjAv on effects evoked by BjV on microcirculatory network and 2) to determine the distribution of AvBt on systemic and microcirculatory network.

METHODS: Internal spermatic fascia of Wistar rats was exposed to visualise the microcirculation vessels. AvBj was i.v. injected (2.5 ml/kg) 15 min before, simultaneously or 15 after topical application of BjV (10 µg/10µl sterile saline). Control animals received equivalent amount of horse normal serum. AvBt was also fluorescein-isothiocyanate (FITC) labelled before injection to visualise the dynamic of its distribution on microcirculation. FITC-albumin labelled was i.v. injected to determine vascular permeability increase. Immunoenzimatic assay was carried out in rat serum to determine the clearance of AvBt.

RESULTS: Data obtained showed that the three AvBj treatment protocols did not completely neutralise the local blood coagulation, vascular permeability, haemorrhage and leukocyte-endothelial interactions induced by topical application of BjV. Differently, in vitro incubation of AvBj/BjV previously to application on the microcirculatory network neutralised all effects. Images obtained after FITC-AvBt labelled injection showed that the AvBt remains inside the vessel until the rapid action of BjV. Also, the complete microcirculatory stasis induced by the venom impairs the AvBt distribution. ELISA assay showed that AvBt is present at the circulating blood until 4 hours after its application.

CONCLUSIONS: Our data show that AvBt has antibodies against toxins responsible for the local effects induced by BjV and suggest that AvBt does not completely reverse these effects because the interaction BjV/AvBt only takes place after BjV action. Also, the impaired microcirculation blood flow evoked by BjV prevents the adequate AvBt distribution.

Financial support: FAPESP- 96/10315-1.

Bradykinin-potentianting peptides precursor from Crotalus durissus terrificus venom gland

Lameu, C.; Baptista, G.R.; Barbosa, S.R.; Yamane, T.; Camargo, A.C.M.; Hayashi, M.A.F.

^{Correspondence}

The bradykinin-potentiating peptides (BPPs) were the first naturally occurring ACE inhibitors described. Characteristically in Bothrops jararaca, they contain 5 to 13 amino acid residues that have a pyroglutamyl residue at the N-terminal and a proline at the C-terminal. Six BPPs isoforms were identified in a single precursor isolated from a Bothrops jararaca venom gland. This precursor encodes, not only seven BPPs isoforms aligned in tandem but, surprisingly, it also contains the sequence of a 22 amino acid residues C-type natriuretic peptide (CNP) at the C-terminus of the molecule (Murayama et al., PNAS, 1997). Except for one, the pentapeptide BPP-Va, all the other BPPs identified share similar features including a high content of proline residues and the tripeptide Ile-Pro-Pro at the C-terminus. Several BPPs precursors isolated from the venom gland of other members belonging to Crotalinae subfamily has been described. Despite the differences observed in the BPPs coding region, all isolated precursors contain tandemly repeated BPPs at the N-terminus and a CNP at the C-terminus.

The present study reports the isolation of a cDNA encoding a BPP precursor from the venom gland of another serpent belonging to Crotalinae subfamily Crotalus durissus terrificus. Northern blot and primary sequence analysis allowed us to observe unique features of the BPP-CNP precursor expressed in this specimen.

Financial support by FAPESP.

A heat-stable hemolysin from clinical Enterobacter cloacae strain

Simi, S.^I; Carbonell, G.V.^I; Darini, A.L.^III; Gatti, M.S.V.^I; Falcón, R.^I; Toyama, M.H.^II; Marangoni, S.^II; Yano, T.^I

^IDepto de Microbiologia e Imunologia, Instituto de Biologia. UNICAMP, SP

^IIDepto de Bioquímica, Instituto de Biologia. UNICAMP, SP

^IIIDepto de Análises Clínicas, Toxicológicas e Bromatológicas3, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, USP, SP

^{Correspondence}

INTRODUCTION:Enterobacter spp. have been recognized as increasingly important pathogens in recent years. Toxins of E. cloacae has been described, however the relevance of these toxins remains to be established. In this study, we describe some properties of a hemolysin produced by clinical E. cloacae.

METHODS:E. cloacae was cultured in TSB with 0.5% glucose with stationary incubation. The culture supernatant was assayed for hemolytic activity with different species of erythrocytes. Crude hemolysin was partially purified by ultrafiltration, acetone precipitation, and reversed phase chromatography (mBondapak C18), HPLC. Physicochemical properties of hemolysin such as heat and pH stability, effect of chemical reduction and proteolytic enzymes were determined. Biological assays including rat intestinal loop, suckling mouse test, skin permeability test in rabbits were done with hemolysin.

RESULTS:E. cloacae hemolysin was active on horse, sheep, guinea pig and human erythrocytes. The hemolysin presented an estimated molecular mass smaller than 10 kDa, was resistant to proteolic enzymes and 2-mercaptoethanol, was heat stable, soluble in organic solvents and stable to acid pHs but not above pH 6. The hemolysin caused hemorrhage and fluid accumulation in the rat intestinal loop test, however gave a negative reaction in the suckling mouse assay and in the rabbit skin permeability test.

CONCLUSIONS:E. cloacae hemolysin showed some physicochemical properties similar to other heat-stable Enterobacteriaceae enterotoxins. This study, is the first description of a low molecular mass heat-stable hemolysin that causes hemorrhagic fluid secretion in ligated rat ileal loops, which may contribute to our understanding of the pathogenesis of this species.

Cytotoxic enterotoxin of Aeromonas hydrophila induces cytoskeletal changes and perturbation mitochondrial on intestinal epithelial cells

Falcón, R.^{I, II}; Martins, L.M.^I; Pimentel, S.^III; Carvalho, H.F.^III; Gatti, M.S.V.^I; Yano, T.^I

^IPedro Kouri Institute, Havana, Cuba

^IIDept of Microbiology & Immunology. Institute of Biology. University (UNICAMP), Campinas, SP, Brasil

^IIIDept ofCell Biology. Institute of Biology. University (UNICAMP), Campinas, SP, Brasil

^{Correspondence}

INTRODUCTION: The cytotoxic enterotoxin secreted by Aeromonas hydrophila (Ent-ctx) possesses the ability to evoke a fluid secretory response in a ligated intestinal loop model, amongst other biological activities. The purpose of the current study was to evaluate the alterations induced by Ent-ctx on intestinal epithelial cells and its association with the pathogenesis of the gastrointestinal infections.

METHODS: The enterotoxic toxin purified was assayed in the rat intestinal loop test. Intestinal epithelial cells treated with Entx-ctx were included in historesin BJ4 and stained with blue toluidine for microscopic analyses. The structural organization of epithelial cells was analyzed by fluorescence actin staining. The morphological and intracellular changes were accompanied by scanning and transmission electron microscopy.

RESULTS AND DISCUSSION: Gross morphology showed a dilation and collapse of the intestinal villi, apparently provoked by edema. Ultrastructural analyses showed intracellular alterations such as mitochondrial swelling with obliterated cristae and membrane accumulation as mielin-bodies. The prominent features of the epithelial cells were a disruption of the basal lamina and disalignment the microvilli leading to cytoskeletal disorganization.

CONCLUSIONS: Ent-ctx acts on the intestinal epithelial cell cytoskeleton to alter actin microfilaments. The cytopathogenic response of this enterotoxin was irreversible and progressive, causing perturbation of the mitochondrial structure and function. These alterations induced by the cytotoxic enterotoxin of A. hydrophila can be correlated with the capacity of this toxin to induce cell death by apoptosis in HT29 (Falcón et al. Biochem Cell Biol 79: 525, 2001).

Detection of putative metalloproteinases in elapid venoms

Checchi, P.^I; Della-Casa, M.S.^I; Kosmiskas, L.O.C.^II; Nascimento, N.^II; Moura da Silva, A.M.^I; Mirtschin, P.^III; Fernandes, I.^I; Spencer, P.J.^{I, II}

^ILaboratório de Imunopatologia, Instituto Butantan

^IILaboratório de Biologia Molecular, Instituto de Pesquisas Energéticas e Nucleares, São Paulo, Brazil

^IIIVenom supplies, Tanunda, Austrália

^{Correspondence}

Toxins affecting haemostasis are well known to occur in viperid venoms. In these venoms the major systemic effects can be ascribed to these proteins. Among these toxins, a particularly important group is the snake venom metalloproteinases (SVMP) responsible for the often-massive haemorrhage. Elapid venoms are better known for their neurotoxic action and few haemorhagins have been described in this family. Our aim was to evaluate the presence of SVMP-related proteins in elapid venoms using antibodies against jararhagin, a P3-SVMP from the pit viper Bothrops jararaca.

Equal concentrations (3mg) of Hoplocephalus stephensis, Naja melanoleuca, Naja mossambica, Notechis ater niger, Notechis scutatus, Oxyuranus microlepidotus, Oxyuranus scutellatus, Pseudechis australis, Pseudechis colleti, Pseudechis guttatus, Pseudechis porphyriacus and Pseudonaja textilis venoms were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was incubated with mouse polyclonal antibody against jararhagin. The membrane was developed with a rabbit anti-mouse IgG conjugate. In parallel, all the venoms were tested in mice for haemorrhagic activity.

Our results indicate that all venoms presented immunoreactive bands. According to their apparent size, a vast majority of the identified proteins appear to belong to the P3 and P4 SVMPs. The only exception we could observe was the N. mossambica venom where a lower molecular weight band was detected, suggesting the presence of a P1 SVMP. Most of the venoms presented haemorrhagic activity when assayed in vivo.

According to our observations, different components of the SVMP group are present in the venom of the elapid family, however, in much lower concentration than in viperids. The SVMP genic family appears to be present in both families, with variations of the expression rate. Finally, although mainly neurotoxic, the presence of putatives SVMPs in elapids might be important from the clinical point of view.

Local treatment of experimental envenoming by Bothrops alternatus in dogs with the aqueous extract of Kalanchoe brasiliensis

Silva Junior, P.G.P.^I; Melo, M.M.^I; Cardoso, R.R.^I; Ferreira, K.M.^I; Fonseca, F.V.^I; Lago, L.A.^I; Habermehl, G.G.^II

^IDepartamento de Clínica e Cirurgia, Escola de Veterinária, UFMG, Brasil

^IIChemisches Institut, Tierärztliche Hochschule, Hannover, Germany

^{Correspondence}

The bothropic venom acts locally causing edema, hemorrhagic halo and necrosis around the affected area. The traditional treatment with anti-serum is only effective to antagonize the systemic effects, but it does not neutralize local lesions. Bothropic envenoming may have severe consequences such as loss of the limb due to necrosis. Auxiliary treatments must be developed in order to antagonize these local effects. The aim of this study was to verify the effectiveness of the aqueous extract of Kalanchoe brasiliensis (100%) compared to placebo (saline). Desiccated venom of Bothrops alternatus (0.3mg/kg) was diluted in saline and injected IM in ten dogs. These dogs were divided into two groups: Group I was treated with Saline and Group II received topic treatment with Kalanchoe brasiliensis (100%). These treatments began 30’ after venom inoculation, and were repeated at 3, 6, 12, 24 hours after inoculation. After 24 hours the treatment was repeated twice a day for 14 days. The lesions were evaluated once a day by measuring the skin thickness (edema) with a pachymeter, the hemorrhagic halo with a special ruler and by observing the appearance of ulcers in dermis (necrosis). Immediately after the inoculation, local edema was observed and all dogs presented signs of pain. Decrease of edema and the hemorrhagic halo was observed in dogs treated with Kalanchoe brasiliensis, this group had a better recovery when compared to the Control group (Saline). Our results support the notion that the aqueous extract of Kalanchoe brasiliensis is effective in antagonizing the local effects against Bothrops alternatus venom.

Experimental envenomation by Bothrops moojeni in dogs

Silva Junior, P.G.P.^I; Cardoso, R.R.^I; Alzamora Filho, F.^I; Lucia, M.^II; Habermehl, G.^III

^IDepartamento de Clínica e Cirurgia, Escola de Veterinária, UFMG, Brasil

^IIEscola de Veterinária, UNIFENAS, Brasil

^IIIChemisches Institut, Tierärztliche Hochschule, Hannover, Germany

^{Correspondence}

Envenoming caused by snakes of Bothrops genus is an important issue in Veterinary Medicine, due to its high incidence, seriousness and sequels. Envenoming caused by Bothrops moojeni is poorly studied in animals. “Caiçaca” is the popular name of B. moojeni, which is largely distributed in Brazil, being present in the following States: Paraná, São Paulo, Minas Gerais, Mato Grosso, Mato Grosso do Sul, Goiás and Maranhão. B. moojeni envenoming is associated with intense and immediate local reaction, which is characterized by edema and pain as the earliest signs. In order to determine the clinical aspects, Bothrops moojeni desiccated venom was diluted in saline and injected IM (0.3mg/kg) in five dogs. All the animals were examined before and 2, 12, 36, 48, 96, 144 and 192 hours after inoculation. Intensity of edema was estimated by pachymeter measurement and the hemorrhage development by measuring the diameter of the hemorrhagic halo with a special ruler. Blood was collected from the cephalic vein, with and without EDTA, and the hematological parameters and blood-clotting time were assessed. Immediately after the venom inoculation, pain and decrease of rectal temperature were observed. Cardiac frequency was increased, returning to normal levels after a week. Large local edema was observed, affecting thoracic limbs, and pectoral and cervical areas. Three dogs showed hemorrhagic halo without necrosis. No blood coagulation was observed within four days after inoculation, with blood-clotting time returning to normality after that. Leucocytosis, decreased red blood cell count, decreased hemoglobin concentration, decreased packed cell volume, and thrombocytopenia were also observed and could be explained by the hemorrhage.

Inhibition of 3H-Gaba and 3H-Dopamine uptake into rat brain synaptosomes by Tityus serrulatus venom and its toxin TSTX-V

Cecchini, A.L.^{I, II, III}; Vasconcelos, F.^I; Giglio, J.R.^II; Amara, S.G.^III; Arantes, E.C.^I

^IDepartamento de Física e Química, FCFRP-USP

^IIDepartamento de Bioquímica e Imunologia, FMRP-USP/Ribeirão Preto-SP, Brazil

^IIIVollum Institute, HHMI, Oregon Health & Science University, Portland-Oregon, USA

^{Correspondence}

OBJECTIVES: Scorpion venoms contain toxins that have well-established actions on the function of ionic channels of excitable cells. However, the effects of these toxins on neurotransmitter transport have not yet been examined. This study evaluates the effects of Tityus serrulatus scorpion venom (TsV) and TsTX-V, an a-toxin, on GABA and dopamine (DA) uptake in isolated rat brain synaptosomes.

METHODS AND RESULTS: TsTX-V was purified from TsV by a combination of ion exchange chromatography and reverse phase HPLC. Homogeneity of TsTX-V was evidenced by a single band by PAGE, SDS-PAGE (Mr=7230) and isoelectric focusing (pI=8.0). Both the venom and its toxin were assayed for their effects on ³H-GABA uptake into cortical synaptosomes and on ³H-DA uptake into striatal synaptosomes. TsV(0.43ng/mL) inhibited both GABA and DA uptake (~50%). TsTX-V showed IC50s= 9.37nM and 22.2nM for GABA and DA uptake, respectively. In contrast, maximal concentrations of TsV(1300ng/mL) and TsTX-V(2770nM) had no effect on ³H-L-glutamate uptake. The inhibitory effects of TsV and TsTX-V were still observed when external Ca²⁺ was replaced by Sr²⁺(7.6mM) and EGTA(1mM), but not when synaptosomes were pre-treated with TTX(1mM). TsTX-V did not inhibit ³H-GABA uptake in COS-7 cells expressing GAT-1 or GAT-3, the major brain GABA transporters, suggesting that the toxin indirectly inhibits transport.

CONCLUSIONS: TsV and TsTX-V appear not to affect transporters expressed in heterologous expression systems, including COS-7 cells, but can inhibit GABA and DA transport activity in a Ca²⁺-independent manner in native systems such as synaptosomes.

Support: Fapesp, Capes

Stability of bothropic antivenoms under different conditions of temperature

Silva, D.G.; Clemente, C.A.; Barbosa, C.F.; Lima, S.L.; Velarde, D.T.

Fundação Ezequiel Dias – Belo Horizonte – MG

^{Correspondence}

OBJECTIVE: To evaluate the stability of the bothropic antivenoms produced in the Fundação Ezequiel Dias, providing data of their behavior through time, under different storage temperatures

METHODOLOGY: Four conditions of study of stability were delineated with different storage temperatures and sampling periods: study of shelf life (5+3°C) for 36 months, intermediate study (25+3°C) for 30 months, accelerated study (37+2°C) for eight months and stress study (45+2°C) for eighteen months. The first condition seeks to validate the stability of the product, confirming the expiry period and in the last two conditions we intend to study the chemical degradation or physical modification of the product. Three lots of bothropic antivenoms were used in this study, and the following characteristics were evaluated: aspect, pH, protein content, concentration and efficiency of the preservative (phenol), in vivo and in vitro activities and electrophoretic and chromatographic analyses.

RESULTS AND CONCLUSIONS: After twelve months of the study the results indicated that the appearance was the first factor to be altered by drastic temperatures (37 and 45°C). The pH, protein content, phenol concentration and electrophoretic pattern did not change during the study, even under the most drastic conditions for twelve months. As was anticipated, the in vivo and in vitro activities showed a reduction at the end of the accelerated and stress studies. No significant modification was observed in these activities with the lower temperatures used in the study. This indicates the stability of the products for twelve months. A variability should be considered in the results of those activities, that it is inherent to the biological and immunochemical assays. Antivenoms kept at 45°C for twelve months showed variations in Superose 12 chromatograms.

Intracerebral morphological and ultrastructural alterations after intravenous administration of Lonomia obliqua spicule extract in rats

Silva, G.H.^I; Hyslop, S.^I; Cruz-Höfling, M.A.^II

^IDepartamento de Farmacologia, F.C.M.

^IIDepartamento de Histologia e Embriologia, I.B., Universidade Estadual de Campinas (UNICAMP), Campinas, SP, Brasil

^{Correspondence}

OBJECTIVE: Clinical reports have shown cerebral damage, including edema and intracerebral hemorrhage, after envenoming by Lonomia obliqua caterpillars. In this work, we investigated the neurotoxicity of L. obliqua spicule extract injected intravenously in rats.

METHODS AND RESULTS: Morphological and ultrastructural analyses were done using light and transmission electron microscopy 6 h, 18 h, 24 h and 72 h after i.v. injection of L. obliqua spicule extract (200 mg/kg). For ultrastructural analysis, lanthanum nitrate was used as an extracellular tracer to assess blood-brain barrier (BBB) breakdown. Six hours after envenoming, light microscopy showed cerebellar edema which decreased by 72 h. Intracerebral hemorrhage occurred in only one rat 6 h after extract injection. BBB breakdown was assessed by transmission electron microscopy based on the passage of extracellular tracer between brain capillary endothelial cells. BBB breakdown was observed in the cerebellum and hippocampus 18 h post-injection. The cerebellum was more sensitive to the venom than the hippocampus, as shown by the greater number of leaky capillaries. After 72 h, the number of capillaries showing BBB breakdown was lower than after 18 h.

CONCLUSION: The morphological and ultrastructural alterations observed indicate that Lonomia obliqua spicule extract has a neurotoxic action. Assays in vivo with purified fractions of the venom are necessary to identify the component responsible for these effects.

Financial support: CNPq, CAPES, FAPESP, FAEP.

Effects caused by myotoxin I and myotoxin II from Bothrops moojeni venom in rat isolated kidney

Barbosa, P.S.F.; Havt, A.; Toyama, M.H.S.; Martins, A.M.C.; Nobre, A.C.L.; Soares, T.F.C.; Fonteles, M.C.; Monteiro, H.S.A.

UNICAMP and Federal University of Ceará-UFC

^{Correspondence}

Acute renal failure is one the most common systemic complications after snakebite, however, its pathogenesis remains obscure. In this study we evaluated the renal effects of B. moojeni myotoxins (Bmtx-I and BmtxII) in rat isolated perfused kidneys. The B. moojeni myotoxins were purified by ion-exchange chromatografy and reverse phase HPLC. Wistar rats (240 - 280 g) were anesthetized with sodium pentobarbitone (50 mg/kg, i.p.) and the right renal artery was cannulated as described by Fonteles et al., (Am. J. Physiol. 244, 235-346, 1983). Bothrops moojeni myotoxins (5 mg/mL) were added to the perfusion system 30 min after the beginning of each perfusion. The renal effects were compared with a control group (CG), analyzed by ANOVA (p< 0,05). Bmtx-I increased the perfusion pressure (CG120 = 113.5 ± 0.833, Bmtx-I120 = 169.2 ± 5,0), renal vascular resistance (CG120 = 5.216 ± 0.16, Bmtx-I120 = 7.60 ± 0.31), urinary flow (CG120 = 0.150 ± 0.002, Bmtx-I120 = 0.296 ± 0.037), and glomerular filtration rate (CG120 = 0.617 ± 0.022, Bmtx-I120 = 0886 ± 0.149). Sodium, potassium and chloride tubular transport (%TNa+, %TK+, %TCl-) decreased after administration of Bmtx-I. Bmtx-II had no effect on renal physiology, except for a transient decrease in potassium tubular transport (CG90 = 75.22 ± 1.41, Bmtx-II = 66.61 ± 2.29). In conclusion, BmTx-I may have exerted its renal effects by indirectly increasing calcium influx after damage to the cell membrane. Apparent inability of Bmtx-II to induce the renal effects similarly to Bmtx-I should be explained by the absence of the C-terminal lysine rich region in Bmtx-II.

Purchased by FUNCAP/FAPESP

Preliminary results from renal alterations promoted by Bothrops erythromelas venom analyzed in the isolated perfused rat kidney method

Sousa, F.C.M.; Havt, A.; Barbosa, P.S.F.; Martins, A.M.C.; Angelim, E.V.; Nobre, A.C.L.; Facó, P.E.G.; Guarnieri, M.C.; Fonteles, M.C.; Monteiro, H.S.A.

Federal University of Ceará – Brazil

^{Correspondence}

Renal alterations promoted by ophidian accidents promote severe damages that can be cause death. Our goal is identify the possible nephrotoxic effects promoted by Bothrops eryhtromelas venom in the isolated perfused rat kidney method. Wistar rats were anesthetized with sodium pentobarbitone (50mg/mL), and their kidneys were surgically removed following Fonteles and coworkers (Am. J. Physiol. 244, 235-346, 1983) technique. We used a Krebs-Henseleit perfusion solution modified by serum bovine albumin (6g%). Each experiment (n=2) lasted 120 minutes. The first 30 minutes of each perfusion were used as internal control (IC) and compared (Student t Test, *p<0,05) to other 3 treated (10 mg/mL) periods (T) of 30 minutes, that were named 60, 90 and 120 minutes. The most intense effects were seen in the 120 minutes period. Bothrops erythromelas venom caused intense decreases in the perfusion pressure (IC30 = 109.9 ± 0.44, T90 = 72.03 ± 3.65, T120 = 84.53 ± 0.60 mmHg) and renal vascular resistance (IC30 = 4.54 ± 0.017, T90 = 2.97 ± 0.15, T120 = 3.50 ± 0.024 mmHg/g/min/mL). Urinary flow was reduced at the 60 minutes period but at 120 minutes we found an intense increase. Glomerular filtration rate was also reduced at the 60 minutes period. Sodium tubular transport (IC30 = 80.90 ± 0.56, T90 = 58.96 ± 2.10, T120 = 58.68 ± 2.32 %) and potassium tubular transport (IC30 = 62.99 ± 1.64, T90 = 52.67 ± 1.44, T120 = 53.36 ± 1.44 * mmHg) were reduced in the periods named 90 and 120 minutes. The Bothrops erythromelas venom caused an intense decrease in the vascular parameters which was independent of the increase of urinary flow. Maybe the reductions for sodium and potassium tubular transport had intensified the alterations for urinary flow in the end of each perfusion.

Supported by CAPES

Neuroethological analysis of Wistar rats injected I.C.V. with venom and isolated fractions of the social wasp Agelaia vicina

Oliveira, L^I; Ribeiro, A.M^I; Pizzo, A.B.^I; Guizzo, R.^I; Miranda, A.^II; Santos, W.F.^I

^INeurobiology and Venoms Laboratory, FFCLRP/USP

^IIBiophysics Dept. UNIFESP/SP

^{Correspondence}

OBJECTIVES: To evaluate the behavioral effects caused by i.c.v. injection in rats, of the venom from the social wasp Agelaia vicina and the fractions isolated by reverse phase HPLC in Wistar rats.

MATERIALS AND METHODS: Cannules were implanted in the lateral ventricle of male Wistar rats (200-250g), using stereotaxic determined coordinates. Animal behavior was registered in VHS tapes for 30 min. in intervals of 180 sec 5, 10 and 15 min after injection. The rats (n=6) were injected with 3 mL of denatured venom (15,30 and 60 glands) and with crude venom (30 glands). To analyze the animals’ behavior open field, rotarod and bar models were used.

RESULTS: The rats (n=6) had their locomotor activity reduced after intracerebroventricular injection (90%). This could be seen as a significant decrease of the number of line crossings in the open field. The animals treated with 60 glands did not equilibrate on the Rotarod (100%) in the periods of 5, 10, 20 and 30 min after injection demonstrating an ataxic frame. In suspended bar experiments rats injected with haloperidol and 60 glands were able to stay stained in the periods of 5 and 30 min indicating catalepsy (83%) .The injection of 3mL from the fractions (AvTx 1-4) purified by HPLC reverse-phase in acetonitrile/water/TFA gradient also reduced significantly the animal’s locomotor activity as well as the number of line crossings (85%). Concluding, the venom of A. vicina and the fractions purified by HPLC may contain components that could be helpful in understanding the neurotransmission system involved in locomotor activity, catalepsy and ataxia.

Financial support: CAPES, CNPq and FAPESP

Isolation and partial characterization of a fibrin(ogen)olytic metalloprotease and a plasminogen activating serine protease from the venom of Bothrops brazili

Modesto, J.C.A.^I; Ventura, J.S.^I; Oliva, M.L.V.^II; Chudzinski-Tavassi, A.M.^I

^ILaboratório de Bioquímica e Biofísica, Instituto Butantan, São Paulo, SP

^IIDepartamento de Bioquímica, Universidade Federal de São Paulo (UNIFESP), São Paulo, SP, Brasil

^{Correspondence}

Snake venoms contain a complex mixture of proteins that participate and interfere with the control and regulation of a number of important biological processes of their prey. Some of these proteases, inhibitors and toxins directly affect the haemostatic system. Systematic disturbances observed in victims of snake bites by Bothrops sp. are hemorrhage and blood incoagulability caused by a coagulopathy consumption. Metalo and serine proteases are responsible for procoagulant and fibrino(geno)lytic activities of Bothrops snake venoms. In Brazil, the Bothrops brazili snake is found in a few specific regions of the Amazon Forest and the proteins isolated from this venom have not been purified and characterized.

We present here the isolation and partial characterization of two proteins presents in the Bothrops brazili venom, one is a fibrin(ogen)olytic enzyme and the second is a plasminogen activator.

The enzymes were isolated using ion-exchange chromatography (DEAE Sephadex A-50), followed by two steps in the FPLC system: molecular exclusion (Superdex 200) and ion-exchange (Resource Q). The homogeniety was evaluated by SDS PAGE (10%). The fibrin(ogen)olytic activity was tested based on fibrinogen degradation in fibrin and fibrin-agarose plates. The plasminogen activator activity was measured indirectly by the formation of plasmin using the Solid Fibrin Assay (SOFIA) and S-2251 as substrate.

The fibrin(ogen)olytic enzyme was characterized as a metalloprotease (approximate molecular mass 25 kDa) that degrades both the Aa and Bbchains of fibrinogen and fibrin by a path independent of plasminogen activation. Fibrinogen and fibrin products are different from those induced by plasmin. The fibrin(ogen)olytic enzyme did not hydrolyse quenched substrates based on the b-chain of insulin. The plasminogen activator protein (approximate molecular mass 30 kDa) is a serine protease which requires fibrin fragments, so it can be classified as a t-PA like protein.

Supported by grants from FAPESP.

Inhibitory effect of Crotalus durissus terrificus snake venom (CdtV) of macrophage phagocytosis. Contribution of crotoxin

Sampaio, S.C.^I; Brigatte, P.^I; Santos, A.C.R.^II; Santos, EC.^II; Sousa e Silva, M.C.C.^I; Curi, R.^III; Cury, Y.^I

^ILaboratory of Pathophysiology; Butantan Institute

^IILaboratory of Immunopathology; Butantan Institute

^IIIInstitute of Biomedical Sciences/USP, Brazil

^{Correspondence}

Previous work from our laboratory has shown that, in addition to the opioid-mediated analgesic effect, CdtV inhibits phagocytic function of macrophages. The present work investigated: (a) the mechanisms responsible for the inhibitory effect of CdtV, and (b) the involvement of crotoxin (CTX), the main toxin of CdtV, in this effect. The CdtV, administered by sc route (30µg) to male Wistar rats or incubated with rat peritoneal resident macrophages inhibited phagocytosis of opsonized zymosan by these cells. The percentage of inhibition in the in vivo experiments was 46% and in the in vitro assays was: 0,25µg/ml-41%, 0,5µg/ml-44% and 1,0µg/ml-44%. Naloxone (1mg/kg) did not modified the inhibitory effect of CdtV. On the other hand, neutralization of CdtV by specific antivenin blocked venom effect. Crotoxin, (19µg, sc) also inhibited (46%) the phagocytic activity of macrophages. This was a long lasting effect since it persisted for 14 days after CdtV administration. The inhibitory effect was also observed in vitro for CTX incubated (0,3, 0,016, 0,008 0,004µg/ml) with macrophages (30%, 33%, 47%, 53% of inhibition, respectively). The venom or CTX effect was not a consequence of a toxic effect, since FACS analysis did not show alterations of cell viability. These data suggest that the inhibitory effect of CdtV is not mediated by its opioid effect and demonstrate that CTX is involved with these inhibitory response.

Financial support: FAPESP, CNPq

Molecular cloning and sequence analysis of cDNAs coding for venom toxin inhibitors from the opossum Didelphis marsupialis

Trugilho, M.R.O.^I; Junqueira-de-Azevedo, I.L.M.^II; Neves-Ferreira, A.G.C.^I; Rocha, S.L.G.^I; Ho, P.L.^II; Domont, G.B.^III; Perales, J.^I

^IDepto. Fisiologia e Farmacodinâmica, IOC-FIOCRUZ

^IICentro de Biotecnologia, I. Butantan

^IIIDepto. Bioquímica, IQ-UFRJ

^{Correspondence}

Some animals, such as snakes and a few mammals present natural resistance to snake envenomation. Most of the times, this resistance is due to the presence of soluble neutralizing proteins in their sera. The South Americam opossum D.marsupialis resists to several Viperidae snake venoms. From its serum two proteins with antihaemorragic activity (DM40 and DM43) and one with antimyotoxic activity (DM64) were previously isolated. The aim of this work was to clone and sequence the complete cDNA coding for these anti-toxic proteins. Total RNA was extracted from the liver of D.marsupialis and a cDNA library was constructed using the pGEM11Zf(+) plasmid. Based on the sequences of internal peptides from the three DMs obtained by Edman chemistry and on the partial cDNA sequence of oprin (an antihaemorragic protein isolated from D.virginiana serum), we synthesized the specific primer DM130F complementary to a region of highly conserved amino acid sequence (¹³⁰FDLYQE¹³⁵). The cDNA library was screened by PCR with the primers DM130F and NotI oligo(dT), resulting in the amplification of two DNA fragments of approximately 500 and 800 bp. These fragments were cloned, sequenced and confirmed as cDNAs coding for DM64 and for an unknown protein homologous to DM43, named DM43b. Using other specific oligonucleotides and the cDNA library as template, the nucleotide sequences were extended by PCR in order to obtain the N-terminal sequence, signal peptide and the 5´UTR region of DM64 and DM43b. The complete cDNA sequences were obtained by superposing all sequenced fragments. In addition, a RT-PCR was performed resulting in the amplification of a partial cDNA from DM43. A search on the GenBank database showed that the amplified sequence was identical to DM43. The other sequences, cloned from the library, were homologous to DM43, oprin and also to a1B-glycoprotein, a human plasma protein of unknown function, and a member of the immunoglobulin supergene family. These natural inhibitors can be useful as tools in the study of the physiopathological effects of venoms and also as a new alternative for the treatment of snake envenomations.

Interactions between lefaxin and factor X molecules

Faria, F.^I; Bezeaud, A.^II; Guillin, M.C.^II; Anglés-Cano, E.^III; Sampaio, M.U.^IV; Chudzinski-Tavassi, A.M.^I

^ILaboratório de Bioquímica e Biofísica – Instituto Butantan (Center for Applied Toxinology CAT/CEPID – FAPESP), São Paulo, Brazil

^IIE9907 – INSERM, Paris, France

^IIIU143 - INSERM, Paris, France

^IVDepartamento de Bioquímica – UNIFESP-EPM, São Paulo, Brazil

^{Correspondence}

The salivary glands of Haementeria depressa leech produce some proteins that induce unclotable blood. The purification and partial characterization of a Factor Xa inhibitor, named lefaxin (leech factor Xa inhibitor), was previously described by FARIA et al (1999). Lefaxin is a single chain glycoprotein, Mr 30 kD, pI 5.7, the inhibition of FXa was determined first by inhibition of FXa amidolytic activity determined with chromogenic substrate (S2765) with a Ki about 4nM and second by reduction of FXa ability to activate prothrombin, in the prothrombinase complex (EC50 10nM).

The aim of this study is to characterize the inhibition by the Dixon plot, and to determine the interaction site of FX molecule.

Lefaxin is a competitive FXa inhibitor with a Ki about 2nM, this inhibitor was able to bind to FX molecule immobilized on the CM5 chip in a BIACORE system, with a Kd about 4nM. The interaction with FXa using the FX (zymogen) as competitor showed that lefaxin is able to bind to the zymogen (Kd about 6nM). Binding to a FXa active site blocked (FXa-GGACK) was observed (Kd about 30nM).

These results indicate that lefaxin, as well as rTAP (JORDAN et al, 1992) and antistasin (LAPATTO et al, 1997), can bind to FX via an exosite, although lefaxin isn’t a FXa inhibitor antistasin-like.

Financial Support: FAPESP, INSERM, and CAPES

Biological activity and purification of a neurotoxin from Averrhoa carambola star fruit (Oxalidaceae)

Carolino, R.O.G.^I; Beleboni, R. O.^I; Moyses-Neto, M.^III; Cairasco, N. G.^II; Coutinho-Netto, J.^I

^IBiochemistry and Immunology - FMRP/USP - SP - Brazil

^IIPhysiology Departments - FMRP/USP - SP - Brazil

^IIINephrology Divisions (HCRP) - FMRP/USP - SP - Brazil

^{Correspondence}

OBJECTIVES: The consumption of star fruit (A. carambola) is very widespread in Brazil. However, the consumption of star fruit can be hazardous to patients suffering of renal failure. The symptoms vary from nausea, vomiting, intractable hiccups, insomnia, psychomotor agitation and several degrees of conscious alterations, persistent convulsions and death. The aim of this work was to isolate the active fraction, responsible for convulsions from star fruit, as well to study its action on GABAergic and glutamatergic systems.

METHODS AND RESULTS: Fruits were collected from trees non treated with pesticides, homogenized in distilled water 1:1 (w/v) and sequentially treated with lead acetate and ammonium sulfate. This extract was submitted to DEAE, CM-cellulose and reverse phase HPLC chromatography. The active fraction (referred as to AcTx) was able to induce convulsion and death after intracisternal administration in mice. L-glutamate and GABA uptake as well as glutamate release in synaptosomes from rat cerebral cortex were not altered in presence of AcTx. On the other hand, AcTx displaced ³H-GABA binding from its receptors in synaptic membranes, but did not alter glutamate binding. Acid hydrolysis and amino acids analysis revealed no amino acids in its structure.

CONCLUSIONS: The star fruit has a neurotoxic compound, not a peptide, acting through GABA receptors. Further studies are on its way to better explain the intoxication symptoms and the complete chemical structure.

Finantial support: CAPES e FAPESP.

Expression and biological activity of the disintegrin domain of bothrostatin (D-BTT) from the venom gland of Bothrops jararaca

Silva, C.A.^I; Mentele, R.^II; Camargo, A.C.M.^I; Serrano, S.M.T.^I

^ILaboratório de Bioquímica e Biofísica, Instituto Butantan, CEP 05503-900, São Paulo, Brasil

^IIDepartment of Clinical Chemistry and Clinical Biochemistry, University Hospital of Surgery, Ludwig-Maximilians University, Munich, Germany

^{Correspondence}

Disintegrins form a group of RGD containing peptides isolated from the venom of Viperidae and Crotalidae snakes. A large number of disintegrins has been isolated and characterized because of their clinical potential as antithrombotic agents. They possess both a high sequence homology and a notable variability in potency and selectivity in their interactions with integrin receptors. Venom disintegrins bind to the fibrinogen receptor aIIbb3 on the surface of platelets resulting in the inhibition of fibrinogen-dependent platelet aggregation, and more recently they have been shown to competitively inhibit the adhesive functions of a variety of integrins on cell surfaces. Bothrostain represents a precursor of the N-II class of the Reprolysins, isolated from a cDNA library derived from the venom glands of B. jararaca. Its cDNA has 2045 base pairs in length and encodes a pro-domain, a metalloproteinase domain with the characteristic zinc binding sequence and a disintegrin domain with the RGD sequence. The objective of this work was to express and analyze the biological activity of the disintegrin domain on human platelets. For this purpose the disintegrin domain was subcloned in pGEX-4T1 and expressed as a fusion protein with glutathione S-transferase (GST) in E. coli BL21. The recombinant disintegrin domain of bothrostatin (D-BTT) was obtained after cleavage of the fusion protein with thrombin and its authenticity was confirmed by N-terminal protein sequencing. Both D-BTT and GST/D-BTT proteins inhibited collagen-induced platelet aggregation in a dose-dependent fashion with estimated IC50 values of 12 nM and ~1mM, respectively.

Financial Support: CAT-Cepid/FAPESP.

Covalent structure and some pharmacological features of native and cleaved a-KTx_12-1, a four disulfide-bridged toxin from Tityus serrulatus venom

Pimenta, A.M.C.^{I, III}; Mansuelle, P.^I; Diniz, C.R.^II; de Lima, M.E.^III; Martin-Eauclaire, M.F.^I

^ILaboratoire de Biochimie - Ingénierie des Protéines, UMR 6560 - IFR Jean Roche, Marseille, France

^IICentro de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias, Belo Horizonte, MG, Brazil

^IIILaboratório de Venenos e Toxinas Animais, Depto. de Bioquímica e Imunologia, ICB, UFMG, Belo Horizonte, Minas Gerais, Brasil

^{Correspondence}

In searching for new molecules able to block voltage-activated K+-channels currents, a screening was carried out on rat brain synaptosomal preparations using ¹²⁵I-Kaliotoxin (¹²⁵I-KTx) in competition tests as an activity marker. A toxin with four disulfide bridges from Tityus serrulatus venom, named a-KTx12-1, was found able to compete with ¹²⁵I-KTx with an IC50 of 46 nM. The obtained amino-acid sequence and molecular mass are identicals to previously described butantoxin and differs by only one residue at C-terminal region to TsTX-IV, a toxin that was found able to inhibit Ca²⁺-activated K+ channels of high conductance. Enzymatic cleavages in native peptide followed by mass spectrometry peptide mapping analysis were used to determine the disulfide bridges pattern of a-KTx12-1. Also, after the cleavage of the first six N-terminal residues, including the unusual disulfide bridge which forms a N-terminus ring, the potency of the cleaved peptide was found to decrease at least 50 fold compared to the native toxin on the same preparation. Although activity on voltage-activated K+-channels is found toward the C-terminal portion of KTx, it is clear, in light of these results, that at least in case of a-KTx12-1 the N-terminal portion plays an important role on its potency.

Financial support: CAPES/COFECUB, CNPq, INSERM, CNRS and FAPEMIG.

Individual variability in Tityus serrulatus (Scorpiones, Buthidae) venom elicited by MALDI-TOF mass spectrometry

Pimenta, A.M.C.^{I, II}; Almeida, F.M.^{I, II}; de Lima, M.E.^II; Bougis, P.E.^I; Martin-Eauclaire, M.F.^I

^ILaboratoire de Biochimie, Ingènierie des Protèines, UMR6560, IFR Jean Roche, 15, Bd Pierre Dramard, 13916, Marseille, France

^IILaboratório de Venenos e Toxinas Animais, Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627, 31270-901, Belo Horizonte, Minas Gerais, Brazil

^{Correspondence}

Venom variability in Tityus serrulatus specimens, a Brazilian parthenogenic scorpion, was assessed by mass spectrometry (MALDI-TOFMS) analyses. An expanded time lag venom extraction protocol was carried out using ten scorpions to verify individual variations that might happen due different rates in protein expression and/or processing. First extraction event took place after 20 days of animals starvation to allow the venom gland to be filled up. The second and third extraction events were carried out 24 hours and 7 days, respectively, after the first extraction. By means of MALDI-TOF analyses, important variations were observed in venoms of a single specimen extracted at different times, specially in latter extraction events. These variations are most probably related to expression and processing of mature toxins. Since T. serrulatus is a parthenogenetic species, gender variations are naturally excluded and we did not expected intra-specific variations, which was confirmed. Knowledge of individual venom variability is extremely important to avoid misunderstandings in the use of venom proteomic analysis as a taxonomic tool.

New methodology for the isolation of an antimyotoxic protein from opossum serum and a relative protein from human plasma

Chermont, S.A.^I; Rocha, S.L.G.^I; Jurgilas, P.B.^I; Neves-Ferreira, A.G.C.^I; Domont, G.B.^II; Perales, J.^I

^IDepto. Fisiologia e Farmacodinâmica, IOC, FIOCRUZ, Rio de Janeiro

^IIDepto. Bioquímica, IQ, UFRJ, Rio de Janeiro

^{Correspondence}

Bothropic venom toxins are known to produce mainly local tissue damage, such as haemorrhage and myonecrosis. Natural resistance to snake venom toxic effects is observed in some animals and, in most cases, is due to the presence of soluble neutralizing proteins in their sera. From the opossum (Didelphis marsupialis) serum, we have previously isolated an acidic glycoprotein named DM64 using ion exchange, hydrophobic interaction and size exclusion chromatographies. DM64 inhibits both the myotoxicity and cytotoxicity of myotoxins I and II from Bothrops asper venom. The cDNA coding for DM64 was cloned and its deduced protein sequence showed homology to a1B-glycoprotein, a human plasma protein of unknown function, member of the immunoglobulin supergene family. The aim of this study was to purify DM64 from opossum serum using affinity chromatography with myotoxin. A similar protocol was used to attempt the isolation of a1B-glycoprotein from human plasma. Opossum serum was submitted to an affinity column coupled with myotoxin I from Bothrops asper venom. Homogeneous DM64 was obtained after eluting the column with 0.1 M glycine/HCl pH 2.7. Human plasma was initially submitted to Blue-Sepharose chromatography. The bound fraction was eluted with 1 M NaCl and applied to the affinity column coupled with myotoxin I. A single protein was eluted with glycine/HCl. It was homogeneous by native PAGE and presented 103 kDa on Superdex 200, in accordance with the molecular mass reported for the dimer of a1B-glycoprotein. In conclusion, we purified DM64 from opossum serum using affinity chromatography with myotoxin. The identification of the protein isolated from human plasma using this same methodology is under way.

Neurotoxin-like protein from the Triatoma infestans kissing bug salivary gland

Correa, P.S.^I; D´Souza-Ault, M.^I; Martins, N.F.^II; Feijó, G.^I; Santana, J.M.^I; Lozzi, S.P.^I; Teixeira, A.R.L.^I

^ILaboratório Multidisciplinar de Pesquisa em Doença de Chagas, Universidade de Brasília

^IILaboratório de Bioinformática, EMBRAPA - Recursos Genéticos e Biotecnologia, Brasília, DF, Brasil

^{Correspondence}

INTRODUCTION: Salivary proteins from several blood-sucking arthropods have revealed an unusual evolutionary relationship. Most induce vasodilation, inhibit blood coagulation, and reduce inflammation by different mechanisms (Montfort et al, 2000). Recently, it was found a Triatominea salivary protein with cytolytic and pore-forming activity (Amino et al, 2002). Our goal is to study the relationship between structure and function of these toxins. For this purpose the saliva from T. infestans, the major vector of Chagas disease, was submitted to a molecular screaning to identify pharmacological properties. This work presents study on the biology, biochemistry, molecular biology and modeling of a new cytolytic salivary gland protein.

METHODOLOGY: The T. infestans salivary gland cDNA library that was constructed, was screened to select the clones. Twenty clones were analysed. The most functional clone was selected for heterologous expression in insect cells and E.coli. The sequences were submitted to several bioinformatics analysis and molecular modeling procedures.

RESULTS AND CONCLUSION: the sequence analysis using BLAST and FASTA indicated 88% similarity with a neurotoxin from the scorpion Buthus occitanus. Its secondary structure was predicted using the TITO server and PROSPECT program and a proper template was found. The three dimensional structure was built using MODELLER 4.0. A theoretical model that was validated by PROCHECK and PROSAII programs, revealed a globular protein mainly with alpha-helix . The BLOCKS+ search revealed a phosphatase domain in the protein core. Active site prediction showed a possible binding cavity for the substrate docking.

DM43, a snake venom metalloproteinase inhibitor, induces cellular death on fibroblast cell line (3T3)

Jurgilas, P.B.^I; De Meis, J.^II; Mendes-da-Cruz, D.^II; Farias-de-Oliveira, D.^II; Savino, W.^II; Perales, J.^I

^IDepto. Fisiologia e Farmacodinâmica, Lab. Toxinologia, IOC, FIOCRUZ, Rio de Janeiro, Brasil

^IIDepto. Imunologia, Lab. Pesquisas sobre o timo, IOC, FIOCRUZ, Rio de Janeiro, Brasil

^{Correspondence}

Natural resistance of marsupials against the toxic effects of venoms is due to the presence of soluble neutralizing proteins in their sera. We have isolated from opossum serum (Didelphis marsupialis) an acidic glycoprotein with 43 kDa (DM43), with an effective inhibition action to metalloproteinases snake venoms, by non-covalent complex formation (Neves-Ferreira et al., 2000). Recently, it was showed that, the tissue inhibitors of metalloproteinases (TIMPs), are multifunctional factors which, besides their inhibitory effect, have others activities including regulation of proliferation, pro-MMP-2 activation, inhibition of angiogenesis, cell transformation, and apoptosis (Brew et al., 2000). The aim of this work was to study the ability of DM43 to produce some cellular effect, as TIMPs do. Celularity, viability and proliferation were carried out on 3T3 fibroblasts, incubating three doses of DM43 (10, 250 e 1000ng) for 20 hours at 37°C. Apoptosis and necrosis were detected using flow cytometry by light scatter and analyzed on a FACScan. Cells were also reviewed microscopically for morphological evidence. Our results demonstrated a celularity decrease, in all doses used of DM43. This decrease could be promoted by the increase of cellular death and/or change of cellular cycle. Cell cycle analysis demonstrated similar results in control and in DM43 treated cells. The measured of the cellular death by 7-Actinomicin D (7AAD) labeling showed a significative increase of cell death in all doses analyzed, but preferentially with 10ng of DM43. Interestingly, DM43 in this lowest dose seems to be responsible for the lower celularity and the higher cell death. Our data suggests that the snake-venom metalloproteinase inhibitor (DM43) promotes cell death in fibroblast (3T3). Studies are under way to understand the mechanisms involved in this phenomenon.

Supported by: CAPES, CNPQ, FIOCRUZ.

Evaluation of cutaneous lesions experimentally inducted by Bothrops jararaca snake venom

Araujo, L.F.^I; Macera, J.M.P.^II; Melo, P.A.^III

^ICurso de Pós-Graduação em Dermatologia do Departamento de Clínica Médica

^IIServiço de Anatomia Patológica, HUCFF

Departamento de Farmacologia Básica e Clínica , ICB, CCS, UFRJ, Rio de Janeiro, Brazil

^{Correspondence}

The development of malignant tumors has been subject of study in many different animal models. Previous report (Mello et al., Skeletal Radiol. 2000, 29(5):298-301) related a case about a man bitten accidentally by a snake of the genus Bothrops, which causes 90.4% of bites in Brazil. After complete recuperation from ulcerated lesion, it kept as assymptomatic scaling area. It turned into differentiated squamous cell carcinoma in 23 years. Due to lack of studies about inducted lesions by ophidian toxins non- related to acute phases, we tested protocol with histopathological study to characterise the inducted chronic morphological alterations. Swiss male mice and the B. jararaca venom were used in this study. The lesion was induced on the intermediate portion of the tail by venom intradermic injection (100 mg in 100 ml for each animal). The animals were sacrificed in several intervals (1, 2, 3, 7, 12, 14 and 23 days) under general anesthesia in order to remove the tail for histological processing. Macroscopic alterations showed evolution from acute reaction to healing. Some animals showed persistent ulceration, necrosis and keratosis. The microscopy revealed epidermal and dermal necrosis, acute inflammatory infiltrate, progressing to pseudoepitheliomatous hyperplasia, acanthosis, basal layer loss, disarrangement, pleomorphism and parakeratosis. Our experiments demonstrated the B. jararaca venom injection can produce difficult healing lesions in some animals whose histopathological studies revealed dysplastic alterations seen in pre-malignant lesions. The experiments also suggest this venom is instrument to study the epithelial regeneration.

Supported by CAPES, FAPERJ, FUJB and PRONEX

Actions of gyroxin, isolated from Crotalus durissus cascavella venom, in the isolated rat kidney

Martins, A.M.C.; Toyama, M.H.; Oliveira, D.G.; Marangoni, S.; Almeida, A.C.P.; Havt, A.; Nobre, A.C.L.; Barbosa, P.S.F.; Facó, P.E.G.; Fonteles, M.C.; Monteiro, H.S.A.

Unicamp and Federal University of Ceará-UFC

^{Correspondence}

OBJECTIVES: Envenomation by Crotalus genus leads to coagulation disorders, myotoxicity, neurotoxicity and acute renal failure, which is the principal cause of death. Thus we investigate the actions of the gyroxin isolated from Crotalus durissus cascavella on renal function.

METHODS AND RESULTS: The isolated rat kidneys were perfused with Krebs-Henseleit solution containing 6% of bovine serum albumin in absence and in presence of toxins. The parameters studied included perfusion pressure (PP), renal vascular resistance (RVR), glomerular filtration rate (GFR), urinary flow (UF), percent of sodium tubular transport (%TNa+), percent of potassium tubular transport (%TK+) and percent of chloride tubular transport (%TCl-). Maximum effects were seen in the last 30 minutes of each perfusion named 120 minutes. Treated group (gyr) was compared to a control group (ct) analyzed by Student t Test (*p<0,05). Gyroxin (5 mg/ml) increased significantly the PP (ct120= 114.3 ± 3.2 mmHg, gyr120= 144.5 ± 4.5 mmHg*) and RVR (ct120= 5.3 ± 0.05 mmHg/mL.g-1.min-1, gyr120= 7.0 ± 0.5 mmHg/mL.g-1.min-1*) and UF in the last 30 minutes of each perfusion, but GFR (ct120= 0.65 ± 0.01mL.g-1.min-1, gyr120= 0.67 ± 0.04 mL.g-1.min-1) remained stable during the 120 min. of perfusion. The %TNa+ (ct120= 81.2 ± 0.3, gyr120= 69.9 ± 1.9*) %TK+(ct120= 69.9 ± 0.9, gyr120= 63.7 ± 0.9*) and %TCl- (ct120= 78.9 ± 0.9, gyr120= 72.0 ± 1.1*) decreased significantly after the infusion of gyroxin.

CONCLUSION: These results indicate that gyroxin participates in the toxic effect of Crotalus durissus cascavella venom on isolated rat kidney.

Supported by FAPESP, CNPq

Determination of primary structure and its bactericidal activity of new CB isolated from Crotalus durissus collilineatus

Oliveira, D.G.^I; Toyama, M.H.^I; Carneiro, E.M.^II; Boschero, A.C.^II; Joazeiro, P.P.^III; Beriam, L.O.S. ^IV; Marangoni, S.^I

^IDepartamento de Bioquímica, Universidade Estadual de Campinas (UNICAMP), Campinas, Brasil

^IIDepartamento de Fisiologia e Biofísica, Universidade Estadual de Campinas (UNICAMP), Campinas, Brasil

^IIIDepartamento de Histologia e Embriologia Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Campinas, Brasil

^IVLaboratório de Bacteriologia Vegetal, Centro Experimental do Instituto Biológico de Campinas, São Paulo, S.P, Brasil

^{Correspondence}

OBJECTIVES: In this work we determined the amino acid sequence of new crotalic PLA2 from the C. d. collilineatus venom and its antibacterial activities on Xanthomonas axonopodis pv. passiflorae and C. m. sp michiganesnis.

METHODS AND RESULTS: The amino acid sequence of novel PLA2 was determined in the procise f (Applied Biosystem) and its amino acid sequence was: HLLQFNKMIKFETRRNAIPFYAFYGCYCGWGGRPKDATDRDRCCFVHDCCYEKLTGCNYKWDFYRY

SLRSGYFQCGKGTWCEQQICECDRVAAECLRRSLSTYRYGYMIYPKSRCRKPSETC. Cdcolli F6 shows higher antibacterial effect on the Gram-positive than Gram-negative bacterial strains. Thus, the inhibition of growth was of 95% and 85% for Claribacter ssp (Gran positive) and for Xanthomonas ssp, (Gran negative), respectively. Cdcolli F6 to produce cell membrane disruption allowing the penetration of the toxin into the cells (Xanthomonas ssp), and also induced several modifications principally in the LPS composition as suggested by electrophoresis analysis.

CONCLUSION: In conclusion, the bactericidal efficacy of PLA2(s), in general, and of Cdcolli F6, in particular, is dependent on both the presence of positive amino acid residue that allows electrostatic interaction between the Cdcolli F6 molecule and the bacterial membrane and their catalytic activity that hydrolysis the bacterial cell membrane.

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

Toyama, M.H.^I; Oliveira, D.G.^I; Santos, L.M.B.^II; Pereira, R.^III; Antunes, E.^III; Monteiro, H.S.A.^IV; Martins, A.M.C.^IV; Beriam, L.O.S.^V; Novello, J.C.^I; Marangoni, S.^I

^IDepartamento de Bioquímica, Faculdade de Ciências Médicas, UNICAMP

^IIDepartamento de Imunologia e Microbiologia, Inst. de Biologia, Faculdade de Ciências Médicas, UNICAMP

^IIIDepartamento de Farmacologia, Faculdade de Ciências Médicas, UNICAMP

^IVDepartamento de Fisiologia e Farmacologia, Universidade Federal do Ceará

^VLaboratório de Bacteriologia Vegetal, Centro Experimental do Instituto Biológico de Campinas, São Paulo, S.P., Brasil

^{Correspondence}

OBJECTIVES: We purified a novel L-amino acid oxidase (LAO) from Crotalus durissus cascavella snake venom and molecular, platelet aggregating, bactericidal and cytotoxic characterization was made.

METHOD AND RESULTS: This novel LAO was purified by two chromatographic steps: molecular mass and SP 5PW HPLC. The purified enzyme (CDCE-LAO) is present as a dimer on gel filtration, with an apparent M(r) of 60 kDa for the monomer as estimated by Tricine SDS-PAGE. CDCE-LAO showed high enzymatic specificity agains L-leucine (56U/mg) and induce platelet aggregation of whole and washed platelets using doses of 5,3 and 1mg/ ml. This novel protein inhibited around 85% and 35% of gram-positive and gram-negative bacterial growth rate, respectively and induced a significant decrease of viable B-cell number after stimulation with Con A in vitro. This novel protein shows high content of acidic amino acids and its n-terminal amino acid sequence was determined to be ADDRNPLAECFQENDYEEFLEIARNGLKATSNPKHVVIVGA...

CONCLUSION: This novel LAO represents around 7.5% of whole Crotalus durissus cascavella venom. In the three other samples of Crotalus durissus cascavella venom this LAO represent around 2.3 - 3.5% of whole venom. This novel protein showed significant bactericidal, platelet and haemagglutination activities and this suggests that this protein plays an important role in of blood homeostasis.

Partial purification of metalloprotease from Bothrops jararacussu snake venom

Mazzi, M.V.; Esmeraldino, L.E.; Buckeridge, Y.M.; Veronese, E.L.G.; Costa, S.M.C.; Cintra, A.C.O.; Sampaio, S.V.

Department of Clinical, Toxicological and Bromatological Analyses, School of Pharmaceutical Sciences of Ribeirão Preto-University of São Paulo. Avenida do café s/n. 14040-903, Ribeirão Preto, SP, Brazil

^{Correspondence}

OBJECTIVE: Isolation of metalloprotease for further biochemical and pharmacological characterization.

METHODS AND RESULTS: Crude B. jararacussu venom (CV) was fractionated on a Sephadex G-75 column, equilibrated and eluted with 0,05 M, pH 8,0 ammonium bicarbonate buffer at 25°C. The hemorrhagic fraction was rechromatographed on a DEAE-Sepharose column equilibrated and initially eluted with a 0,01 M, pH 7,2 phosphate buffer, followed by a NaCl concentration gradient which varied from 0.0 to 0.3 M in the same buffer. Hemorrhagic and clotting activities of the resulting subfractions were assayed according to Theakston and Reid, 1983. For the hemorrhagic assay the fractions were injected i.d. in 18-22g Swiss mice and the halos analyzed by the computer program “Image Tool”. For clotting assays, the fractions (25 mL) were added to 200mL of plasma and the time for first signs of fibrin formation recorded. From gel filtration four fractions (SI to SIV) were obtained and only SI was hemorrhagic. Rechromatography of SI yielded seven subfractions (SIDI to SIDVII) among which SIDV, SIDVI and SIDVII were hemorrhagic, while SIDIV and SIDV were coagulant.

CONCLUSION: SIDV revealed the highest hemorrhagic activity, which was lost at low pH (3,5).

Financial support: CNPq and FAPESP.

New insights on the mechanism of action of w-Phonetoxin-IIA

Gouvea dos Santos R ^{I, II}; Van Renterghem C^I; Martin-Moutot N^I, Mansuelle P^III; Sampieri F^III; Cordeiro MN^IV; Diniz CR^IV; de Lima ME^V; Seagar M^I

^IINSERM U464, Marseille, France

^IIRadiobiologia, CDTN, BH, Brazil

^IIICNRS UMR 6560, Marseille, France

^IVFUNED, BH, Brazil

^VDepto Bioquimica ICB/UFMG, Brazil

^{Correspondence}

w-phonetoxin IIA (w-PtxIIA) is a potent neurotoxin from Phoneutria nigriventer venom evoking flaccid paralysis and death after intracerebroventricular injection in mouse. In order to shed more light on its mechanism of action, we have used patch clamp methods in cell lines expressing recombinant Cav2.1, Cav2.2 and Cav2.3 channels and studied the biochemical parameters of binding of the radiolabeled w-PtxIIA on native and recombinant Ca⁺² channels.

P24C4 fraction, obtained from the C4 column RP-HPLC, was fractionated by reverse phase HPLC. Analysis of the purified fraction was recorded on a MALDI-TOF Perseptive Voyager Elite spectrometer and a 8363 kDa peptide was detected. After amino acid sequencing the purity of the peptide was confirmed and it was identified as w-PtxIIA. Calcium currents generated by Cav2.1 and Cav2.2 were blocked totally and almost irreversibly by 3 nM w-PtxIIA (toff > 230 and > 330 min, respectively), while Cav2.3 showed partial and readily reversible inhibition (toff= 85 s). w-PtxIIA was radiolabeled with ¹²⁵I using lactoperoxidase method and its binding on native (rat brain synaptosomal membranes) and recombinant calcium channels was studied. Binding assays demonstrated that rat brain synaptosomes display multiple classes of binding sites for ¹²⁵I- w-PtxIIA. High affinity binding of ¹²⁵I-w-PtxIIA (Kd= 50 pM) was totally displaced by w-PtxIIA (Ki = 100 pM), but only partially by w-conotoxin GVIA (25% inhibition) and w-conotoxin MVIIC (50% inhibition at 0.3 mM). ¹²⁵I- w-PtxIIA bound to the recombinant Cav2.1 and Cav2.2 channels, expressed on BHK cells, with high affinity (160 and 50 pM, respectively). The non-saturable (0.4 nM) interaction of ¹²⁵I- w-PtxIIA with Cav2.3 channels is in good agreement with the low affinity observed in the eletrophysiological experiments (Kd= 67 nM).

To summarize, w-PtxIIA binds to a single high affinity binding site on rat brain synaptosomes and this interaction results in the Cav2.1, Cav2.2 and Cav2.3 calcium currents blockade.

Supported by: CAPES/COFECUB, CDTN/CNEN

Antimicrobial effect induced by the Bothrops jararacussu and Bothrops alternatus venom in Staphylococcus aureus and Escherichia coli

Carlos, G. B.^I; Soares, A. M.^II; Marcussi, S.^I; Pietro, R. C. L. R.^III

^IMestrado em Biotecnologia de Plantas Medicinais e Microrganismos, UNAERP

^IIDepartamento de Biotecnologia, UNAERP

^IIIDepartamento de Farmácia, Universidade de Ribeirão Preto, UNAERP

^{Correspondence}

Group-II phospholipases had been described present bactericidal properties, specially the mammalian enzymes of inflammatory fluids and the isolated from snake venom from genus Bothrops. This study investigates the antimicrobial activity of Bothrops jararacussu and Bothrops alternatus crude venom on Staphylococcus aureus and Escherichia coli. The minimum inhibitory concentration (MIC) was visually determined through the macrodilution method using 4 x 10⁵ CFU (Colony-forming units)/mL and venom diluted in saline. MIC was defined as the lowest venom concentration that prevented the growth. The minimal bactericidal concentration (MBC) was estimated by subculturing the incubations in Müller-Hinton solid medium after different times: 20 minutes, 1 and 24 hours, and MBC was expressed in that concentration that reduces 90-100% of growth. The MIC and MBC were 1 and 2 mg/mL to B. jararacussu and B. alternatus crude venom to S. aureus. To E. coli the results of MIC and MBC were higher than these values. Our results show that the crude venom of Bothrops has antimicrobial activity especially to Gram-positive bacteria. This effect suggests that the crude venom of Bothrops genus presents biotechnologic importance to the research of new antimicrobial agents. Further studies should clarify the mechanism of antimicrobial activity of venom peptides.

Financial Support: UNAERP

Structural and functional analysis of an acidic platelet aggregation inhibitor and hypotensive phospholipase A₂ from Bothrops jararacussu snake venom

Soares, A.M.^{I, II}; Andrião-Escarso, S.H.^I; Carlos, G.B.^II; Marcussi, S.^II; Fontes, M.R.M.^III; Fuly, A.L.^IV; Côrrea, F.M.A.^V; Rosa, J.R.^VI; Greene, J.L.^VI; Giglio, J.R.^I

^IDepartamento de Bioquímica e Imunologia, FMRP/USP, Ribeirão Preto-SP

^IIDepartamento de Biotecnologia, UNAERP, Ribeirão Preto-SP

^IIIDepartamento de Física e Biofísica, IB, UNESP, Botucatu-SP

^IVDepartamento de Bioquímica Médica, UFRJ, Rio de Janeiro-RJ

^VDepartamento de Farmacologia, FMRP/USP, Ribeirão Preto-SP

^VICentro de Química de Proteínas, USP, Ribeirão Preto-SP, Brazil

^{Correspondence}

An acidic (pI ~ 4.3) phospholipase A2 (BthA-I-PLA2) was isolated from Bothrops jararacussu snake venom by ion-exchange chromatography on CM-Sepharose followed by rechromatography by RP-HPLC C-18 column. It is a 14 kDa single chain Asp49 PLA2 with approximately 122 amino acid residues, 7 disulfide bonds and the following N- and C-termini sequences (85 residues): ¹SLWQFGKMINYVM-GESGVLQYLSYGCYCGLGGQGQPTDATDRCCFVHDCC⁵¹-----⁸⁴QIC ECDRVATT CFRDNKDTYDIKYWFYGAKNCQEK¹¹⁸-----. Crystals of this acidic protein were obtained which diffracted beyond 2.0 Å resolution using a rotating anode source. These crystals are monoclinic and have unit cell dimensions a = 33.9, b = 63.8, c = 49.1 Å and b =104.0°. Although not myotoxic, cytotoxic or lethal, the enzyme is catalytically 3 to 4 times more active than basic myotoxic PLA2s from the same venom (BthTX-I and II) and other Bothrops venoms. Although devoid of toxic effects, it was able to induce time-independent edema, these activity being inhibited by ethylenediaminetetraacetic acid (EDTA). In addition, it showed hypotensive response and inhibited platelet aggregation. The catalytic, desintegrin and pharmacological activities were abolished after chemical modification by 4-bromophenacyl bromide which covalently binds to His48 of the catalytic site. Antibodies raised against crude B. jararacussu venom recognized this acidic PLA2, whilst Asp49 anti-BthTX-II recognized it partially and Lys49 anti-BthTX-I showed the least cross-reaction. These data show that myotoxicity is not necessarily correlated with catalytic activity in native PLA2s homologues and that any of these two activities may even exist alone.

Financial support: FAPESP and CNPq.

Analysis of expressed sequence tags (EST) of Thalassophryne nattereri fish venom glands using a cDNA library constructed in pGEM11ZF+

Magalhães, G.S.^I; Junqueira de Azevedo, I.M.J.^II; Lopes-Ferreira, M.^I; Ho, P.L.^II; Moura-da-Silva, A.M.^I

^ILab. Imunopatologia-Instituto Butantan

^IICentro de Biotecnologia-Instituto Butantan, Brazil

^{Correspondence}

Envenomation by Thalassophryne nattereri fishes is an important medical problem in North and Northeast of Brazil, causing in human victims intense pain and edema followed by necrosis. The effects of T. nattereri venom in inflammation and blood coagulation include peculiar mechanisms, completely distinct from other animal venoms. These observations suggest that new categories of highly potent toxins might be present in this venom, and to assess this possibility a representative cDNA library obtained from T. nattereri venom gland mRNA was used. The mRNA was extracted after gland stimulation by collecting its venom 4 days previously. Library was plated and 1,300 clones were isolated, of which a hundred have already been partially sequenced and searched for homology in the DDBJ/GENBANK with blastN and blastX programs. Clones matching known sequences were classified according to their function, showing similarity with proteins involved in cell structure/motility, gene/protein expression and metabolism. Some clones provided sequences showing low significant similarities with known gene products. Amongst this group, 4 sequences did not find any hit in the database, suggesting the presence of entirely new genes. Interesting enough 14 sequences showed similarity, yet low, with ADAM family of proteins, which are usually present in mammals and snake venoms representing important components for cell-matrix interactions. These data suggest that this library is representative of the venom gland constitution and further sequencing will lead us to isolate the clones coding for the components of this venom responsible for its toxicity.

Financial support: FAPESP

A survey of gene expression and diversity in the venom glands of the pit viper Bothrops insularis through the generation of expressed sequence tags (ESTs)

Azevedo, I.L.M.J.; Ho, P.L.

Centro de Biotecnologia, Instituto Butantan - São Paulo SP – [e-mail]

^{Correspondence}

In order to produce a global panorama of the transcriptional activity of snake venom glands and to correlate this with its venom composition, we constructed a plasmidial cDNA library from B. insularis mRNA, which was validated by restriction analysis of sample clones and used to generate an Expressed Sequence Tags (ESTs) database. Good quality sequences were obtained from 610 independent clones, clustered in unique genes and further analyzed by similarity comparison with molecular databases. These analyses revealed the putative identification of 210 distinct gene products. Toxin sequences correspond to 56% of all transcripts (85 clusters), metalloproteinases (23%) and the bradykinin-potentiating peptides (BPPs) (11%) being the major components. This approach revealed a new highly expressed toxin similar to vascular endothelial growth factor (VEGF), which was recently reported (Junqueira-de-Azevedo et al., (2001) J. Biol. Chem. 276 pp. 39836). Among the 125 clusters matching cellular proteins, the major part represents molecules involved in gene and protein expression, notably in disulfide bond assembly, reflecting a high specialization of this tissue for toxin synthesis. An unusual representation of retrotransposon-like sequences was also found and could be related to the occurrence and diversity of many paralogous forms of toxins in venom gland. In conclusion, our B. insularis dbEST allowed the identification of the most common classes of toxins present in Viperidae venoms, which parallels the complex hemorrhagic effects evoked by the venom on the prey. In addition, it provides the first comprehensive set of reptilian gene sequences described so far.

Support: FAPESP, CNPq and Fundação Butantan.

Production and characterization of monoclonal antibodies against bothropstoxin-1, a K49 phospholipase A2

Campos, L.A.^I; Smith, T.J.^II; Nascimento, N.^I; Smith, L.A.^II; Spencer, P.J.^I

^IGrupo de Química de Proteínas, Laboratório de Biologia Molecular, IPEN/CNEN-SP

^IIToxinology Division, US Army Medical Research Institute of Infectious Diseases, USA

^{Correspondence}

Myotoxins are components of venom of many snakes from different genera. These toxins belong to two major groups, one that includes short chains peptides like crotamine and myotoxin A, while the other group is composed of molecules with a phospholipase A2 structure. Many of the latter myotoxins display critical mutations within the calcium-binding loop and, therefore, are practically inactive when assayed in the usual mixed micelles enzyme tests. These mutations include Q4/E, Y28/Q and D49/K. Toxins bearing these features are commonly referred to as K49 myotoxins. Although enzymatically inactive, the K49 toxins induce severe myonecrosis by a mechanism not yet fully understood. Evidences indicate a role for the cationic region of the c-terminal domain. Also, site directed mutagenesis studies allowed a good mapping of the protein surface. However, it is not clear whether these described regions are involved in binding domain recognition or in toxicity. Our goal was to produce and characterize monoclonal antibodies against non-overlapping epitopes of bothropstoxin-1, a K 49 myotoxin.

The antibodies were screened by surface plasmon resonance aiming to isolate antibodies against non overlapping epitopes. In our primary screening, 11 clones were selected for ascitic fluid production and subsequent IgG purification . The ascitic fluids were tested in vitro, after a pre-incubation step, against differentiated C2C12 myotubes. Our data suggest that 4 of our antibodies are able to neutralize the cytolytic effect of the toxin. Whether these antibodies bind to topologically close sites or to different sites involved in the toxic activity has yet to be investigated .

Mapping antigenic sites of toxin VII (g-toxin) from the scorpion Tityus serrulatus with the use of synthetic peptides

Nunes, K. P; Andrez, R. M. A; Hermógenes, A. L. N; Bohórquez, K; Granier, C.; Chávez-Olórtegui, C.

Fundação Ezequiel Dias, Belo Horizonte, MG, Brasil and Institute de Biotechnologia et Pharmacie, Université de Montpellier-France

^{Correspondence}

Toxin VII (TsVII), a b-type toxin also known as Ts g is the most potent neurotoxin in the venom of scorpion Tityus serrulatus. The immunization of animals with such toxic substances for the production of antibodies is a difficult problem. As peptides derived from the sequence of scorpion toxins are nontoxic themselves, but might be immunogenic, the generation of anti-peptides antibodies able to recognize the parent toxin appeared to be an alternative strategy. With the aim of localising immunogenic areas, we have measured the ability of anti-TsVII neutralizing antibodies to bind to sets of immobilized synthetic peptides. Twenty four overlapping pentadecapeptides of uniform size, frameshifted by 2 residues, covering the complete amino acid sequences of TsVII were prepared on cellulose membranes according to the protocol previously desribed by Molina et al.,1996 (Peptide Res. 9, 151-155). The results of immunoassay with cellulose-bound peptides demonstrated that one main antigenic region was located in the N-terminal part of TsVII. Anti- TsVII antibodies also cross-reacted with the N-terminal part of Tsll, another b-type toxin and TsIV toxin (Tityus toxin of Diniz), a representative a-type toxin. As residues in the N-terminal region are conserved in scorpion toxins it is probable that residues 1-15 in the protein sequence are the key residues, for antibody recognition and neutralizing properties.

The results obtained in the present study indicate that the Spot synthesis method using the synthetic peptides covalently bound to the cellulose membrane should be used as the technique for the precise localization of linear functional epitopes.

Supported by: CNPq, INSERM, FAPEMIG.

Actions of convulxin, isolated from Crotalus durissus cascavella venom, in the isolated rat kidney

Martins, A.M.C.; Toyama, M.H.; Nobre, A.C.L.; Havt, A.; Facó, P.E.G.; Barbosa, P.S.F.; Almeida, A.C.P.; Bezerra, G.P.; Fonteles, M.C.; Monteiro, H.S.A.

UNICAMP and Federal University of Ceará- UFC – Brazil

^{Correspondence}

The most serious systemic change and primary cause of death after snakebite accidents is acute renal failure. In this work, we studied the action of convulxin, isolated from Crotalus durissus cascavella venom on renal function in the isolated rat kidneys perfused with Krebs-Henseleit solution containing 6% of bovine serum albumin. The parameters studied included perfusion pressure (PP), renal vascular resistance (RVR), glomerular filtration rate (GFR), urinary flow (UF), percent of sodium tubular transport (%TNa+), percent of potassium tubular transport (%TK+) and percent of chloride tubular transport (%TCl-). Maximum effects were seen in the last 30 minutes of each perfusion named 120 minutes. Treated group (cvx) was compared to a control group (ct) analyzed by Student t Test (*p<0,05). Convulxin (5 mg/ml) did not cause any alterations on the renal parameters studied. After the infusion of convulxin we observed that PP (ct120= 114.3 ± 3.2 mmHg, cvx120= 122.1 ± 5.4 mmHg*), RVR (ct120= 5.3 ± 0.05 mmHg/mL.g^-1.min^-1, cvx120 = 5.7 ± 0.6 mmHg/mL.g^-1.min^-1*), FU (ct120= 0.15 ± 0.002mL.g^-1.min^-1, cvx120= 0.14 ± 0.01 mL.g^-1.min^-1), GFR (ct120= 0.65 ± 0.01mL.g^-1.min^-1, cvx120= 0.67 ± 0.04 mL.g^-1.min^-1), %TNa+ (ct120= 81.2 ± 0.3, cvx120= 81.3 ± 2.1*), %TK+(ct120= 69.9 ± 0.9, cvx120= 73.5 ± 1.8) and %TCl- (ct120= 78.9 ± 0.9, cvx120= 79.3 ± 1.8) remained stable during the 120 min. of perfusion. These results showed that convulxin do not participate of the toxic effect of Crotalus durissus cascavella venom in isolated rat kidney model.

Financial supported by CAPES/FAPESP

Acute effects of microcystin-LR on the intestinal function in rats: a preliminary study

Nobre,A.C.L.; Nunes-Monteiro,S.M.; Monteiro, M.C.S.A.; Havt, A.; Facó, P.E.G.; Barbosa, P.S.F.; Angelim, E.V.; Martins, A.M.C.; Lima, A.A.M.; Monteiro, H.S.A.

Department of Physiology and Pharmacology, Federal University of Ceará, Ceará, Fortaleza – Brazil

^{Correspondence}

OBJECTIVE: Microcystins, the cyclic heptapeptide toxins produced by freshwater cyanobacteria like Microcystis aeruginosa. The most common and potently toxic of these toxins is microcystin-LR (MCLR). The aim of the present investigation was to examine the intestinal functional effect caused by microcystin-LR.

METHODOLOGY: Adult both sexes Wistar rats weighing 180-200 g were anesthetized with ketamine (60 mg.kg^-1 body weight, IM) and xylazine (10 mg.kg^-1 body weight, IM). Animals were fasted prior experimentation for 24 hours with access to water ad libitum. The 30 cm of proximal and distal of intestine was cannulated. The solution with Ringer or MCLR (1mg/ml) was administrated by proximal cannula, using a Holter Roller Pump with reduced flow (0,16 ml/min). The perfusate was collected each 20 minutes during 100 minutes. Then, the samples were analyzed for sodium, potassium, chloride and osmolarity. Data were analyzed by unpaired Student t Test (*p<0,05).

RESULTS: MCLR inhibited significantly the intestinal transport (mEq/min/g) of chloride (Control: 4,011±0,046 vs MCLR:-1,850±0,1848*), potassium (Control:-0,418±0,039 vs MCLR:-0,225±0,011*), sodium (Control:-4,637±0,565 vs MCLR:-1,360±0,064*), osmolarity (Control:-8,467±0,863 vs MCLR:-0,473±0,447*), but did not changed water transportation (Control: -0,006±0,0006 vs MCLR: 0,004±0,0007 mL/min/g).

CONCLUSIONS: MCLR increase the osmolarity and decrease ions transport but did not altered water transport in the isolated intestinal loop. Maybe microcystin is able to increase intestinal ions reabsorption.

Financial support: CNPq, CAPES.

Hemostatic system alterations induced by neuwiedase: a metalloproteinase isolated from snake venom Bothrops neuwiedi

Rodrigues, V.M.; Izidoro, L.F.M.; Rodrigues, R.S.; Hamaguchi, A.; Homsi-Brandeburgo, M. I.

Institute of Genetic and Biochemistry, Universidade Federal de Uberlândia, Minas Gerais, Brazil

^{Correspondence}

Snake venoms of several species cause changes in the hemostatic system, by interfering with platelet function, damaging blood vessels and activating coagulation factors such as prothrombin, factor IX and fibrinogen. Neuwiedase is a 22 kDa class P-I metalloproteinase isolated from the venom of the B.neuwiedi. It is able to induce myotoxicity, edema and bleeding pulmonary. In this work we analysed the disturbance induced by the whole venom and neuwiedase on hemostatic system. Mice male (n=5, 25g) received 0,6 LD50 of neuwiedase (LD50= 5,0mg/kg) or 0,6 LD50 of whole venom (LD50=1,66mg/kg) by the i.p. route. Venom and neuwiedase were dissolved in 100ml PBS. Control mice received PBS alone. After 6 hours each animal was sacrificed and blood collected by puncture cardiac in presence or ausence of anticoagulant for quantitative analysis of red cells, hemoglobin, platelets (at equipment Coulter T-890), prothrombin and thrombin time, plasmatic fibrinogen concentration (at equipment Thrombolizer Compact) and whole blood coagulation time (at room temperature). The neuwiedase induced a significant decrease only of platelets (p<0,05 test t Student) and whole venom caused a reduction of platelets, red cells and hemoglobin (p<0,05 test t Student). An significant increase of prothrombin, thrombin and blood coagulation time was observed when the whole venom or neuwiedase were injected by i.p route at different inoculations time. Neuwiedase degraded 83% of plasmatic fibrinogen, however whole venom was capable to degraded only 35% when compared with control (PBS). The neuwiedase is a important metalloproteinase from the B. neuwiedi snake venom, by interfering with the normal hemostatic mechanisms. It showed action on proteins and cellular elements in blood, suggesting its potential use as an therapeutical agent for the treatment of human diseases.

Chemical and pharmacological characterization of the alkaloids of the skin of Epipedobates Flavopictus (Anura, Dendrobatidae)

Mortari, M.R.; Schwartz, C.A.; Schwartz, E.F.; Santos, M.M.; Sebben, A.

Laboratório de Toxinologia, Departamento de Ciências Fisiológicas – Instituto de Ciências Biológicas – Universidade de Brasília, Brasília, Brasil

^{Correspondence}

OBJECTIVES: The objectives of the present work were to isolate and to characterize, chemical and pharmacologically the most abundant alkaloids present in the cutaneous secretion of Epipedobates flavopictus.

METHODS: The specimens were collected in Pirenópolis (Goias State, Brazil), their skins were removed and the alkaloids extracted with methanol. The alkaloids were purified by HPLC and each fraction was identified by gas chromatography and mass spectrometry. The crude extract and the identified fractions were tested on isolated frog sciatic nerve by the technique of "sucrose gap" and on isolated guinea pig ileum muscle.

RESULTS: Pumiliotoxin 251Da, histrionicotoxin 285Da and two decahydroquinolines: 219A and 243A were identified in E. flavopictus cutaneous secretion. The crude extract and the pumiliotoxin 251Da produced similar effects on frog sciatic nerve such as a prolongation of the potential and an elevation of the relative potential. Both produced rhythmic contractions and increased the muscular tension on isolated guinea pig ileum. Histrionicotoxin 285Da and decahydroquinolines did not produced any effect on sciatic nerve or guinea pig ileum probably due to their low toxicity and also to the small amount obtained by purification.

CONCLUSIONS: In the present work we demonstrated the presence of Pumiliotoxin 251Da, histrionicotoxin 285Da and two decahydroquinolines: 219A and 243A in the cutaneous secretion of the dendrobatidae frog Epipetobates flavopictus.

First occurrence of 11-oxotetrodotoxin in a terrestrial vertebrate

Pires Jr.,O.R.^I; Morales, R.A.V.^{I, II}; A. Sebben^I; Bloch Jr, C.^II; Schwartz, C.A.^I

^ILaboratório de Toxinologia, Departamento de Ciências Fisiológicas-UnB,

^IIEmbrapa-Recursos Genéticos e Biotecnologia-DF

^{Correspondence}

11-oxotetrodotoxin is a TTX analogue 3 to 4 four times more potent than TTX itself. It was discovery in the southern Pacific puffer fish Arothron nigropunctatus and, up to now, it was only described in two marine vertebrates. In this work we described the occurrence of this analogue in Brachycephalus ephippium from Atlantic rain forest using LC-FLD and LC-MS/MS systems.

Eight adults specimens of B. ephippium were collected and brought alive to laboratory and killed by cooling up to a frozen state and submitted to toxin extraction with methanol 70%-acid acetic1%, evaporated and ressuspended in deionized water. The semipurified extract was submitted to a HPLC system using a post-column fluorescent detection system (LC-FLD) and to a LC-MS/MS system carried out in selection ions monitor mode (SIM) for m/z 320Da and 336Da corresponding to M+H+ of TTX and 11-oxoTTX respectively. These were chosen as precursor ions to be fragmented by collision-induced dissociation (CID).

The comparison with retention time of synthetic 11-oxoTTX, produced by oxidation of TTX with Fenton’s reagent, in the LC-FLD system, and with SIM data confirm its identity. The CID data supports the identification of M+H+ 336Da as 11-oxoTTX showing some common guanidine ions of TTX family (162Da, 150Da and 178Da) and particular ions of 318Da, 300Da and 282Da corresponding to continuous loss of water from its structure.

It is the first occurrence of 11-oxoTTX in an terrestrial vertebrate. The origin of this molecule in B. ephippium extracts is not yet understood but can be suggested that this formation occurs by transformation of some derivative present with TTX or even itself in this animal.

The first evidence of bacterial TTX-producing in amphibians

Schwartz, C. A.; Pires Jr., O. R.; Sebben, A.

Laboratório de Toxinologia, Departamento de Ciências Fisiológicas, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, Brazil

^{Correspondence}

For marine animals is assumed an exogenous origin of TTX, many works report several bacteria genus producing TTX and related substances The same has been suggested for amphibians, although, there is no support to this hypothesis.

Seven bacterial strains isolated of B. ephippium intestinal contents, were cultured in ISP-4 liquid media (for 5 days) and substituted for modified liquid media ISP-4 low phosphate concentration, Gallacher and Birkbeck, 1993 (Appl. Env. Microb. 59(11), 3981-3983) demonstrated that the stress induced for depletion of phosphate, in the culture media, increases the TTX production in marine bacteria. The cultures were centrifugated to separate bacteria from culture media. To the cells were added 100 ml of 0.1 % acetic acid. The mixture was heated to boiling and filtered. All bacteria extracts and culture media were semi-purified by a ion-exchange column (Amberlite GC-50, NH4+) and active charcoal treatment (Norit-A). The semi-purified extracts were submitted to an HPLC system using a post-column fluorescent detection system (LC-FLD).

Neither bacteria extracts or ISP-4 culture media showed fraction similar to TTX. Only low phosphate ISP-4 culture media presented a fraction with retention time equal to 14,793 and 14,839 minutes, similar to of TTX standard, 14,425 minutes. Bacteria, as TTX source in amphibian was suggested by analysis in LC-FLD system, however it is not discarded the origin of this kind of compounds from diet. Although the mechanism of absorbing and accumulating this toxin or related substances remains indefinite, the identification of TTX-producing strains in intestinal microbiota in other amphibians must be done to elucidated the amount of bacteria involved in TTX production in terrestrial environment.

Guanidine compounds of a Brazilian pufferfish Sphoeroides spengleri

Morales, R.A.V.^{I, II}; Pires Jr, O.R.^I; Oliveira, J.S.^III; Freitas, J.C.^III; Schwartz, C.A.^I; Bloch Jr, C.^II

^ILaboratório de Toxinologia, Departamento de Ciências Fisiológicas-UnB

^IIEmbrapa-Recursos Genéticos e Biotecnologia-DF

^IIIDepartamento de Fisiologia, Instituto de Biociências e Biologia Marinha-USP

^{Correspondence}

Puffer fish toxicity derives from the presence of guanidine compounds such as TTX and analogs probably acquired from food chain or from symbiotic bacterial strains present in their skin or digestive tract. In this work we analysed the guanidine compounds present in Sphoeroides spengleri, a brazilian pufferfish from São Sebastião Channel using LC-FLD and LC-MS/MS.

85 specimens of S. spengleri were submitted to toxin extraction with methanol 70%-acid acetic1%, evaporated and ressuspended in deionized water. The semipurified extract was submitted to an HPLC system using a post-column fluorescent detection system (LC-FLD) and to an LC-MS/MS system, carried out under a selection ion mode (SIM). The m/z values of 320Da, 290Da, 302Da, 304Da and 336Da were chosen as precursor ions, fragmented by collision-induced dissociation.

The retention time data from the LC-FLD system showed the presence of TTX, 4-epiTTX, 6-epi-TTX, 4,9-anidroTTX and TDA while the SIM data confirmed the presence of these compounds and revealed 4 other analogs 11-norTTX-6(S)-ol, 5-deoxyTTX, 11-deoxyTTX, 11-oxoTTX in puffer extracts.

The identity of the 320Da, 302Da and 304Da ions was achieved by their fragmentation pattern and the formation of characteristic daughter ions such as TTX(302Da, 284Da, 256Da,178Da and 262Da), 4,9-anhydroTTX(256Da, 210Da, 195Da, 162Da and 135Da) and 5-deoxyTTX(286Da, 176Da, 162Da, and 146Da), according to Shoji, Y. et al., 2001(Analytical Biochem.290,10-17).

The identification of puffer toxins is somewhat limited in an LC-FLD detection system, due to C9-base analog different fluorescent intensities and conversion efficiencies. LC-MS/MS is thus a powerful tool, capable of determining guanidine compounds previously gone undetected by current used methods.

Futher report of occurrence of tetrodotoxin (TTX) and new analogues in the Anura family Brachycephalidae

Pires Jr, O.R.^I; Morales, R.A.V.^{I, II}; A. Sebben^I; Bloch Jr, C.^II; Schwartz, C.A.^I

^ILaboratório de Toxinologia, Departamento de Ciências Fisiológicas-UnB,

^IIEmbrapa-Recursos Genéticos e Biotecnologia-DF

^{Correspondence}

Tetrodotoxin (TTX) is a potent neurotoxin that occurs in a wide range of animals, among terrestrial organisms, TTX has been reported only in certain amphibians. In this work, the occurrence of TTX and analogues was examined in three braquicephalid species: Brachycephalus ephippium, B. nodoterga and B. pernix using LC-FLD and LC-MS/MS.

The methanolic extracts were evaporated and ressuspended in deionized water. The semipurified extract was submitted to an HPLC system using a post-column fluorescent detection system (LC-FLD) and to an LC-ESI/MS/MS system, carried out under a selection ion mode (SIM).

The retention time data from the LC-FLD system showed the presence of TTX, 4-epiTTX, 4,9-anidroTTX and TDA while the SIM data confirmed the presence of these compounds and revealed other analogs 11-norTTX-6(S)-ol, 5-deoxyTTX, 11-deoxyTTX, 11-oxoTTX, 6-epiTTX. Two new mass components were also identified by mass analysis, 348Da and 330Da.

The identity of the 320Da, 302Da and 304Da ions was achieved by their fragmentation pattern and the formation of characteristic daughter ions such as TTX(302Da, 284Da, 256Da,178Da and 262Da), 4,9-anhydroTTX(256Da, 210Da, 195Da, 162Da and 135Da) and 5-deoxyTTX(286Da, 176Da, 162Da, and 146Da), according to Shoji, Y. et al., 2001(Analytical Biochem.290,10-17).

The two unknown compounds showed daughter ions similar to TTX, suggesting new TTX analogues. The fragmentation of TTX and analogues prove to be a powerful tool to characterize this compound family.

There are almost 4.000 species of amphibians in 26 recognized families, among these, only six were described containing TTX (Urodela: Salamandridae and Ambystomatidae, Anura: Bufonidae, Dendrobatidae, Brachycephalidae and Rhacophoridae), probable TTX are not limited to these few families of amphibians.

Effect of denervation on the myotoxicity of Bothrops jararacussu venom on the fast- and slow-twitch muscles in mice

Tomaz, M.A.; Calil-Elias, S.; Fernandes, F.F.A.; Moraes, R.A.M.; Drusano, M.F.; Melo, P.A.

Departamento de Farmacologia Básica e Clínica, Instituto de Ciências Biomédicas, CCS, Cidade Universitaria – UFRJ, 21941-560, Rio de Janeiro, Brazil

^{Correspondence}

We examined the effect of denervation, by the sectioning of the sciatic nerve, on myotoxic effect of B. jararacussu venom on isolated EDL (Extensor digitorum longus – a fast- twitch muscle) and SOL (Soleus – a slow – twitch muscle) from mouse, as well as their total content of the enzyme creatine kinase (CK). Myotoxicity was assessed in vitro by loss of CK from muscles continuously perfused with physiological saline solution (PSS) alone or with crude B. jararacussu venom (25mcg/ml). The increase of rate of CK released (above basal level) induced by venom was measured. EDL and SOL had the same range of basal rate of CK release (0.25-0.36 ±0.04 U.g^-1.h^-1 (N =12), weight (7-12 mg) . The rate of CK release after 60 minutes of exposure to the venom, from EDL control, 7, 14 and 21 days after denervation were 13.67 ± 2.5, 9.55 ± 2.1, 5.10 ±1.4 and 3.12 ± 0.7 U.g^-1.h^-1 (N =4-6), respectively. While on SOL muscle were and 3.02 ± 0.9, 2.28 ±0.3, 0.13 ± 0.02 and 0.05 ± 0.05 U.g^-1.h^-1 (N =4-6 ), respectively. The CK content decreased circa of 40% (EDL) and 60% (SOL) on the day 7 and decreased of 48% and 60% on day 14, for EDL and SOL respectively. These data show that EDL is more sensitive to B. jararacussu venom than is SOL, despite the difference in total CK content.

Financial Support: CNPq-PIBIC, FAPERJ, FUJB-UFRJ, CAPES, FOGART-MIRT

Mice skeletal muscle regeneration after being damaged by Bothrops jararacussu venom: effect of heparin

Calil-Elias, S.^I; Oliveira, J.S.^I; Tomaz, M.A.^I; Fernandes, F.F.A.^I; Martinez, A.M.B^I; Melo, P.A.^II

^IDepartamento de Farmacologia Básica e Clínica, ICB, UFRJ. 21941-560, Rio de Janeiro, RJ, Brasil

^IIDepartamento de Histologia e Embriologia, ICB, UFRJ. 21941-560, Rio de Janeiro, RJ, Brasil

^{Correspondence}

We have previously shown that heparin antagonizes the myotoxic effect of Bothrops jararacussu venom on mice EDL (Extensor digitorum longus) muscle (Calil-Elias, et. al., 2002). We analyzed the effect of treatment, by the intravenous route, with either heparin (H – 10 mg/Kg), low molecular weight heparin (LMWH – 10 mg/Kg), or specific antivenom (AV – 1 mL/Kg) between 15 and 240 min. after the venom injection. Assessment was performed by electronic microscopy and the measurement of creatine kinase (CK) content. 21 days after the injection of the venom, the animals were killed under anesthesia with ether, and the EDL muscles were removed by dissection. A group of isolated muscles was homogenized for measurement of total CK content while another group was immersed in fixative solution and processed for electronic microscopy. The venom induced a 38% decrease in CK content compared to normal, and after treatment with H and LMWH the decrease was 30.5% and 1.3%, respectively. Our observations in electronic microscopy showed that the treatment with H and LMWH caused a complete regeneration of myofibers and other elements of intracellular organization. On the other hand, the animals that received only the venom injection showed regenerated cells that were completely disorganized. These data indicated that heparin improves the regeneration of EDL muscle damaged by B. Jararacussu venom.

Supported by: CAPES, FAPERJ, CNPq-PRONEX, FUJB-UFRJ.

Dart-poison frogs of Colombia

Angel, R.

Instituto Colombiano de Medicina Tropical, Medellín, Colombia

^{Correspondence}

Colombia is one of the richest countries of the world in biodiversity. It has the first place in amphibia, both in species number and in toxicity. Dendrobatidae family is represented by the Phyllobates, Dendrobates, Epipedobates and Minyobates genera. Among the five species of the Phyllobates genus, there are three in Colombia: P.aurotaenia, P. bicolor, P. terribilis. The Dendrobates genus has 7 species, the Epipedobates 8 species and the Minyobates genus has 7 species. The mayority of the total species are distributes in the Colombian West and along the Pacific Coast.

The natives Embera tribe are in the Colombian West and they use poisoned darts with skin extracts from the Phyllobates species, through the “bodoquera or cerbatana” (blowgun), in order to capture animals for their feeding and also for self-defence and revanche against their enemies. This poison is stored in the cutaneous glands that cover the dorsal skin and are two types: serouses and mucouses. The principal component of the poison from Phyllobates species, is batrachotoxin (BTx- C24H33N05-), and steroidal alkaloid wich acts as an activator of the Na+ ion conducts of the neurons and muscle cells, wich increases the permeability to Na+ and induce permanent depolarization.

From D. histrionicus it has been isolated histrionicotoxins , potents blockers non competitives of nicotinic channels. From D. lehmani it has been isolated a new alkaloid by the name of lehmizidine. From some species of Epipedobates genus it has been isolated epibatidine, a potent analgesic in experimentation.

The poison study of frogs could conduct to new tools for biological investigation and perhaps as medicaments.

From the ecological point or view, the deforesting accelerated rhythm, and illegal commerce of the frogs towards other countries, are carrying factors to the extinction of these beautiful jewels of nature.

Study of comparative anesthesic reactions with ketamine and ketamine/midazolan in serpents of the species Crotalus durissus terrificus

Silva, A.M.^I; Ribeiro, W.^I; Lopes Martins, R.A.B^I; Prianti A.C.G^I; Cruz, M.L.^II; Cogo, J.C.^I

^IUniversidade do Vale do Paraíba - UNIVAP, S.J. Campos - S.P

^IIUniversidade Estadual Paulista, UNESP -Botucatu -S.P

^{Correspondence}

Experimental scientific studies involving serpents demand research of alternatives to offer more safety in the manipulation. Equally, the present work is aimed at obtaining safe manipulation for serpents of the Crotalus durissus terrificus species, using anesthesic substances ketamine (KTM) and its association ketamine/midazolan (KTM/MDZ). Ten adult specimens of Crotalus durissus terrificus were used (492 ± 123 gr.), collected in the river Paraíba valley area, maintained in the Serpentarium of the University of the Valley of Paraíba - UNIVAP, kept at a constant temperature of 26ºC and divided in 2 groups of 5 animals each. Group I – intramuscular injection KTM (100mg/ml), in doses of 80 mg/kg. Group II – Intramuscular injection KTM (100mg/ml), in doses of 80mg/kg, in the same syringe with MDZ (5mg/ml), in the dose of 0,2 weight mg/kg. To evaluate the pharmacological answer during the anesthesic process, different physiologic parameters (breathing frequencies, heart rate, pain sensitivity, time of apnea and rectal temperature) and behavior (displacement, exhibition of the tongue, coilability, rattle, bite, and escape) were observed.

In relation to the physiologic parameters we can conclude that there were not significant differences between KTM and KTM/MDZ, except when evaluated the intensity of pain sensitivity parameter after mechanical stimulation. As, we can conclude that the association KTM/MDZ showed a mean of 1,4 (n=5) on a scale of 0 - 4,0 time vs intensity (240 min) (p<0,001), although when compared with KTM showed a mean of 3,0 (n=5). It is believed that this is related to the rapid effect of this chemical class.

Based on the data moderated in the literature, we didn't observe the same pharmacological profile for KTM. This way the job of KTM/MDZ presents an action profile that makes it possible to have safe manipulation, resulting from the muscle relaxation effect promoted by Benzodiazepinic and analgesic/anesthesic of KTM.

An epileptogenic peptide from the sea anemone Bunodosoma cangicum

Campêlo, P.A.; Santana, A.N.C.; Paiva, C.N.; Cavalheiro, E.A.; Ricart, C.A.O.; Cunha, R.B.; Treptow, W.L.; Sousa, M.V.; Cardi, B.A.; Carvalho, K.M.

Laboratório de Neurofarmacologia, Universidade Estadual do Ceará, Fortaleza, CE, Universidade de Brasília, DF, Brazil

^{Correspondence}

OBJECTIVES: Sea anemones tentacles release toxins which may be used in the capture of prey or to protect then from predators. Several polypeptides with action on the voltage-sensitive sodium channel have been isolated from anemones. In this study, we demonstrated the isolation, the epileptogenic effects and the sequence of a new peptide (cangitoxin, CGX) from the anemone Bunodosoma cangicum.

METHODS AND RESULTS: The specimens were immersed in distilled water (1:2, w:v) (30 min) and the suspension containing the discharged nematocysts was fractionated by HPLC using a C18 column (25x250 mm)(5 ml/min)(acetonitrile 0-40%, 30 min). The effluent containing the peptide was lyophilized and stored at -80°C. The amino acid sequence (Edman, 1950) of CGX, performed by an Applied Biosystems sequencer, was GVACRCDSDGPTVRGNSLSGTLWLTGGCPSGWH NCRGSGPFIGYCCKK. Only CGX sequence and those of BgII and BcIII, contain Arg-05, Tre-12, Arg-35, Ser-37 and Fen-40, constituting a new subclass of Type 1 peptides. It is the only that joins Thr-12 and Val-13 in the region that interact with the sodium channel suggesting new peculiarities in the mechanism of action of CGX. The electroencephalographic recording (EEG) alterations following an intrahippocampal injection of CGX into the rat brain showed important seizure periods that evolved to status epilepticus that lasted 8-12 hr.

CONCLUSION: The epileptogenic effects of CGX are very similar to those of the acute phase of the pilocarpine model of temporal lobe epilepsy and they suggest that it may be a tool to develop a new experimental model of status epilepticus, opening new perspectives in the field of the sea anemone toxin study.

Prospection of venom peptidase activities via fluorescence-based assay of naphthylamide derivatives

Gasparello-Clemente, E.; Olivo, R.A.; Silveira, P.F.

Laboratory of Pharmacology, Instituto Butantan

^{Correspondence}

The fluorometry of naphthylamide derivatives was employed for revealing representative peptidase activities in whole venoms of the snakes Bothrops jararaca, Bothrops alternatus, Bothrops atrox, Bothrops moojeni, Bothrops insularis, Crotalus durissus terrificus and Bitis arietans, of the scorpion Tityus bahiensis, and of the spiders Phoneutria nigriventer and Loxosceles intermedia. As a result, neutral aminopeptidase (APN) and prolyl-dipeptidyl aminopeptidase IV (DPP IV) were presented in all snake venoms (highest levels in Bothrops alternatus). All examined peptidases showed relatively low levels in arthropod venoms, except basic aminopeptidase (APB) from Phoneutria nigriventer venom. Relatively high levels of acid aminopeptidase (APA) were restricted to Bitis arietans venom. Bitis arietans also exhibited a prominent content of APB which was lower in the other venoms. Prolyl endopeptidase and proline iminopeptidase activities were respectively detectable only at low levels in Tityus bahiensis and Bothrops insularis while pyroglutamate aminopeptidase activity was undetectable in all venoms. In conclusion, the difference between snake and arthropod venoms was mainly represented by APN and also by DPP IV activities. The contents of APA and APB activities constituted the difference between Bitis arietans and Brazilian snake venoms. The distribution of the peptidase activities investigated reflected an interesting trend of evolutionary divergence in the different processing of peptides in different venoms and/or in different abilities of the venoms examined to hydrolyze different peptides during envenomation. Our data also provide evidence that the measurement of venom peptidase activities may be useful for the purposes of ecophysiological studies, for taxonomic classification of venomous animals, and to elucidate the evolutive history of their venoms.

FAPESP/FUNDAP fellowship. Research Grant 00/10023-8 from FAPESP

Cytotoxic effects in marine dinoflagellates extract

Naves, J.L. ; Freitas, J.C.

Biosciences Institute and CEBIMar, University of São Paulo, Brazil

^{Correspondence}

Filter feeding animals, as clams and fishes, can accumulate dinoflagellates toxins and when eaten by men, cause paralytic shellfish poisoning (PSP) and diarrheic shellfish poisoning(DSP) intoxications. Among the species that occurs in the coast of São Paulo States, Prorocentrum gracile was isolated from phytoplankton samples and Prorocentrum sp is a benthic species epiphytic on the seaweed Galaxaura marginata. They were being isolated and cultivated to identify eventual toxins.

These organisms were kept in constant temperature chamber at 12/12h photoperiod and maintained in multiplate wells with F/2 culture broth at 21°C. At the exponential phase of growth, the cells were transferred to bigger wells. The polar (EPs) (methanol) and apolar (EAs) (methylene chloride) extracts of the cultivated species are being tested, after solvent removal, for cytotoxicity in the embryo development of sea urchin eggs and in mouse erythrocytes. The EAs were tested in the hemolytic assay and the hemolysis was analyzed by the erythrocytes suspension transparence.

P. gracile EP exhibited no antimitotic activity while Prorocentrum sp EP and EA induced cells anomalies and cell division inhibition at CE50 78.75 ug/mL (IC 32.56–190.5) and CE50 22.5 ug/mL (IC 2.96-170.8) respectively (n=3). EAs induced hemolysis in the following concentrations: P. gracile - CE50 218,5 ug/mL (IC 115.8-412.4), Prorocentrum sp - CE50 28.25 ug/mL (IC 11.48-69.50). The Prorocentrum sp EP also induced hemolysis (CE50 200.2 ug/mL, IC 97.89 – 409.2ug/mL).

These results suggest the presence of cytotoxins in these dinoflagellates species.

Financial support: FAPESP.

Structural studies of Bothroinsularin, a thrombin inhibitor protein, from B. insularis venom

A L Oliveira-Carvalho; M. Assafim; D.L.S. Dutra; H.C. Castro; R.B. Zingali

Departamento de Bioquímica Médica, ICB, CCS, UFRJ

^{Correspondence}

Members of the protein family named C-type lectins have been purified of venoms from Viperidae snakes. These proteins display high amino acid sequence similarity although presenting diverse biological functions. Bothrojaracin (bjc), purified from Bothrops jararaca, a thrombin-binding protein (27 KDa) is a member of this family. Western blots analysis of Bothrops insularis crude venom show a variety of bands recognized by the polyclonal antibodies (anti-bjc), including a ~30 KDa protein.

METHODS AND RESULTS: Purification of crude venom by gel filtration followed by affinity chromatography on a PPACK-a-thrombin sepharose resulted in a protein consisting of a dimer formed by two 15 KDa chains as observed by SDS PAGE under reducing condition. This protein was recognized in western-blot assays using polyclonal antibodies raised against bothojaracin. Its N-terminal sequences (25aa), showed high similarity with bothrojaracin (90%) and others members of c-type lectin family. Bothroinsularin formed a complex with a-thrombin with a molar rate of 1/1. Similar data was obtained with prothrombin. The carboxymethylated a and b subunits were separated by a reversed- phase chromatography (Sephasil C8 Pharmacia) in an HPLC System.

CONCLUSION AND PERSPECTIVES: Here we describe the purification and characterization of a thrombin-binding protein named bothroinsularin. This protein shows high similarity with bjc. The sequence of the separated chains will be determined for further cloning and expression.

Finantial support: CNPq, CEE, Finep, Faperj

Phospholipase A2, procoagulant and fibrinolytic activities from Lonomia obliqua venom

Veiga, A.B G.; Pinto, A.F.M.; Dobrovolski, R.; Guimarães, J. A.

Laboratório de Bioquímica Farmacológica, Centro de Biotecnologia, UFRGS

^{Correspondence}

INTRODUCTION: Venom from Lonomia obliqua caterpillar is responsible for a haemorrhagic disorder reported in Southern Brazil. The clinical profile – characterized by haematuria, severe bleeding, acute renal failure and even death of untreated patients – probably results from the action of several and distinct active principles, present in different caterpillar secretions that enter the blood system during accidental contact.

OBJECTIVES: In this work we analyse phospholipase A2, procoagulant and fibrinolytic activities in different materials from L. obliqua: secretion obtained by thermal (-20 ºC) stress, tegumental extract, spicule extract and haemolymph.

MATERIALS: The materials were assayed in order to: a) indicate phospholipase A2 activity by indirect haemolysis of rabbit erythrocytes using egg yolk as substrate, b) analyse procoagulant activity upon recalcification time of human plasma in an ELISA-like system monitored by SpectraMax, c) analyse fibrinolytic activity by SDS-PAGE.

RESULTS: Procoagulant activity was found in all the samples: spicule extract (45.0 U/mg), secretion obtained by thermal stress (11.5 U/mg), tegumental extract (11.0 U/mg), haemolymph (6.0 U/mg). Fibrinolytic activity was observed after 24h of incubation in the haemolymph, in the tegumental extract and in the secretion obtained by thermal stress, the latter being the highest one, even after 10min of incubation. Phospholipase A2 activity was only found in the spicule extract (80.0 U/mg).

CONCLUSION: The activities studied in this work are not homogenously distributed throughout the caterpillar´s body. Results strongly indicate that the haemorrhage observed in this envenomation seems to be due to the combination of these activities.

Supported by: CAPES and FAPERGS.

Occurrence of bufotenine in the Osteocephalus genus

Costa, T.O.G.^{II, III}; Brito, J.P.^I; Morales, R.A.V.^I; Gordo, M.^III; Pinto, A.C.^II; Bloch Jr.,C.^I

^IEmbrapa–Recursos Genéticos e Biotecnologia

^IIUFRJ–Universidade federal do Rio de Janeiro

^IIIUA–Universidade do Federal do Amazonas

^{Correspondence}

Bufotenine (5-hydroxy-N,N-dimetyltryptamine) is a tryptophan derived indole alkylamine widely distributed in Leguminosae plant family and vertebrate animals, especially mammals and amphibians from genus Bufo. In humans, this molecule is associated with brain diseases and acts as a potent hallucinogenic factor, showing activity similar to LSD upon interaction with the 5HT2 receptor. This study investigates the presence of bufotenine in the skin secretion of three amphibians’ species (O. taurinus, O. oophagus and O. langsdorf) from the Amazon and Atlantic rain forests using RP-HPLC, ESI-MS/MS and NMR.

The skin extracts were obtained by mild electrical stimulation, collected in Milli-Q water, and were later frozen, and lyophilized. The water-soluble fractions from the crude extract were fractionated by RP-HPLC, using a semi-preparative C18 column in a linear acetonitrile gradient with 0,1% of trifluoracetic acid. Bufotenine fractions eluted at 21,5 min and were submitted to a further purification step using analytical RP-HPLC.

ESI-MS analysis of the purified fractions produced a single ion of 205,16 Da (M+H+) and a fragmentation pattern, characterized by the daughter ions: 160,1 Da (M+H+) corresponding to the loss of C2H7N, and 58,2 Da (M+H+) corresponding to C3H8N, obtained upon cleavage of the amine ring substitution between methylenes. NMR (400MHz) spectra in conjunction with HSQC experimental data confirmed the bufotenine identity.

This is the first description of bufotenine in the Osteocephalus genus. These data associated with the geographical distribution of the studied species, so far strongly supports the theory that indole alkaloids are widely spread among anuran families as a chemical defense system.

Insertion in lipid monolayers of St II, a pore-forming toxin, is modulated by the presence of cholesterol and sphingomyelin

Martinez, D.^I; Alvarez, C.^I; Lanio, M.E.^I; Pazos, F.^I; Tejuca, M.^I; Gutierrez-Aguirre, I.^II; Gonzalez-Manas, J.M.^II

^IUniversity of Havana, Cuba

^IIThe University of Basque Country, Spain

^{Correspondence}

Sticholysin II (St II) is a polypeptide toxin produced by the sea anemone Stichodactyla helianthus characterized by forming pores in membranes and its activity is inhibited by sphingomyelin. The interaction of this toxin with lipid monolayers was determined by measuring changes in surface pressure as a function of the lipid composition, particularly the influence of phosphatidylcholine (PC), sphingomyelin (SM), cholesterol (Cho) and their mixtures were thoroughly studied. Association of St II to single lipids was analyzed in monolayers as well as in an ELISA with lipid coating, revealing in both systems a remarkable preference for SM, two fold higher than that obtained for PC. Using monolayers comprising binary lipid mixtures, the highest critical pressure values (pc) were reached for SM:Cho mixtures close to equimolarity while the lowest values were attained for mixtures that did not contain SM. Ternary mixtures containing 50% SM and more than 30% cholesterol resulted optimal for the toxin, considering their high pc values and the slope of the curves Dp/p0. Insertion kinetics in monolayers, at the same starting pressure, as a function of lipid composition evidenced differences in the extreme composition cases (PC, PC:Cho and and all the ternary mixtures containing 50 % SM and 30 % Cho) both in terms of the process extension as well as rate. This phenomenon is coincident with that observed in permeabilization assays of LUV loaded with fluorophores. This behavior could be explained by the formation of lipid domains facilitating the toxin insertion even though a better accommodation of the protein can not be ruled out.

Cardiac troponin I release after severe sorpion envenoming by T. serrulatus

Cupo, P.; Hering, S.E.

Department of Pediatrics, Faculty of Medicine of Ribeirão Preto, University of São Paulo

^{Correspondence}

OBJECTIVES: The objective of the present study was to determine the presence of cardiac injury in patients with severe scorpion envenoming by serial measurements of cardiac troponin I (TnI), currently considered the gold standard for diagnosis of acute myocardial infarction.

PATIENTS AND METHODS: Eight children aged 2 to 9 years, with signs and symptoms of severe scorpion envenoming by Tityus serrulatus were studied. All patients were submitted to ECGs at admission, and every 12 hours on subsequent days after the sting. M-mode, two-dimensional color-flow Doppler transthoracic echocardiograms (ECHO) were performed in all patients at intervals after the sting. Blood was collected upon admission, before antivenom therapy and at intervals after the sting for the determination of glucose, CK-MB (activity and mass), AST, myoglobin, TnI, blood count, electrolytes, and circulating antigen venom.

RESULTS: All patients showed clinical manifestations of cardiac dysfunction, with ECG and echocardiographic alterations and 5 developed pulmonary edema. CK-MB mass and myoglobin were elevated in all patients but one on admission and TnI was normal in all children, except for two patients who arrived later. These values increased subsequently, followed by normalization within 4 to 5 days, with myoglobin being the first marker that returned to normal.

CONCLUSIONS: The detection of TnI in patients with severe scorpion envenoming, and the observed temporal pattern and serum levels meet the criteria established for the diagnosis of acute myocardial infarction. The rapid reversibility of cardiac dysfunction, together with the normalization of the enzymatic, ECG and echocardiographic data, indicates the occurrence of an acute myocardial lesion without underlying or associated coronary disease.

Analysis of anti-ache factors in Cyanobacterial bloom samples

Barros, L. P.^I; Romero, A.H.^I; Yunes, J.S.^I; Monserrat, J.M.^II

^IUnidade de Pesquisas em Cianobactérias, FURG, RS, Brazil

^IIDepartamento de Ciências Fisiológicas, FURG, RS, Brazil

^{Correspondence}

Cyanobacteria of the genera Anabaena can produce an organophosphorous-like toxin (anatoxina-a(S)). Previous works (Monserrat et al, 2000, Barros, 2002) demonstrated the anticholinesterasic action of aqueous extracts containing the cyanobacterium Anabaena spiroides from South Brazilian waters. The same authors also developed an optimized protocol based on Ellman (1961) to be applied in cyanobacterial bloom samples. The present work tested that protocol on the extraction of anticholinesterasic factors of environmental samples from Brazil and other countries containing predominantly Anabaena spiroides cells. Samples taken directly from blooms and scums were identified and quantified in cell numbers, lyophilized at -30°C and sent to UPC-FURG, RS. Lyophilized powders were dissolved in ethanol, sonicated and filtered. The final solution was evaporated at 40°C, ressuspended in chloroform and washed in distilled ater pH 3,3. The extract was incubated during one hour at 25°C with the enzyme acetylcholinesterase, AChE (Sigma, USA). The enzyme activity was measured by spectophotometry. Sixteen samples from public drinking-water reservoirs, were tested. Samples produced an AChE inhibition from 2,57 to 11,12% at variable number of A. spiroides cells from 428 to 10,132 cells mL^-1. However, in a single bloom sample with the number of cells higher than 100,000 cells. mL^-1 AChE inhibition reached up to 31%. Environmental samples containing Planktothrix agardhi produced AChE inhibitions from 6,49 to 7,56% while the number of cells varied from 15,666 to 137,000 cells mL^-1. Bloom samples containing single or predominant species of Anabaena circinalis produced a 0% AChE inhibition. The present work suggested that the increase on AChE inhibition observed may be directly related to the number of cells of the especies A . spiroides or P. aghardii . However, further analysis will be necessary to draw a possible linearity between those parameters.

Effects of Crotalus durissus terrificus venom on mitochondrial activity

Silva, N.S.; Prianti, A.C.; Ribeiro, W.; Lopes-Martins, R.A.; Cogo, J.C.; Pacheco-Soares, C.

Serpentário do Centro de Estudos da Natureza (CEN), Instituto de Pesquisa & Desenvolvimento, Universidade do Vale do Paraíba (UNIVAP), São José dos Campos, SP, Brasil

^{Correspondence}

Snake venoms can affect a variety of intracellular metabolic pathways. In this work, we examined the effects of Crotalus durissus terrificus (South American rattlesnake) venom on the mitochondrial activity of cultured cells. CHO-K1 cells (1x10⁶ cells/ml) were cultured overnight on sterile coverslips in 24-well plates (NUNC) containing minimum essential medium supplemented with 5% (v/v) fetal calf serum (FCS), 100 U penicillin/ml and 100 mM streptomycin (Gibco) at 37ºC in a humidified 5% CO2 atmosphere. Venom diluted in PBS was added to the cells (final conc. 5, 10 and 20 mg/ml) which were then incubated as above for up to 60 min. Subsequently, the cells were washed with PBS and then incubated with the fluorescent probe JC-1 (5,5´,6,6´-tetrachloro-1,1´,3,3´,-tetraethyl-benzimidazol-carbocyanine iodide, 0.1 mM) for 10 min. After washing with PBS, the cells were fixed in 4% paraformaldehyde in 0.1 M PHEM buffer, pH 6.8, rinsed with PHEM buffer and the coverslips then mounted on slides containing N-propyl-gallate. The slides were analyzed by epi-fluorescence using a Leica DMLB microscope and photographed with a Leica MPS30 camera. After incubation with 5 mg of venom/ml for 60 min, the cells showed high mitochondrial activity with no change in numbers compared to non-treated cells. At concentrations of 10 and 20 mg/ml, the venom altered mitochondrial activity and caused time-dependent cell death.

Financial support: FVE-UNIVAP, FAPESP.

The 3D homology model of the first neurotoxic peptide (TF4) from the scorpion Tityus fasciolatus

Barbosa, J.A.R.G.^I; Wagner, S.^II; Castro, M.S.^{II, III}; Fontes, W.^III; Sousa, M.V.^III; Schwartz, E.F.^II; Pires Jr., O.R.^II; Sebben, A.^II; Schwartz, C.A.^II

^ICentro de Biologia Molecular Estrutural - Laboratório Nacional de Luz Síncrotron (CeBiMe-LNLS), Campinas/SP, Brasil

^IILaboratório de Toxinologia, CFS/IB, Universidade de Brasília, Brasília/DF, Brasil

^IIICentro Brasileiro de Serviços e Pesquisas em Proteínas, Universidade de Brasília, Brasília/DF, Brasil

^{Correspondence}

The scorpion Tityus fasciolatus is endemic to the central region of Brazil, where human envenomation has been reported. One neurotoxic peptide, named Tf4, was purified from this venom and sequenced. Tf4 has 6.6 kDa and delays frog sodium channel inactivation reversibly.

The complete sequencing of Tf4 allowed us to search the protein data bank (PDB) for similar sequences of proteins that had their 3D structure known. There were 18 hits in total and identities of the 18 sequences with the Tf4 sequence were between 31 and 67%. Four structures were used to build the 3D homology model of Tf4: Ts1, PDB code 1B7D; Cse-v5, PDB code 1I6F; Cse-v3, PDB code 2SN3 and BmK-M8, PDB code 1SNB. The program O was used to superimpose the Ts1, Cse-v5, Cse-v3 and BmK-M8 structures and build the Tf4 model.

Most of the model followed the Ts1 structure that has the highest identity with Tf4. No insertions or deletions are found between Tf4 and Ts1, except for the additional glycine N-terminal residue in Tf4. This longer N-terminus and the other 20 residues that are different between the two sequences contribute to the weaker toxic activity presented by Tf4 when compared to Ts1. A study based on the Tf4 model showing possible candidates for this loss of activity, focusing on the differences shared with Ts1, will be presented.

Cloning, expression analysis and heterologous production of snake venom vascular endothelial growth factors (svVEGF) from three bothropic snake species

Junqueira-de-Azevedo, I.L.M.; Silva, M.B.; Farsky, S.H.P.; Ho, P.L.

Centro de Biotecnologia, Instituto Butantan – São Paulo SP – [e-mail]

^{Correspondence}

The analysis of abundant Expressed Sequence Tags (ESTs) from the Viperidae snake Bothrops insularis venom glands revealed the occurrence of a cDNA coding for a putative Vascular Endothelial Growth Factor-like (VEGF-like) protein. The deduced primary sequence, after complete sequencing of the longest snake venom VEGF (svVEGF) cDNA, displayed similarities with vertebrate VEGFs, placental growth factors (PlGF), platelet-derived growth factor (PDGF) and with the hypotensive factor (HF) from Vipera aspis venom. To investigate its possible role in the snake venoms, the cDNA was subcloned, expressed in E. coli with a 6X His-tag as an insoluble monomer and was purified by a Ni2+-affinity chromatography after 8M urea extraction. Antiserum against the recombinant svVEGF was generated in mice and tested in Western-blot against proteins from various snake venoms and cellular extracts of snake venom glands and other tissues, showing that the mature svVEGF is ubiquitously distributed throughout snake venoms, even from Elapidae family, but not expressed in other cellular extracts. This result was also confirmed by Northern Blot studies of RNAs from other related Viperidae species and by cloning and complete sequencing of svVEGF cDNAs from B. jararaca and B. erytromelas pitviper. The recombinant protein was also dimerized after refolding processes and biologically characterized, showing ability to increase vascular permeability. These results indicate that svVEGF is a novel and important active toxin during the early stages of bothropic snake bite envenoming and represents a new member of the VEGF family of proteins [J. Biol. Chem. (2001) 276 (43) pp 39.836].

Supported by FAPESP, CNPq and Fundação Butantan.

Isolation of a new serine protease (uruprot) from Bothrops alternatus snake venom

Ribeiro, D. A.; Silva, J. A.; Marangoni, S.; Novello, J. C.

Departaments of Biochemistry, Institute of Biology State University of Campinas (UNICAMP) Campinas, SP Brazil

^{Correspondence}

OBJECTIVE: Isolation and partial biochemical and biological characterization of a Uruprot serine protease from the venom of Bothrops alternatus.

METHODS AND RESULTS: The serine protease named Uruprot was isolated using a combination of molecular exclusion and ion-exchange HPLC.

The approximate molecular mass of the reduced (DTT-treated) protein was approximately 32 kDa as determined by Tricine SDS-PAGE 12.5%. Two dimensional (2D) electrophoresis of uruprot showed an isoelectric point of around 4.6 and confirming the molecular mass in SDS-PAGE.

Amino acid analysis showed Asx/12, Glx/22, Ser/30, Gly/54, His/1, Arg/2, Thr/45, Ala/13, Pro/14, Tyr/6, Val/10, Met/0, half Cys/6, Ile/31, Leu/10, Phe/33 and Lys/9. Uruprot showed a high content of acidic residues (Asp and Glu), and a high content of hydrophobic amino acids (Ser, Thr, Pro, Ile, Leu and Phe). These data corroborate the acidic nature of the protein as well as its hydrophobicity. The N-terminal sequence of Uruprot was VVGGDECNINEHRSLVAIFDS. N-terminal sequence of uruprot showed high homology sequence with the N-terminal sequences of other serine proteases as catroxobin, crotalase and bilineobin. Uruprot had hemolytic activity and inhibited ristocetin-induced aggregation of washed platelets at a minimum concentration of 10 mg/ml.

CONCLUSION: In this work, we purified and characterized a serine protease from the venom of a South American pit viper, the “urutu” (Bothrops alternatus).

Thus, uruprot is a serine protease acid with a only band and showed hemolytic activity and inhibit the platelet aggregation induced for ristocetin.

Financial Support: CAPES-CNPq, FAPESP

Low energy laser effects in myonecrosis caused by Bothrops jararacussu crude venom

Silva, R.D.A.^I; Leite, G.B.^I; Rodrigues-Simioni, L.^I; Cruz-Höfling, M.A.^II

^IDept of Pharmacology, UNICAMP - Campinas, SP, Brazil

^IIDept. of Histology and Embryology, UNICAMP - Campinas, SP, Brazil

^{Correspondence}

OBJECTIVE: The severity of the local effects caused by B. jararacussu (Bjssu) snake accidents prompted us to investigate these rapidly developing biological phenomena and also to follow the time course of the injurious process as well as to search for new approach to neutralize the severe muscle damage that occurs in the snakebites victims. The present innovating work investigates the possible benefits of irradiating the region affected by B. jararacussu venom with low energy He-Ne laser (KLD – Biosistemas Equip. Eletron. Ltda, Amparo, SP, l = 632.8 nm, 5 mW). Histopathological and myographical analysis were done to assess the laser treatment.

METHODOLOGY: The tibialis anterior muscle of adult anesthetized Wistar rats (250–350 g) were prepared for myographic records. After stabilization of the in vivo preparation the muscle was injected in the medium third either with (Bjssu) (60 µg/0.02 ml) or sterile saline (controls). Irradiated and unirradiated groups were investigated (n=3/group). Three laser doses were administered (30 mJ/cm² -9 sec, 90 mJ/cm² - 27 sec and 30 mJ/cm² - 9 sec at each 20 min) after 60 min post-venom or post-saline injection and the effect was observed during 60-120 min. After myographic recording the muscles were rinsed and fixed with Bouin’s fixative for histopatological studies.

RESULTS: The results revealed that the dose of 30 mJ/cm² produced a significant decrease of the neuromuscular blockade compared to unirradiated groups. In addition, smaller percentage of myonecrosis per muscle sections were detected with this same dose.

CONCLUSION: We concluded that the irradiation with low energy laser can prevent the progress of the myonecrotic process caused by the inoculation of the B. jararacussu venom in in vivo preparation.

Support: CAPES

Relationship between the localization of epitopes in the toxins of Tityus serrulatus scorpions and the neutralizing potential of therapeutic antivenoms

Maria, W.S.; Granier, C.; Velarde, D.T.; Chávez-Olórtegui, C.

Fundação Ezequiel Dias (FUNED), Belo Horizonte, MG, Brazil and Institut de Biotechnologie et Pharmacologie, Faculté de Pharmacie, Montpellier, France

^{Correspondence}

Administration of antivenom, prepared from hyperimmunized horse plasma is a recognized therapeutic means of circumventing poisoning by Tityus serrulatus (Ts) scorpion venom. The neutralizing potential of antivenoms has been assessed by studying neutralization of lethality, a procedure that causes the death of a large number of mice. It has therefore become increasingly important to develop alternative methods for assaying antivenoms. Here we address the issue of the identification of epitopes, by assessing the capacity of overlapping peptides derived from the three T. serrulatus toxins, Ts IV (a-type), Ts II and Ts VII (toxin-g both b-type) to react with horse anti-Ts antivenoms of different potency.

Anti-Ts venom plasmas were obtained following conventional immunization schedules of the FUNED. Mice were used in the in vivo neutralization assays following the official resolution of Health Department from Brazil. Results were analyzed by the Probit test and neutralization is expressed as 50% effective dose (ED50). Overlapping pentadecapeptides (24 for Ts VII, 25 for Ts II and 26 Ts IV toxins) frameshifted by 2 residues were prepared according to the protocol previously described by Molina et al.,1996 and bound to cellulose membranes. These membranes were probed by incubation with sera of immunized horses .

The results showed that the same peptides are strongly recognized by the sera with high neutralizing potential and indicate that the Spot-technique using synthetic peptides covalently bound to cellulose membrane should be used as a in vitro technique to evaluate the potency of Brazilian Tityus antivenoms.

Supported by: CNPq, INSERM.

Differential ELISA for Bothrops atrox and lachesis muta muta accidents using monoclonal antibodies

Colombini, M.; Cardoso, D.F.; Fernandes, I.; Moura-da-Silva, A.M.

Laboratório de Imunopatologia, Instituto Butantan, SP

^{Correspondence}

B. atrox and L. m. muta snakes are responsible for most accidents occurring in Brazilian Amazon. The clinical features of the accidents are similar, and there are still controversies about efficacy of Bothrops antivenoms for treating L. m. muta accidents. In this work, we evaluated the efficacy of B. atrox and L. m. muta experimental antivenoms for cross-neutralisation of haemorrhagic and coagulant activities of the venoms, which were preferentially neutralised by homologous antivenoms. B. atrox antivenom failed completely in neutralising L. m. muta-induced coagulophaty, thus indicating the necessity of a differential diagnosis and the use of specific therapy. The differential diagnosis was accessed by ELISA, using polyclonal antibodies raised in rabbits (reference method) and a new assay using biotin-labelled monoclonal antibodies. Monoclonal antibodies were obtained using as antigens the non-variable specific 50 kDa B. atrox venom band and 24 kDa specific antigen for L. m. muta venom. MoAbs were screened using homologous or heterologous venoms and 4 clones were selected for B. atrox and 6 clones for L. m. muta venoms. ELISA standardisation was carried out using venom standard curves diluted in normal human serum. Sensitivity was similar in both methods reaching venom concentrations of 0.1 ng/ml. Specificity was much higher in MoAbs assays since no reaction was detected with normal sera used as control, while cut-off levels of reference assay was around 10 ng/ml venom. The MoAb assay is under evaluation using serum samples of patients from Pará State. This test will be important both for diagnosis and epidemiological surveys, providing a rational for production and distribution of antibothropic, antilachetic or polyvalent antivenoms.

Supported by FAPESP 00/02396-9

Datas de Publicação

• Publicação nesta coleção
  09 Dez 2003
• Data do Fascículo
  2003