Conferences

CONFERENCES

Toxinologist: to be or not to be?

Prado-Franceschi, J.

Departamento de Farmacologia, FCM, UNICAMP

^{Correspondence}

Looking back to 1964, I can still feel the emotions evoked by my first contact with toxinology in the laboratory of Dr. Oswaldo Vital Brazil, who was studying the venom of Crotalus durissus terrificus at UNICAMP, and of my expectations on becoming a toxinologist. Under Dr. Vital Brazil's supervision, I started my career with studies on the pharmacology of crotoxin, crotamine and fraction I, from which I isolated convulxin, the pharmacological characterization of which resulted in my doctoral thesis in 1970. In later work with Dr. Bernardo B. Vargaftig at the Institut Pasteur, we showed that convulxin could be a very useful tool for studying platelet aggregation. At UNICAMP, I have collaborated with Dr. Lea Rodrigues-Simioni on studies of the neuromuscular actions of Bothrops venoms, following her initial description in 1983 of pool IV (now known as Bothropstoxin) of Bothrops jararacussu venom. These studies included not only B. jararacussu but also B. insularis, B. neuwidii, and B. lanceolatus (with Dr. Albetiza Lôbo de Araújo). In the 1990s, we became interested in the venom of the colubrid Philodryas olfersii. Using fractions obtained by Dr. Marina Assakura (Instituto Butantan), we characterized a neurotoxic fraction and identified a myotoxin (the first described for a colubrid) in P. olfersii venom. In 1988, during a Symposium on Toxic Proteins, I proposed the creation of the Brazilian Society on Toxinology (Sociedade Brasileira de Toxinologia or SBTx). Prof. Vital Brazil was its first President and I, the vice-president. Shortly after its formation, the Society hosted the fourth Pan-American Symposium of the International Society on Toxinology in 1992, of which I was the chairwoman. Reflecting again on my experience in this field, I realize that many people have been involved in my career. To all of them, I say a heartfelt 'Thank-you'.

Tribute to Dr. Julia Prado-Franceschi

Simioni, L.R.

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

^{Correspondence}

I am privileged to have this opportunity to present Dr. Julia Prado-Franceschi who is to be honored at this VI Symposium of the Brazilian Society of Toxinology (SBTX). For our contemporaries, this presentation is unnecessary, so I would like to present Julia to the younger toxinologists.

The Brazilian Society of Toxinology originated in Julia’s mind and heart. In 1988, the Academy of Sciences of the State of São Paulo, in the person of the then president Prof. Watanabe, requested Julia’s help and that of our Department in organizing a symposium in honor of Prof. Oswaldo Vital Brazil. At lunch during this event, Julia put forward the idea of organizing a Brazilian branch of the International Society on Toxinology (IST). The proposal was accepted enthusiastically by those present. During the symposium, Prof. Dr. Vital Brazil and Julia were nominated president and vice-president, respectively, of the newly formed SBTX. Initially, there was some resistance to the creation of this society on the part of some leading members of the IST. However, in 1992, four years after the founding of the society, the IV Panamerican Symposium of the IST and the II Symposium of the SBTX were held jointly in Campinas, São Paulo state. The organization and success of this event were in large part due to Julia’s ability to present her arguments convincingly. Those present at the event remember how productive and pleasing the meeting was. Since that event, the number of Brazilian toxinologists, which until then had involved only a few individuals, has risen markedly, with an ever increasing participation in world congresses organized by the IST. As foreseen by Julia, the number of young Brazilian toxinologists contributing to the field has also increased. We wish to say to our honored colleague that her effort and enthusiasm in founding the SBTX have paid off. The Society has grown and is now in the hands of a group of competent and interested individuals. In addition, the quality of toxinological research done by Brazilian toxinologists is continuously improving and is a source of satisfaction and pride for us as Brazilians.

Sea-animals toxins as useful targets for molecular modeling and drug-design

Maigret, B.

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

^{Correspondence}

Sea animal species are, with those coming from tropical rain forests, the largest reservoir for natural products finding. Considering the impact of such natural products in drug discovery for a large variety of diseases, attention will be presented on the use of sea animals venom for drug design. Several examples coming from sea anemone toxins will be used in this presentation, highlighting the necessary combination of multidisciplinary approaches. Computer modelling investigations will be described complementing successfully structural and biochemical experiments.

Clinical aspects of accidents caused by aquatic animals

Haddad JR., V.

Faculdade de Medicina de Botucatu - Universidade Estadual Paulista - Brasil

^{Correspondence}

The accidents by venomous and poisonous aquatic animals may provoke serious problems, including important morbidity and occasional death of the patients. The cnidarians (jellyfishes, especially Cubomedusas, and Portuguese-man-of-war) caused nearly 25% of the accidents observed in a sequence of 283 accidents by aquatic animals registered by the author in Brazil (1). The sea urchins provoked about 50% of the accidents and diverse species of venomous fishes nearly 25%. The more important venomous fishes observed in Brazilian marine waters were catfishes (more than 50% of the accidents by fishes), stingrays and scorpionfishes, the same fishes that cause the majority of the accidents caused by venomous fishes around the world, but there are many others species of Brazilian fishes that have venom in their spines or stings and cause accidents in a minor frequency.

In freshwater ambient, the stingrays and catfishes provoke severe accidents, with observation of intense pain and necrosis. The mechanism of the poisoning and the effects of the toxins are very alike the marine species of stingrays and catfishes.

The author presents the first measures for the control of the severe pain observed mainly in the accidents caused by cnidarians and venomous fishes.

(1) Haddad Jr, V. Atlas de animais aquáticos perigosos do Brasil. São Paulo, Editora Roca, 2000. 145 p.

Phycotoxins in the coast of Santa Catarina, Southern Brazil

Proença, L.A.O.

Centro de Ciências Exatas da Terra e do Mar, CTTMar, Universidade do Vale do Itajaí

^{Correspondence}

Until a few years ago, documentation on toxins produced by algae in Brazilian waters was rare in the literature and mostly regarded to fresh water environment. Following the growth of shellfish aquaculture off the Santa Catarina coast, in the beginning of 1990´s, a pilot monitoring program to study harmful algae and phycotoxins was set in 1997 at Amação do itapocoroy bight (26°36´ S. 48° 36´ W). Basically, methods used in this program included phytoplankton identification, phytoplankton cell culture, mouse and invertebrate bioassays and HPLC analysis from mussel tissue and plankton samples. Several toxins were found both in mussel tissue and in the water column. Among then, okadaic acid, saxitoxin and congeners and domoic acid were found for the first time in the region. These toxins cause the well documented: diarrhetic, paralytic and amnesic shellfish poisonings syndromes, DSP, PSP and ASP, respectively. Until now, toxic algae species related were the dinoflagellate Dinophysis acuminata, D. acuta, Gymnodinium catenatum and the diatom Pseudonitzschia spp. Other potentially toxin-producing algae found were naked flagellates, such as the raphidophytes Heterosigma akashiwo, Fibrocapsa japonica and Chattonella sp. and the haptophyte Phaeocystis cf globosa. Potentially toxic cyanobacteria, Trichodesmium spp, also occurred in the region. These findings show that Brazilian waters are not free phycotoxins producing species and indicate the necessity to establish a continuous monitoring program at mussel culturing areas. The chemical and biological diversity observed makes this a complex issue, which must be accessed, as it may cause serious harm.

Cyanobacterial toxins - a problem for drinking water in South Brazilian reservoirs

Yunes, J.S.

Unidade de Pesquisas em Cianobactérias, FURG, RS, Brazil

^{Correspondence}

Cyanobacteria are prokaryotic photoautothrophic microorganisms remanescent from precambrian primitive filamentous forms. Some cyanobacterial genera are able to fix atmospheric N2 and some genera develop massive growth (>10⁶ cells.mL^-1) in water reservoirs, among those, some cyanobacteria are also able to produce potent toxins (neuro or hepatotoxins). In South Brazil the occurrence of toxic blooms have become frequent in reservoirs for drinking-water purposes. The majority of blooms includes cyanobacteria of the genera Microcystis, Anabaena and/or Cylindrospermopsis. Peptide toxins named microcystins are produced by Microcystis and Anabaena species and are common in the euthrophic waters of dams near urban centers. Alternatively, in the same environments coiled Anabaena species produced anatoxin-a (S)-like neurotoxins. Other neurotoxins, with paralytic effect, named saxitoxins, are produced in turbid waters of shallow lakes and dams by Cylindrospermopsis and Planktothrix species. In general all cyanobacterial blooms are causing serious concern to the Health Authorities and staff of Water treatment Plant Units. Brazil has established a temporary regulation for three cyanotoxins in potable waters and their analysis in water are subject of a constant effort of the “Unidade de Pesquisas em Cianobacterias” of FURG, Southern-Brazil. Following the present cenario of problems with cyanotoxins in potable waters in Brazil, a broader discussion on the matter is required in order to gather efforts and further capabilities to overcome this foreseen worst difficulties.

Thalassophryne nattereri (niquim): from toxins to therapy

Lopes-Ferreira, M.

Laboratório de Imunopatologia do Instituto Butantan, São Paulo, Brasil

^{Correspondence}

The accidents caused by the fish T. nattereri represent a serious medical, economic and social problem in the North and Northeast of Brazil. The venom induces severe edema and pain followed by a fast settling necrosis, both in human victims and experimental models. Analysis of its local effects showed a myotoxic effect with muscle damage and difficult regeneration. Blood flow at microvessels was also impaired with stasis concomitantly with the presence of thrombi in venules, focal transient constrictions in arterioles, and increase in vascular permeability. Venom action was locally restricted and no alteration on systemic blood coagulation was observed. Venom lacked a direct pro-coagulant activity but exerted a strong cytolytic effect on platelets and endothelial cells in vitro. Local inflammatory response was discrete since T. nattereri venom stimulate the release of low levels of IL-1b, IL-6 and TNF-a cytokines, and diminished infiltrate of cells was observed in damaged tissue 24 hours after injection. The mechanisms involved in venom action were evaluated. Nociceptive and edematogenous activities were not reduced neither by treatment with inhibitors of serotonin and histamine nor by non-steroidal and steroidal anti-inflammatory drugs, but by the administration of a kallikrein specific inhibitor demonstrating that tissue kallikrein-dependent pathways are involved in local effects. The toxin responsible for T. nattereri venom effects was characterized as a 48 kDa fraction, able to convert LMW kininogen into Lys-BK, in a metal-dependent proteolysis. T. nattereri antivenom produced in horses was able to recognize all venom bands including the 48 kDa fraction and neutralized venom-induced edema, nociception and necrosis in experimental models. These results indicate that the use of serumtherapy possibly associated with local administration of kalikrein inhibitors is a promising treatment for accidents caused by the fish T. nattereri.

Financial support: FAPESP (01/02937-2)

Comparative studies among the fresh water (Potamotrygon sp.) and marine (Aetobatus sp. and Dasyatis sp.) stingray venoms

Barbaro, KC^I; Cardoso, JLC^II; Haddad, JR, V^III

^ILab Imunopathology

^IIVital Brazil Hospital, Instituto Butantan, São Paulo

^IIIDermatology Department, UNESP, Botucatu

^{Correspondence}

Stingrays are cartilaginous fishes found along Brazilian coast and in some rivers of Northern and Middle-Western Brazil. The venom apparatus is located in their tail, comprised of bilaterally retroserrate spines covered by glandular and integument tissues. Pain is the most conspicuous symptom observed in accidents wounded by stingrays, whereas cutaneous necrosis is commonly observed in accidents by fresh water stingray. The aim of this work was to characterize some aspects of fresh water and marine stingray venoms. By SDS-PAGE, the Aetobatus sp. and Dasyatis sp. venoms showed a similar electrophoretic profile which two major bands located around 10 and 15 kDa. Fresh water stingray (Potamotrygon) venom presented a major band of approximately 12 kDa. Several minor components distributed between 18 and 130 kDa were also detected in the three venoms. Lethal (mice) and dermonecrotic activities (rabbits) were not detected. Edematogenic activity among all venoms was similar and dose-dependent. The presence of nociceptive activity was verified in all three venoms. Antigenic cross-reactivity was observed among the three venoms studied using anti-Dasyatis and anti-Potamotrygon sera produced in mice. These sera recognized different components above 50 kDa of each venom by Western blotting. The enzymatic activity was performed only with Potamotrygon sp. and Dasyatis sp. venoms. Both venoms presented gelatinolytic and caseinolytic activities which was much more evident in fresh water stingray venom. Hyaluronidase activity was detected only in a component around 84 kDa in Potamotrygon sp. venom. Our results demonstrated some differences among fresh water and marine stingray venoms which could explain partially the different clinical pictures presented by patients wounded by fresh water and marine stingrays.

The interaction of snake venom LYS49-phospholipases A₂ with membranes studied by protein engineering

Ward, R.J.

Departamento de Química, FFCLRP-USP, Av. Bandeirantes 3900, Ribeirão Preto, SP, Brazil

^{Correspondence}

Bothropstoxin-I (BtxTx-I) is a myotoxic, homodimeric PLA2 homologue from B. jararacussu venom which shows a Asp49Lys substitution. Although lacking detectable hydrolytic activity, BtxTx-I causes rapid Ca²⁺-independent release of liposome entrapped markers. Flexibility at the dimer interface results in "open" and "closed" dimer conformations, and a transition between these two forms suggests a mechanism for the Ca²⁺-independent membrane damaging activity. Site-directed BthTx-I mutants expressed as inclusion bodies in E. coli, refolded and purified by reverse phase chromatography, were used to evaluate the Ca²⁺-independent membrane damaging and myotoxic activities. Phospholipid hydrolysis, myotoxic activity and entrapped fluorescent marker release from liposomes were measured for several active site mutants. No catalytic activity was detected with the native, wild type recombinant and His48Gln proteins, yet myotoxic and membrane damaging activities were unaffected. These results demonstrate that neither the myotoxic nor the Ca²⁺-independent membrane damage by BtxTx-I involves phospholipid hydrolysis. In contrast, substitution of positively charged amino acids between positions 115-122 abolished or significantly reduced membrane damaging activity, although no effect was observed by substitution of the lysines between positions 123-133. This suggests a purely physico-chemical explanation for the membrane damaging activity which involves alternative functions for the distal and proximal regions of the C-terminal loop. The myotoxicity of these mutants were also altered, although in a different pattern, suggesting overlapping yet distinct structural determinants in the C-terminus. Finally, the model predicts that the stability of the homodimer is important in the Ca²⁺-independent membrane damaging mechanism. Therefore, the monomer/dimer equilibrium constant of the native BthTx-I was compared to dimer interface mutants, and which revealed a direct correlation between dimer stability and membrane damaging activity.

Supported by FAPESP and CNPq.

Basic phospholipases A₂ from Bothrops sp venoms: understanding a complex pharmacological profile involved in local tissue damage

Gutiérrez, J.M.^I; Chacur, M.^II; Zuliani, J. P.^III; Longo, I.^II; Lomonte, B.^I; Picolo, G.^II; Teixeira, C.F.P.^III; Cury, Y.^II

^IInstituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica

^IILaboratorios de Fisiopatología

^IIIFarmacología, Instituto Butantan, Sao Paulo, Brasil

^{Correspondence}

Many Bothrops sp venoms contain basic phospholipases A₂ (PLA₂). Some of them are catalytically-active Asp-49 variants, whereas others are Lys-49 PLA₂s devoid of enzymatic activity. Although these proteins were originally described as ‘myotoxins’, further studies have documented a more complex pharmacological profile. Besides myotoxicity, they induce pain and promote a complex inflammatory response. Moreover, they exert a broad cytolytic activity in vitro, including bactericidal activity, and some of them are highly lethal when injected into the brain, also inducing an anticoagulant effect. The mechanisms involved in myotoxic, hyperalgesic and proinflammatory activities of basic PLA₂s isolated from the venom of B. asper have been investigated. Myotoxicity depends on the ability of these toxins to interact with acceptor sites in the sarcolemma, followed by disorganization of the bilayer and prominent Ca²⁺ influx. Catalytic activity is not a strict requirement for myotoxicity and, in the case of Lys-49 PLA₂s, membrane damage is due to a cationic-hydrophobic region located at the C-terminus. Catalytic activity of Asp-49 PLA₂s potentiates such membrane-damaging effect. Hyperalgesia induced by Asp-49 and Lys-49 PLA₂s is mediated by histamine, bradykinin, TNF-a and IL-1. In the case of Lys-49 variants, sympathomimetic amines and cyclooxygenase-derived eicosanoids are also involved. Catalytic activity is required in Asp-49 PLA₂-induced hyperalgesia, whereas the molecular region responsible for cytotoxicity is involved in Lys-49 PLA₂-induced algogenic effect. Both types of PLA₂s elicit an inflammatory infiltrate associated with the expression of a number of adhesion molecules. They also promote the synthesis of leukotriene B₄, thromboxane B₂, IL-6 and matrix metalloproteinases. Incubation of B. asper PLA₂s with macrophages in vitro stimulates phagocytosis and the production of H₂O₂ and NO. Basic PLA₂s from Bothrops sp venoms play a prominent role in the local tissue alterations characteristic of these envenomations.

Supported by FAPESP, Vicerrectoría de Investigación (Universidad de Costa Rica) and NeTropica.

Exogenous secretory phospholipases A₂ induce experimental acute pancreatitis and lung injury in rats. Effect of crotapotin on the acute pancreatitis induced by caerulein

Landucci, E.C.T.^II; Camargo, E.^I; Esquisatto, L.C.M.^I; De Nucci, G.^I; Antunes, E.^I

^IDepartment of Pharmacology, UNICAMP

^IIDepartament of Biochemistry, UNICAMP

^{Correspondence}

INTRODUCTION: Phospholipases A₂ (PLA₂) are important mediators in the acute pancreatitis (AP) and consequent lung injury. We have investigated the AP induced by exogenous PLA₂ from Naja naja venom and the PLA₂ homologue Piratoxin I (a toxin from B.pirajai venom) and the effect of crotapotin (a non-toxic and non-enzymatic acid polypeptide naturally complexed with PLA₂ in the venom of Crotalus durrissus terrificus) on AP induced by caerulein.

METHODS: Acute pancreatitis was induced by two methods: (1) the i.v. infusion of caerulein (a cholecystocinin analogue, 5 µg/kg/h, 1 mL/h) or PLA₂ from Naja naja venom (30 µg/kg/h, 1 mL/h) for 4 h, (2) the injection into the pancreatobiliary duct of sodium taurocholate 5%, PLA₂ from Naja naja venom or Piratoxin I (100 and 300 mg/kg) in a steady manual pressure over a period of 60 sec. Plasma protein extravasation was measured by ¹²⁵I-human serum albumin. Crotapotin was concomitant infused with caerulein at the doses of 100 and 200 µg/rat. Neutrophil infiltration was assessed by evaluation of the MPO activity in the rat lung tissues.

RESULTS: PLA₂ from Naja naja venom did not cause a pancreatic plasma extravasation when infused i.v., although promoted elevated levels of neutrophil infiltration in rat lungs. Both PLA₂ from Naja naja venom and Piratoxin I when injected into the pancreatobiliary duct induced significantly pancreatic plasma extravasation when compared with saline group, but only the higher dose of both substances was able to evoke significant neutrophil infiltration in rat lung. Crotapotin was able to reduces the pancreatic plasma extravasation and the sPLA₂ activity.

CONCLUSIONS: When infused intravenously PLA₂ from Naja naja venom may be inactivated by pancreatic juice, but when injected into the pancreatobiliary duct either PLA₂s used are able to induce pancreatic plasma extravasation and lung neutrophil infiltration, confirming the key role of PLA₂s in this disease. The inhibitory effect of crotapotin on AP induced by caerulein suggesting that the crotapotin interacts with binding to PLA₂ inespecific sites.

Financial support: FAPESP and CNPq

Phospholipase A₂ isolated from Micrurus lemniscatus venom

Casais e Silva, L.L.^I; Lebrun, I.^III; Gutiérrez, J.M.^IV; Lomonte, B.^IV; Alape-Giron, A.^IV; Teixeira, C.F.P.^II

^IEscola Bahiana de Medicina e Saúde Pública (EBMSP)

^IILaboratório de Farmacologia

IIILaboratório de Bioquímica, Instituto Butantan, São Paulo, SP, Brasil

IVInstituto Clodomiro Picado, San Jose,1000, Costa Rica.

^{Correspondence}

In Brazil, Micrurus snakes are responsable by severe and fatal envenomation. Despide of their clinical importance only few studies have been carried about their venom. Phospholipase A₂ (PLA₂) enzymes are among the main toxic components present in this venom. We report the isolation, characterization and amino acid sequence of PLA₂ from the venom of M. lemniscatus from Bahia State, Brazil. Crude venom was fractionated using RP-HPLC system coupled to a C8 wide pore octyl column. The amino acid sequence of the select fraction (ML6) was determined by Edman sequencing procedure derived from digests utilizing trypsin and chymotrypsin. The phospholipase consists of a single polypeptide chain with a monomeric molecular weight of ~14 Kd, estimate by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Purified ML6 consists of 118 amino acid residues and revealed a high degree of homology with other PLA₂s from several sources, mainly with other elapidic myotoxic phopholipases. ML6 has anticoagulant and PLA₂ activities determined by indirect hemolysis. It produces myonecrosis in mice upon intramuscular injection with peak at 3 hours, as observed by increase of serum CK activity and histological analysis. Futhermore, an in vitro cytolytic effect, evaluated by lactic dehydrogenase enzyme release, was observed on muscular differentiated cells (myotubes) but not on murine skeletal muscle (C2C12), endothelial (tEnd) of inflammatory cells. In addition, ML6 induces increase of vascular permeability, degranulates mast cells and induces paw edema suggesting inflammatory effects. However, ML6 was not lethal after i.v. injection but it was toxic when injected into brain.

Epidemiology of deaths caused by poisonous animals in Brazil

Wen, F.H.

Hospital Vital Brazil, Instituto Butantan

^{Correspondence}

One of the main objectives of antivenom therapy is preventing the occurrence of deaths. This objective has been partially achieved in the 1980's by the time the Ministry of Health had implemented the National Program of Snakebites Control, lately named National Program of Control of Accidents Caused by Poisonous Animals. The lethality has been dramatically dropped with the decentralized distribution of antivenom. Nevertheless, about a hundred of deaths have still been registered in whole country, most of them presumably preventable with adequate antivenom therapy and supportive treatment. The causes of mortality are poorly known since the information system of accidents does not emphasize description of complications that may lead to fatal evolution. In parallel, difficulties to perform adequate necropsy also obscure the study of pathophysiological conditions in which patients die.

Snakebites are far the most common cause of death among the poisonous animals, followed by scorpion stings. Among the snakes, Crotalus is far the commonest cause of death (lethality = 1.87%), although the absolute number of Bothrops fatal cases is twice higher. Scorpion stings are known to be responsible for systemic severe envenoming in children under 14 years old: the lethality in this age group is about 1.8% while in adults the lethality is less than 0.01%. Spiders seem to be rarely associated with deaths in Brazil.

Recently Lonomia caterpillars have been described as an important agent of severe envenomation in southern region of Brazil, and consequently of systemic complications that my lead to fatal outcome in consequence of intracranial hemorrhage and acute renal failure. Before the production of Lonomia antivenom, the calculated lethality was 1.7%, dropping to about 0.3% in patients had received the specific treatment.

In conclusion, although lethality by poisonous animals is relatively low, the causes of deaths should be more precisely documented, for a better understanding of the mechanisms of venoms activity and, perhaps most importantly, to be used as a tool for development of public politics of health.

Causes of deaths in snake bites in Brazil

França, F.O.S.

Hospital Vital Brazil, Instituto Butantan, São Paulo, Brazil

Hospital das Clínicas- Faculty of Medicine- University of São Paulo,Brazil

^{Correspondence}

The National Program for Surveillance and Control of Snake Bites in Brazil indicates that 20,000 accidents occur yearly (incidence rate = 15 accidents/100,000 population per year) with more than 100 deaths per year. Bothrops genus causes almost 90% of accidents, followed by Crotalus (8%), Micrurus (1%) and Lachesis (1%). The overall case-fatality rate is 0.4% but varies according to the genus of snake, ranging from 0.4% for Bothrops to 2.5% for Crotalus envenoming. To determine the causes of death we have to know continuously where this accidents occur and the epidemiological, clinical and therapeutic data to characterize the real situation of this health problem in our country. But unfortunately we don´t know the cause of death in the majority of this cases. Researchers has related many factors that can affect the prognosis of snakes accidents. Many variables probably has influence on snake bites severity. Basically, we can divide this factors in three groups: 1) Epidemiological: Time between bite and treatment: patients who had received treatment after 6 hours after the bite, presents an important risk factor for severity of envenoming; Use of tourniquet: increase the probability of local complications, including local infections that could progress to septicemia; Site of bite: face and intravascular are more severe; Patient’s age: patients aged with less than 7 and more than 70 years presents, in general, major chance to develop complications; Length of snake: adult snakes could cause more commonly severe cases; 2.) Clinical: Probably almost all deaths by Bothrops and Lachesis occurs in consequence of acute renal failure, massive hemorrhage, shock and septicemia. In Crotalus and Micrurus accidents the presence of signs of respiratory distress and in Crotalus the presence of renal failure define the severity; 3) The quality of treatment: Correct evaluation of the severity in the admission; Use the especific antivenom in adequate dosage intravenously; Precocity in detection and treatment the complications in severe envenoming and; Transference to ITU in severe cases.

Finally, in order to contribute to decrease the lethality of snake bites we have to keep continuous up dating of the national data, maintain and increase the decentralization of distribution of the antivenom, references services to treat severe cases, improve the systematic training of the health care workers, disseminate information on prevention and first aid after snake bites to the population and stimulate the research to improve the treatment.

Scorpion envenomation - Fatal cases

Cupo, P.

Depto. de Puericultura e Pediatria - Faculdade de Medicina de Rib. Preto - USP

^{Correspondence}

Scorpion envenomation represents an important and serious public health problem in Brazil and also in others parts of the world. It may cause severe hemodynamic and cardiorespiratory alterations, particularly in children, being the cardiac failure and pulmonary edema the causes of death. Cardiac damage has been attributed to sympathetic hyperstimulation caused by the venom, by direct action on the myocardium or through hemodynamic effects, to local action of the toxin in the heart and, as side effect, to increased levels of cytokines.

Evidence of cardiac injury includes in addition to clinical manifestations, electro and echocardiographic changes, increased levels of creatinokinase, lactatodesydrogenase, and its fractions CKMB and LD1. Recently it was also described an increased levels of troponin I, the gold standard for the diagnosis of myocardial injury, which support the cardiac origin of pulmonary edema.

Unfortunately the epidemiological data of scorpion poisoning as well anathomopathological data of fatal cases are scarce, limited mainly to short communications of isolated cases.

During 1982-1998, 7880 victims of scorpion bites were managed at the Emergency Unity of Clinical Hospital of Faculty of Medicine of Ribeirão Preto. Most of the accidents were caused by T. serrulatus and were classified as mild in 7639 (97%) patients, moderate in 105 (1.3%), and severe in 136 (1.7%), occurring 7 deaths (0.09%). Most of the severe and moderate poisoning (91% and 84%) occurred in children under 15 years, as well as the deaths (5 under 7 years and 2 under 12). The pulmonary edema/shock developed in the first 10 h and most of the patients died within the 24 h of the bite.

Anathomopathological data of 6 patients revealed lung with increased size, diffuse alveolar edema, areas with hemorrhage, and inflammatory infiltrate. The coronary arteries were pervious in all the patients and the cardiac chambers were increased in 4. Microscopy was carried out in 5 patients and showed vacuolar degeneration of cardiac fibers, focal areas with myocitolysis and necrosis, interstitial edema with inflammatory infiltrate.

Pathological aspects of human deaths due to venomous animals in Brazil

Benvenuti, L.A.

Lab. Anat. Patol. INCOR

^{Correspondence}

Human death due to venomous animals is rare. In Brazil, the most dangerous species are the snakes from the genera Bothrops, Crotalus and Micrurus, and the scorpion Tytius serrulatus. The viscerocutaneous form of loxoscelism, the stings of honey bees and the contact with Lonomia caterpillars can produce severe clinical compromise and death. Bothrops venom induces local lesions characterized by acute inflammatory infiltrate, hemorrhage and necrosis, which can secondarily infect, evolving the patient to septic shock and death. Meanwhile, death is usually due to systemic actions of the venom, which promotes hemorrhage, thrombosis and disseminated intravascular coagulation. The venom of the tropical rattlesnake (Crotalus durissus terrificus) induces direct myotoxicity and systemic rhabdomyolysis. In severe cases, myoglobinuria is prominent and its toxic action on the renal tubular ephitelium leads to acute tubular necrosis and renal failure. The venom from the snakes of the genus Micrurus (coral snakes) produces neuromuscular blockade due to direct action of its neurotoxins on the end-plate receptors. Death is usually due to acute respiratory failure and asphyxia. Severe envenomation by the scorpion Tytius serrulatus produces cardiac failure, pulmonary edema, shock and may evolve to death, particularly in children. Coagulative myocytolysis is present in the myocardium, probably secondary to the deleterious effect of large amounts of circulating cathecolamines. Death due to the stings of honey bees can be caused by severe anaphylactic reactions or the direct action of the venom. In massive envenomation, rhabdomyolyis, myoglobinuria, renal acute tubular necrosis, hepatocellular necrosis, focal myocardial necrosis, and histopathological features of acute respiratory distress syndrome were described. Viscerocutaneous form of loxoscelism is characterized by intravascular hemolysis, disseminated coagulation and acute tubular necrosis, which may evolve to acute renal failure and death. Contact with Lonomia caterpillars can induce severe coagulopathy, systemic hemorrhagic syndrome and death due to cerebral hemorrhage.

Antimicrobial peptides in amphibian skin secretions

Mangoni, M.L.; Miele, R.; Mignogna, G.; Borro, M.; Fiocco, D.; Simmaco, M.; Barra, D.

Dipartimento di Scienze Biochimiche ‘A. Rossi Fanelli’, Università La Sapienza Piazzale Aldo Moro 5, 00185 Roma

^{Correspondence}

Amphibian skin secretions contain many biologically active compounds, among which a number of antimicrobial peptides has been isolated and characterized in our laboratory. These peptides are synthesized by ribosomes as inactive precursors and proteolytically processed in several steps to the active forms, which are then stored in the granular glands of the skin. Some of these molecules contain, as result of a post-translational modification, a D-amino acid in their sequence. Antimicrobial peptides are considered the effector molecules of innate immunity, acting as a first line of defense against bacterial infections.

We have studied the biological activity of purified peptides and their analogues against Gram-positive and Gram-negative bacteria as well as fungi. We have also used frogs as model systems for the study of factors controlling both natural infections and the natural flora. We have demonstrated that glucocorticoids (GC), known to suppress immune functions in mammalian cell cultures, influence the peptide-mediated innate immunity in amphibia. In particular, GC-treatment of Rana esculenta results in the almost complete inhibition of the de novo synthesis of the antimicrobial peptides normally present in the skin secretion, as monitored by both HPLC and Northern blot analyses. In addition, immunoblot analysis of skin proteins shows a clear rise of IkBa. These results suggest that frogs in vivo regulate their peptide antibiotic synthesis by an NF-kB/IkBa-like mechanism. The determination of the structure of a number of genes coding for antimicrobial peptides in Bombina orientalis has actually shown that their promoter regions contain recognition sites for nuclear factors. Experiments performed in vivo demonstrate for the first time the inducibility of defence peptides in a vertebrate and that the absence of natural flora prevents the production of antimicrobial peptides in skin secretion.

Neuropharmacological effects of the bufodienolides isolated from Bufo paracnemis skin

Carvalho, K.M.

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

^{Correspondence}

Amphibians have undergone profound evolutionary changes to survive to predators and microorganisms, and their skin secretions containing toxins and several biological active substances seems to be a paramount importance. In this work, we shown new neuropharmacological effects of bufodienolides isolated from Bufo paracnemis parotoid secretion. After extraction, the secretion was dissolved in ethanol (1:4,w,v), centrifuged at 5000g/20min and submitted to HPLC chromatography using a C₁₈ column (25x250mm,5ml/min) eluted with acetonitrile (0-40%,30min). Fractions eluted with 38% and 40% of acetonitrile were pooled and lyophilized. High resolution NMR analysis by application of 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 their identification as bufodienolides, known as marinobufagin and telecinobufagin. Telecinobufagin induced a strong local anesthetic activity assayed by: a) infiltration anesthesia: telecinobufagin(0.5%), 181±15min, bupivacaine(0.5%), 110±10min, and lidocaine(2%), 49±5min, b) mouse tail flick test: telecinobufagin(0.5%), 152±12min, bupivacaine(0.5%), 52±8min, and lidocaine(2%), 18.4±4min, c) cornea test: telecinobufagin(0.5%), 49.4±6min, bupivacaine(0.5%), 46.8±5min and lidocaine(2%), 11.5±3min, d) tooth pulp stimulation: telecinobufagin(0.5%), 160±12,5min, bupivacaine(0.5%), 150±11,9min and lidocaine(2%), 50±4,5min. These results suggest that telecinobufagin may be a prototype of a potent new class of local anesthetic. Marinobufagin induced a strong convulsant activity with seizures in the electrographic recordings in mice (5mg/kg,i.p.) and in rats (5mg/kg,i.p.), resulting in status epilepticus (>1h). Seizures decreased 100% in the animals pretreated with phenitoin (50mg/kg,i.p.) and 80% with diazepam (10mg/kg,i.p.), but they were not affected by phenobarbital (50mg/kg,i.p.). These results suggest that marinobufagin may be used to develop an experimental status epilepticus model for the pharmacological evaluation of anticonvulsant drugs used in epilepsy treatment.

The presence of tetrodotoxin in amphibians

Schwartz, C. A.

Laboratório de Toxinologia, Universidade de Brasília, Brasília, DF, Brasil

^{Correspondence}

Tetrodotoxin (TTX) is one of the most potent marine toxin already isolated. TTX occurs in a wide range of marine animals. Among terrestrial animals TTX is known only in the Amphibia class, occurring in a few families of salamanders and anurans. Recently, the occurrence of tetrodotoxin and analogues was examined in three braquicephalid species: Brachycephalus ephippium, B. nodoterga and B. pernix.

In toxicity assay with intra-peritonial injection in mice, B. nodoterga extract shown poisonless, while B. pernix extract had the highest toxicity among the studied species.

The tissues extracts showed differences in their toxicities. The skin showed be the most toxic tissue, followed by the liver. The presence of TDA, TTX, 4-epiTTX and anhydroTTX were suggested by comparison of retention time in LC-FLD system. The presence of 6-epiTTX, 11-oxoTTX, 11-norTTX(S)-ol and 5-deoxiTTX were identified by LC-ESI/MS system.

Mass spectrometry MALDI-TOF and LC-ESI/MS, confirm the family Brachycephalidae as the fourth anuran family to contain TTX.

The fragmentation spectra obtained by LC-ESI/MS/MS of TTXs showed a fragmentation patterns that can be used to characterize this family of compounds.

Bacteria, as TTX source in amphibian was suggested by analysis in LC-FLD system of the semi-purified bacteria extracts and its culture medium. Peaks with the same time of retention of TTXs, suggest the presence of these compounds. However it is not discarded the diet origin.

Since the first detection of TTX in terrestrial vertebrates, the number of identified TTX-containing amphibian species increased. There are almost 4.000 species of amphibians in 26 recognised 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.

Peptides and proteins of Brazilian amphibians

Bloch Jr., C.

Laboratório de Espectrometria de Massa; Embrapa Recursos Genéticos e Biotecnologia. SAIN Parque Rural, Av. W3 Norte 70 770-900, Brasília, DF

^{Correspondence}

Amphibian skin secretion is a somewhat unique source of bioactive material. Ranging from simple sugars, alkaloids, amines, opioid substances, hormones, neurotransmitters to clearly established antibiotics (1), antimicrobial peptides (2) and protease inhibitors (3). As a rule, a given amphibian species secretes a unique repertoire of small sized antimicrobial peptides through specialized epithelial cells. Most of them share overlapping structural and sequence features, such as cationic residues, an amphiphilic a-helix, and a sequence of 10 to 46 residues long. The presence of these related peptides in the frog skin plays a primary role in a primitive but elaborated innate host defense mechanism (4). It is a chemical shield, against a wide range of invaders at their first point of contact, the skin, mucous membranes or other surfaces.

This work reports the results of a systematic screening, functional investigation and structural analysis of a number of bioactive molecules found in the water-soluble skin secretions of amphibians from the Brazilian biodiversity. Among the studied genera are: Phyllomedusa, Physalaemus, Osteocephalus, Hyla, Brachycephalus and Trachycephalus.

References

1. HANCOCK, R.E.W. and LEHRER, R.I., (1998) Trends in Biotechnology 16:82-88.

2. HANCOCK, R.E.W. and DIAMOND, G., (2000) Trends in Microbiology 8,9:402-410.

3. CONLON, J.M. and KIM,J.B. (2000) Biochem. Biophys. Res. Comm. 279 (3):961-964.

4. GURA, T., (2001) Science 291:2068-2071.

Bioactive peptides from Brazilian frog species included in the genera Leptodactylus, Hyla and Odontophrynus

Matsushita, R.H.^I; Nascimento, A.C.C.^I; Sena, G.¹; Zanotta, L.C.^I; Sousa, M.V.^II; Schwartz, C.A.^I; Schwartz, E.F.^I; Sebben, A.^I; Fontes, W.^II; 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 emergence of multi-drug resistant microorganims has stimulated a search for new antibiotic compounds with clinical applications. Over the past several decades numerous biologically active components (biogenic amines, alkaloids, steroids, peptides and proteins) have been isolated from anurans skin secretions. These secretions contain a large array of bioactive peptides, including antimicrobial cationic peptides.

Nowadays many potent antimicrobial peptides have been isolated from several species and these peptides exhibited lytic activity against bacteria, protozoa and yeasts, as well as against filamentous fungi. Recently it was also demonstrated that amphibian antimicrobial peptides inactivate frog virus 3, playing a role in maintaining amphibian health.

Different families of peptides have been described according to their biological activities, structural features and genomic organization. Some examples include: bombinins and type H bombinins isolated from Bombina species; magainins from Xenopus laevis; dermaseptins isolated from Phyllomedusa genus; brevinins from the Japanese frog Rana porsa brevipoda; ranateurins from Rana catesbeiana; temporins isolated from Rana temporaria and caerins and caeridins from the Australian genus Litoria.

Very little information is available about the purification and characterization of bioactive peptides isolated from amphibian species of South America. In the present study we described the structural and biological characterization of bioactive peptides isolated from the skin secretions of brazilian frog species, included in the genera Hyla, Leptodactylus and Odontoprhynus.

Among snakes, venoms and public health

Melgarejo, A.R.

Divisão de Animais Peçonhentos, Instituto Vital Brazil. Rua Vital Brazil Filho, 64, 24230–340, Niterói, RJ

^{Correspondence}

In the beginning of the twentieth century, the problem of snakebite in Brazil determined an interdisciplinary effort due to the severity of the accidents or their incidence. The main participant of this effort was the scientist Vital Brazil. In order to discuss the problem, the development of simultaneous scientific research in biology, biochemistry, pharmaceutics and therapeutics was necessary, together with an educational campaign, that still continue to nowadays. The main product, the serum–therapy development, solved the medical problems, but there are still some nebulous points in which the biological study of the snakes can bring some significant contributions. Good examples are our studies on two Viperidae snake species and their management in the serpentarium. Particularly in the case of the bushmaster, Lachesis muta, the biggest of our poisonous snakes, this represents a great challenge because of problems in captivity adaptation. Besides, the small production of venom -around 200 mg for extraction– and some particularities, as having the highest LD50 of the native pitvipers, difficult the production and control of the antivenom, it makes impossible biochemical and pharmacological researches. Oviparous species, and extremely dependent of the preserved forest environment, this snake will need high controlled environmental and sanitary conditions for the successful of the breeding. A high well directed capture effort in the nature will be needed. Another situation, difficult as well, we have with another snake, the jararacussu (Bothrops jararacussu), far away the biggest venom producer (up to 1.670 mg in our serpentarium). However, your venom is not well neutralized by the specific antivenom, as Vital Brazil had already shown in 1904. The particular diet and feeding behavior of this species can explain your venom gland hypertrophy.

Ecological and phylogenetic correlates of feeding habits in neotropical pitvipers of the genus Bothrops

Marques, O.A.V.

Laboratório de Herpetologia, Instituto Butantan

^{Correspondence}

The neotropical pitviper genus Bothrops occurs from Mexico to Argentina. Data on the feeding habits of 21 forms indicate that most Bothrops are diet generalists and show ontogenetic shift in prey types. Four species apparently became mammal specialists. Two island species have distinct diets: one in which adults feeds heavily on birds and another whose diet to be restricted to ectothermic prey. Mammal specializations may be related to life history strategies and the shifts observed in island species are a consequence of prey availability (there is no small mammals in the islands). Generalist species of Bothrops incorporate endotherms in their diets at a narrow range of sizes and drop ectotherms at variable sizes. Ontogenetic shifts from ectotherms to mammals in Bothrops is plesiomorphic and may be largely a consequence of ontogenetic shifts in body size. Caudal luring was observed in seven species and may occur in juveniles of all diet generalists. Feeding frequency is relatively low in Bothrops, perhaps due to sedentary habits and other factors related to inhabiting warmer climates. Mean relative prey size is moderate in Bothrops, although very large prey is occasionally eaten. Mammal specialists are stouter and feed on larger prey than generalists. Relative prey size decreases during ontogeny in some species. A high diversity of macrohabitat use occurs in Bothrops and the more arboreal the species, the more frequently it feeds on anurans. Lanceheads occur in several kinds of forests and open habitats and their diets seem to reflect local prey availability. Although a few apomorphies appeared in some taxa, in general feeding habits in Bothrops are conservative, as most characters we analysed were already present in early crotalines.

Comparative study of venoms of genera Bothrops: phylogenetic analysis

Furtado, M.F.D.

Laboratório Herpetologia – Instituto Butantan

^{Correspondence}

The Neotropical pitviper genus Bothrops comprises about 20 species in Brazil, occurring in all main ecosystems. This taxon is responsible for most snakebite in human. There is no consensus in the systematics of this group. Morphology, habits and MtDNA have been extensively used as characters to subdivide the genus Bothrops. In this study, we use the phylogenetic analysis of activities of venoms from 18 species of Bothrops and Bothrocophias hyoprorus (outgroup). Eighteen activities of venom were determinated, Results obtained were codified in characters and data matrix was constructed. The analysis of data (Hennig 86) revealed 1687 trees. Remotion of amidolytic activity character, produced 4 most parsimonious tree with same topology, with 50 steps and consistency index =38 and retention index =63. B.atrox appears closest to the outgroup, B.bilineatus in an isolated branch, B. brazili and B. marajoensis group together, isolated branches appear with B. moojeni, B. leucurus, B. pradoi, B. neuwiedi, B. jararacussu and B.pirajai. Differences in trees were noticed regarding the position of B. erythromelas, B.alternatus, B. taeniatus, B. cotiara and B. fonsecai . All of them constant in one branch in 2 trees whereas B. itapetiningae joined to that branch in 2 trees and with B. insularis e B. jararaca in another 2 cladograms. Venom activities common for all Bothrops species are: fibrinolytic activity by metalloproteinase and hydrolyse of fibrinogen a-chain. Amidolytic activity presents wide variation among the species. Venom activities such as band of 63 kDa in SDS-PAGE, phospholipase activity and serino-proteinase inhibitor group Bothrops clades in an arrangement presented below. Comparing phylogeny produced with venom data to MtDNA phylogenies (Salomão et al, 1999, Kaupia:127-134), there are similarities in topology concerning: group 1 - B. moojeni, B. leucurus, B. pradoi, group 2 - B. jararacussu, B. pirajai, group 3 - B. jararaca, B. insulares and group 4 - B. cotiara, B. fonsecai, B. alternatus, B. itapetiningae. Clashes were detected when analyzing the grouping of B. atrox, B. marajoensis, B. brazili, B. neuwiedi, B. erythromelas, B. taeniatus and B. bilineatus.

What he confocal microscope can do for you

Ownby, C.L.

Department of Physiological Sciences, College of Veterinary Medicine, Oklahoma State University

^{Correspondence}

The scanning laser confocal microscope is an essential tool for today’s studies of the biology of cells. Basically a light microscope system, the confocal microscope uses lasers to excite specific fluorescent dyes that have been incorporated into cellular molecules of interest to cell biologists and other scientists. In addition to increased sensitivity when compared to a typical epifluorescent microscope, confocal microscopy provides better resolution and also the ability to optically section the sample allowing for 3D reconstruction. Dual and triple labeling studies are also possible with the confocal microscope and digital images are routine.

Immunolabelling experiments permit the localization of one, two or several proteins of interest in the cell either separately or simultaneously. Proteins of interest can be co-localized with fluorescent probes specific for cellular organelles to determine their cellular location and distribution under various conditions. Membrane trafficking in cells can be studied with green fluorescent protein (GFP) and other related proteins and membrane potentials and ion fluxes can be studied using specific fluorescent dyes. Fluorescence in situ hybridization (FISH) allows for gene mapping and gene expression studies. More recently many experiments on live cells can be performed with the confocal microscope and its close relative, the two-photon microscope.

The purpose of this presentation is to explain how the confocal microscope works, to give many examples of the types of studies that can be done using it and to show how it can help you in your research projects.

Proteomic approach applied to the study of peptides from the Brazilian tree-frog Hyla biobeba skin secretion

Fontes, W.^II; Matsushita, R.H.^I; 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}

Amphibian skin secretions have been considered a rich source of bioactive compounds, such as biogenic amines, steroids, alkaloids, peptides and proteins. Biologically active peptides represent a significant part of this repertoire of substances and they act on different physiological processes, including passive defense against microorganisms. In order to evaluate the diversity of peptides present in amphibian skin secretions, we applied a proteomic approach to improve the number of peptides identified from the skin secretion of the tree-frog Hyla biobeba, commonly found in our region. Adult specimens of Hyla biobeba were collected from Distrito Federal region and their skin secretions were obtained by mild electrical stimulation, dissolved in water and lyophilized. Dried secretion aliquots were applied onto a C18 reversed-phase column (Vydac 218TP54, 4.6 x 250 mm, Separations Group, USA) and fractionated using a linear gradient of acetonitrile. Eluted fractions were manually collected, vacuum-dried and submitted to mass spectrometric analysis using a MALDI-TOF spectrometer (Reflex IV, Bruker). The samples were prepared using acyano-4-hydroxy-cinnamic acid (20 mg/mL) as matrix dissolved in acetonitrile:0.1% TFA (1:2) and applied onto the target by dry-droplet method. The m/z used range was 0-3 kDa. This kind of analysis permitted the rapid homogeneity assessment of most chromatographic fractions and also the determination of their molecular masses. Some of these peptides were selected for further purification and structural characterization.

cDNA libraries and transcriptome: unraveling new toxins and understanding the mechanisms of envenomation

Ho, P.L.

Centro de Biotecnologia, Instituto Butantan, 05503-900, São Paulo, SP, Brazil

^{Correspondence}

A global panorama of transcriptional activity in the venom glands would correlate with its venom composition. The comprehension of the venom composition is fundamental for the mechanisms of envenomation. We have constructed a cDNA library from the Bothrops insularis venom gland and 677 expressed sequence tags (ESTs) were generated from 610 aleatory clones. The sequences were clusterized and 297 resulted cluster sequences were compared with the databanks. Toxin sequences corresponded to 56%, being the metalloproteases (23%) and bradykinin-potentiating peptides (11%) the major toxin components. A new toxin structure-related to Vascular Endothelial Growth Factor was also identified and characterized (JBC276:39836, 2001) that might contribute to venom dissemination and to hypotension observed in the envenomed animals. In order to understand the role of metaloproteases, since they are the major transcript components in the venom gland as revealed by this project, we hypothesized that they would be able to act on plasminogen in order to generate the anti-angiogenic factor angiostatin or angiostatin-like molecules. This result was confirmed and may account for the poor and incomplete regenerative response observed in the damaged tissue upon Viperidae snake bite envenomings (BBRC 294:879, 2002), besides the other actions described for this class of enzymes. The results obtained here allowed us to identify the major toxins from B. insularis venom gland, the description of new toxins and also to a better understanding of the mechanisms of envenomation that affects locally and systemically. In collaborative projects, this approach is being applied now to other venom glands, from the spider Loxosceles laeta, the tick Amblyomma cajennense, the fish Thalossopherinae nattereri, the caterpillar Lonomia obliqua and others.

Support: FAPESP, CNPq, Fundação Butantan

Venom proteins as structural model systems

Arni, R. K.

Department of Physics, IBILCE/UNESP

^{Correspondence}

The action and damage caused by snake venom proteins have been extensively studied and provide us with an excellent oppurtunity to correlate structural information with pharmacological and biochemical data to improve our understanding of their function and mode of action. These proteins are involved in a number of key biological processes such as membrane damage, lipid hydrolysis, myotoxicity, hemostasis, fibrinolysis and blood coagulation.

High resolution diffraction data have been collected from a number of snake venom phospholipases both in the native states and bound to fatty acids, amphiphiles, phospholipids and inhibitors. This provides us with a wealth of information about the mode of action and stereochemical requirements of these enzymes.

Structural data is also available for a number of snake venom thrombin-like enzymes currently being studied in our laboratory and structural comparisons with the serine proteases involved in the control and regulation of blood coagulation should be important for the design of novel inhibitors and substrates.

This presentation will focus on the structural aspects of these important venom proteins.

Acknowledgements: Financial support by FAPESP and CNPq is gratefully acknowledged.

Use of toxins in biotechnology

Kalapothakis, E.^I; Almeida, A.P.^II; Chávez-Olórtegui, C.^III; Ribeiro, B.M.^IV; Cruz, I.^V; Mangili, O.C.^VI; Gomez M.V.^I

^IDepartamentos de Farmacologia, UFMG, MG, Brazil

^IIDepartamentos de Fisiologia, UFMG, MG, Brazil

IIIDepartamentos de Bioquímica-Imunologia, UFMG, MG, Brazil

^IVDepartamento de Biologia Celular, UNB, DF, Brazil

^VEmbrapa Milho e Sorgo de Sete Lagoas, MG, Brazil

^VIDepartamento de Fisiologia, Setor de Ciências Biológicas, UFPR, MG, Brazil

^{Correspondence}

Venom from spiders and scorpions, represents a source of natural peptides which could have pharmacological potential similar to traditional sources, particularly in the development of drugs targeted to specific receptors, ion channels or the release of neurotransmitters. Our group has generated, systematically, knowledge about the mode of action of neurotoxins from the spiders Phoneutria nigriventer and Loxosceles intermedia and also from the scorpions Tityus serrulatus and Tityus bahiesis. The research lines of 3 projects, deal with important problems and are innovative in that they utilize peptide neurotoxins in the areas of medicine and agriculture. The first of these projects involves the use of neurotoxins in the treatment of cerebral ischemia, different types of pain (such as neurogenic, neuropathalogic and inflammatory pain), as well as in cardiac arrhythmias. The second idea developed involves the use of neurotoxins in agriculture, with the development of biological agents to control economically important pests at low cost and with a low impact on the environment when compared to conventional (chemical) insecticides. The third and last point is related to the use of recombinant toxins in the production of antiserum and vaccines to protect against the venom of the brown spider Loxosceles intermedia and Tityus serrulatus scorpion, which continues to cause serious social and economic problems in Brazil.

Inhibitory properties of anti-bothropic complex from Didelphis albiventris serum and polyclonal antibodies on pharmacological effects of toxins from Bothrops snake venoms

Soares, A.M.^I; Trento, E.P.^II; Giglio, J.R.^III

^IDepartamento de Biotecnologia, UNAERP, Ribeirão Preto, SP

^IIDepartamento de Bioquímica, UNIC, Cuiabá, MT

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

^{Correspondence}

Anti-bothropic complex (ABC) was isolated from the serum of the South American opossum (Didelphis albiventris) by single-step affinity chromatography using a Sepharose-immobilized metalloprotease (BaP1) from Bothrops asper as the binding protein. Functional studies pointed out that ABC inhibits the hemorrhagic and proteolytic activities on fibrin, fibrinogen, and casein induced by the metalloproteases isolated from B. asper BaP1 and BaH4, B. neuwiedi neuwiedase and B. moojeni MOO3. In addition to the anti-hemorrhagic and anti-proteolytic activities, ABC also showed anti-myotoxic, anti-lethal, and anti-edematogenic effects against myotoxic phospholipases A₂ isolated from B. asper Basp-I, II and III, B. moojeni MjTX-I and II, B. jararacussu BthTX-I and II, B. pirajai PrTX-I and III, and B. neuwiedi BnSP-7. Moreover, it had inhibitory effects on the phospholipase A₂ activity of the crude venom as well as the isolated venom phospholipases A₂. Biochemical characterization of ABC showed the presence of two glycosylated subunits of 43 and 45 kDa, respectively, with an isoelectric point <4. The two subunits were separated by ion-exchange HPLC. The N-terminal sequences of both subunits (LKAMDPTPXLWIETESP, where X is Arg-9 and Pro-9, respectively) showed a high degree of identity with other serum inhibitors isolated from different marsupials. Antibodies to ABC cross-reacted with different snake venoms, 04 isolated metalloproteases and 12 purified class II myotoxic phospholipases A₂ found in snake venoms of the genus Bothrops. In neutralization experiments, pre-incubation of crude venom or isolated toxins with antibodies to the ABC inhibited their hemorrhagic, PLA₂ and myotoxic activities.

Financial supported: FAPESP and CNPq.

Inhibitors of phospholipases A₂ in the blood plasma of snakes

Fortes-Dias, C.L.

FUNED, Belo Horizonte, MG

^{Correspondence}

It has long been noted that several animal species are resistant to envenomation by snake bites. This natural immunity was ascribed, in most cases, to the presence in the blood of neutralizing factors active against distinct pharmacological venom activities. We have been focusing on phospholipase A₂ inhibitors present in the blood plasma of snakes, whose members have been organized into three different classes (alpha, beta and gamma) according to their structural features and specificities (1). A gamma- inhibitor (CNF) from the blood plasma of South American rattlesnake Crotalus durissus terrificus has been taken as our model. CNF was cloned and sequenced from the encoding cDNA (2) and is composed of two structural units known as three finger motifs, similar to those found in the urokinase-type plasminogen activator receptor (u-PAR) and Ly-6 related proteins (1). CNF is a hexameric glycoprotein of 160 kDa that acts by replacing the PLA₂ subunit (CB) in the heterodimeric crotoxin (CACB), thereby neutralizing its toxicity (2,3). Protein-protein interactions in CACB and CACNF complexes have been studied through binding experiments to synaptosomes and SPOT technology (4).

References

1. Ohkura, N, Okuhara, H, Inoue, S, Ikeda, K & Hayashi, K (1997). Biochem. J. 325: 527-531.

2. Fortes-Dias, CL, Lin, Y, Ewell, J, Diniz, CR. & Liu, T-Y (1994) J. Biol. Chem. 269: 15646-15651.

3. Perales, J, Villela, C, Domont, GB, Choumet, V, Saliou, B, Moussatché, H, Bon, C & Faure, G (1995) Eur. J. Biochem. 227: 19-26.

4. Molina, F., Laune, D, Gougat, C, Pau, B & Granier, C (1996) Peptide Res. 9: 151-153.

Natural inhibitors of toxins and snake venoms

Melo, P.A.

Departamento de Farmacologia Básica e Clínica, ICB, UFRJ. Rio de Janeiro RJ. Brazil

^{Correspondence}

Some previous data from our laboratory so far obtained, indicated that extracts or purified constituents of plant Eclipta prostrata (EP) are effective in the prevention of the myotoxicity of crotalid snake venoms. As part of a program aimed at synthesizing biologically active flavonoids, some coumestans bearing different patterns of oxygenation at rings A and D were chosen as target molecules, synthesized for the first time and their action as antimyotoxic compared with that observed for wedelolactone (W) a EP constituent. We mainly tested a coumestan named PCALC36, which 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 mcg/g b.w.). Also, this coumestan at 3 mcg/g neutralized at the same dose the venoms of Agkistrodon contortrix laticinctus and Crotalus viridis viridis by 50% and 70% respectively, and reduced the myotoxicity of BthTX-I and BthTX-II by 35% and 75%. Also we have shown that heparin and other polyanions inhibit the myotoxic effect of Bothrops venoms by formation of acid-base complexes with the basic myotoxins. Electron microscopy studies show that heparin treatments promoted a good muscle regeneration, with the organization of regenerated fibers being similar to the control while the animals that did not receive any treatment exhibited a complete disorganization of muscle cells. These data indicate that heparin improves the regeneration of EDL muscle damaged by B. jararacussu venom.

Financial support: CAPES, FAPERJ, PRONEX (No. 41.96.0888.00), FUJB-UFRJ and CNPq.

DM43 and DM64, two immunoglobulin-like proteins from Didelphis marsupialis serum with antihemorrhagic or antimyotoxic activity

Perales, J.

Department Fisiologia e Farmacodinâmica, IOC, FIOCRUZ, Rio de Janeiro

^{Correspondence}

Natural resistance to snake venom toxic effects is observed in some animals and, in many cases, this is due to the presence of soluble neutralizing proteins in their sera. Our group has isolated several of these neutralizing factors from the sera of some mammals and snakes. From the opossum (Didelphis marsupialis) serum, we have isolated and characterized three of them. Two (DM40 and DM43), with antihemorrhagic activity, and the other one (DM64), with antimyotoxic activity. In spite of these different activities, they have some similar characteristics, they are acidic glycoproteins, homodimeric under native conditions and, the most important, they present immunoglobulin-like structure. The complete amino acid sequences of DM43 and DM64 showed 78% similarity. However, DM43 has three immunoglobulin-like domains, while DM64 has five. DM43 and DM40 inhibited the enzymatic activity of several snake venom metalloproteinases (SVMP), such as jararhagin and bothrolysin and formed stable inactive non-covalent complexes with them. DM64 was able neither to inhibit these SVMPs nor to form complexes with them. On the other hand, DM64 neutralized both the in vivo myotoxicity and the in vitro cytotoxicity of myotoxins I (mt-I/Asp⁴⁹) and II (mt-II/Lys⁴⁹) from B.asper venom. The inhibitor formed non-covalent complexes with both toxins, but did not inhibit the PLA₂ activity of mt-I. Accordingly, DM64 did not neutralize the anticoagulant effect of mt-I nor its intracerebroventricular lethality, effects that depend on its enzymatic activity, which demonstrates the dissociation between the catalytic and toxic activities of this Asp⁴⁹ myotoxic PLA₂. Furthermore, despite its similarity with the metalloproteinase inhibitors DM43 and DM40, these proteins did not present any antimyotoxic activity against mt-I or mt-II and did not form complexes with them. We recently postulated that the difference between the activities of these two homologous proteins is probably consequence of the presence of either three or five immunoglobulin-like domains and/or other important structural differences, such as the presence of gaps and residues’ substitutions.

Venoms from spiders of the genus Phoneutria

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

^IFundação Ezequiel Dias, BH, MG

^IIDepto. Ciências Fisiológicas,UFES, Vitória, ES

^IIIDepto.Bioquímica e Imunologia, ICB, UFMG, BH, MG

^{Correspondence}

Venoms of spiders, such as the genus Phoneutria are an important part of the rich biodiversity found in Brazil. We have shown that they are a source of potentially valuable biologically active proteins, such as lethal neurotoxins acting on Na+, K+ and Ca2+ ion channels and chemical receptors in the neuro-muscular systems of animals, priapic agents, novel proteolytic enzymes and insecticides. Various of these might be developed for use in medicine, or in agriculture (as defensive insecticides genetically engineered into crop plants), and/or as powerful laboratory tools. We are using reverse phase and ion-exchange chromatography (HPLC and FPLC) to purify these proteins from the venoms of Ph. nigriventer , Ph. reidyi and Ph. spp, and determining their biological activities by both in vivo and in vitro tests, using mice and various insects, and also using electro-physiological methods To date we have succeeded in obtaining about 280 of these protein components in a pure state. Our analyses of the venoms from these three different species of Phoneutria found in Brazil have revealed that whilst they are quite similar in overall composition, there are significant quantitative and qualitative differences between them. Most notable is the observation that the venoms from Ph. reidyi and Ph. spp contain much higher concentrations of the proteins with insecticidal activity, and somewhat lower quantities of the proteins toxic for mammals, than the venom of Ph. nigriventer. We have also demonstrated that the forms ofTx1, one of the most neurotoxic and abundant proteins found in the venoms of all three species exhibit slightly different biochemical properties in each species.

Financial support: CNPq, FAPEMIG, FUNED.

Functional expression of toxins from the Brazilian "armed" spider Phoneutria nigriventer in E. coli.

Diniz, M.R.V.

From the Centro de Pesquisa e Desenvolvimento. Fundação Ezequiel Dias, Belo Horizonte, MG, Brazil

^{Correspondence}

The venomous glands of the Brazilian spider Phoneutria nigriventer produce a complex mixture of basic neurotoxic dissulfide-rich polypeptide ranging in size from 2-16 Kda They are associated with distinct effect of the venom. The molecular characterization of the precursor sequence of theses toxins have been performed by integrating information generated from a molecular genetic approach using cDNAs with data provided from primary sequence of native toxins. The likely mechanisms by which the toxins precursors peptides are processed to their mature forms have been deduced. A particularly interesting feature of the prepropeptides are the occurrence of the glutamate rich sequence interposed between the signal sequence and mature toxin peptide. Excision of the functional toxin appears to be signalled by flanking arginine residue and probably occurs only after the dissuphides linkages have been formed. The cDNA coding region for matured toxin of Tx1 and Tx3-2 have been amplified, cloned and expressed as thioredoxin fusion product in cytoplasm of Escherichia coli. After affinity purification and cleavage, the recombinant toxins displayed the same properties as the native toxins at least in their neurotoxic biological activity after intracerebral ventricular injection.

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

Phoneutria nigriventer venom: novel mechanisms for the activation of sensory nerves via 5-HT₄ receptors

Costa, S.K.P.

Centre for Cardiovascular Biology & Medicine and Centre for Neuroscience, King's College, London SE1 1UL, UK

^{Correspondence}

OBJECTIVES: The expression of 5-HT4 receptor has been demonstrated in rat sensory C-fibres, thus suggesting a role in the activation of sensory neurons. Localised intense pain and neurogenic oedema formation are manifestations associated with bites by the Brazilian spider Phoneutria nigriventer (Costa et al., 1997). We have used the rat isolated vagus nerve which contains sensory fibres in a grease-gap preparation to investigate Phoneutria nigriventer venom (PNV)-induced sensory nerve activation. In addition, neurogenic oedema was assessed in the skin of Wistar rats (200 g) by extravasation of ¹²⁵I albumin given intravenously.

RESULTS: PNV depolarises vagus nerve in a dose-dependent manner (0.121 ± 0.02, 0.20 ± 0.02 and 0.41 ± 0.06 mV at 1, 3 and 10 µg ml^-1 respectively, n=11). Pre-treatment of the nerves with capsaicin (5 µM) desensitised the nerve to subsequent application of PNV but not KCl. The 5-HT4 receptor antagonist RS39604 significantly inhibited PNV- (from 0.62 ± 0.13 to 0.22 ± 0.08* mV, at 1 µM, n=14) and 5-HT (from 0.67 ± 0.06 mV to 0.46 ± 0.04* mV, at 1 µM, n=13)-induced depolarisation but had no effect on capsaicin-induced response. In the rat skin in vivo, the PNV-induced plasma extravasation dependent on tachykinin NK1 receptor activation was also significantly inhibited by RS39604.

CONCLUSION: Thus, our finding provides evidence that functional 5-HT4 receptors are present at the microvascular level in rat skin and their antagonists can modulate neurogenic oedema. This may contribute to growing arguments that implicates 5-HT4 receptors in mechanisms of sensory nerve activation with respect to peripheral pain and inflammation.

Acknowledgements: British Heart Foundation and FAPESP.

Costa SKP, De Nucci G, Antunes E & Brain SD. Eur J Pharmacol, 339:223-6, 1997.

Mechanisms of action of Phoneutria nigriventer toxins on ion channels

Beirão, P.S.L.

Department of Biochemistry and Immunology, Instituto de Ciências Biológicas da UFMG, Belo Horizonte, MG, Brazil

^{Correspondence}

The Phoneutria nigriventer (family Ctenidae, suborder Labdognatha) venom is rich in polypeptide toxins, with molecular weights that range from 2 to 9 kDa. It can be factionated into three major fractions that exert neurotoxic effects when injected into mouse central nervous system. In our laboratory we have investigated the effects on ion channels of toxins purified from the venom of P. nigriventer, using the patch clamp technique, in the modalities of whole cell, perforated patch and loose patch clamp. The toxins were purified by the group of Professor Carlos Diniz. Fraction PhTx2 has a marked effect on Na⁺ channels, decreasing the rate of inactivation and shifting to hyperpolarizing potentials the voltage-dependences of the conductance and of the steady-state inactivation. Fraction PhTx2 contains 9 toxins, and two of them (toxins Tx2-5 and Tx2-6) reproduce the major effects of the fraction. These toxins are expressed as preprotoxins, and their amino acid sequences (and of their precursors) have no significant identity with any known peptide, except among themselves. P. nigriventer Tx2-6, whose precursor has 82 amino acids, exerts complex effects. Its predominant effect is similar to a-scorpion toxins, but also cause a negative shift of the conductance voltage-dependence and a decrease of Na⁺ current similar to b-scorpion toxins. P. nigriventer Tx1 and fraction PhTx3 have no significant effect on Na⁺ currents.

PhTx3 exerts marked inhibitory effect on Ca²⁺ currents. By using cells that express the different types of Ca²⁺ channels, and using appropriate pharmacological tools to selectively inhibit unwanted currents, it was possible to isolate T-, L-, N-, P/Q- and R-type Ca²⁺ channels. We have observed that toxin Tx3-3 inhibited P/Q- and R-type currents, with high affinity. It also inhibits, albeit less effectively, L- and N-type channels, and has no significant effect on T-type currents. Thus, this toxin can be classified as an w-type toxin. We also observed that toxin Tx3-2 inhibits L-type Ca²⁺ currents. On the other hand, toxin Tx3-1 has no direct effect on Ca²⁺ currents, but inhibits A-type K+ currents, thus affecting Ca²⁺ oscillation frequency in GH3 cells.

Our results show that Phoneutria nigriventer venom is a rich source of neuroactive peptides that act on different types of ion channels.

Supported by FAPEMIG and CNPq.

Pharmacological studies of toxins isolated from the venom of the spider Phoneutria nigriventer

De Lima,M.E.^I; Santos, R.G.^I; Figueiredo, S.G.^II; Cordeiro, M.N.^III; Richardson, M.^III; Pelhate, M.^IV; Seagar, M.^V; Diniz, C.R.^I,III

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

^IIDepto. Ciências Fisiológicas, UFES

^IIIFundação Ezequiel Dias, BH-MG

^IVUniversité D’Angers, France

^VUniversité de la Mediterranée, France.

^{Correspondence}

The P. nigriventer venom contains many toxins acting on ionic channels.

We examined the pharmacological properties of different toxins purified from this venom, including TX4(6-1) with insecticidal activity and w-Phonetoxin IIA which is active on mice. The experimental approach included binding experiments, with the radiolabeled derivatives of toxins, on cockroach (Periplaneta americana) nerve cord synaptosomes, for insect-toxin and both rat brain synaptosomes and BHK cell line membranes, for mammal-toxins. Electrophysiological recordings on cockroach isolated giant axon under current-clamp and voltage-clamp conditions were performed using the double oil-gap single fiber technique. Electrophysiological whole cell currents were also recorded from isolated stable BHK cells expressing Cav2.1, Cav2.2 and Cav2.3 channels (P/Q, N and R-type currents, respectively). Tx4(6-1) is ineffective on mammalian Na_v channels and competes with the a-like toxin ¹²⁵I-Bom IV from scorpion Buthus occitanus mardochei, for binding on the site 3 of insect Nav channel (IC₅₀~25 nM). Physiological consequences of this binding to the insect Na_v channel were shown by electrophysiology: TX4(6-1) prolongs evoked axonal action potentials due to a slowing down of sodium current inactivation. w-Phone IIA inhibits high threshold voltage-dependent calcium currents in neurons. Calcium currents generated by Ca_v 2.1, Ca_v2.2 were blocked almost irreversibly by 3 nM wPhone IIA, whereas Ca_v2.3 showed partial and readily reversible inhibition. Binding assays with mono [¹²⁵I-wPhone IIA] indicated that membranes expressing recombinant Ca_v2.1 or Ca_v2.2 channels showed a single class of sites with similar affinity (KD~50 pM) whereas low affintiy interactions were detectable with Ca_v2.3. Binding assays demonstrated that rat brain synaptosomes display multiple classes of binding sites for ¹²⁵I-wPhone IIA. High affinity binding of ¹²⁵I-wPhone IIA was totally displaced by wPhone IIA (Ki=100 pM), but only partially by w-conotoxin GVIA (25% inhibition) and w-conotoxin MVIIC(50% inhibition at 0.3 µM).

TX4(6-1) and wPhone IIA, among other Phoneutria toxins, exemplify the complex action of this venom on different ionic channels, in both mammals and insects.

Supported by CNPq, CAPES/COFECUB and FAPEMIG.

Entry of protein toxins into the cytosol

Olsnes, S.

Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway

^{Correspondence}

Protein toxins like bacterial diphtheria toxin, cholera toxin, anthrax toxin, Shiga toxin and the plant toxin ricin exert their action in the cytosol by enzymatically modifying vital proteins. The toxin start their route into the cells by binding to cell surface receptors followed by endocytosis of the toxin-receptor complex. Diphtheria toxin and anthrax toxin are equipped with their own translocation devices facilitating transport of the enzymatically active toxin subunit to the cytosol. Cholera toxin, Shiga toxin and ricin lack translocation devices and are dependent on cellular functions for their translocation. They are transported retrograde through the Golgi apparatus to the endoplasmic reticulum where they are translocated to the cytosol by the misfolded protein degradation pathway. Once in the cytosol, the enzymatically active toxin subunit is able to avoid degradation by the proteasomes.

An overview over the field will be given together wit a critical assessment of the evidence. Finally experiments will be described that indicate that also certain growth factors enter the cytosol in related ways.

Enterolobin: toxin and what else?

Sousa, M.V.; Ricart, C.A.O.; Fontes, W.; Lima, C.M.R.; Morhy, l

Brazilian Center for Protein Research, Department of Cell Biology, University of Brasilia, Brasilia, DF, Brazil

^{Correspondence}

Enterolobin was described in the mid 80s as the first cytolytic plant protein. It was purified from Enterolobium contortisiliquum seeds, and was able to lyse red blood cells and other cell types. Its amino acid sequence and oligomerization pattern shared several similarities with aerolysins - pore-forming toxins from Aeromonas bacteria. Further biological characterization demonstrated that enterolobin possesses toxic activity against insect larvae and pro-inflammatory action in rats. The toxin was later shown to be localized in seed cell cytoplasm and nucleus, raising questions on its physiological role in the plant apart from a defensive one. Recent database searches using DoubleTwist engines found that enterolobin sequence has two high score pairs (HSPs) overlapping type III antifreeze protein signatures. Furthermore, preliminary data pointed to a probable protective function of enterolobin against hydric and saline stresses over the seed. Besides its toxicity to possible predators of E. contortisiliquum seeds, enterolobin may also carry a general anti-stress activity.

In the field of biotechnological applications, some blood cells pose resistance to cytolysis by enterolobin, so that a patent request for a new and fast cell isolation process is being prepared for deposit.

Enterolobin DNA cloning, sequencing and expressing are underway. Future site-directed mutagenesis studies associated with biological, biochemical and structural characterization of mutant enterolobins will shed brighter light on the mechanism of action, physiological functions and biotechnological applications of enterolobin.

Acknowledgements: CNPq, PADCT, FAPDF, FUB-UnB (Brazil) and IFS (Sweden) for facilities and financial support, students and collaborators.

Plant ureases: a novel group of multifunctional proteins with insecticidal properties

Carlini, C.R.; Follmer, C.; Pires-Alves, M.; Ferreira da Silva, C.T.

Depto.Biofísica-IB, Universidade Federal do Rio Grande do Sul, Porto, Alegre, RS

^{Correspondence}

Plants produce a wide array of substances including proteins, such as lectins, chitinases, enzyme inhibitors, ribosome-inactivating proteins, etc, in response to a multitude of predators and pathogens. Canatoxin (CNTX)-like proteins and urease constitute a novel group of multifunctional proteins with a putative role in plant defense against insect predation and phytopathogenic fungi. CNTX is a neurotoxic protein (184 kDa) from jackbeans (Canavalia ensiformis), lethal to mice and rats by intraperitoneal route, but inactive if given orally. CNTX is also lethal when ingested by a group of insects with cathepsin-based digestion, in which the protein is proteolitically “activated” to give a 10 kDa entomotoxic peptide. Determination of partial aminoacid sequence indicated a high homology with urease found in the same seed. RT-PCR applied to mRNA isolated from C. ensiformis tissues confirmed the presence of two genes sharing 86% similarity. Further studies have shown that canatoxin is a Zn/Ni hybrid isoform of urease with about 40% of its ureolytic activity. As described for CNTX, urease also displayed platelet aggregating activity, interaction with glycoconjugates, and entomotoxic effects. Studies with p-hydroxy-mercuribenzoate, a classical inhibitor of ureases, suggested at least two distinct domains involved in the biological properties of the proteins, as it abolished the ureolytic activity of both isoenzymes without interfering in their interaction with glycoconjugates or the effects on platelets. Our data thus indicate that canatoxin and urease belong to a novel group of multifuncional plant proteins related to defense against insect predation.

CNPq-PRONEX-FAPERGS

From a scientific discovery to a pharmaceutical product. The evasin project

Camargo, A.C.M.

Center for Applied Toxinology (CAT/CEPID-Fapesp)

^{Correspondence}

A Research Cooperation Relationship has been established between Coinfar (Brazilian Pharmaceutical Industries Consortium – Biolab/Sanus, Biosintetica and União Química), Uniemp (Instituto Universidade Empresa), Fapesp and the CAT (Center for Applied Toxinology – CEPID-Fapesp) in order to develop an anti-hypertensive product named EVASIN (Endogenous Vasopeptide Inhibitor), which derived from scientific research on the Bothrops jararaca venom.

The Parties agreed on a first project to be carried out “Project EVASINs”, which relates to advanced results and related research based on the patent number PI0101088-3, filed at the Brazilian Institute of Industrial Property – INPI.

This project gives us the chance to discuss the potential of animal and microbial toxins for the development of pharmaceutical products, the interests of the Brazilian Pharmaceutical Industries, the gaps existing for the development of a drug from natural substances, the need for specialized management and the strategic steps to be undertaken. We will be talking also about the establishment of an agency, named AGIF (Agência de Gestão da Inovação Farmacêutica), its general profile and aims.

Toxinology as a subject in undergraduate and graduate courses

Barraviera, B.

Professor Titular do Departamento de Doenças Tropicais da Faculdade de Medicina de Botucatu – UNESP

^{Correspondence}

Clinical Toxinology is the study of envenoming by bacterial, plant, and animal toxins. It is, therefore, a new concept in Public Health. For many years, it has been exclusive to specialist Institutes and not part of most medical schools curricula in this country. It has recently been introduced as a discipline in Tropical Diseases. In this way, Toxinology has gained relevance in some medical schools, in both undergraduate and graduate courses.

Here in Botucatu, Toxinology has been part of the curriculum since 1965, it is taught from the fourth year onwards. In 1993, it was also included in the Tropical Diseases graduation course. Students who graduate from Botucatu Medical School have been prepared to treat envenomed patients.

Nowadays, with the continuing expansion of scientific knowledge, some topics are being omitted, and once again, Toxinology could be one of the first subjects to go from these graduation courses.

The only alternative to make all the subjects fit into the current curriculum is to build faster systems for knowledge transference. This requires a simple unique solution - the combination of printed matter, video, CD-ROM, and Internet.

Signaling mechanisms involved in the interaction of disintegrins with leukocytes and tumor cells

Barja-Fidalgo, C.; Coelho, A.L.J.; Freitas, M.S.; Oliveira, A.M.; Coelho, R.M.; Zingali, ^IR.B.

Departamento de Farmacoloia, Instituto de Biologia, Universidade do Estado do Rio de Janeiro

^IDepto de Bioquímica, CCS, Universidade Federal do Rio de Janeiro

^{Correspondence}

Integrin-mediated adhesions between cells or cell and extracellular matrix (MEC) transduce to intracellular signals, inducing activation of tyrosine kinase-mediated pathways, mobilization of cytoskeleton, modulating an array of cellular functions and, in some cases, the gene expression. Disintegrins have been reported to inhibit integrin-related functions like adhesion of tumor cell, angiogenesis, platelet aggregation and leukocyte migration. This family of cystein rich peptides, mono or dimeric, often present an RGD motif in a cystein-holding “hair-pin” looping , are able to be selectively recognized by integrins. We have been studying the effects of monomeric RGD-disintegrins Kistrin (KR-av), Flavoridin (FL-a5>av) and Jarastatin (JT-av>a5, am) and a non-RGD heterodimeric disintegrin, EC3 (-a9, -a4), on human neutrophil (PMN) chemotaxis and apoptosis and also on melanoma cells proliferation. Moreover we have investigated the intracellular signals transduced by the interactions of the DIS with those cells. Our results show that all DIS inhibited PMN migration in vitro, although only JT and EC3 were directly chemotactic to these cells. PMN interaction with JT, KR and EC3, but not FL, induces actin polymerization, tyrosine kinase and focal adhesion kinase (FAK) activation. While JT and KR have activated MAP kinase pathway, FL and EC3 inhibited this signaling pathway involved in the modulation of apoptosis in PMN. In parallel interaction of RGD-disintegrins in melanoma murine cells (B16F10) inhibited proliferation in vitro. KR and FL, but not JT, were able to induce reorganization of actin cytoskeleton and MAPK activation pathway in melanoma cells. However, cel interactio with KR and JT, but not FL, stimulated c-Fos transcription factor which is involved in the modulation of proliferation. Taken together our data suggest that the snake venoms disintegrins are putative prototypes for the study and the therapeutic control of inflammation process and tumor cells proliferation.

(FAPERJ, CNPq, UERJ-SR2, Brazil)

Bothrojaracin as antithrombotic agent

Zingali, R.B.; Monteiro, R.Q.; Castro, H.C.

Departamento de Bioquímica Médica, ICB/CCS, UFRJ, Rio de Janeiro Brazil

^{Correspondence}

Bothrojaracin (BJC) first isolated from Bothrops jararaca venom is a 27 kDa heterodimer. It binds specifically to a-thrombin with a K_D=0.7 nM and influences but does not block the proteinase catalytic site, although it inhibits thrombin biological activities such as fibrinogen clotting, platelet aggregation, protein C activation. BJC also binds the inactive thrombin precursor, prothrombin. Gel filtration chromatography (Superose 12) showed that fluorescein-labeled BJC ([5F]BJC) forms a 1:1 Ca²⁺-independent, non-covalent complex (100,000 Da) with prothrombin. On the other hand, [5F]BJC did not interact with prothrombin. fragments 1 or 2. Isothermal titration calorimetry (ITC) showed that the binding of prothrombin to BJC is endothermic, indicating that the binding is entropically driven with an K_D = 76 nM. BJC efficiently displaced fluorescein-labeled Hir^54-65(SO^3-) from complexes formed with a-thrombin or prothrombin, suggesting that both BJC and Hir^54-65(SO^3-) compete for the same binding sites in those molecules. These data indicate that BJC binds the anion-binding exosite I precursor (proexosite I) on human prothrombin. BJC also greatly inhibited thrombin formation induced by O. scutellatus venom, suggesting a direct interference in the prothrombin activation. BJC strongly inhibited the zymogen activation by factor Xa in the presence but not absence of factor Va. The same effect was observed in the presence of phospholipids or platelets, suggesting a specific interference on the cofactor activity. It is proposed that BJC has two independent mechanisms for anticoagulation: 1) inhibition of exosite I-dependent activities on a-thrombin, and 2) inhibition of prothrombin activation through interaction with proexosite I.

Supported by FAPERJ, CNPq and Finep IFS contract F3156.

Cloning and primary structure analysis of bradykinin-potentianting peptides precursors

Hayashi, M.A.F.; Murbach, A.F.; Ianzer, D.; Silva, C.A.; Pires, R.S.; Silveira, P.F.; Sarai, A.; Britto, L.R.G.; Portaro, F.C.V.; Dive, V.; Camargo, A.C.M.

^{Correspondence}

The bradykinin-potentiating peptides (BPPs) present in Bothrops jararaca (Bj) venom were the first naturally occurring angiotensin-converting enzyme (ACE) inhibitor described. The study of the structure and activity of these BPPs and analogues were essential for the development of the ACE inhibitors employed in the medicine for the treatment of cardiovascular problems. Five years ago, we have described the cloning and sequence analysis of a cDNA from snake venom gland coding for both BPPs and C-type natriuretic peptide (CNP), both present in a single precursor protein. Northern blot analysis indicated the presence of a homologous mRNA in the Bj CNS, and the respective cDNA was cloned from the snake brain. A 1.7 Kb cDNA clone encoding a 265 amino acids precursor was isolated and seven BPPs and a CNP could be recognized in its deduced amino acid sequence. Surprisingly, although the brain precursor sequence shows a very high similarity to the venom gland one, their sequences are not identical to each other, showing some substitutions and deletions, that determine the expression of different BPPs isoforms in each snake tissue. Evolutionary analyses were performed based on the comparison of the several BPPs precursors isolated till now.

Thus, the question that we address in the present study is whether the snake venom BPPs are merely toxins that coincidentally display strong inhibitory action on the mammalian ACE or whether they represent orthologous compounds of endogenous ACE inhibitors or, moreover, of endogenous bioactive molecules which perform physiological functions through either modulation of the ACE activity or by acting toward not yet determined receptors.

Financial support by FAPESP.

Proteomic visualization of viperid venoms: complexity, diversity and similarity

Serrano, S.M.T.^I,II; Shannon, J.D.^I; Fox, J.W.^I

^IDepartment of Microbiology, University of Virginia School of Medicine, Charlottesville, VA 22908, USA

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

^{Correspondence}

The complexity of viperid venoms has long been appreciated in the field of toxinology and medicine. However, it is only recently that the depth of that complexity has become quantitatively accessible. With the resurgence of 2D PAGE and the advances in mass spectrometry essentially all venom components can be visualized and identified given the effort and resources. Here we present the use of various technologies for examining venom complexity and demonstrate their associated advantages and disadvantages. 2D PAGE comparisons between different genera of viperid venoms; different species of the same genus and specimens of the same genus and species demonstrate the similarity as well as the apparent diversity among these venoms. Finally, we have developed techniques to examine subpopulations of venom proteins (metalloproteinase proteome; serine proteinase proteome; and the glycoproteome). These tools will allow for a better understanding of venom complexity and toxic properties as well as enabling investigators with particular interests to focus on these subpopulations of proteins for further study.

Coevolution between mechanical patterns of predation and the venomous function in scorpions of the super-family Buthoidea

Lourenço, W.R.

Laboratoire de Zoologie (Arthropodes), Muséum National d’Histoire Naturelle, 61 rue de Buffon 75005 Paris, France

^{Correspondence}

All of the extant scorpion species, which are assigned to 6 super-families and 16 to 20 families, possess venom glands. The presence of a telson + aculeus, and possibly tegumental glands, are also evident in fossil scorpions from the Palaeozoic, Mesozoic and Cenozoic (Lourenço, 2001, Lourenço & Weitschat, 2001, Jeram, 2001).

At present the following points can be suggested:

1. Originally, the telson played a major mechanical role in predation, with the aculeus acting rather as a “spear-head” (Lourenço, 2001, 2002).

2. Over the course of evolutionary time, tegumental glands evolved in the telson’s vesicle, however, their primitive role was certainly only digestive.

3. Many, if not most, non-buthid scorpions (i. e. ischnurids, scorpionids), evolved more sophisticated mechanical techniques of predation (e.g. development of strong pedipalps). The use of venom (toxins) for the capture of prey is rather facultative in these groups.

4. Buthoids are the most complex group of scorpions, with 3 families and about 80 genera. These cannot be considered as a homogeneous (monophyletic) unit, but instead represent 4 or 5 different evolutionary levels (Lourenço, 2000).

5. Within buthoids, only a few species of buthids possess venoms formed of complex mixtures of highly specific toxins. These are represented by species of genera such as Androctonus, Buthus, Centruroides, Leiurus, Mesobuthus and Tityus, all of which can be placed in the highest evolutionary level within buthoids. These groups have been the subject of intensive biochemical and molecular research (Loret & Hammock, 2001).

To interpret the evolution of buthoid scorpion toxins, studies must be conducted with the venoms of primitive extant lineages. These are represented by species of the Malagasy microcharmids and some relictual buthids such as Ananteris, Anomalobuthus, Akentrobuthus, Birulatus, Himalayotityobuthus, Lychasiodes, Microtityus, Pseudouroplectes, Sabinebuthus and Tityobuthus.

The sub-family of K⁺-channel erg-like toxins isolated from scorpion venoms

Possani, L.D.^I; Corona, M.^I; Gurrola, G.B.^I; García-Gómez, B.I.^I; Pardo, L.^I; Merino, E.^I; Enzo, W.^I,II

^IInstitute of Biotechnology, Universidad Nacional Autonoma de Mexico, Av. Universidad, 2001 Apartado Postal 510-3, Cuernavaca 62210 - MEXICO

^IIDepartment of Biotechnology and Biosciences, University of Milano-Bococca, I-20126 Milano, Italy

^{Correspondence}

Ergtoxin-1 (ErgTx1) was shown to inhibit specifically K⁺-channels of the ether-a-go-go-related genes (Gurrola et al., FASEB J. 13:953-962, 1999). This peptide contains 42 amino acid residues compacted by four disulfide bridges (Scaloni et al., FEBS Lett. 479:156-157, 2000). At least 23 distinct peptides and/or genes were obtained from venomous glands of Mexican scorpions of the species: Centruroides (C.) noxius, C. limpidus limpidus, C. sculpturatus and C. gracilis. They all contain four disulfide bridges, having between 42 to 47 amino acid residues. A phylogenetic tree constructed with the primary structure of these peptides shows that they might conform four different structural groups, and according to the international classification proposed (Tytgat et al., Trends Pharm. Sci. 20:444-447 1999) these scorpion peptides constitute a new sub-family (g-KTx). The physiological effect of ErgTx1 was thoroughly studied recently using side-directed mutants of the human hypothalamic ERG-channels expressed in Xenopus laevis oocytes (Pardo et al. J.Biol. Chem. 277:16403-16411, 2002). ErgTx1 is a true channel blocker, interacting directly with amino acids situated in between the S5 and S6 segment of the channel. The effect of ErgTx2 was recently assayed on K⁺-currents of lactrotropic cells (Lecchi et al., J. Neurosci. 22:3414-3425, 2002). The slowly deactivating long-lasting component (I_ERGS) is inhibited by ErgTx2. Some of the other peptides belonging to this sub-family also affect ERG-channels, with different affinities. However, most of the sequences obtained are still at the stage of research, for which we know the coding gene, but thus far have not obtained the pure peptide, for direct assay.

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

Lonomia obliqua, a biochemical and molecular view

Chudzinski-Tavassi, A.M.

Lab. de Bioquímica e Biofísica, Instituto Butantan

^{Correspondence}

Accidental contact with Lonomia obliqua induces an acquired hemorrhagic diathesis. Prolongation of coagulation times with significant reduction of fibrinogen, decrease of factors V, VIII and protein C, and an increase of fragments 1+2, TAT were found in 105 plasma patients. A prominent increase in D-dimer levels occured without alterations of t-PA or U-PA. A moderate reduction of plasminogen and AP were also observed. These data suggested a consumption coagulopathy and a secondary fibrinolysis. Lonomia obliqua bristles extract exerted procoagulant activity in human plasma but is not able to clot purified fibrinogen and shows FX and FII activation activity in purified systems. In addition, neither inhibition of FXIII nor degradation occured. Cross-linked fibrin were not hydrolysed by the extract, however fibrinogen a chain degradation was observed after long time pré-incubation. The generated products are not similar to fragments D and E, plasmin derived. Plasminogen activation was not observed in SOFIA system. Furthermore, bristles extract not affected platelet aggregation. From the extract, we purified Lopap, (Lo) a serine-protease of 69 kDa prothrombin activator. In vivo, Lo induces thrombin formation, neutrophil margination in pulmonary rat microvasculature, fibrinogen depletion and 30% platelet reduction, human platelet aggregation Lopap was able to activate prothrombin independently of prothrombinase complex components but Ca++ , Va and PL increase its activity. On endothelial cells (EC) from HUVECs, flow cytometric analysis showed that the incubation with Lo (5 ug/ml) during 1 h produced an increased expression of ICAM-1 (7±1 vs. 19±3 arbitrary units of fluorescence (UAF), p<.05, n=7) and E-Selectin (4±1 vs. 24±5 UAF, p<.05, n=3) without modifying VCAM-1 levels (3±1 vs. 4±1 UAF, n/s, n=6). The E-selectin-increased expression was specific since EC pretreatment with serum against L. obliqua almost completely inhibited this response. It was not associated with thrombin formation, once hirudin did not modify the ICAM-1 upregulation. An increase of IL-8 (1.4±0.2 x 5.3±1.7 ng/ml) and PGI2, time dependent, is also observed (0.46± 0.05 control x 4.9 ±1.1 induced by 30 min) and (2.8 ± 0.9 control x 15.6 ± 2.7 induced by 4h). ELISA studies showed that Lo was not able to induce von Willebrand factor release nor its synthesis (n=5). Lo had no effect on washed platelets aggregation, which were induced by several agonists, suggesting that it does not contribute for platelet function in vivo. Also, AT was able to inhibit amidolytic activity of thrombin generated by incubation of Lo with prothrombin. Lo hydrolysed the fluorescent peptide, a prothrombin sequence derived, having the hydrolysis site recognized by thrombin (Arg284-Thr285) (Km 0,3269 mM and Kcat 2,91 s-1). Recombinat Lopap was obtained and DNA from aleatory clones are being sequenced. In conclusion, the direct action of Lo on EC could have an important role in the pathogenesis of inflammatory and coagulation disorders observed during the Lo infusion in rats or in human accidental contact with L. obliqua. Probably, during prothrombin activation, Lo induces prethrombin 2 formation and possible meizothrombin.

(Supported by FAPESP, CNPq)

Molecular cloning and expression of a functional dermonecrotic and complement-dependent haemolytic factor from Loxosceles laeta spider venom gland

Fernandes-Pedrosa, M.F.^I; Junqueira-de-Azevedo, I.L.M.^II; Gonçalves-de-Andrade, R.M.^I; Van Den Berg, C.W.^III; Ramos, C.R.R.^II; Ho, P.L.^II; Tambourgi, D.V.^I

^ILaboratório de Imunoquímica

^IICentro de Biotecnologia, Instituto Butantan, Brazil

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

^{Correspondence}

The bite of spiders of the genus Loxosceles can induce a variety of biological effects, including dermonecrosis and complement dependent haemolysis. The aim of this study was to generate recombinant proteins from the Loxosceles spider gland to facilitate structural and functional studies in the mechanisms of loxoscelism. Using “Expressed Sequencing Tag” (EST) strategy of aleatory clones from L. laeta venom gland cDNA library we have identified clones containing inserts coding for proteins with significant similarity with previously obtained N-terminus of sphingomyelinases P1 and P2 from L. intermedia venom. The longest cDNA insert from clone H17, which shared a high percentage of identity with these

, was further subcloned and expressed in a bacterial system. The final construct encoded for a 33-kDa-fusion protein containing a 6xHis-tag at its N-terminus. The recombinant L. laeta spider venom protein was endowed with all biological properties ascribed for the whole L. laeta venom and purified toxins P1 and P2 from L. intermedia, including dermonecrotic and complement-dependent haemolytic activities and ability of hydrolysing sphingomyelin. Considering its lipase activity, the recombinant L. laeta toxin expressed from clone H17 was named L. laeta Smase I. Antiserum raised against L. laeta Smase I recognised a 32-kDa protein in crude L. laeta venom and gland extract and was able to block the dermonecrotic reaction caused by the whole L. laeta venom. This is the first report of the complete primary structure of the dermonecrotic and complement dependent haemolytic factor from Loxosceles spider venom glands that was obtained through cDNA cloning and functionally expressed in E. coli. This study also demonstrates conclusively that the sphingomyelinase activity in the whole venom is responsible for the major pathological effects of Loxosceles spider envenomation.

Supported by The Wellcome Trust and CAPES.

Loxosceles spider venom induces metalloproteinase mediated cleavage of MCP/CD46 and MHCI and induces protection against C-mediated lysis

Van Den Berg, C.W.^I,II; Andrade, R.M.G.^II; Magnoli, F.C.^II; Marchbank, K.J.^III; Tambourgi, D.V.^II

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

^IILaboratório de Imunoquímica, Instituto Butantan, Brazil

^IIIDepartment of Medical Biochemistry, University of Wales, College of Medicine, UK

^{Correspondence}

We have recently shown that sphingomyelinase D toxins from the spider Loxosceles intermedia induce Complement (C) dependent haemolysis of autologous erythrocytes by induction of cleavage of cell surface glycophorins through activation of a membrane bound metalloproteinase. The aim of this study was to investigate the effects of these toxins on C-regulator expression and the C-resistance of nucleated cells. Cells were incubated with Loxosceles venom/toxins and the expression of C-regulators was assessed by flow cytometry. A reduced expression of membrane cofactor protein (MCP) was observed, while expression of decay accelerating factor (DAF) and CD59 was not affected. Analysis of other cell surface molecules showed a reduced expression of MHCI. Western blotting showed that a truncated form of MCP was released into the supernatant. Release could be prevented by inhibitors of metalloproteinases of the adamalysin family but not by inhibitors specific for matrix metalloproteinases. Cleavage of MCP was induced close to or in the membrane as demonstrated by the cleavage of transmembrane chimeras of CD59 and MCP. Although the venom/toxins induced a release of MCP, the C-susceptibility was decreased. The mechanism of this induction of resistance may involve a change in membrane fluidity induced by the sphingomyelinase activity of the toxin/venom and/or involvement of membrane bound proteases. The identity of the metalloproteinase(s) activated by the spider venom and the role in pathology of Loxoscelism remains to be established.

Supported by the Wellcome Trust as a Collaborative Research Initiative Grant to DVT and CWB and a Royal Society Travel Grant to CWB

Publication Dates

• Publication in this collection
  09 Dec 2003
• Date of issue
  2003