Insights into the role of oligomeric state on the biological activities of crotoxin: crystal structure of a tetrameric phospholipase A(2) formed by two isoforms of crotoxin B from Cratalus durissus terrificus venom

Crotoxin B (CB or Cdt PLA(2)) is a basic Asp49-PLA(2) found in the venom of Crotalus durissus terrificus and it is one of the subunits that constitute the crotoxin (Cro). This heterodimeric toxin, main component of the C. d. terrificus venom, is completed by an acidic, nontoxic, and nonenzymatic component (crotoxin A, CA or crotapotin), and it is related to important envenomation effects such as neurological disorders, myotoxicity, and renal failure. Although Cro has been crystallized since 1938, no crystal structure of this toxin or its subunits is currently available. In this work, the authors present the crystal structure of novel tetrameric complex formed by two dimers of crotoxin B isoforms (CB1 and CB2). The results suggest that these assemblies are stable in solution and show that Ser1 and Glu92 of CB1 and CB2, respectively, play an important role in the oligomerization. The tetrameric and dimeric conformations resulting from the association of the isoforms may increase the neurotoxicity of the toxin CB by the creation of new binding sites, which could improve the affinity of the molecular complexes to the presynaptic membrane.

Crystallization and preliminary X-ray diffraction analysis of crotoxin B from Crotalus durissus collilineatus venom

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

Crystallization and preliminary X-ray crystallographic studies of a Lys49-phospholipase A(2) homologue from Bothrops pirajai venom complexed with rosmarinic acid

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

Crystallization and preliminary X-ray diffraction studies of BmooPLA(2)-I, a platelet-aggregation inhibitor and hypotensive phospholipase A(2) from Bothrops moojeni venom

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

Crystallization and preliminary X-ray diffraction analysis of three myotoxic phospholipases A2 from Bothrops brazili venom

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

Orpotrin: A novel vasoconstrictor peptide from the venom of the Brazilian Stingray Potamotrygon gr. orbignyi

Characterization of the peptide content of venoms has a number of potential benefits for basic research, clinical diagnosis, development of new therapeutic agents, and production of antiserum. In order to analyze in detail the peptides and small proteins of crude samples, techniques such as chromatography and mass spectrometry have been employed. The present study describes the isolation, biochemical characterization, and sequence determination of a novel peptide, named Orpotrin from the venom of Potamotrygon gr. orbignyi. The natural peptide was shown to be effective in microcirculatory environment causing a strong vasoconstriction. The peptide was fully sequenced by de novo amino acid sequencing with mass spectrometry and identified as the novel peptide. Its amino acid sequence, HGGYKPTDK, aligns only with creatine kinase residues 97-105, but has no similarity to any bioactive peptide. Therefore, possible production of this peptide from creatine kinase by limited proteolysis is discussed. Taken together, the results indicate the usefulness of this single-step approach for low molecular mass compounds in complex samples such as venoms. (c) 2006 Elsevier B.V. All rights reserved.

Anti-hemorrhagic Activity of Four Brazilian Vegetable Species Against Bothrops jararaca Venom

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

Kinetics of venom and antivenom serum levels, clinical evaluation and therapeutic effectiveness in dogs inoculated with Crotalus durissus terrificus venom

This work evaluated the clinical and therapeutic aspects as well as serum levels of venom and antivenom IgG by enzyme-linked immunosorbent assay (ELISA) in experimental envenomation of dogs with Crotalus durissus terrificus venom. Twenty-eight mixed breed adult dogs were divided into four groups of seven animals each, Group I: only venom; Group II, venom + 50 ml of anti-bothropic-crotalic serum (50mg) + fluid therapy; Group III, venom + 50 ml of anti-bothropic-crotalic serum + fluid therapy + urine alkalination; Group IV, 50 ml of anti-bothropic-crotalic serum. The lyophilized venom of Crotalus durissus terrificus was reconstituted in saline solution and subcutaneously inoculated at the dose of 1mg/kg body weight. The dogs presented clinical signs of local pain, weakness, mandibular ptosis, mydriasis, emesis and salivation. The venom levels detected by ELISA ranged from 0 to 90ng/ml, according to the severity of the clinical signs. Serum antivenom ranged from 0 to 3ug/ml and was detected for up to 138h after treatment. ELISA results showed the effectiveness of the serum therapy for the venom neutralization.

Co-60 gamma irradiation prevents Bothrops jararacussu venom neurotoxicity and myotoxicity in isolated mouse neuromuscular junction

The ability of gamma radiation from Co-60 (2000 Gy) to attenuate the toxic effects of Bothrops jararacussu venom was investigated on mouse neuromuscular preparations in vitro. A comparative study between the effects of native and irradiated venoms was performed on both phrenic-diaphragm (PD) and extensor digitorum longus (EDL) preparations by means of myographic, biochemical and morphological techniques. Native venom (10 and 20 mug/ml) induced a concentration-dependent paralysis of both directly and indirectly evoked contractions on PD preparations. At 20 mug/ml, it also caused a pronounced myotoxic effect on the EDL muscle preparation that was characterized by an increase of creatine kinase release and by several morphological changes of this preparation. By contrast, irradiated venom, even at concentrations as high as 40 mug/ml, induced neither paralyzing nor myotoxic effects. It was concluded that Co-60 gamma radiation is able to abolish both the paralyzing and the myotoxic effects of B. jararacussu venom on the mouse neuromuscular junction. These findings support the hypothesis that gamma radiation could be an important toot to improve antisera production by reducing toxicity while preserving immunogenicity. (C) 2002 Elsevier B.V. Ltd. All rights reserved.

Comparative biochemical studies of myotoxic phospholipase A(2) from Bothrops venom

Venoms from Bothrops jararacussu, Bothrops asper, Bothrops atrox, Bothrops pirajai, Bothrops moojeni, Bothrops alternatus and Bothrops (Bothriopsis) bilineata were fractionated using a simplified procedure based on ion-exchange chromatography on CM-Sepharose at pH 8.0 or reverse phase HPLC. The resulting elution profiles showed important differences in the myotoxin content of these venoms. The venoms from B. alternatus, B. atrox and Bothriopsis bilineata did not contain the major myotoxin found in the other venoms. The amino acid sequence of the first 50 residues of the N-terminal region of the PLA(2)-like myotoxins showed a homology of 90-96% with other bothropic myotoxins. All of the myotoxins isolated induced rat paw edema, increased the level of plasma creatine kinase and produced myonecrosis together with polymorphonuclear cell infiltration.

Changes in the morphology and ultrastructure of the Dufour's gland during the life cycle of the bumble bee queen, Bombus terrestris L-(Hymenoptera : Bombini)

The Dufour's gland is found closely associated with the sting apparatus of all female hymenopterans, playing multiple roles among bees. In some species of Bombus the gland may be involved in production of nestmate recognition pheromones, but in B. terrestris its function is not certain yet. The morphology of the :Dufour's gland of B. terrestris queens and the ultrastructural features of its cells were studied in different ages and behavioural stages using routine transmission electron microscopy. Measurements of the length and the diameter of the gland in the same conditions were also made. The Dufour's gland of the queen increases significantly in size (both in length and in diameter) with age and reproductive activity the ultrastructural features of the gland show electrondense material that comes from the haemolymph. This material is also present in the intercellular spaces, and is conducted to the subcuticular space, to be released directly into the glandular lumen. Hence at least part of the secretion is probably taken up directly from the haemolymph. The ultrastructural features indicate a more active phase of the gland corresponding to the period of egg-laying of the queen, and a decrease in activity when the queen is in hibernation as well as after the competition point. In conclusion, the gland is probably involved in reproduction, more specifically, in the marking off eggs.

Two new bradykinin-related peptides from the venom of the social wasp Protopolybia exigua (Saussure)

Two bradykinin-related peptides (Protopolybiakinin-I and Protopolybiakinin-II) were isolated from the venom of the social wasp Protopolybia exigua by RP-HPLC, and sequenced by Edman degradation method. Peptide sequences of Protopolybiakinin-I and Protopolybiakinin-II were DKNKKPIRVGGRRPPGFTR-OH and DKNKKPIWMAGFPGFTPIR-OH, respectively. Synthetic peptides with identical sequences to the bradykinin-related peptides and their biological functions were characterized. Protopolybiakinin-I caused less potent constriction of the isolated rat ileum muscles than bradykinin (BK). In addition, it caused degranulation of mast cells which was seven times more potent than BK. This peptide causes algesic effects due to the direct activation of B-2-receptors. Protopolybiakinin-II is not an agonist of rat ileum muscle and had no algesic effects. However, Protopolybiakinin-II was found to be 10 times more potent as a mast cell degranulator than BK. The amino acid sequence of Protopolybiakinin-I is the longest among the known wasp kinins. (c) 2006 Elsevier B.V. All rights reserved.

Insect Venom Peptides

The insects of the order Hymenoptera ( bees, wasps, and ants) are classified in two groups, based on their life history: social and solitary. The venoms of the social Hymenoptera evolved to be used as defensive tools to protect the colonies of these insects from the attacks of predators. Generally they do not cause lethal effects but cause mainly inflammatory and/or immunological reactions in the victims of their stings. However, sometimes it is also possible to observe the occurrence of systemic effects like respiratory and/or kidney failure. Meanwhile, the venoms of solitary Hymenoptera evolved mainly to cause paralysis of the preys in order to permit egg laying on/within the prey's body; thus, some components of these venoms cause permanent/transient paralysis in the preys, while other components seem to act preventing infections of the food and future progenies. The peptide components of venoms from Hymenoptera are spread over the molar mass range of 1400 to 7000 da and together comprise up to 70% of the weight of freeze-dried venoms. Most of these toxins are linear polycationic amphipatic peptides with a high content of alpha-helices in their secondary structures. These peptides generally account for cell lysis, hemolysis, antibiosis, and sometimes promote the delivery of cellular activators/mediators through interaction with the G-protein receptor, and perhaps some of them are even immunogenic components. In addition to these peptides, the Hymenopteran venoms also may contain a few neurotoxins that target Na+ and/or Ca+2 channels or even the nicotinic ACh receptor. This review summarizes current knowledge of the biologically active Hymenoptera venoms.

Behavioral responses evoked in honey bee workers by Dufour gland extracts (Hymenoptera : Apidae)

A series of experiments was performed to assess possible roles of the honey bee Dufour gland secretion. Bioassays with extracts of queen and worker glands from two colonies were made under artificial conditions, in which nestmate and non-nestmate forager workers were tested. The results demonstrate that forager workers display behavioral responses when exposed to Dufour gland extracts of nestmates, but remain indifferent when exposed to non-nestmate extracts. Also, the results demonstrate that forager workers are attracted by virgin queen gland extracts, and repelled by forager worker extracts. The data demonstrate that the Dufour gland secretion is colony- and caste-specific. The attractant remains in the nest. The repellent effect of forager worker extracts is interpreted as an alarm-like pheromone. The attractant effect of virgin queen extracts could be useful in the swarming process to attract scout bees to the eventual founder virgin queen or to attract old foragers to the virgin queen that remains in the nest.