Isolation and chemical characterization of PwTx-II: A novel alkaloid toxin from the venom of the spider Parawixia bistriata (Araneidae, Araneae)

Brazil has many species of spiders belonging to Araneidae family however, very little is known about the composition, chemical structure and mechanisms of action of the main venom components of these spiders. The main objective of this work was to isolate and to perform the chemical characterization of a novel beta-carboline toxin from the venom of the spider Parawixia bistriata, a typical species of the Brazilian 'cerrado'. The toxin was purified by RP-HPLC and structurally elucidated by using a combination of different spectroscopic techniques (UV, ESI-MS/MS and H-1 NMR), which permitted the assignment of the molecular structure of a novel spider venom toxin, identified as 1-4-guanidinobutoxy-6-hydroxy-1,2,3,4-tetrahydro-beta-carboline, and referred to here as PwTx-II. This compound is toxic to insects (LD50 = 12 +/- 3 eta g/mg honeybee), neurotoxic, convulsive and lethal to rats (LD50 = 9.75 mg/kg of male Wistar rat). (c) 2005 Elsevier Ltd. All rights reserved.

Assignment of the disulfide bridges in bothropstoxin-I, a myonecrotic Lys49 PLA(2) homolog from Bothrops jararacussu snake venom

Bothropstoxin-I (BthTX-1), a Lys49 phospholipase A(2) homolog with no apparent catalytic activity, was first isolated from Bothrops jararacussu snake venom and completely sequenced in this laboratory. It is a 121-amino-acid single polypeptide chain, highly myonecrotic, despite its inability to catalyze hydrolysis of egg yolk phospholipids, and has 14 half-cystine residues identified at positions 27, 29, 44, 45, 50, 51, 61, 84, 91, 96, 98, 105, 123, and 131 (numbering according to the conventional alignment including gaps, so that the last residue is Cys 131). In order to access its seven disulfide bridges, two strategies were followed: (1) Sequencing of isolated peptides from (tryptic + SV8) and chymotryptic digests by Edman-dansyl degradation; (2) crystallization of the protein and determination of the crystal structure so that at least two additional disulfide bridges could be identified in the final electron density map. Identification of the disulfide-containing peptides from the enzymatic digests was achieved following the disappearance of the original peptides from the HPLC profile after reduction and carboxymethylation of the digest. Following this procedure, four bridges were initially identified from the tryptic and SV8 digests: Cys50-Cys131, Cys51-Cys98, Cys61-Cys91, and Cys84-Cys96. From the chymotryptic digest other peptides were isolated either containing some of the above bridges, therefore confirming the results from the tryptic digest, or presenting a new bond between Cys27 and Cys123. The two remaining bridges were identified as Cys29-Cys45 and Cys44-Cys105 by determination of the crystal structure, showing that BthTX-1 disulfide bonds follow the normal pattern of group II PLA(2)s.

Eumenitin, a novel antimicrobial peptide from the venom of the solitary eumenine wasp Eumenes rubronotatus

A novel antimicrobial peptide, eumenitin, was isolated from the venom of the solitary eumenine wasp Eumenes rubronotatus. The sequence of eumenitin, Leu-Asn-Leu-Lys-Gly-Ile-Phe-Lys-Lys-Val-Ala-Ser-Leu-Leu-Thr, was mostly analyzed by mass spectrometry together with Edman degradation, and corroborated by solid-phase synthesis. This peptide has characteristic features of cationic linear a-helical antimicrobial peptides, and therefore, can be predicted to adopt an amphipathic a-helix secondary structure. In fact, the CD spectra of eumenitin in the presence of TFE or SDS showed a high content of alpha-helical conformation. Eumenitin exhibited inhibitory activity against both Gram-positive and Gram-negative bacteria, and moderately stimulated degranulation from the rat peritoneal mast cells and the RBL-2H3 cells, but showed no hemolytic activity against human erythrocytes. This antimicrobial peptide in the eumenine wasp venom may play a role in preventing potential infection by microorganisms during prey consumption by their larvae. (c) 2006 Elsevier B.V. All rights reserved.

Decoralin, a novel linear cationic alpha-helical peptide from the venom of the solitary eumenine wasp Oreumenes decoratus

A novel peptide, decoralin, was isolated from the venom of the solitary eumenine wasp Oreumenes decoratus. its sequence, Ser-Leu-Leu-Ser-Leu-Ile-Arg-Lys-Leu-Ile-Thr, was determined by Edman degradation and corroborated by solid-phase synthesis. This sequence has the characteristic features of linear cationic a-helical peptides; rich in hydrophobic and basic amino acids with no disulfide bond, and accordingly, it can be predicted to adopt an amphipathic a-helix secondary structure. In fact, the CD spectra of decoralin in the presence of TFE or SDS showed a high a-helical conformation content. In a biological evaluation, decoralin exhibited a significant broad-spectrum antimicrobial activity, and moderate mast cell degranulation and leishmanicidal activities, but showed virtually no hemolytic activity. A synthetic analog with C-terminal amidation showed a much more potent activity in all the biological assays. (c) 2007 Elsevier B.V. All rights reserved.

Crytallization and high-resolution X-ray diffraction data collection of an Asp49 PLA(2) from Bothrops jararacussu venom both in the presence and absence of Ca2+ ions

Snake venom PLA(2)s have been extensively studied due to their role in mediating and disrupting physiological processes such as coagulation, platelet aggregation and myotoxicity. The Ca2+ ion bound to the putative calcium-binding loop is essential for hydrolytic activity. We report the crystallization in the presence and absence of Ca2+ and X-ray diffraction data collection at 1.60 Angstrom (with Ca2+) and 1.36 Angstrom (without Ca2+) of an Asp49 PLA(2) from Bothrops jararacussu venom. The crystals belong to orthorhombic space group C222(1). Initial refinement and electron density analysis indicate significant conformational. changes upon Ca2+ binding. (C) 2004 Elsevier B.V. All fights reserved.

CRYSTALLIZATION AND PRELIMINARY DIFFRACTION DATA OF BOTHROPSTOXIN-I ISOLATED FROM THE VENOM OF BOTHROPS-JARARACUSSU

The myotoxic Lys-49 phospholipase bothropstoxin I was crystallized, and X-ray diffraction data were collected to 3.5 Angstrom resolution. Preliminary analysis reveals the presence of four molecules in the asymmetric unit.

Isolation, characterization and biological activity of acidic phospholipase A(2) isoforms from Bothrops jararacussu snake venom

Acidic phospholipase A(2) (PLA(2)) isoforms in snake venoms, particularly those from Bothrops jararacussu, have not been characterized. This article reports the isolation and partial biochemical, functional and structural characterization of four acidic PLA(2)s (designated SIIISPIIA, SIIISPIIB, SIIISPIIIA and SIIISPIIIB) from this venom. The single chain purified proteins contained 122 amino acid residues and seven disulfide bonds with approximate molecular masses of 15 kDa and isoelectric points of 5.3. The respective N-terminal sequences were: SIIISPIIA-SLWQFGKMIDYVMGEEGAKS; SIIISPIIB-SLWQFGKMIFYTGKNEPVLS; SIIISPIIIA-SLWQFGKMILYVMGGEGVKQ and SIIISPIIIB-SLWQFGKMIFYEMTGEGVL. Crystals of the acidic protein SIIISPIIIB diffracted beyond 1.8 Angstrom resolution. These crystals are monoclinic with unit cell dimensions of a = 40.1 Angstrom, b = 54.2 Angstrom and c = 90.7 Angstrom. The crystal structure has been refined to a crystallographic residual of 16.1% (R-free = 22.9%). Specific catalytic activity (U/mg) of the isolated acidic PLA(2)s were SIIISPIIA = 290.3 U/mg; SIIISPIIB = 279.0 U/mg; SIIISPIIIA = 270.7 U/mg and SIIISPIIIB = 96.5 U/mg. Although their myotoxic activity was low, SIIISPIIA, SIIISPIIIB and SIIISPIIIA showed significant anticoagulant activity. However, there was no indirect hemolytic activity. SIIISPIIIB revealed no anticoagulant, but presented indirect hemolytic activity. With the exception of SIIISPIIIB, which inhibited platelet aggregation, all the others were capable of inducing time-independent edema. Chemical modification with 4-bromophenacyl bromide did not inhibit the induction of edema, but did suppress other activities. (C) 2003 Editions scientifiques et medicales Elsevier SAS. All rights reserved.

Distribution of I-125-labeled crotamine in mice tissues

Crotamine is a strong basic polypeptide from Crotalus durissus terrificus (Cdt) venom composed of 42 amino acid residues tightly bound by three disulfide bonds. It causes skeletal muscle spasms leading to spastic paralysis of hind limbs in mice. The objective of this paper was to study the distribution of crotamine injected intraperitoneally (ip) in mice. Crotamine was purified from Cdt venom by gel filtration, followed by ion exchange chromatography, using a fast-performance liquid chromatography (FPLC) system. Purified crotamine was irradiated at 2 kGy in order to detoxify. Both native and irradiated proteins were labeled with 125, using chloramine T method, and separated by get filtration. Male Swiss mice were injected ip with 0.1 mL (2 x 10(6) cpm/mouse) of I-125 native or irradiated crotamine. At various time intervals, the animals were killed by ether inhalation and blood, spleen, liver, kidneys, brain, lungs, heart, and skeletal muscle were collected in order to determine the radioactivity content. The highest levels of radioactivity were found in the kidneys and the liver, and the lowest in the brain. (c) 2006 Elsevier Ltd. All rights reserved.

Calcium-Independent Membrane Damage by Venom Phospholipases A(2)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Anoplin, a novel antimicrobial peptide from the venom of the solitary wasp Anoplius samariensis

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

The analgesic effect of crotoxin on neuropathic pain is mediated by central muscarinic receptors and 5-lipoxygenase-derived mediators

Crotoxin (CTX). a neurotoxin isolated from the venom of the South American rattlesnake Crotalus durissus terrificus. induces analgesia. In this study, we evaluated the antinociceptive effect of CTX in a model of neuropathic pain induced by rat sciatic nerve transection. Hyperalgesia was detected 2 h after nerve transection and persisted for 64 days. Immersion of proximal and distal nerve stumps in CTX solution (0.01 mM for 10 s), immediately after nerve transection, blocked hyperalgesia. The antinociceptive effect of CTX was long-lasting, since it was detected 2 h after treatment and persisted for 64 days. CTX also delayed, but did not block, neurectomy-induced neuroma formation. The effect of CTX was blocked by zileuton (100 mg/kg, p.o.) and atropine (10 mg/kg. i.p.), and reduced by yohimbine (2 mg/kg, i.p.) and methysergide (5 mg/kg, i.p.). on the other hand. indomethacin (4 mg/kg, i.v.). naloxone (1 mg/kg, i.p.). and N-methyl atropine (30 mg/kg, i.p.) did not interfere with the effect of CTX. These results indicate that CTX induces a long-lasting antinociceptive effect in neuropathic pain, which is mediated by activation of central muscarinic receptors and partially, by activation of alpha-adrenoceptors and 5-HT receptors. Eicosanoids derived from the lipoxygenase pathway modulate the action of crotoxin. (C) 2008 Elsevier B.V. All rights reserved.

Understanding the in vitro neuromuscular activity of snake venom Lys49 phospholipase A(2) homologues

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

Proteome analysis of snake venom toxins: pharmacological insights

Snake venoms are an extremely rich source of pharmacologically active proteins with a considerable clinical and medical potential. To date, this potential has not been fully explored, mainly because of our incomplete knowledge of the venom proteome and the pharmacological properties of its components, in particular those devoid of enzymatic activity. This review summarizes the latest achievements in the determination of snake venom proteome, based primarily on the development of new strategies and techniques. Detailed knowledge of the venom toxin composition and biological properties of the protein constituents should provide the scaffold for the design of new more effective drugs for the treatment of the hemostatic system and heart disorders, inflammation, cancer and consequences of snake bites, as well as new tools for clinical diagnostic and assays of hemostatic parameters.