Functional characterization on invertebrate and vertebrate tissues of tachykinin peptides from octopus venoms

It has been previously shown that octopus venoms contain novel tachykinin peptides that despite being isolated from an invertebrate, contain the motifs characteristic of vertebrate tachykinin peptides rather than being more like conventional invertebrate tachykinin peptides. Therefore, in this study we examined the effect of three variants of octopus venom tachykinin peptides on invertebrate and vertebrate tissues. While there were differential potencies between the three peptides, their relative effects were uniquely consistent between invertebrate and vertebrae tissue assays. The most potent form (OCT-TK-III) was not only the most anionically charged but also was the most structurally stable. These results not only reveal that the interaction of tachykinin peptides is more complex than previous structure–function theories envisioned, but also reinforce the fundamental premise that animal venoms are rich resources of novel bioactive molecules, which are useful investigational ligands and some of which may be useful as lead compounds for drug design and development.

Potamotrygon tigrina, a new species of freshwater stingray from the upper Amazon basin, closely related to Potamotrygon schroederi Fernandez-Yepez, 1958 (Chondrichthyes: Potamotrygonidae)

A new species of Neotropical freshwater stingray, family Potamotrygonidae, is described from the Rio Nanay in the upper Rio Amazonas basin of Peru. Potamotrygon tigrina, n. sp., is easily distinguished from all congeners by its conspicuous dorsal disc coloration, composed of bright yellow to orange vermiculations strongly interwoven with a dark-brown to deep-black background. Additional features that in combination diagnose P. tigrina, n. sp., include the presence of a single angular cartilage, low and not closely grouped dorsal tail spines, and coloration of tail composed of relatively wide and alternating bands of creamy white and dark brown to black. Potamotrygon tigrina is closely related to Potamotrygon schroederi Fernandez-Yepez, 1958, which occurs in the Rio Negro (Brazil) and Rio Orinoco (Venezuela, Colombia). Both species are very similar in proportions and counts, and share features hypothesized to be derived within Potamotrygonidae, related to their specific angular cartilage morphology, distal tail color, dorsal tail-spine pattern, and ventral lateral-line system. To further substantiate the description of P. tigrina, n. sp., we provide a redescription of P. schroederi based on material from the Rio Negro (Brazil) and Rio Orinoco (Venezuela). Specimens from the two basins differ in number of vertebral centra and slightly in size and frequency of rosettes on dorsal disc, distinctions that presently do not warrant their specific separation. Potamotrygon tigrina is frequently commercialized in the international aquarium trade but virtually nothing is known of its biology or conservation status.

BIOCHEMICAL VARIABILITY BETWEEN VENOMS FROM DIFFERENT HONEYBEE (APIS-MELLIFERA) RACES

1. The comparison of molecular exclusion cromatography profiles of venoms from sting apparatuses of Apis mellifera ligustica, Apis mellifera adansonii and Africanized honey-bees in Sephadex G-100 revealed both qualitative and quantitative differences.2. The venoms from A.m. ligustica and A.m. adansonii presented, respectively, three and two peaks characteristic of each sub-species, while Africanized honey-bee was characterized by the absence of eight peaks common to the former.3. The polypeptides with M(r) in the range from 100,000 to 7500 da correspond respectively to 62.0%, 66.6% and 68.7% of total proteins from the venon of A.m. ligustica, A.m. adansonii and Africanized honey-bees, while the peptidic fraction with M(r) range from 4100 to 2000 da corresponds to 11.4%, 32.4% and 10.2% of venom protein, respectively.

Testicular structure and spermatogenesis of Amazonian freshwater cururu stingray Potamotrygon cf. histrix

The cururu stingray Potamotrygon cf. histrix, a new and endemic Amazonian freshwater species, presents appropriate characteristics for fish keeping and is exploited from its natural environment. The present study identified the testicular structure and spermatogenesis of this species. Gonads from adult male specimens were dissected, fixed and processed for histological analysis. The testes were of testicular/epigonial type. The presence of germinal papillae was observed in the upper portion of organ with primordial germ cells and Sertoli cell precursors. The testis was lobular with zonal organization and cystic gametogenesis, with the occurrence of spermatoblasts. The Sertoli cells underwent morphological modifications over the course of gamete formation. The spermatozoids had long heads and were spiraled on their own axis. Information on the reproductive biology will serve as basis for studies on the reproduction and phylogeny of this peculiar group of cartilaginous fish.

Intraspecific Variation of Biological Activities in Venoms from Wild and Captive Bothrops jararaca

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

New Nanostructured Silica Adjuvant (SBA-15) Employed to Produce Antivenom in Young Sheep Using Crotalus durissus terrificus and Apis mellifera Venoms Detoxified by Cobalt-60

Equine antivenom is considered the only treatment for animal-generated envenomations, but it is costly. The study aimed to produce Apis mellifera (Africanized honeybee) and Crotalus durissus terrificus (C.d.t.) antivenoms using nanostructured silica (SBA-15) as adjuvant and cobalt-60 (60Co)-detoxified venoms utilizing young sheep. Natural and 60Co-irradiated venoms were employed in four different hyperimmunization protocols. Thus, 8 groups of 60- to 90-d-old sheep were hyperimmunized, enzyme-linked immunosorbent assay (ELISA) serum titers collected every 14 d were assessed clinically daily, and individual weight were measured, until d 84. Incomplete Freund's (IFA) and nanostructured silica (SBA15) adjuvants were compared. The lethal dose (LD50) for both venoms was determined following intraperitoneal (ip) administration to mice. High-performance liquid chromatography on reversed phase (HPLC-RP) was used also to measure the 60Co irradiation effects on Apis venom. At the end of the study, sheep were killed in a slaughterhouse. Kidneys were histologically analyzed. LD50 was 5.97 mg/kg Apis and 0.07 mg/kg C.d.t. for native compared to 13.44 mg/kg Apis and 0.35 mg/kg C.d.t. for irradiated venoms. HPLC revealed significant differences in chromatographic profiles between native and irradiated Apis venoms. Native venom plus IFA compared with SBA-15 showed significantly higher antibody titers for both venoms. Apis-irradiated venom plus IFA or SBA-15 displayed similar antibody titers but were significantly lower when compared with native venom plus IFA. Weight gain did not differ significantly among all groups. 60Co irradiation decreased toxicity and maintained venom immunogenic capacity, while IFA produced higher antibody titers. SBA-15 was able to act as an adjuvant without producing adverse effects. Hyperimmunization did not affect sheep weight gain, which would considerably reduce the cost of antiserum production, as these sheep were still approved for human consumption even after being subjected to hyperimmunization.

Phospholipase A(2) myotoxins from Bothrops snake venoms: Structure-function relationship

Phospholipases A(2) constitute the major components from Bothrops snake venoms and have been extensively investigated not only because they are relatively very abundant in these venoms but mainly because they display a range of many relevant biological effects, including: myotoxic, cytotoxic, edema-inducing, artificial membrane disrupting, anticoagulant, neuromuscular, platelet aggregation inhibiting, hypotensive, bactericidal, anti-HIV, anti-tumoural, anti-malarial and anti-parasitic. The primary structures of several PLA(2)s have been elucidated through direct amino acid sequencing or, inderectly, through the corresponding nucleotide sequencing. Two main subgroups were thus described: (i) Asp49 PLA(2)s, showing low (basic, highly myotoxic) to relatively high (acidic, less or non myotoxic) Ca++-dependent hydrolytic activity upon artificial substrates; (ii) Lys49 PLA(2)s (basic, highly myotoxic) , showing no detectable hydrolytic activity on artificial substrates. Several crystal structures of Lys49 PLAs from genus Bothrops have already been solved, revealing very similar fold patterns. Lack of catalytic activity of myotoxic Lys49-PLA(2)s, first related solely with the fact that Lys49 occupies the position of the calcium ion in the catalyticly active site of Asp49 PLA(2)s, is now also attributed to Lys122 which interacts with the carbonyl of Cys29 hyperpolarising the peptide bond between Cys29 and Gly30 and trapping the fatty acid product in the active site, thus interrupting the catalytic cycle. This hypothesis, supported for three recent structures, is also discussed here. All Asp49 myotoxins showed to be pharmacologically more potent when compared with the Lys49 variants, but phospholipid hydrolysis is not an indispensable condition for the myotoxic, cytotoxic, bactericidal, anti-HIV, anti-parasitic, liposome disrupting or edema-inducing activities. Recent studies on site directed mutagenesis of the recombinant Lys49 myotoxin from Bothrops jararacussu revealed the participation of important amino acid residues in the membrane damaging and myotoxic activities.

Geographic variations in the composition of myotoxins from Bothrops neuwiedi snake venoms: biochemical characterization and biological activity

(1) Venom pools from Bothrops neuwiedi (Bn) and from two subspecies, namely Bothrops neuwiedi pauloensis (Bnp) and Bothrops neuwiedi urutu (Bnu), collected in the States of São Paulo (SP) and Minas Gerais (MG), Brazil, were electrophoretically examined. Basic toxins with different isoelectric points were identified in the venom collected in São Paulo (BnSP). These toxins were absent in the corresponding pools from Minas Gerais (BnMG, BnpMG and BnuMG). (2) BnSP, but not BnMG, BnpMG or BnuMG, showed two myotoxins (pI congruent to 8.6 and 8.8, respectively) which were isolated by ion-exchange chromatography on CM-Sepharose. (3) From BnMG, three myotoxic isoforms (pI congruent to 8.2 and M-r = 13600) were isolated by chromatography on CM-Sepharose followed by reversed-phase high-performance liquid chromatography. (4) the chemical and biological characterization of these toxins showed a high similarity with the Lys-49 myotoxins from other bothropic venoms. (5) Doses up to 5 LD50 (i.p.) of p-bromophenacyl bromide alkylated BnSP-7 caused a total loss of lethality in 18-22-g mice, thus indicating that the LD50 was increased by greater than 5-fold. At this dose myotoxicity was also not detectable, but the edematogenic activity on the rat paw apparently did not change. (C) 1998 Elsevier B.V. All rights reserved.

Influence of Quaternary Conformation on the Biological Activities of the Asp49-phospholipases A(2)s from Snake Venoms

One of the main components of snake venoms are the Asp49-phospholipases A(2), also known as svPLA(2)s. The study of these toxins is a matter of great scientific interest due to their wide variety of biological effects. In this work we present strong evidences found in literature and other aspects which strengthen the importance of quaternary assembly for understanding the activities and molecular evolution of svPLA(2)s.

Crystallization and preliminary X-ray diffraction analysis of a Lys49-phospholipase A(2) complexed with caffeic acid, a molecule with inhibitory properties against snake venoms

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

In vitro effects of Crotalus durissus terrificus and Bothrops jararaca venoms on Giardia duodenalis trophozoites

Considering the snake venoms' pharmacological properties and chemotherapeutic potential as well as the need for new alternatives for Giardia infection treatment, the present study was carried out aiming to evaluate the in vitro effects of crude Crotalus durissus terrificus and Bothrops jararaca venoms on the growth and adherence of Giardia duodenalis trophozoites. Trophozoites (10(6)) were exposed to serial twofold dilutions of C. durissus terrificus and B. jararaca venoms that ranged from 3.125 to 200 mu g/ml and from 5 to 320 mu g/ml, respectively. The two venoms inhibited the growth of trophozoites, and the level of inhibition varied according to the assayed concentrations and incubation times. The highest reduction of parasite growth was observed with increasing concentrations of the venoms. No effect on parasite adherence was observed. Light microscope observations revealed changes of the pear-shape aspect of the cell and reduction of flagellar beating frequency in the great part of the trophozoites. This is the first attempt to evaluate the in vitro effects of snake venoms on Giardia trophozoites. The findings stress the need for more investigations and prompt us to perform further studies to identify active principles with antigiardial effect.

Comparative study on extracts from the tissue covering the stingers of freshwater (Potamotrygon falkneri) and marine (Dasyatis guttata) stingrays

Stingrays are elasmobranchs found along the seacoast and in some rivers of Brazil. Pain is the most conspicuous symptom observed in patients wounded by the bilaterally retroserrate stingers located in the tail, which are covered by glandular and integument tissues. In addition, cutaneous necrosis is commonly observed in injuries caused by freshwater stingrays. The aim of this work was to characterize and compare certain properties of tissue extracts obtained from the glandular tissues covering the stinger apparatus of Potamotrygon falkneri and Dasyatis guttata stingrays. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), tissue extracts have similar bands above 80kDa, but most differences were observed below this molecular mass. Lethal, dermonecrotic and myotoxic activities were detected only in P. falkneri tissue extract. Edematogenic activity was similar and dose dependent in both tissue extracts. Nociceptive activity was verified in both tissue extracts, but P. falkneri presented a two-fold higher activity than D. guttata tissue extract. No direct hemolysis, phospholipase A(2) and coagulant activities were observed in both tissue extracts. Antigenic cross-reactivity was noticed by ELISA and Western blotting, using antisera raised in rabbits. Species-specific sera reacted with several components of both tissue extracts, noticeably above 22 kDa. Both tissue extracts presented gelatinolytic, caseinolytic and fibrinogenolytic activities, which were not caused by the action of metalloproteinases. Hyaluronidase activity was detected only in P. falkneri tissue extract. Our experimental observations suggest that P. falkneri tissue extract is more toxic than D. guttata tissue extract. These results may explain why injuries caused by freshwater stingrays are more severe in human accidents. (c) 2007 Elsevier Ltd. All rights reserved.

Preliminary data on the feeding habits of the freshwater stingrays Potamotrygon falkneri and Potamotrygon motoro (Potamotrygonidae) from the Upper Paraná River basin, Brazil

As raias da família Potamotrygonidae representam um grupo singular da ictiofauna Neotropical. Apesar de serem antigos os relatos sobre o grupo, ainda são muitas as questões que permanecem sem resposta, sobretudo no que diz respeito à biologia das espécies que ocorrem na Bacia do Paraná-Paraguai. No presente trabalho foi analisada a dieta de Potamotrygon falkneri e Potamotrygon motoro, capturadas no Alto Rio Paraná, a jusante da Usina Hidrelétrica Engenheiro Souza Dias (UHE Jupiá). As duas espécies de raias apresentaram dieta diversificada, ingerindo 14 itens, entre moluscos, crustáceos, insetos e peixes, porém com predominância de insetos aquáticos em diversidade e abundância. Somente um indivíduo de cada espécie ingeriu peixe. Potamotrygon motoro consumiu principalmente Ephemeroptera, enquanto P. falkneri, principalmente Mollusca, Hemiptera e Trichoptera. Os dados aparentemente indicam uma dieta mais especializada de P. motoro, com maior consumo de Ephemeroptera (Baetidae), e uma dieta mais generalizada de P. falkneri. A análise dos indivíduos capturados em três micro-hábitats, que diferem quanto ao tipo de substrato e presença de vegetação marginal, sugere diferenças nos tipos de alimentos consumidos.