First identification of interspecies hybridization in the freshwater stingrays Potamotrygon motoro and P-falkneri (Myliobatiformes, Potamotrygonidae)

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

An intriguing model for 5S rDNA sequences dispersion in the genome of freshwater stingray Potamotrygon motoro (Chondrichthyes: Potamotrygonidae)

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

Neuromuscular effects of venoms and crotoxin-like proteins from Crotalus durissus ruruima and Crotalus durissus cumanensis

A myographic study was performed to compare the neuromuscular effects of venoms and crotoxin-like proteins from Crotalus durissus ruruima and Crotalus durissus cumanensis in mice phrenic-diaphragm preparation. It was concluded that both venoms present neurotoxic activity as a consequence of their crotoxin content. Furthermore, crotoxin from C.d. cumanensis is more potent than that from C.d. ruruima venom. At the concentration range in which both venoms express neurotoxic activity, only C.d. cumanensis venom also manifest a direct myotoxic effect that probably involves the synergic participation of other components than crotoxin. (C) 2015 Elsevier Ltd. All rights reserved.

Production, characterization and structural analysis of proteins from Corynebacterium pseudotuberculosis and snake venoms

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Peptidomics of Three Bothrops Snake Venoms: Insights Into the Molecular Diversification of Proteomes and Peptidomes

Snake venom proteomes/peptidomes are highly complex and maintenance of their integrity within the gland lumen is crucial for the expression of toxin activities. There has been considerable progress in the field of venom proteomics, however, peptidomics does not progress as fast, because of the lack of comprehensive venom sequence databases for analysis of MS data. Therefore, in many cases venom peptides have to be sequenced manually by MS/MS analysis or Edman degradation. This is critical for rare snake species, as is the case of Bothrops cotiara (BC) and B. fonsecai (BF), which are regarded as near threatened with extinction. In this study we conducted a comprehensive analysis of the venom peptidomes of BC, BF, and B. jararaca (BJ) using a combination of solid-phase extraction and reversed-phase HPLC to fractionate the peptides, followed by nano-liquid chromatography-tandem MS (LC-MS/MS) or direct infusion electrospray ionization-(ESI)-MS/MS or MALDI-MS/MS analyses. We detected marked differences in the venom peptidomes and identified peptides ranging from 7 to 39 residues in length by de novo sequencing. Forty-four unique sequences were manually identified, out of which 30 are new peptides, including 17 bradykinin-potentiating peptides, three poly-histidine-poly-glycine peptides and interestingly, 10 L-amino acid oxidase fragments. Some of the new bradykinin-potentiating peptides display significant bradykinin potentiating activity. Automated database search revealed fragments from several toxins in the peptidomes, mainly from L-amino acid oxidase, and allowed the determination of the peptide bond specificity of proteinases and amino acid occurrences for the P4-P4' sites. We also demonstrate that the venom lyophilization/resolubilization process greatly increases the complexity of the peptidome because of the imbalance caused to the venom proteome and the consequent activity of proteinases on venom components. The use of proteinase inhibitors clearly showed different outcomes in the peptidome characterization and suggested that degradomic-peptidomic analysis of snake venoms is highly sensitive to the conditions of sampling procedures. Molecular & Cellular Proteomics 11: 10.1074/mcp.M112.019331, 1245-1262, 2012.

A Transcriptomic View of the Proteome Variability of Newborn and Adult Bothrops jararaca Snake Venoms

Background: Snake bite is a neglected public health problem in communities in rural areas of several countries. Bothrops jararaca causes many snake bites in Brazil and previous studies have demonstrated that the pharmacological activities displayed by its venom undergo a significant ontogenetic shift. Similarly, the venom proteome of B. jararaca exhibits a considerable variation upon neonate to adult transition, which is associated with changes in diet from ectothermic prey in early life to endothermic prey in adulthood. Moreover, it has been shown that the Brazilian commercial antibothropic antivenom, which is produced by immunization with adult venom, is less effective in neutralizing newborn venom effects. On the other hand, venom gland transcripts of newborn snakes are poorly known since all transcriptomic studies have been carried out using mRNA from adult specimens. Methods/Principal Findings: Here we analyzed venom gland cDNA libraries of newborn and adult B. jararaca in order to evaluate whether the variability demonstrated for its venom proteome and pharmacological activities was correlated with differences in the structure of toxin transcripts. The analysis revealed that the variability in B. jararaca venom gland transcriptomes is quantitative, as illustrated by the very high content of metalloproteinases in the newborn venom glands. Moreover, the variability is also characterized by the structural diversity of SVMP precursors found in newborn and adult transcriptomes. In the adult transcriptome, however, the content of metalloproteinase precursors considerably diminishes and the number of transcripts of serine proteinases, C-type lectins and bradykinin-potentiating peptides increase. Moreover, the comparison of the content of ESTs encoding toxins in adult male and female venom glands showed some genderrelated differences. Conclusions/Significance: We demonstrate a substantial shift in toxin transcripts upon snake development and a marked decrease in the metalloproteinase P-III/P-I class ratio which are correlated with changes in the venom proteome complexity and pharmacological activities.

N-glycome profiling of Bothrops jararaca newborn and adult venoms

Glycosylation is an important post-translational modification of snake venom proteins and contributes to venom proteome complexity. Many snake venom components are known to be glycosylated, however, very little is known about the carbohydrate structures present in venom glycoproteins. Previous studies showed that the ontogenetic shift in diet, from ectothermic prey in early life to endothermic prey in adulthood, and shift in animal size are associated with changes in the venom proteome of the snake Bothrops jararaca. In this study we explored the composition of the N-glycome released from newborn and adult B. jararaca venom proteins. We used an ion trap mass spectrometer (IT-MS) to disassemble glycan structures based on the use of several pathways of MS (MSn) and demonstrate the presence of some structural isomers in both newborn and adult venom B. jararaca N-glycans. The main N-glycans identified in both venoms are of the hybrid/complex type however some mannose-rich type structures were also detected. The N-glycan composition of newborn and adult venoms did not vary indicating that differences in the utilization of the N-glycosylation motif could be the explanation for the differences in the glycosylation levels indicated by the differential electrophoretic profiles previously reported for B. jararaca newborn and adult venoms. (C) 2011 Elsevier B.V. All rights reserved.

Snake venoms and hemostasis

Snake venoms are complex mixtures of biologically active proteins and peptides. Many of them affect hemostasis by activating or inhibiting coagulant factors or platelets, or by disrupting endothelium. Based on sequence, these snake venom components have been classified into various families, such as serine proteases, metalloproteinases, C-type lectins, disintegrins and phospholipases. The various members of a particular family act selectively on different blood coagulation factors, blood cells or tissues. For almost every factor involved in coagulation or fibrinolysis there is a venom protein that can activate or inactivate it. Venom proteins affect platelet function by binding or degrading vWF or platelet receptors, activating protease-activated receptors or modulating ADP release and thromboxane A2 formation. Some venom enzymes cleave key basement membrane components and directly affect capillary blood vessels to cause hemorrhaging. L-Amino acid oxidases activate platelets via H2O2 production.

Spider Venoms Potentially Lethal to Humans

Spiders have one pair of venom glands, and only a few families have reduced them completely (Uloboridae, Holarchaeidae) or modified them to another function (Symphytognathidae or Scytodidae, see Suter and Stratton 2013). All other 42,000 known spider species (99%) utilize their venom to inject it into prey items, which subsequently become paralysed or are killed. Spider venom is a complex mixture of hundreds of components, many of them interacting with cell membranes or receptors located mainly in the nervous or muscular system (Herzig and King 2013). Spider venom, as it is today, has a 300-million-yearlong history of evolution and adaptation and can be considered as an optimized tool to subdue prey. In Mesothelae, the oldest spider group with less than 100 species, the venom glands lie in the anterior part of the cheliceral basal segment. They are very small and do not support the predation process very effectively. In Mygalomorphae, the venom glands are well developed and fill the basal cheliceral segment more or less completely. Many of these 3,000 species are medium- to large-/very large-sized spiders, and they have created the image of being dangerous beasts, attacking and killing a variety of animals, including humans. Although this picture is completely wrong, it is persistent and contributes considerably to human arachnophobia. The third group of spiders, Araneomorphae or “modern spiders”, comprises 93% of all spider species. The venom glands are enlarged and extend to the prosoma; the openings of the venom ducts are moved from the convex to the concave side of the cheliceral fangs and enlarged as well. These changes save the chelicerae from the necessity of being large, and hence, on the average, araneomorph spiders are much smaller than mygalomorphs. Nevertheless, they possess relatively large venom glands, situated mainly in the prosoma, and may also have rather potent venom.

Main Components of Spider Venoms

Venom glands are alreadypresent in theoldes t spider group, the Mesothelae. Theglands lie in the anterior portion of the cheliceral basal segment but are very small, and it is doubtful how much the venom contributes to the predatory success. In mygalomorph spiders, the well-developed venom glands are still in the basal segment of the chelicerae and produce powerful venom that is injected via the cheliceral fangs into a victim. In all other spiders (Araneomorphae), the venom glands have become much larger and reach into the prosoma where they can take up a considerable proportion of this body part. Only a few spiders have reduced their venom glands, either partially or completely (Uloboridae, Holarchaeidae and Symphytognathidae are usually mentioned) or modified them significantly (Scytodidae, see Suter and Stratton 2013). As well as using venom, spiders may also use their chelicerae to overwhelm an item of prey. It is primarily a question of size whether a spider chews up small arthropods without applying venom or if it injects venom first. Very small and/or defenceless arthropods are picked up and crashed with the chelicerae, while larger, dangerous or well-defended items are carefully approached and only attacked with venom injection. Some spiders specialize on prey groups, such as noctuid moths (several genera of bola spiders among Araneidae), web spiders (Mimetidae), ants (Zodarion species in Zodariidae, aphantochiline thomisids, several genera among Theridiidae, Salticidae, Clubionidae and Gnaphosidae) or termites (Ammoxenidae). However, these more or less monophagous species amount only to roughly 2 % of all known spider species, while 98 % are polyphagous. From these considerations, it follows that the majority of spider venoms are not tailored to any given invertebrate or insect group but are rather unspecialized to be effective over a broad spectrum of prey types that spiders naturally encounter.

The toxins and venoms, and their antibodies,

Mode of access: Internet.

Spatial memory and orientation strategies in the elasmobranch Potamotrygon motoro

We investigated whether juvenile freshwater stingrays (Potamotrygon motoro) can solve spatial tasks by constructing a cognitive map of their environment. Two experimental conditions were run: allocentric and ego-allocentric. Rays were trained to locate food within a four-arm maze placed in a room with visual spatial cues. The feeding location (goal) within the maze (room) remained constant while the starting position varied for the allocentrically but not for the ego-allocentrically trained group. After training, all rays solved the experimental tasks; however, different orientation strategies were used within and between groups. Allocentrically trained rays reached the goal via novel routes starting from unfamiliar locations, while ego-allocentrically trained rays primarily solved the task on the basis of an egocentric turn response. Our data suggest that P. motoro orients by constructing a visual cognitive map of its environment, but also uses egocentric and/or other orientation strategies alone or in combination for spatial orientation, a choice which may be governed by the complexity of the problem. We conclude that spatial memory functions are a general feature of the vertebrate brain.

Post-translational modification accounts for the presence of varied forms of nerve growth factor in Australian elapid snake venoms

The Australian elapid snakes are amongst the most venomous snakes in the world, but much less is known about the overall venom composition in comparison to Asian and American snakes. We have used a combined approach of cDNA cloning and 2-DE with MS to identify nerve growth factor (NGF) in venoms of the Australian elapid snakes and demonstrate its neurite outgrowth activity While a single 730 nucleotide ORF, coding for a 243 amino acid precursor protein was detected in all snakes, use of 2-DE identified NGF proteins with considerable variation in molecular size within and between the different snakes. The variation in size can be explained at least in part by Winked glycosylation. it is possible that these modifications alter the stability, is necessary to activity and other characteristics of the snake NGFs. Further characterisation delineate the function of the individual NGF isoforms.