Paratrygon aiereba irradiated anti-mucus serum reduce edematogenic activity induced in experimental model.

Accidents by freshwater stingrays are common in northern Brazil, there is no specific therapy for high morbidity and local tissue destruction. The irradiation of venoms and toxins by ionizing radiation has been used to produce appropriate immunogens for the production of antisera. We planned to study the efficacy of stinging mucus irradiation in the production of antisera, with serum neutralization assays of edematogenic activity and quantification of cytokines performed in animal models of immunization with native and irradiated mucus of Paratrygon aiereba, a large freshwater stingray. Antiserum potency and its cross-reactivity with mucus from other freshwater stingrays were detected by ELISA. Immunization models demonstrated the ability to stimulate a strong humoral response with elevated levels of serum IgG detectable by ELISA, and both native and irradiated mucus were immunogenic and capable of recognizing mucus proteins from other freshwater neotropical stingrays. Mucus P. aiereba causes cellular and humoral adaptive immune responses in cells of immunized mice producing antibodies and cytokines such as TNF-α, IL-6 and IL-17. Rabbit antisera immunized with mucus from P. aiereba irradiated at 2 kGy showed a significant reduction of mucus-induced edematogenic activity in mice. Our data suggest that the use of antisera against freshwater stingray mucus show the possibility of specific therapy for these accidents.

Proteomic analysis of soluble proteins retrieved from Duttaphrynus melanostictus skin secretion by IEx-batch sample preparation.

Amphibians display a toxic secretion that works as chemical defenses against predators and/or microorganisms that is stored in specialized glands located in the tegument. For some animals, such glands have accumulated in specific regions of the body and formed prominent structures known as macroglands. The Bufonidae family displays conspicuous macroglands in a post-orbital position, termed parotoids, which secretions are known to be extremely viscous and rich in alkaloids and steroids. Few proteins have been described in this material, though. Mainly, because of the difficulties to handle such biological matrix. In this context, we have performed a proteomic study on the parotoid macrogland secretion of the Asian bufonid Duttaphrynus melanostictus. By employing the Ion-Exchange (IEx)-batch chromatography (anionic, cationic and both) we obtained six fractions - bound and unbound - that were submitted to an in solution-trypsin digestion followed by LC-MS/MS. Proteins related to: antioxidant activity, binding processes (carbohydrate/lipid/protein), energy metabolism, hydrolases, lipid metabolism and membrane traffic were identified. Moreover, IEx was able to preserve the biological activity of the retrieved proteins (peptidasic). The current study increases the knowledge on the proteins present in the bufonids parotoid macrogland secretion, providing a better understanding of the physiological role played by such molecules. SIGNIFICANCE: The current approach allowed a detailed proteomic analysis of the several proteins synthesized in the D. melanostictus parotoid macrogland (Bufonidae) that are secreted into the skins, but embedded within a complex viscous biological matrix. Moreover, our results aim to increase the knowledge on the biological role played by such proteins at the skin.

Proteomic analysis of soluble proteins retrieved from Duttaphrynus melanostictus skin secretion by IEx-batch sample preparation

Amphibians display a toxic secretion that works as chemical defenses against predators and/or microorganismsthat is stored in specialized glands located in the tegument. For some animals, such glands have accumulated inspecific regions of the body and formed prominent structures known as macroglands. The Bufonidae familydisplays conspicuous macroglands in a post-orbital position, termed parotoids, which secretions are known to beextremely viscous and rich in alkaloids and steroids. Few proteins have been described in this material, though.Mainly, because of the difficulties to handle such biological matrix. In this context, we have performed a pro-teomic study on the parotoid macrogland secretion of the Asian bufonidDuttaphrynus melanostictus. By em-ploying the Ion-Exchange (IEx)-batch chromatography (anionic, cationic and both) we obtained six fractions -bound and unbound–that were submitted to an in solution-trypsin digestion followed by LC-MS/MS. Proteinsrelated to: antioxidant activity, binding processes (carbohydrate/lipid/protein), energy metabolism, hydrolases,lipid metabolism and membrane traffic were identified. Moreover, IEx was able to preserve the biological ac-tivity of the retrieved proteins (peptidasic). The current study increases the knowledge on the proteins present inthe bufonids parotoid macrogland secretion, providing a better understanding of the physiological role played bysuch molecules.Significance:The current approach allowed a detailed proteomic analysis of the several proteins synthesized intheD. melanostictusparotoid macrogland (Bufonidae) that are secreted into the skins, but embedded within acomplex viscous biological matrix. Moreover, our results aim to increase the knowledge on the biological roleplayed by such proteins at the skin

Proteomic analysis of soluble proteins retrieved from Duttaphrynus melanostictus skin secretion by IEx-batch sample preparation

Amphibians display a toxic secretion that works as chemical defenses against predators and/or microorganismsthat is stored in specialized glands located in the tegument. For some animals, such glands have accumulated inspecific regions of the body and formed prominent structures known as macroglands. The Bufonidae familydisplays conspicuous macroglands in a post-orbital position, termed parotoids, which secretions are known to beextremely viscous and rich in alkaloids and steroids. Few proteins have been described in this material, though.Mainly, because of the difficulties to handle such biological matrix. In this context, we have performed a pro-teomic study on the parotoid macrogland secretion of the Asian bufonidDuttaphrynus melanostictus. By em-ploying the Ion-Exchange (IEx)-batch chromatography (anionic, cationic and both) we obtained six fractions -bound and unbound–that were submitted to an in solution-trypsin digestion followed by LC-MS/MS. Proteinsrelated to: antioxidant activity, binding processes (carbohydrate/lipid/protein), energy metabolism, hydrolases,lipid metabolism and membrane traffic were identified. Moreover, IEx was able to preserve the biological ac-tivity of the retrieved proteins (peptidasic). The current study increases the knowledge on the proteins present inthe bufonids parotoid macrogland secretion, providing a better understanding of the physiological role played bysuch molecules.Significance:The current approach allowed a detailed proteomic analysis of the several proteins synthesized intheD. melanostictusparotoid macrogland (Bufonidae) that are secreted into the skins, but embedded within acomplex viscous biological matrix. Moreover, our results aim to increase the knowledge on the biological roleplayed by such proteins at the skin

Biochemical Analyses of Proteins from Duttaphrynus melanostictus (Bufo melanostictus) Skin Secretion: Soluble Protein Retrieval from a Viscous Matrix by Ion-Exchange Batch Sample Preparation.

A crucial step in scientific analysis can be sample preparation, and its importance increases in the same rate as the sensitivity of the following employed/desired analytical technique does. The need to analyze complex, viscous matrices is not new, and diverse approaches have been employed, with different success rates depending on the intended molecules. Solid-phase extraction, for example, has been successfully used in sample preparation for organic molecules and peptides. However, due to the usual methodological conditions, biologically active proteins are not successfully retrieved by this technique, resulting in a low rate of protein identification reported for the viscous amphibian skin secretion. Here we describe an ion-exchange batch processing sample preparation technique that allows viscous or adhesive materials (as some amphibian skin secretions) to be further processed by classical liquid chromatography approaches. According to our protocol, samples were allowed to equilibrate with a specific resin that was washed with appropriated buffers in order to yield the soluble protein fraction. In order to show the efficiency of our methodology, we have compared our results to classically prepared skin secretion, i.e., by means of filtration and centrifugation. After batch sample preparation, we were able to obtain reproductive resolved protein chromatographic profiles, as revealed by SDS-PAGE, and retrieve some biological activities, namely, hydrolases belonging to serine peptidase family. Not only that, but also the unbound fraction was rich in low molecular mass molecules, such as alkaloids and steroids, making this sample preparation technique also suitable for the enrichment of such molecules.

Biochemical analyses of proteins from Duttaphrynus melanostictus (Bufo melanostictus) skin secretion: soluble protein retrieval from a viscous matrix by ion-exchange batch sample preparation

A crucial step in scientific analysis can be sample preparation, and its importance increases in the same rate as the sensitivity of the following employed/desired analytical technique does. The need to analyze complex, viscous matrices is not new, and diverse approaches have been employed, with different success rates depending on the intended molecules. Solid-phase extraction, for example, has been successfully used in sample preparation for organic molecules and peptides. However, due to the usual methodological conditions, biologically active proteins are not successfully retrieved by this technique, resulting in a low rate of protein identification reported for the viscous amphibian skin secretion. Here we describe an ion-exchange batch processing sample preparation technique that allows viscous or adhesive materials (as some amphibian skin secretions) to be further processed by classical liquid chromatography approaches. According to our protocol, samples were allowed to equilibrate with a specific resin that was washed with appropriated buffers in order to yield the soluble protein fraction. In order to show the efficiency of our methodology, we have compared our results to classically prepared skin secretion, i.e., by means of filtration and centrifugation. After batch sample preparation, we were able to obtain reproductive resolved protein chromatographic profiles, as revealed by SDS-PAGE, and retrieve some biological activities, namely, hydrolases belonging to serine peptidase family. Not only that, but also the unbound fraction was rich in low molecular mass molecules, such as alkaloids and steroids, making this sample preparation technique also suitable for the enrichment of such molecules.

Biochemical analyses of proteins from Duttaphrynus melanostictus (Bufo melanostictus) skin secretion: soluble protein retrieval from a viscous matrix by ion-exchange batch sample preparation

A crucial step in scientific analysis can be sample preparation, and its importance increases in the same rate as the sensitivity of the following employed/desired analytical technique does. The need to analyze complex, viscous matrices is not new, and diverse approaches have been employed, with different success rates depending on the intended molecules. Solid-phase extraction, for example, has been successfully used in sample preparation for organic molecules and peptides. However, due to the usual methodological conditions, biologically active proteins are not successfully retrieved by this technique, resulting in a low rate of protein identification reported for the viscous amphibian skin secretion. Here we describe an ion-exchange batch processing sample preparation technique that allows viscous or adhesive materials (as some amphibian skin secretions) to be further processed by classical liquid chromatography approaches. According to our protocol, samples were allowed to equilibrate with a specific resin that was washed with appropriated buffers in order to yield the soluble protein fraction. In order to show the efficiency of our methodology, we have compared our results to classically prepared skin secretion, i.e., by means of filtration and centrifugation. After batch sample preparation, we were able to obtain reproductive resolved protein chromatographic profiles, as revealed by SDS-PAGE, and retrieve some biological activities, namely, hydrolases belonging to serine peptidase family. Not only that, but also the unbound fraction was rich in low molecular mass molecules, such as alkaloids and steroids, making this sample preparation technique also suitable for the enrichment of such molecules.

Proteomic analysis of the rare Uracoan rattlesnake Crotalus vegrandis venom: Evidence of a broad arsenal of toxins.

The investigation of venoms has many clinical, pharmacological, ecological and evolutionary outcomes. The Crotalus spp. venom can cause hemorrhage, neurotoxicity, myotoxicity, coagulopathy and hypotension. Although neurotoxicity and hemorrhage usually does not occur for the same species, the rare Venezuelan species Crotalus vegrandis presents both characteristic. Different from the other species it has a restricted ecological niche and geographical distribution. Nevertheless, it has a raising medical importance as this rattlesnake population is increasing. Few works describe its neurotoxic and hemorrhagic features, but other toxins might play an important role in envenomation. We combined proteomic methods to identify for the first time the main components of it venom: 2D SDS-PAGE and gel-filtration chromatography for protein mixture decomplexation; LC-MS(2) of low molecular mass fractions and tryptic peptides; bioinformatic identification of toxin families and specific protein species based on unique peptide analysis and sequence database enriched with species-specific venom gland transcripts; and finally polyclonal anti-crotamine Western-blotting. Our results point to a broad arsenal of toxins in C. vegrandis venom: PIII and PII metalloproteases, crotoxin subunits, other phospholipases, isoforms of serine proteases and lectins, l-amino-acid oxidase, nerve growth factor, as well as other less abundant toxins.

Mast cells and histamine play an important role in edema and leukocyte recruitment induced by Potamotrygon motoro stingray venom in mice.

This work aimed to investigate mechanisms underlying the inflammatory response caused by Potamotrygon motoro stingray venom (PmV) in mouse paws. Pre-treatment of animals with a mast cell degranulation inhibitor (cromolyn) diminished edema (62% of inhibition) and leukocyte influx into the site of PmV injection. Promethazine (histamine type 1 receptor antagonist) or thioperamide (histamine type 3 and 4 receptor antagonist) also decreased edema (up to 30%) and leukocyte numbers, mainly neutrophils (40-50 %). Cimetidine (histamine type 2 receptor antagonist) had no effect on PmV-induced inflammation. In the RBL-2H3 lineage of mast cells, PmV caused proper cell activation, in a dose-dependent manner, with release of PGD2 and PGE2. In addition, the role of COXs products on PmV inflammatory response was evaluated. Indomethacin (COX-1/COX-2 inhibitor) or etoricoxib (COX-2 inhibitor) partially diminished edema (around 20%) in PmV-injected mice. Indomethacin, but not etoricoxib, modulated neutrophil influx into the site of venom injection. In conclusion, mast cell degranulation and histamine, besides COXs products, play an important role in PmV-induced reaction. Since PmV mechanism of action remains unknown, hindering accurate treatment, clinical studies can be performed to validate the prescription of antihistaminic drugs, besides NSAIDs, to patients injured by freshwater stingrays.

Local inflammatory reaction induced by Scolopendra viridicornis centipede venom in mice.

Centipede envenomation is generally mild, and human victims usually manifest burning pain, erythema and edema. Despite the abundance and ubiquity of these animals, centipede venom has been poorly characterized in literature. For this reason, the aim of this work was to investigate local inflammatory features induced by Scolopendra viridicornis centipede envenomation in mice, evaluating edema formation, leukocyte infiltration, production of inflammatory mediators, and also performing histological analysis. The highest edematogenic activity induced by the venom, determined by plethysmometry, was noticed 0.5 h after injection in mice footpad. At 24 h, edema was still detected in animals that received 15 and 60 µg of venom, and at 48 h, only in animals injected with 60 µg of venom. In relation to leukocyte count, S. viridicornis venom induced cell recruitment, mainly neutrophils and monocytes/macrophages, in all doses and time periods analyzed in comparison with PBS-injected mice. An increase in lymphocytes was detected especially between 1 and 24 h at 60 µg dose. Besides, eosinophil recruitment was observed mainly for 15 and 60 µg doses in early time periods. Edema formation and cell recruitment were also confirmed by histological analysis. Moreover, S. viridicornis venom stimulated the release of IL-6, MCP-1, KC, and IL-1ß. Conversely, S. viridicornis venom did not induce the release of detectable levels of TNF-α. We demonstrated that the edematogenic activity induced by S. viridicornis venom was of rapid onset, and the venom stimulated secretion of pro-inflammatory mediators which contribute to the inflammatory reaction induced by S. viridicornis venom in an experimental model.

Inflammatory mediators generated at the site of inoculation of Loxosceles gaucho spider venom.

Patients bitten by Loxosceles spiders generally manifest marked local inflammatory reaction and dermonecrosis. This report evaluated edema formation, leukocyte infiltration and release of inflammatory mediators at the injection site of Loxosceles gaucho venom. BALB/c mice were i.d. injected with venom and thereafter paws were disrupted and homogenized to obtain differential counts of migrated cells, as well to assay the levels of cytokines, chemokines and lipid mediators. Increased footpad thickness was detected as soon as 30 min after venom injection, and 24h later was similar to that of the control group. Loxosceles venom mildly augmented the recruitment of leukocytes to the footpad in comparison with PBS-injected mice. Moreover, it stimulated the release of IL-6, MCP-1 and KC at 2 and 24h after venom injection. In addition, higher levels of PGE(2) were detected 30 min after venom injection in comparison with control group. However, the venom failed to increase levels of IL-1 beta, TNF-alpha, TXB(2) and LTB(4). Our results demonstrate that L. gaucho venom evokes an early complex inflammatory reaction, stimulating the secretion of pro-inflammatory cytokines and lipid mediators (PGE(2)), and recruiting leukocytes to the footpad which contribute to the local reaction induced by L. gaucho venom.

Inflammatory mediators generated at the site of inoculation of Loxosceles gaucho spider venom

Patients bitten by Loxosceles spiders generally manifest marked local inflammatory reaction and dermonecrosis. This report evaluated edema formation, leukocyte infiltration and release of inflammatory mediators at the injection site of Loxosceles gaucho venom. BALB/c mice were i.d. injected with venom and thereafter paws were disrupted and homogenizedto obtain differential counts of migrated cells, as well to assay the levels of cytokines, chemokines and lipid mediators. Increased footpad thickness was detected as soon as30 min after venom injection, and 24 h later was similar to that of the control group. Loxosceles venom mildly augmented the recruitment of leukocytes to the footpad in comparison with PBS-injected mice. Moreover, it stimulated the release of IL-6, MCP-1 and KC at 2 and 24 h after venom injection. In addition, higher levels of PGE2 were detected30 min after venom injection in comparison with control group. However, the venom failed to increase levels of IL-1b, TNF-a, TXB2 and LTB4. Our results demonstrate that L. gaucho venom evokes an early complex inflammatory reaction, stimulating the secretionof pro-inflammatory cytokines and lipid mediators (PGE2), and recruiting leukocytes to the $footpad which contribute to the local reaction induced by L. gaucho venom.