Sebastián Estrada-Gómez

Intraspecific Variation of Centruroides Edwardsii Venom from Two Regions of Colombia

We report the first description studies, partial characterization, and intraspecific difference of Centruroides edwardsii, Gervais 1843, venom. C. edwardsii from two Colombian regions (Antioquia and Tolima) were evaluated. Both venoms showed hemolytic activity, possibly dependent of enzymatic active phospholipases, and neither coagulant nor proteolytic activities were observed. Venom electrophoretic profile showed significant differences between C. edwardsii venom from both regions. A high concentration of proteins with molecular masses between 31 kDa and 97.4 kDa, and an important concentration close or below 14.4 kDa were detected. RP-HPLC retention times between 38.2 min and 42.1 min, showed bands close to 14.4 kDa, which may correspond to phospholipases. RP-HPLC venom profile showed a well conserved region in both venoms between 7 and 17 min, after this, significant differences were detected. From Tolima region venom, 50 well-defined peaks were detected, while in the Antioquia region venom, 55 well-defined peaks were detected. Larvicidal activity was only detected in the C. edwardsii venom from Antioquia. No antimicrobial activity was observed using complete venom or RP-HPLC collected fractions of both venoms. Lethally activity (carried out on female albino swiss mice) was detected at doses over 19.2 mg/kg of crude venom. Toxic effects included distress, excitability, eye irritation and secretions, hyperventilation, ataxia, paralysis, and salivation.

Characterization and cDNA sequence of Bothriechis schlegelii l-amino acid oxidase with antibacterial activity

Snake venoms are complex mixtures of proteins including l-amino acid oxidase (lAAO). A lAAO (named BslAAO) with a mass of 56kDa and a theoretical Ip of 5.79, was purified from Bothriechis schlegelii venom through size-exclusion, ion exchange and affinity chromatography. The entire protein sequence of 498 amino acids, was determined from cDNA using reverse-transcribed mRNA isolated from venom gland. The enzyme showed dose-dependent inhibition of bacterial growth. BslAAO showed inhibitory effect against S. aureus with a MIC of 4μg/mL and a MBC of 8μg/mL. Against Acinetobacter baumannii, showed a MIC of 2μg/mL and MBC of 4μg/mL, No effect was observed in Escherichia coli. This antibacterial activity was inhibited by catalase, indicating that antimicrobial activity was due to H2O2 production. BslAAO did not show any cytotoxic activity toward mouse myoblast cell line C2C12 or peripheral blood mononuclear cells. The enzyme oxidated l-Leu, with a Km of 16.37μM and a Vmax of 0.39μM/min. Snake venoms lAAOs, are potential frames of different therapeutics molecules since these enzymes exhibit low MICs and MBCs and show to be harmless to human cells due to microorganisms being generally several fold more sensitive to reactive oxygen species than human tissues.

Extraction and partial characterization of venom from the Colombian spider Pamphobeteus aff. nigricolor (Aranae:Theraphosidae).

We report the first studies of characterization and extraction of the Pamphobeteus aff. nigricolor (Pocock, 1901) (Aranae:Theraphosidae) venom done in Colombia using the electro-stimulation technique previous anesthesia with isofluorane. After each extraction process, a low viscosity, colorless venom was obtained. This venom showed a 1.01 mg/μl density and a pH of 5. The humidity percentage did not show a significance difference between males and females (P > 0.05) with a general media of 77.49 ± 1.74%. In all cases the venom yielded was variable between males and females, with a media of 22.45 ± 5.17 mg (wet weight) and 4.58 ± 0.94 mg (dry weigh), obtaining larger amounts in females, 28.34 ± 7.49 mg and 5.69 ± 1.36 (wet and dry weight respectively). Venom showed a hemolytic activity dependent of enzymatic active phospholipase and neither coagulant nor proteolytic activities were observed. Electrophoretic profile showed a main protein content with a molecular mass below 14 kDa. RP-HPLC venom profile revealed a difference among male and female venoms content where 17 and 21 main fractions were obtained respectively. Three peptides, Theraphotoxin-Pn1a, Theraphotoxin-Pn1b and Theraphotoxin-Pn2a, were identified using HPLC-nESI-MS/MS. These peptides showed a high identity with other peptides found on Theraphosides which are proved to affect voltage-gated calcium channels.

Partial Characterization of Venom from the Colombian Spider Phoneutria Boliviensis (Aranae:Ctenidae)

We report on the first studies on the characterization of venom from Phoneutria boliviensis (Aranae:Ctenidae) (F. O. Pickard-Cambridge, 1897), done with Colombian species. After the electrostimulation extraction process, the venom showed physicochemical properties corresponding to a colorless and water-soluble liquid with a density of 0.86 mg/mL and 87% aqueous content. P. boliviensis venom and RP-HPLC fractions showed hemolytic activity and hydrolyzed the synthetic substrate 4-nitro-3-octanoyloxy-benzoic acid, indicating the presence of phospholipases A2 enzymes. The electrophoretic profile showed an important protein content with molecular masses below 14 kDa, and differences between male and female protein content were also revealed. The RP-HPLC venom profile exposes differences between males and female content consistent with the electrophoretic profile. Five fractions collected from the RP-HPLC displayed significant larvicidal activity. Mass analysis indicates the presence of peptides ranging from 1047.71 to 3278.07 Da. Two peptides, Ctenitoxin-Pb48 and Ctenitoxin-Pb53, were partially identified using HPLC-nESI-MS/MS, which showed a high homology with other Ctenitoxins (family Tx3) from Phoneutria nigriventer, Phoneutria keyserlingi and Phoneutria reidyi affecting voltage-gated calcium receptors (Cav 1, 2.1, 2.2 and 2.3) and NMDA-glutamate receptors.

Aguapanela, a new tarantula genus from the Colombian Andes (Araneae, Theraphosidae).

A new monotypic genus of Theraphosidae is described from Colombia: Aguapanela Perafán & Cifuentes gen. nov. with only the type species Aguapanela arvi Perafán, Cifuentes & Estrada sp. nov., from Caldas and Medellin, Antioquia, Colombian Andes. The new genus differs from other theraphosid spiders mainly in the presence of stridulatory setae on the palps and legs I and II, together with the presence of type III and IV urticating setae. Males lack a tibial apophysis on leg I and have a simple palpal bulb with the subtegulum less extended than usual in Theraphosinae, elongated curved embolus, ventrally concave, and with two prolateral keels very flat and developed on the dorsal edge. The female spermathecae have two digitiform elongated and granulated seminal receptacles attached to a semicircular wide membranous base. We describe, diagnose and illustrate the new genus and give some biological remarks. Morphological, systematic and biogeographic aspects are discussed. Chromatographic and electrophoretic profiles of its venom are analyzed.

Characterizing the biological and biochemical profile of six different scorpion venoms from the Buthidae and Scorpionidae family

&NA; The objective of this study was to characterize six different scorpion venoms using biological and biochemical methods, including a preliminary MS/MS and a post‐translational modifications analysis. Despite the diversity of scorpion species of medical importance in Africa and Colombia, the venoms of these arachnids have been poorly studied in these two regions. We report the biochemical, electrophoretic, chromatographic profile, internal peptide sequences with a post‐translational modification report, and a preliminary antitumor activity of five different scorpions of the Buthidae family, Androctonus amoreuxi, Babycurus jacksoni, Grosphus grandidieri, Hottentotta gentili and Tityus fuhrmanni, and one of the Scorpionidae family Pandinus imperator. No L‐amino oxidase activity was detected in the evaluated venoms. Proteolytic activity using azocasein was detected only in G. grandidieri and P. imperator, indicating the possible presence of metalloproteinases in these two venoms. Proteolytic activity using NOBA was detected in all venoms indicating the possible presence of serine‐proteinases. Phospholipase A2 activity was detected in the venoms of P. imperator, G. grandidieri, H. gentili and A. amoreuxi, with P. imperator venom being the most active. All venoms analyzed contained defensin‐like proteins, alpha toxins, metalloproteinases, neuropeptides, DBP affecting ion channels, DBP with antimicrobial activity, among others. Venoms from P. imperator, G. grandidieri and T. fuhrmanni showed a dose‐dependent cytotoxic activity over MCF‐7 cells. Only two isolated RP‐HPLC fractions from P. imperator and T. fuhrmanni showed cytotoxic activity over MCF‐7. No cytotoxic activity was found in the venoms from A. amoreuxi, B. jacksoni, and H. gentili. Graphical abstract Figure. No caption available. HighlightsVenoms showed the presence of proteolytic and phospholipase activity.Venoms contained defensin‐like proteins, metalloproteinases, neuropeptides, DBP, among others.Venoms from P. imperator, G. grandidieri and T. fuhrmani showed a dose‐dependent cytotoxic activity over MCF‐7 cells.Two isolated RP‐HPLC fractions from P. imperator and T. fuhrmanni showed cytotoxic activity over MCF‐7.

Venom from Opisthacanthus elatus scorpion of Colombia, could be more hemolytic and less neurotoxic than thought

We report the first biochemical, biological, pharmacological and partial proteomic characterization studies of the Opisthancanthus elatus venom (Gervais, 1844) from Colombia. The Reverse Phase High-Performance Liquid Chromatography venom profile showed 28 main well-defined peaks, most eluting between 20 and 45min (18-30% of acetonitrile, respectively). High-resolution mass analysis indicates the presence of 106 components ranging from 806.59742Da to 16849.4139Da. O. elatus venom showed hemolytic activity and hydrolyzed the specific substrate BapNa suggesting the presence of proteins with serine-protease activity. Collected RP-HPLC fractions eluting at 52.6, 55.5, 55.8, 56.2, and 63.9min (PLA2 region between 33 and 40% of acetonitrile), showed hemolytic activity and hydrolyzed the synthetic substrate 4-nitro-3-octanoyloxy-benzoic acid, indicating the presence of compounds with phospholipases A2 activity. These RP-HPLC fractions, showed molecular masses values up to 13978.19546Da, corroborating the possible presence of the mentioned enzymes. Tryptic digestion and MS/MS analysis showed the presence of a phospholipase like fragment, similar to on described in other Opisthacanthus genus studies. No coagulant activity was observed. No larvicidal or antimicrobial activity was observed at concentrations evaluated. Lethal and toxic activity is expected at doses above 100mg/kg, no neurotoxic effects were detected at lower doses. In conclusion, O. elatus exhibits a venom with a predominant phospholipase A2 activity than thought; mammals neurotoxic activity is expected above the 100mg/kg, which is very high compared to the venom from other neurotoxic scorpions.

Target-Specificity in Scorpions; Comparing Lethality of Scorpion Venoms across Arthropods and Vertebrates

Scorpions use their venom in defensive situations as well as for subduing prey. Since some species of scorpion use their venom more in defensive situations than others, this may have led to selection for differences in effectiveness in defensive situations. Here, we compared the LD50 of the venom of 10 species of scorpions on five different species of target organisms; two insects and three vertebrates. We found little correlation between the target species in the efficacy of the different scorpion venoms. Only the two insects showed a positive correlation, indicating that they responded similarly to the panel of scorpion venoms. We discuss the lack of positive correlation between the vertebrate target species in the light of their evolution and development. When comparing the responses of the target systems to individual scorpion venoms pairwise, we found that closely related scorpion species tend to elicit a similar response pattern across the target species. This was further reflected in a significant phylogenetic signal across the scorpion phylogeny for the LD50 in mice and in zebrafish. We also provide the first mouse LD50 value for Grosphus grandidieri.

Characterization of the Venom of C. d. cumanesis of Colombia: Proteomic Analysis and Antivenomic Study

The Colombian rattlesnake Crotalus durissus cumanensis is distributed in three geographic zones of the country: the Atlantic Coast, the upper valley of the Magdalena River, and the eastern plains of the Colombian Orinoquía. Its venom induces neurological symptoms, such as eyelid ptosis, myasthenic facies, and paralysis of the respiratory muscles, which can lead to death. Identification and analysis of C. d. cumanensis showed nine groups of proteins responsible for the neurotoxic effect, of which the crotoxin complex was the most abundant (64.71%). Immunorecognition tests of C. d. cumanensis showed that the use of a commercial antivenom manufactured in Mexico resulted in immunoreactivity.

Identifying different transcribed proteins in the newly described Theraphosidae Pamphobeteus verdolaga

Abstract Theraphosidae spider venoms are well known for possess a complex mixture of protein and non‐protein compounds in their venom. The objective of this study was to report and identify different proteins translated from the venom gland DNA information of the recently described Theraphosidae spider Pamphobeteus verdolaga. Using a venom gland transcriptomic analysis, we reported a set of the first complete sequences of seven different proteins of the recenlty described Theraphosidae spider P. verdolaga. Protein analysis indicates the presence of different proteins on the venom composition of this new spider, some of them uncommon in the Theraphosidae family. MS/MS analysis of P. verdolaga showed different fragments matching sphingomyelinases (sicaritoxin), barytoxins, hexatoxins, latroinsectotoxins, and linear (zadotoxins) peptides. Only four of the MS/MS fragments showed 100% sequence similarity with one of the transcribed proteins. Transcriptomic analysis showed the presence of different groups of proteins like phospholipases, hyaluronidases, inhibitory cysteine knots (ICK) peptides among others. The three database of protein domains used in this study (Pfam, SMART and CDD) showed congruency in the search of unique conserved protein domain for only four of the translated proteins. Those proteins matched with EF‐hand proteins, cysteine rich secretory proteins, jingzhaotoxins, theraphotoxins and hexatoxins, from different Mygalomorphae spiders belonging to the families Theraphosidae, Barychelidae and Hexathelidae. None of the analyzed sequences showed a complete 100% similarity. Graphical abstract Figure. No caption available. HighlightsTranscriptomic and proteomic analysis allowed the first complete description of seven different protein sequences of Pamphobeteus verdolaga.Transcriptomic analysis showed proteins with sequence similarity with EF‐hand, cysteine rich secretory proteins, theraphotoxins and hexatoxins.Proteomic analysis showed fragments matching sphingomyelinase, barytoxins, hexatoxins, latroinsectotoxins, linear peptides.Three of the seven translated proteins did not match any known conserved protein domain.