Tityus serrulatus venom peptidomics: Assessing venom peptide diversity

MALDI-TOF-TOF and de novo sequencing were employed to assess the Tityus serrulatus venom peptide diversity. Previous works has shown the cornucopia of molecular masses, ranging from 800 to 3000 Da, present in the venom from this and other scorpions species. This work reports the identification/sequencing of several of these peptides. The majority of the peptides found were fragments of larger venom toxins. For instance, 28 peptides could be identified as fragments from Pape proteins, 10 peptides corresponded to N-terminal fragments of the TsKβ (scorpine-like) toxin and fragments of potassium channel toxins (other than the k-beta) were sequenced as well. N-terminal fragments from the T. serrulatus hypotensins-I and II and a novel hypotensin-like peptide could also be found. This work also reports the sequencing of novel peptides without sequence similarities to other known molecules.     

Two volatiles from the venom gland of the Samsum ant, Pachycondyla sennaarensis

Allergy and anaphylactic reactions after the stings of the Samsum ant Pachycondyla sennaarensis (Mayr, 1862) (Hymenoptera: Formicidae) have been reported from several countries along the Persian Gulf coast, but no analysis has been yet carried out on the ant's venom gland secretions. This study is focused on the identification of volatiles from the venom gland of Pachycondyla sennaarensis using gas chromatography-mass spectrometry, which showed the presence of The main volatile components of the venom gland were phenol-2,4-bis(1,1 dimethylethyl) and trimethyl pyrazine. This is the first record of the occurrence of phenol-2,4-bis(1,1 dimethylethyl) in insects. The venom gland secretions of Pachycondyla species are known contain a variety of volatiles, making the members of this genus distinctive among the ponerine ants.     

Inflammation induced by Bothrops asper venom

Inflammation is a major characteristic of envenomation by snakes from viperine and crotaline species. Bothrops asper snake venom elicits, among other alterations, a pronounced inflammatory response at the site of injection both in humans and experimental animals. This review describes the current status of our understanding of the inflammatory reaction, including pain, triggered by Bothrops asper venom. The experimental studies on the action of this venom as well as the complex network of chemical mediators involved are summarized. Moreover, aspects of the molecular mechanisms orchestrating this important response to envenomation by Bothrops asper are presented. Considering that isolated toxins are relevant tools for understanding the actions of the whole venom, studies dealing with the mechanisms of inflammatory and nociceptive properties of phospholipases A₂, a metalloproteinase and serine-proteases isolated from Bothrops asper venom are also described.     

General biochemical and immunological characteristics of the venom from Peruvian scorpion Hadruroides lunatus

This communication describes the general biochemical properties and some immunological characteristics of the venom from the Peruvian scorpion Hadruroides lunatus, which is the most medically relevant species in Peru. The soluble venom of this scorpion is toxic to mice, the LD₅₀ determined was 0.1 mg/kg and 21.55 mg/kg when the venom was injected intracranial or intraperitoneally, respectively. The soluble venom displayed proteolytic, hyaluronidasic, phospholipasic and cardiotoxic activities. High performance liquid chromatography of the soluble venom resulted in the separation of 20 fractions. Two peptides with phospholipasic activity were isolated to homogeneity and their molecular masses determined by mass spectrometry (MALDI TOF). Anti-H. lunatus venom sera were produced in rabbits. Western blotting analysis showed that most of the protein content of this venom is immunogenic. H. lunatus anti-venom displayed consistent cross-reactivity with venom antigens from the new World-scorpions Tityus serrulatus and Centruroides sculpturatus venoms; however, a weaker reactivity was observed against the venom antigens from the old World-scorpion Androctonus australis Hector.     

In vitro and in vivo studies on some toxic effects of the venom from Hemiscorpious lepturus scorpion

The aim of this laboratory-based study was to investigate some of the toxic effects induced by the venom from Hemiscorpious lepturus (H. lepturus). For this aim, pharmacological, histological, biochemical methods as well as complete blood cell count were used to assess these toxic actions. In addition, in vitro haemolysis studies on human washed blood suspension and cytotoxicity on cultured fibroblasts were also undertaken. In vitro pharmacological test was made on rat isolated ileal segment. To this end, the effects of the venom on the contractile responsiveness to acetylcholine were recorded using F30 transducer and Darco chart recorder. For assessment of the haemolytic potency, varying concentrations (2, 10, 20 and 40 μg/ml) of the venom were added to 0.5 ml of 5% washed human blood and after 30 min, 2, 4, 8, 12 and 24 h of exposure, the degree of lysis (extent of redness developed in the supernatant solution after centrifugation) were measured by ELISA method. Cytotoxicity potential of the venom was assessed by trypan blue exclusion test. The venom (0.1, 1 and 10 μg/ml) was mixed with confluent fibroblast cell culture and the extent cytotoxicity was assessed microscopically. In vivo studies were conducted by a subcutaneous administration of sub-lethal dose (10 μg) of the venom and after 7 days the skin, at the site of injection, and kidney samples were stained by H & E method and examined microscopically. In addition, biochemical assessments including measurement of serum aspartate aminotransferase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP) and amylase levels and urine analysis were made. The results showed that the venom prevented the relaxation phase of the acetylcholine-induced contractions on the isolated ileal segments and finally produced sustained spasmodic contractions. This spasmodic action was abolished by 1 μM atropine. The venom produced haemolysis of red blood cells in a concentration-dependent and duration-of-exposure manner, with 100% of haemolysis produced after 24 h following exposure to 40 μg/ml of venom. While cultured fibroblasts cells were more sensitive and disintegrated after 15 min of exposure to 1 μg/ml of the venom. Histological findings showed evidences of excessive inflammatory responses accompanied with signs of necrosis in the skin at the site of injection as well as structural damage in the nephrones. There was a significant rise in the serum enzymes. In addition, the number of the RBCs were reduced. The urine showed positive readings for proteinuria, blood and intact RBCs. The overall results suggest that the venom from H. lepturus primarily is a cytotoxic agent and has haemolytic, nephrotoxic and to some extent hepatotoxic activity.     

Identification and characterization of venom proteins of two solitary wasps, Eumenes pomiformis and Orancistrocerus drewseni

Secretory proteins were identified in the venoms of two solitary hunting wasps, Eumenes pomiformis and Orancistrocerus drewseni, by SDS-PAGE in conjunction with mass analysis. More than 30 protein bands (2-300 kDa) were detected from the crude venom of each wasp. With the aid of the previously constructed venom gland/sac-specific EST libraries, a total of 31 and 20 proteins were identified from 18 to 20 distinctive protein bands of E. pomiformis and O. drewseni venoms, respectively. Arginine kinase was the most predominant protein in both wasp venoms. Along with the full-length arginine kinase, a truncated form, which was known to have paralytic activity on a spider, was a common predominant protein in the two wasp venoms. Insulin/insulin-like peptide-binding protein was abundantly found only in E. pomiformis venom, which might be due to its unique behaviors of oviposition and provision. The presence of various immune response-related proteins and antioxidants suggested that wasps might use their venom to maintain prey fresh while feeding wasp larvae by protecting the prey from microbial invasion and physiological stresses. It seemed that some venom proteins are secreted into venom fluid from venom gland cells via exosomes, not by signal sequence-mediated transport processes.     

Antinociceptive properties of Micrurus lemniscatus venom

The therapeutic potential of snake venoms for pain control has been previously demonstrated. In the present study, the antinociceptive effects of Micrurus lemniscatus venom (MlV) were investigated in experimental models of pain. The antinociceptive activity of MIV was evaluated using the writhing, formalin, and tail flick tests. Mice motor performance was assessed in the rota rod and open field tests. In a screening test for new antinociceptive substances - the writhing test - oral administration of MlV (19.7-1600 μg/kg) produced significant antinociceptive effect. The venom (1600 μg/kg) also inhibited both phases of the formalin test, confirming the antinociceptive activity. The administration of MlV (1600 μg/kg) did not cause motor impairment in the rota rod and open field tests, which excluded possible non-specific muscle relaxant or sedative effects of the venom. The MIV (177-1600 μg/kg) also increases the tail flick latency response, indicating a central antinociceptive effect for the venom. In this test, the MlV-induced antinociceptive effect was long-lasting and higher than that of morphine, an analgesic considered the gold standard. In another set of experiments, the mechanisms involved in the venom-induced antinociception were investigated through the use of pharmacological antagonists. The MlV (1600 μg/kg) antinociceptive effect was prevented by naloxone (5 mg/kg), a non-selective opioid receptor antagonist, suggesting that this effect is mediated by activation of opioid receptors. In addition, the pre-treatment with the μ-opioid receptor antagonist CTOP (1 mg/kg) blocked the venom antinociceptive effect, while the k-opioid receptor antagonist nor-BNI (0.5 mg/kg) or the δ-opioid receptor antagonist naltrindole (3 mg/kg) only partially reduced the venom-induced antinociception. The present study demonstrates, for the first time, that oral administration of M. lemniscatus venom, at doses that did not induce any motor performance alteration, produced potent and long-lasting antinociceptive effect mediated by activation of opioid receptors.     

Intra-specific variation in venom of the African Puff Adder (Bitis arietans): Differential expression and activity of snake venom metalloproteinases (SVMPs)

Bitis arietans is considered one of the most medically significant snakes in Africa, primarily due to a combination of its extensive geographical distribution, common occurrence and highly potent haemorrhagic and cytotoxic venom. Our investigation has revealed a remarkable degree of intra-species variation between pooled venom samples from different geographical origins across sub-Saharan Africa and Arabia, and within a group of individual specimens from the same origin in Nigeria as determined by a combination of immunological, biochemical and proteomic assays. We demonstrate significant quantitative and qualitative differences between B. arietans venom in terms of protein expression, immunogenicity and activity of snake venom metalloproteinases (SVMPs); toxins with a primary role in the haemorrhagic and tissue-necrotic pathologies suffered by envenomed victims. Specifically, we have identified a processed PII SVMP that exhibits striking inter-specimen variability.     

Pharmacokinetic studies of scorpion venom before and after antivenom immunotherapy.

The improvement of the immunotherapeutic treatment of envenomations requires a better knowledge of the pharmacological actions of the scorpion venom and of the mechanism of its in vivo neutralization by antivenom. In the present work, we determined the toxicokinetic parameters of the toxic fraction of Androctonus australis garzonii venom in the absence and after antivenom immunotherapy, in experimentally envenomed rabbits. After subcutaneous injection of the scorpion venom, toxins showed a fast and complete resorption from the site of injection associated with a simultaneous distribution in a large extracellular compartment and with an important body clearance. The precocious intravenous injection of an appropriate antivenom dose was shown to induce an immediate, complete and durable neutralization of toxins, as well as their rapid redistribution from the peripheric compartment to the vascular one. On the contrary, the intramuscular injection of the same antivenom dose produced a slower and partial redistribution of toxins, leading to a delayed neutralization of the venom. The intravenous injection of smaller antivenom doses induced transient decreases of circulating toxins, indicating that a minimal antivenom dose has to be administered to allow an efficient and durable neutralization of the venom. We concluded also that this minimal effective dose of antivenom has to be injected precociously, by intravenous route, to achieve an efficient immunotherapy.     

The neutralizing capacity of Androctonus crassicauda antivenom against Mesobuthus eupeus scorpion venom

Scorpion envenomations are considerable health problem in tropical and subtropical regions. There are approximately 1500 species of scorpions worldwide. The number of dangerous species in the Buthidae family is significantly higher than in other families of scorpions. Mesobuthus eupeus is a member of Mesobuthus genus, Buthidae family. In this study, the potency and para-specific activities of Androctonus crassicauda antivenom were investigated against M. eupeus scorpion venom. The median lethal dose of M. eupeus and A. crassicauda scorpion venoms were found to be 0.18mg/kg, 15.45mug/kg by i.c.v injection route. The antivenom showed neutralization effect against the venoms of M. eupeus. One milliliter of A. crassicauda antivenom neutralizes 464LD(50) of M. eupeus and 940LD(50)A. crassicauda venom on mice. Western blotting demonstrated immunologic reaction with the venoms. The monovalent antivenom has immunoactivity and neutralizing capacity to the scorpion venoms. This study indicates that the antivenom produced by Refik Saydam Hygiene Center could be used for the treatment of M. eupeus stings in Turkey.     

Milking and partial characterization of venom from the Brazilian spider Vitalius dubius (Theraphosidae)

The theraphosid spider genus Vitalius contains several species found in southeastern Brazil. In this work, we used electrostimulation to obtain venom from Vitalius dubius and examined its general composition. Male spiders yielded significantly less (p < 0.05) venom (12.5 ± 0.7 mg of liquid/spider, n = 16; mean ± S.E.M.) than female spiders (25.5 ± 2.0 mg of liquid/spider, n = 11). However, when corrected for spider weight, males yielded slightly more venom (2.89 ± 0.16 mg/g vs. 2.45 ± 0.76 mg/g for males and females, respectively, p < 0.05). Venom yield correlated linearly with spider weight for spiders weighing up to ∼12-13 g, but decreased in very heavy females. There was a marked decrease in venom yield after the first milking. The protein concentration of pooled venom was 18.3 ± 2.4 mg/ml (n = 4) and accounted for 16.6 ± 4.7% of the dry venom weight. The venom contained high hyaluronidase activity (275 ± 24 TRU/mg of protein, n = 4), with a molecular mass of ∼45 kDa estimated by zymography. SDS-PAGE revealed a few proteins with molecular masses >14 kDa but showed two staining bands of peptides <14 kDa. The venom reacted in ELISA with affinity-purified IgG from commercial arachnidic antivenom. Immunoblotting with this IgG detected proteins of 30-140 kDa only. Fractionation of the venom by reverse-phase chromatography resulted in five major and eight minor peaks.     

Isolation and molecular cloning of beta-neurotoxins from the venom of the scorpion Centruroides suffusus suffusus

This communication reports the identification and characterization of two new toxins from the venom of the scorpion Centruroides suffusus suffusus, named: CssVIII and CssIX, according to the original nomenclature of toxins previously described for this scorpion. The isolation was obtained by means of two chromatographic steps, and a cDNA library was used to fully identify their precursors. CssVIII and CssIX contain signal peptides of 19 and 17 amino acid residues, and mature peptides of 66 and 65 residues, respectively. Intracranial injections into mice of both purified toxins showed toxicity results similar to those found for toxins CssII and CssIV. Additionally, they compete with the parent toxin CssIV, in binding and displacement experiments, conducted with brain synaptosomes showing nanomolar affinities. These results strongly support the conclusion that they are new β-neurotoxins and certainly would be of the interest of researchers in the field of venomics for studying sodium channels.     

Physiological resistance of grasshopper mice (Onychomys spp.) to Arizona bark scorpion (Centruroides exilicauda) venom

Predators feeding on toxic prey may evolve physiological resistance to the preys' toxins. Grasshopper mice (Onychomys spp.) are voracious predators of scorpions in North American deserts. Two species of grasshopper mice (Onychomys torridus and Onychomys arenicola) are broadly sympatric with two species of potentially lethal bark scorpion (Centruroides exilicauda and Centruroides vittatus) in the Sonoran and Chihuahuan deserts, respectively. Bark scorpions produce toxins that selectively bind sodium (Na(+)) and potassium (K(+)) ion channels in vertebrate nerve and muscle tissue. We previously reported that grasshopper mice showed no effects of bark scorpion envenomation following natural stings. Here we conducted a series of toxicity tests to determine whether grasshopper mice have evolved resistance to bark scorpion neurotoxins. Five populations of grasshopper mice, either sympatric with or allopatric to bark scorpions, were injected with bark scorpion venom; LD50s were estimated for each population. All five populations of grasshopper mice demonstrated levels of venom resistance greater than that reported for non-resistant Mus musculus. Moreover, venom resistance in the mice showed intra- and interspecific variability that covaried with bark scorpion sympatry and allopatry, patterns consistent with the hypothesis that venom resistance in grasshopper mice is an adaptive response to feeding on their neurotoxic prey.     

Identification, cDNA cloning and heterologous expression of a hyaluronidase from the tarantula Brachypelma vagans venom

Hyaluronidases (Hyal) present in the venom of poisonous animals have been considered as “spreading factors” that facilitate a fast penetration of the venom in the prey. We have found that hyaluronidase from the tarantula Brachypelma vagans venom (BvHyal) displays a substrate-specific Hyal activity against hyaluronan. By using a combined strategy based on peptide sequencing and RT-PCR, we have cloned a BvHyal cDNA. Active recombinant BvHyal was efficiently expressed in a baculovirus system in insect cell.     

Characterization of peptide components in the venom of the scorpion Liocheles australasiae (Hemiscorpiidae).

Scorpion venoms are composed of a number of neurotoxic peptides. A variety of toxins have been isolated from the venoms of scorpions of the family Buthidae, however, little interest has been paid to non-Buthidae scorpions. In this study, we examined the toxicity of the venom of Liocheles australasiae (Hemiscorpiidae) to mice and crickets, and characterized the peptide components by HPLC and mass spectrometry. Over 200 components were detected in the L. australasiae venom by LC/MS analysis, with components of molecular masses ranging from 500 to 5000 Da being particularly abundant. A number of peptides contained two to four disulfide bridges, which was estimated based on the mass difference after derivatization of Cys residues. A peptide having a monoisotopic molecular mass of 7781.6 Da and four disulfide bridges was isolated from the venom. The peptide has a primary structure similar in terms of the position of eight Cys residues to those observed in several peptides found from scorpions, ticks and insects, although biological roles of these peptides are unknown.     

Biochemical profile of dogs experimentally envenomed with Tityus serrulatus scorpion venom

The aim of this study was to evaluate the canine blood and urinary profiles after envenomation by Tityus serrulatus venom. Twelve dogs were randomly distributed into two equal groups. Control group animals received 0.5 mL phosphate buffered saline (PBS) injected subcutaneously into the internal portion of the left thigh, whilst dogs in the envenomed group were injected with scorpion venom (250 μg/kg in 0.5 mL PBS). No significant alterations were detected in the urine of envenomed dogs. Levels of plasma glucose and serum urea, creatinine, total protein, potassium, alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK), lactate dehydrogenase (LDH), and amylase were determined. Semi-quantitative analysis of serum cardiac troponin I (cTnI) was performed using an immunochromatographic test. The concentrations of cortisol and insulin were determined using commercial radioimmunoassay kits. Increases in serum cortisol levels in experimental group animals coincided with hyperglycaemia and was probably a response to pain. Increased insulin levels were observed during the hyperglycaemic peaks. Envenomed dogs presented discreet increases in ALT, AST and CK, but no alterations in LDH, amylase, cTnI, urea, creatinine and potassium levels were observed. It was concluded that the venom of T. serrulatus induces blood and urinary biochemical changes in dogs.     

Re-investigation of venom chemistry of Solenopsis fire ants. I. Identification of novel alkaloids in S. richteri

Dialkylpiperidines are characteristic of fire ants in the genus Solenopsis (Hymenoptera: Formicidae). Workers of the black imported fire ant, S. richteri produce cis and trans stereoisomers of 2,6-dialkylpiperidines with the trans isomer predominating. We used silica gel short column chromatography to separate both stereoisomers (cis and trans) of S. richteri venom alkaloids and coupled gas chromatography mass spectrometry (GC-MS) to identify novel minor components. The identities of various peaks in GC-MS analyses of the venom fractions were based on relative retention times and mass spectral data. GC profiles verified the presence of both cis and trans stereoisomers of C_15:1 and C₁₅ in S. richteri. The GC trace of the cis stereoisomers of S. richteri alkaloids was presented for the first time. In addition to the previously described components of S. richteri venom, seven novel 2,6-dialkyl-Δ^1,2-piperideines and 2,6-dialkyl-Δ^1,6-piperideines were detected. The chemical identities of these minor components were determined by comparing with fragmentations of known compounds. Possible biosynthetic pathways for the production of cis and trans solenopsins by S. richteri are discussed.     

Preclinical testing of Peruvian anti-bothropic anti-venom against Bothrops andianus snake venom

Bothrops andianus is a venomous snake found in the area of Machu Picchu (Peru). Its venom is not included in the antigenic pool used for production of the Peruvian anti-bothropic anti-venom. B. andianus venom can elicit many biological effects such as hemorrhage, hemolysis, proteolytic activity and lethality. The Peruvian anti-bothropic anti-venom displays consistent cross-reactivity with B. andianus venom, by ELISA and Western Blotting and is also effective in neutralizing the venom's toxic activities.