Venom Fractions Research Articles

Calcium channel subtypes – another layer of complexity to an already intricate story

Calcium channel subtypes – another layer of complexity to an already intricate story

Comparison of the medium molecular weight venom fractions from five species of common social wasps by MALDI‐TOF spectra profiling

The average spectral profiles and the exact mass weight (MW) of biomolecules present in the medium fraction (from 900 to 3000 Da) of the venom of five social wasps (three European and one North American Polistes and the European hornet Vespa crabro) were determined by matrix assisted laser desorption ionization time of flight (MALDI-TOF) MS. Data were obtained analyzing the venom of single specimens (N = 46) and elaborated with the ClinProTools 2.0 (CPT) software to search for differences among the five species examined. Interesting differences in the spectral profiles were found, allowing the discrimination of venoms belonging to the different species, and their possible use as a quality control method in venom immunotherapy (VIT) for allergic patients.

Effects of Russell's viper venom fractions on systemic and renal hemodynamics

Changes in systemic and renal hemodynamics induced by Russell's viper venom are well established. The component of the venom responsible for hemodynamic alteration has not been identified. By Sephadex column chromatography five fractions of Russell's viper ( Daboia russellii siamensis) venom were isolated. Each venom fraction consisted of phospholipase A 2, proteolytic enzyme, phosphomonoesterase, phosphodiesterase, arginine ester hydrolase and hyaluronidase of varying activities. Hemodynamic effects of each venom fraction including mean arterial pressure, cardiac output, systemic and renal vascular resistance, renal blood flow and glomerular filtration rate were studied in five groups of dogs; each group had four dogs. Minimal hemodynamic changes were observed in dogs receiving venom fraction I. Increased renal vascular resistance with diminution of renal blood flow and glomerular filtration rate was observed in dogs receiving venom fractions II, III, IV and V. A markedly increased renal vascular resistance with maximal decrease in renal blood flow and glomerular filtration rate was caused by fraction III of the venom with highest PLA 2 and proteolytic enzyme activities. However, renal hemodynamic changes appeared to correlate better with proteolytic enzyme activity than PLA 2 activity. The findings suggested the proteolytic enzyme as an important determinant of hemodynamic alteration. Fractional excretion of Na was increased in dogs injected with venom fraction IV, and is presumed to be due to the inhibition of tubular reabsorption of Na by a natriuretic factor in this venom fraction.

A novel depolarizing activity of scorpion venom fraction M1 due to activation of skeletal muscle nicotinic receptors

A depolarizing activity following interaction with nicotinic acetylcholine receptors (nAchRs) in skeletal muscle cells, was observed for the first time in the non-toxic venom fraction (M1) of the yellow scorpion Buthus occitanus tunetanus (Bot). The effects of M1 fraction were tested on cultured rat myotubes by recording changes in [Ca 2+]i. When applied, M1 (10 μg/mL) induced a transient increase of [Ca 2+] i which could be blocked by a prior application of α-Bungarotoxin ( α-Bg-Tx).

Depression of cardiac L-type calcium current by a scorpion venom fraction M1 following muscarinic receptors interaction involving adenylate cyclase pathway

The effects of a non-toxic fraction, called M1, from Buthus occitanus tunetanus (Bot) scorpion were studied on rat cardiac contraction and calcium transient and current. A decrease in both rate and tension on isolated intact hearts as well as in calcium transient induced by depolarizing 100 K + solution on isolated ventricular cardiomyocytes was firstly observed. Studies with the whole cell patch clamp method showed that M1 decreased the L-type calcium current ( ICa(L)) in a dose-dependent manner with an IC50 of 0.36 μg/mL and a Hill coefficient of 0.95. This effect was blocked and reversed by the specific muscarinic receptors antagonist atropine, 1 μM, and was completely prevented when cardiomyocytes were pretreated with Pertussis toxin, 1 μg/mL, to block the α subunit of the PTX-sensitive G proteins. These results show that M1 fraction of Bot inhibits basal calcium current by interacting with muscarinic receptors and suggest that this inhibition could be attributed to inhibition of adenylate cyclase activity by a mechanism involving PTX-sensitive G proteins.

Neuroprotective effect on brain injury by neurotoxins from the spider Phoneutria nigriventer

The role of calcium channels blockers in ischemic condition has been well documented. The PhTx3 neurotoxic fraction of the spider Phoneutria nigriventer venom is a broad-spectrum calcium channel blocker that inhibits glutamate release, calcium uptake and also glutamate uptake in synaptosomes. In the present study we describe the effect of PhTx3 (1.0 μg/mL), ω-conotoxin GVIA (1.0 μmol/L) and ω-conotoxin MVIIC (100 nmol/L) on neuroprotection of hippocampal slices and SN56 cells subjected to ischemia by oxygen deprivation and low glucose insult (ODLG). After the insult, cell viability in the slices and SN56 cells was assessed by confocal microscopy and epifluorescence, using live/dead kit containing calcein-AM and ethidium homodimer. Confocal images of CA1 region of the rat hippocampal slices subjected to ischemia insult and treated with ω-conotoxin GVIA, ω-conotoxin MVIIC and PhTx3 showed a percentage of dead cells of 68%, 54% and 18%, respectively. The SN56 cells subjected to ischemia were almost completely protected from damage by PhTx3 while with ω-conotoxin GVIA or ω-conotoxin MVIIC the cell protection was only partial. Thus, PhTx3 provided robust ischemic neuroprotection showing potential as a novel class of agents that targets multiple components and exerts neuroprotection in in vitro model of brain ischemia.

A novel peptide from the ACEI/BPP-CNP precursor in the venom of Crotalus durissus collilineatus

In crotaline venoms, angiotensin-converting enzyme inhibitors [ACEIs, also known as bradykinin potentiating peptides (BPPs)], are products of a gene coding for an ACEI/BPP-C-type natriuretic peptide (CNP) precursor. In the genes from Bothrops jararaca and Gloydius blomhoffii, ACEI/BPP sequences are repeated. Sequencing of a cDNA clone from venom glands of Crotalus durissus collilineatus showed that two ACEIs/BPPs are located together at the N-terminus, but without repeats. An additional sequence for CNP was unexpectedly found at the C-terminus. Homologous genes for the ACEI/BPP-CNP precursor suggest that most crotaline venoms contain both ACEIs/BPPs and CNP. The sequence of ACEIs/BPPs is separated from the CNP sequence by a long spacer sequence. Previously, there was no evidence that this spacer actually coded any expressed peptides. Aird and Kaiser (1986, unpublished) previously isolated and sequenced a peptide of 11 residues (TPPAGPDVGPR) from Crotalus viridis viridis venom. In the present study, analysis of the cDNA clone from C. d. collilineatus revealed a nearly identical sequence in the ACEI/BPP-CNP spacer. Fractionation of the crude venom by reverse phase HPLC (C 18), and analysis of the fractions by mass spectrometry (MS) indicated a component of 1020.5 Da. Amino acid sequencing by MS/MS confirmed that C. d. collilineatus venom contains the peptide TPPAGPDGGPR. Its high proline content and paired proline residues are typical of venom hypotensive peptides, although it lacks the usual N-terminal pyroglutamate. It has no demonstrable hypotensive activity when injected intravenously in rats; however, its occurrence in the venoms of dissimilar species suggests that its presence is not accidental. Evidence suggests that these novel toxins probably activate anaphylatoxin C3a receptors.

Novel non-enzymatic toxic peptide of Daboia russelii (Eastern region) venom renders commercial polyvalent antivenom ineffective

The snake venoms are typically complex mixtures of enzymes and non-enzymatic peptides. Regional variation in the non-enzymatic fraction of Russell's viper venom from three regions of India studied. The eastern, western and southern regional venom upon gel permeation chromatography on sephadex-G-75 column resolved into three peaks. All the three overlapping peaks differ in their lethality and enzymatic potency. Peak III of all the regional venom found to be non-enzymatic, Western and southern regional venom has trypsin inhibitory activity with varying potencies. Interestingly, the peak III of eastern region is devoid of trypsin inhibitory activity. But it is highly lethal with a LD 50 0.7 mg/kg body weight and also it exhibited post-synaptic neurotoxicity. On the other hand southern and western regional venom's non-enzymatic peak is non-lethal and did not induce neurotoxic symptoms in experimental model. The antibodies developed against the eastern regional venom cross-reacted with the peaks I and II of other regional venom, but failed to cross-react with the peak III of western and southern regional Russell's viper venom. Commercial anti-venom prepared to neutralize the toxic effects of common poisonous snakes of India, showed positive cross-reaction against peaks I, II and III of all three regional venom tested, except peak III of eastern regional venom. Commercial anti-venom neutralized the lethal toxicity of both western and southern regional Russell's viper venom, and failed to neutralize the lethal effects of eastern regional Russell's viper venom.

Partially purified Grammostola spatulata venom inhibits stretch activated calcium signaling in bladder myocytes and improves bladder compliance in an in vitro rat whole bladder model.

Stretch activated nonselective cationic channels (SACs) are present in urinary bladder myocytes and thought to be activated during bladder filling. We investigated the relationship of stretch induced calcium signaling inhibition in bladder myocytes and bladder compliance modulation in an in vitro whole bladder model. Grammostola spatulata venom (SpiderPharm, Yarnell, Arizona) was purified by preparative high performance liquid chromatography. The resulting fractions were examined for their ability to inhibit the swelling activated intracellular free Ca2+ signal in cultured bladder myocytes. An in vitro rat whole bladder model was used to examine the effect of venom fractions on compliance, emptying and spontaneous contractions during bladder filling. The gadolinium ion, a SAC inhibitor, and venom fractions caused concentration dependent inhibition of the swelling activated intracellular free Ca2+ signal in bladder myocytes. When tested in a rat isolated whole bladder model, 0.1 and 0.2 mg./ml. partially purified venom produced a significant improvement in compliance (p <0.05), caused significant inhibition of the frequency of spontaneous bladder contractions (mean +/- SEM 35.8% +/- 3.7% and 62.3% +/- 4.4%, respectively, p </=0.001) and significantly reduced spontaneous bladder emptying, that is emptying in the absence of exogenous stimulation (38.8% +/- 5.6% and 43.9% +/- 2.5%, respectively, p </=0.0008). However, it produced little or no inhibition of carbachol induced bladder emptying. Our data suggest that the activation of stretch induced signaling in bladder myocytes may have an important role in myogenic regulation of bladder contractility during bladder filling. Inhibition of SACs may improve bladder compliance.

Screening for proteolytic activities in snake venom by means of a multiplexing electrospray ionization mass spectrometry assay scheme

A multiplexed mass spectrometry based assay scheme for the simultaneous determination of five different substrate/product pairs was developed as a tool for screening of proteolytic activities in snake venom fractions from Bothrops moojeni. The assay scheme was employed in the functional characterization of eight model proteases. Time-resolved reaction profiles were generated and the relative reaction progress at each time point was determined. These were used to semi-quantitatively sort the catalytic activities of each enzyme towards the respective substrates into six classes. The resulting activity pattern served as an activity fingerprint for each enzyme. The multiplex assay scheme was then applied to a screening for proteolytic activities in fractions of the pre-separated venom from B. moojeni. Activity patterns of each fraction were generated and used to sort the fractions into three different categories of activity. By comparison of the fingerprint activity patterns of the venom fractions and the model enzymes, a compound with proteolytic properties similar to activated protein C was detected.

A low-cost method to test cytotoxic effects of Crotalus vegrandis (Serpentes: Viperidae) venom on kidney cell cultures

The pathogenesis of the renal lesion upon envenomation by snakebite has been related to myolysis, hemolysis, hypotension and/or direct venom nephrotoxicity caused by the venom. Both primary and continuous cell culture systems provide an in vitro alternative for quantitative evaluation of the toxicity of snake venoms. Crude Crotalus vegrandis venom was fractionated by molecular exclusion chromatography. The toxicity of C. vegrandis crude venom, hemorrhagic, and neurotoxic fractions were evaluated on mouse primary renal cells and a continuous cell line of Vero cells maintained in vitro. Cells were isolated from murine renal cortex and were grown in 96 well plates with Dulbecco's Modified Essential Medium (DMEM) and challenged with crude and venom fractions. The murine renal cortex cells exhibited epithelial morphology and the majority showed smooth muscle actin determined by immune-staining. The cytotoxicity was evaluated by the tetrazolium colorimetric method. Cell viability was less for crude venom, followed by the hemorrhagic and neurotoxic fractions with a CT50 of 4.93, 18.41 and 50.22 microg/mL, respectively. The Vero cell cultures seemed to be more sensitive with a CT50 of 2.9 and 1.4 microg/mL for crude venom and the hemorrhagic peak, respectively. The results of this study show the potential of using cell culture system to evaluate venom toxicity.

Management of severe scorpion sting at rural settings: what is the role of scorpion antivenom?

venom is a potent sodium channel activator resulting in autonomic storm (4). Iberitoxin content of venom inhibit the calcium-dependent potassium channels (17). Clinical manifestations, such as vomiting, profuse sweating, priapism in male, cool extremities, hypertension, hypotension, bradycardia, tachycardia, ventricular premature ventricular contraction, transient runs oftachycardia, transient coronary sinus rhythm, pulmonary edema accompanied by marked tented T-waves, ST segment depression, acute myocardial infarction-like pattern, left bundle branch block, and prolongation of QTc interval (400-650 milliseconds). Clinical effects caused by venoms are due to the release of autopharmacological agents into the circulation (4,13). Pharmacokinetic analysis of experimental data on the venom distribution showed that its uptake by the tissue compartment is rather rapid, with estimated half-life of 5.6 minutes; its concentration showed a continuous rise, reaching a peak level within 37 minutes (14). It is interesting to note that, irrespective of different species of scorpions, similar clinical manifestations have been reported (2,11). It confirms that the lethal fraction of venom from different species of scorpion has similar effects on the autonomic nervous system.

Proteins in the high molecular weight fraction of honeybee venom

Proteins in the high molecular weight fraction of honeybee venom

The improvement of the therapeutic anti- Lachesis muta serum production in horses

The main features associated with pit viper envenomations include the intense local lesions such as oedema, necrosis, acute renal failure and other effects. The severity of these reactions to snakebite depends on the degree of envenomation. Lachesis muta venom (LMV) has weak lethal activity, but due to the large amount often inoculated, the effects are extremely severe and demand anti-venom with a high neutralizing capacity. LMV had the lowest neutralizing antibody induction capacity in horses when compared with that of other venoms. For example, Bothrops anti-venom serum neutralizes 180 times the equivalent LD 50 to Bothrops venom; Crotalus anti-venom neutralizes 250 LD 50 of this venom, while Lachesis anti-venom neutralizes only five LD 50 of the Lachesis toxins. To examine the reasons for this low antibody induction, the H GP mouse line, genetically selected for high antibody production received, at different times during immunization with sheep erythrocytes (SE), whole LMV and isolated venom fractions I–VI eluted by gel-filtration chromatography on Superdex ®75. The specific antibody responsiveness showed a partial, but significant suppression of the anti-SE antibody responses during the kinetics of the primary and even the secondary immunizations, after 50–100 μg of fractions IV and V administration 72–48 h before the first antigen injections. Fraction IV was then applied in a Superose ®12 column and three samples were obtained. The peak IVA containing a component of Mr 27 kDa was liable with the immunosuppressive effect as made evident by its effect on the H mice anti-SE responses. Horses receiving the LMV exempt of fractions IV and V produce highly significant anti- Lachesis sera with a 45 LD 50 neutralizing activity, providing, for the first time, an efficient specific therapeutic heterologous serum for human use.

Characterization and biochemical analyses of venom from the ectoparasitic waspNasonia vitripennis (Walker) (hymenoptera: Pteromalidae)

During parasitism, the ectoparasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) induces a developmental arrest in host pupae that is sustained until the fly is either consumed by developing larvae or the onset of death. Bioassays using fluids collected from the female reproductive system (calyx, alkaline gland, acid gland, and venom reservoir) indicated that the venom gland and venom reservoir are the sources of the arrestant and inducer(s) of death. Infrared spectroscopic analyses revealed that crude venom is acidic and composed of amines, peptides, and proteins, which apparently are not glycosylated. Reversed phase high performance liquid chromatography (HPLC) and sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the proteinaceous nature of venom and that it is composed mostly of mid to high molecular weight proteins in the range of 13 to 200.5 kilodaltons (kDa). Ammonium sulfate precipitation and centrifugal size exclusion membranes were used to isolate venom proteins. SDS-PAGE protein profiles of the isolated venom fractions displaying biological activity suggest that multiple proteins contribute to arresting host development and eliciting death. Additionally, HPLC fractionation coupled with use of several internal standards implied that two of the low molecular weight proteins were apamin and histamine. However, in vitro assays using BTI-TN-5B1-4 cells contradict the presence of these agents.

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.

Milleporin-1, a new phospholipase A 2 active protein from the fire coral Millepora platyphylla nematocysts

Stings of fire corals, potent hydroids common in the Red Sea, are known to cause severe pain and they develop burns and itching that lasts few hours after contact. Nematocyst venom of Millepora platyphylla (Mp-TX) was isolated according to a recent method developed in our laboratory to conduct a previous investigation on the nematocyst toxicity of Millepora dichotoma and M. platyphylla. In this study, Mp-TX was fractionated by using both gel filtration and ion exchange chromatography. Simultaneous biological and biochemical assays were performed to monitor the hemolytic (using washed human red blood cells, RBCs) and phospholipase A 2 (using radiolabeled sn-2 C 14-arachidonyl phosphatidylcholine as a substrate) active venom fractions. The magnitude of both hemolysis and phospholipase A 2 activity was found in a fraction rich of proteins of molecular masses ∼30,000–34,000 Daltons. The former fraction was purified by ion exchange chromatography, and a major bioactive protein factor (approx. 32,500 Daltons , here named milleporin-1) was recovered. Milleporin-1 enzymatic activity showed a significant contribution to the overall hemolysis of human RBCs. This activity, however, could not be completely inhibited using phospholipid substrates. Melliporin-1 fraction retained about 30% hemolysis, until totally rendered inactive when boiled for 3 min. The overall mechanism of action of milleporin-1 to impact the cellular membrane was discussed; however, it is pending more biochemical and pharmacological future studies.

Electrospray ionization quadrupole time‐of‐flight and matrix‐assisted laser desorption/ionization tandem time‐of‐flight mass spectrometric analyses to solve micro‐heterogeneity in post‐translationally modified peptides from Phoneutria nigriventer (Aranea, Ctenidae) venom

Previous studies of the fractionated venom of the Brazilian armed spider Phoneutria nigriventer, obtained by gel filtration, have demonstrated the presence of a fraction PhM, a pool of small peptides (up to 2000 Da) that provoke contractions in smooth muscle of guinea pig ileum. Initial attempts to sequence these peptides were largely unsuccessful because of the low purification yield and the fact that the majority seemed to be blocked at their N-termini. In the present work, analysis of this venom fraction by mass spectrometry has revealed the existence of a highly complex mixture of peptides with molecular weights corresponding to those observed for the muscle-active peptides previously described (800-1800 Da). These peptides appear to be a family of isoforms with some particular features. The amino acid sequences of 15 isoforms have been determined by tandem mass spectrometry (MS/MS) using both electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q/ToFMS) and matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-ToF/ToFMS). These molecules contain post-translational modifications such as proteolysis and C-terminal amidation, which combine to generate additional isoforms. All the isoforms sequenced in this study possess an N-terminal pyroglutamic acid residue. A search for sequence similarities with other peptides in databanks revealed that these peptides are structurally related to the tachykinins, a family of neuro-hormone peptides. The data obtained in this study will be essential for the subsequent steps of this research, the synthesis of these peptides and pharmacological characterization of their biological activity.

Lonomia obliqua caterpillar venom increases permeability of the blood–brain barrier in rats

Human envenoming by caterpillars of the saturniid moth Lonomia obliqua in southern Brazil produces a mild local response (erythema, some edema, and pain) and systemic effects that include incoagulable blood, renal failure and in severe accidents intracerebral hemorrhage. In this work, we used light and electron microscopy to investigate the morphological alterations in the brain and blood–brain barrier of rats injected intravenously with venom from L. obliqua spicules (200 μg/kg). Five semi-purified fractions of venom (200 μg/kg each) were also assayed. Quantitative morphological and ultrastructural analyses were done 6, 18, 24 and 72 h after the i.v. injection of venom and its fractions. Light microscopy showed that 6 h after envenoming there was cerebellar edema, which decreased by 72 h. Intracerebral hemorrhage occurred in only one rat 24 h after the injection of venom. Blood–brain barrier (BBB) breakdown, assessed by transmission electron microscopy based on the passage of an extracellular tracer (lanthanum nitrate) between brain capillary endothelial cells, was observed in the cerebellum and hippocampus 18 h after venom injection. At this time, the cerebellum was more sensitive to the venom than the hippocampus, as shown by the greater number of leaky vessels. The number of capillaries showing breakdown was lower after 72 h than after 18 h. None of the semi-purified fractions significantly increased the number of leaky vessels. These results indicate that L. obliqua caterpillar venom has a deleterious action on the rat BBB. The lack of effect of the venom fractions when administered alone suggested that a synergistic action of venom components may be responsible for the damage seen in the central nervous system, but this was not confirmed when three combinations of the fractions were tested.

Morphofunctional Changes in Incubated Mauthner Neurons in Goldfish Treated with Peptides from Scorpion Venom

Electron microscopy with negative contrast showed that direct interaction of one of the peptide fractions of scorpion venom with monomeric chromatographically pure actin led to polymerization of actin, transforming it from the globular form to the fibrillar form. The effects of prolonged orthodromic stimulation on the evoked electrical activity and ultrastructure of Mauthner neurons (MN) were studied in incubated slices of goldfish medulla oblongata in the presence of this actin-polymerizing venom fraction. Peptides in this fraction were found to stabilize the amplitude of the electrical response of MN to exhaustion and to protect the ultrastructure of afferent chemical synapses and the neurons themselves from damage induced by stimulation. Enhancements in morphofunctional resistance were accompanied by stabilization of actin-containing specialized synaptic structures--desmosome-like contacts. The data obtained here provide evidence that peptides of this fraction of scorpion venom have direct actions on the actin component of the MN cytoskeleton and demonstrate potential for its use as a pharmacological tool able to penetrate living cells with value for studying the role of actin in the mechanisms of adaptation and memory.