Scyphozoan jellyfish venom metalloproteinases and their role in the cytotoxicity

The present study, for the first time, comparatively investigated the enzymatic activities (proteases and hyaluronidases) in the venoms of four Scyphozoan jellyfish species, including Nemopilema nomurai, Rhopilema esculenta, Cyanea nozakii, and Aurelia aurita. For this, various zymographic analyses were performed using assay specific substrates. Interestingly, all the four jellyfish venoms showed gelatinolytic, caseinolytic, and fibrinolytic activities, each of which contains a multitude of enzyme components with molecular weights between 17 and 130 kDa. These four jellyfish venoms demonstrated a huge variation in their proteolytic activities in quantitative and qualitative manner depending on the species. Most of these enzymatic activities were disappeared by the treatment of 1,10-phenanthroline, suggesting they might be belonged to metalloproteinases. Toxicological significance of these venom proteases was examined by comparing their proteolytic activity and the cytotoxicity in NIH 3T3 cells. The relative cytotoxic potency was C. nozakii > N. nomurai > A. aurita > R. esculenta. The cytotoxicity of jellyfish venom shows a positive correlation with its overall proteolytic activity. The metalloproteinases appear to play an important role in the induction of jellyfish venom toxicities. In conclusion, the present report proposes a novel finding of Scyphozoan jellyfish venom metalloproteinases and their potential role in the cytotoxicity.     

Presence of peptide inhibitors in rattlesnake venoms and their effects on endogenous metalloproteases

Long-term storage of proteins with retention of biological activity is a concern for many actual and potential protein drugs. A model for stabilization of proteins for long periods could exist in natural systems, particularly among viperid snakes whose venoms are rich in lytic enzymes, because when secreted into the lumen, they are stored in an inactive and competent state for many months. One mechanism inhibiting autolysis is the production of (relatively) low affinity peptide enzyme inhibitors. We investigated the distribution of two of these peptides (pEQW and pENW) in venoms from nine species of rattlesnakes and evaluated the role of these peptides in inhibiting and stabilizing isolated major venom metalloproteases (Cvo Pr V and cromipyrrhin) from Crotalus oreganus oreganus and C. mitchelli pyrrhus venom. We show that two endogenous peptides, pEQW and pENW, are present in venoms from ten taxa of Crotalus and Sistrurus and that pEQW inhibits Cvo PrV and cromipyrrhin. The peptide inhibitor pEQW also stabilizes cromipyrrhin against autoproteolysis under extreme conditions (heat). Using these peptides as models, it may be possible to design similar low affinity peptide inhibitors of protein drugs which will increase their stability and/or allow for storage under less stringently controlled conditions.     

毒蛇的毒器和蛇咬的生物学

本文描述毒蛇的毒器,包括毒腺、毒牙、导管和有关肌群,并讨论毒蛇咬伤的生物学。     

A rapid and repeatable method for venom extraction from cubozoan nematocysts.

Various comparative studies into the biological activity and relative toxicity of cubozoan venoms have been investigated, in particular the venom from the potentially lethal cubozoan Chironex fleckeri. Efficient and reliable extraction of venom from nematocysts is essential before any research into venom toxicity can be conducted and previous cited methods of extraction have varied greatly, each with their own associated problems. A new standardised technique for the recovery of venom from nematocysts of cubozoans is investigated to decrease the variation displayed between authors due to differing extraction techniques. The use of a mini bead mill beater, as investigated in this trial, allows for the rapid extraction of venom from nematocysts and is devoid of the previously isolated problems experienced with other methods of venom isolation, such as excessive heat build up.     

Cytotoxic, thrombolytic and edematogenic activities of leucurolysin-a, a metalloproteinase from Bothrops leucurus snake venom.

Leucurolysin-a (leuc-a), a 23kDa non-hemorrhagic metalloproteinase, is found in venom of the viper Bothrops leucurus. Here, we examine the biological consequences of leuc-a, including thrombolytic activity, direct effects on endothelial cells in culture and edematogenic activity in vivo. We demonstrate fibrinolytic activity of leuc-a, in which the protease specifically degrades alpha, beta, and gamma-gamma chains. While not causing hemorrhaging, leuc-a does cause thrombolytic activities in whole blood clots. Endothelial cells are highly resistant to leuc-a in culture. Cell viability suffered only when cells were exposed to large quantities of the protease. Nevertheless, leuc-a induces changes in cell morphology. The impact of leuc-a on cell adhesion was confirmed by an adhesion assay, in which cell adhesion to fibronectin decreased due to leuc-a. This mild cellular impact is unlike that of crude venom, where lower concentrations triggered cell death and a greater reduction in cell adhesion. Also, leuc-a increased microvessel permeability with marked edema in mice peritoneum and foot pads. These effects are similar to those of other P-I class SVPMs. These in vivo effects were weaker when crude venom was tested. In conclusion, albeit not showing significant hemorrhagic activity, leuc-a can induce a prominent edema which appears to be significant in the local effects observed after B. leucurus venom accidents.     

Neutralization of the haemorrhagic activities of viperine snake venoms and venom metalloproteinases using synthetic peptide inhibitors and chelators.

Envenoming by the West African saw-scaled viper, Echis ocellatus resembles that of most vipers, in that it results in local blistering, necrosis and sometimes life-threatening systemic haemorrhage. While effective against systemic envenoming, current antivenoms have little or no effect against local tissue damage. The major mediators of local venom pathology are the zinc-dependant snake venom metalloproteinases (SVMPs). The high degree of structural and functional homology between SVMPs and their mammalian relatives the matrix metalloproteinases (MMPs) suggests that substrate/inhibitor interactions between these subfamilies are likely to be analogous. In this study, four recently developed MMP inhibitors (MMPIs) (Marimastat, AG-3340, CGS-270 23A and Bay-12 9566) are evaluated in addition to three metal ion chelators (EDTA, TPEN and BAPTA) for their ability to inhibit the haemorrhagic activities of the medically important E. ocellatus venom and one of its haemorrhagic SVMPs, EoVMP2. As expected, the metal ion chelators significantly inhibited the haemorrhagic activities of both whole E. ocellatus venom and EoVMP2, while the synthetic MMPIs show more variation in their efficacies. These variations suggest that individual MMPIs show specificity towards SVMPs and that their application to the neutralization of local haemorrhage may require a synthetic MMPI mixture, ensuring that a close structural component for each SVMP is represented.     

Contribution of mast cells and snake venom metalloproteinases to the hyperalgesia induced by Bothrops jararaca venom in rats

Bothrops jararaca venom (Bjv) is known to induce local inflammation and severe pain. Since, mast cells are able to secrete mediators involved in algesic processes, in this study we examined the putative role of these cells in the hyperalgesia triggered by Bjv in the rat paw. We noted that treatment with mast cell stabilizer sodium cromoglicate as well as with histamine and 5-hydroxytriptamine receptor antagonists meclizine and methysergide, respectively, inhibited the Bjv-induced hyperalgesia. In addition, we showed that stimulation of isolated rat peritoneal mast cells with Bjv in vitro resulted in the release of stored and neo-generated inflammatory mediators such as histamine and leukotriene C₄, respectively. Bjv-induced histamine secretion was clearly sensitive to treatment with sodium cromoglicate and sodium nedocromil. We further observed that metalloproteinase inhibitors 1,10-phenantroline and DM43 inhibited mast cell degranulation in vitro, under conditions where inhibitors of phospholipase A₂ as well as of serine- and cysteine-proteinases were inactive. Altogether, our findings indicate that mast cells seem to contribute to the hyperalgesia caused by Bjv in the rat paw, and also provide evidence that this response might be dependent on the ability of the Bjv to activate directly mast cells.     

Expression of mRNAs coding for VAP1/crotastatin-like metalloproteases in the venom glands of three South American pit vipers assessed by quantitative real-time PCR

Snake venom metalloproteases encompass a large family of toxins, with approximately 200 members already catalogued, which exhibit a diversity of structures and biological functions. From this relatively large number, only a dozen examples of apoptosis-inducing metalloproteases, like VAP1 and 2 from the venom of Crotalus atrox, are known. Since most VAP1-like toxins ever characterized were purified from the venom of Viperidae species inhabiting diverse places on earth, we investigate the expression of VAP-like metalloproteases in the venom gland of three representative pit vipers of the Brazilian territory. By molecular cloning and quantitative real-time polymerase chain reaction, using as calibrator gene the Crotalus durissus terrificus homolog of VAP1, named crotastatin, it is reported here that VAP1/crotastatin-like homologues in the venom gland of Bothrops atrox, C. d. cascavella and Lachesis m. rhombeata are expressed at different levels. Hence, batroxstatins, the crotastatin-like precursors from B. atrox, are expressed 87 times more than crotastatin-1, from C. d. cascavella, and 7.5-fold that lachestatins, from L. m. rhombeata. Moreover, in silico structural analysis of amino acid sequences indicates that batroxstatin-2, crotastatins and lachestatin-1 and -2 which share the archetypal motifs and metal- binding sites of VAP1, are subgrouped in a branch that comprises some apoptosis-inducing toxins.     

A snake venom metalloproteinase that inhibited cell proliferation and induced morphological changes of ECV304 cells.

TSV-DM, a basic metalloproteinase with a molecular weight of 110kDa, was purified from Trimeresurus stejnegeri venom. TSV-DM degraded the Aalpha chain of fibrinogen more rapidly than the Bbeta chain in a dose dependent manner. The cDNA of TSV-DM encoded a polypeptide of 622 amino acid residues, which comprises a signal peptide, proprotein, metalloproteinase domain, spacer, disintegrin-like domain and cysteine-rich domain. The protein sequence deduced from cDNA was confirmed by peptide mass fingerprinting analysis. It is highly homologous to the members of subclass P-IIIb snake venom metalloproteinase, which comprises vascular apoptosis-inducing proteins. TSV-DM inhibited cell proliferation and induced cell morphologic changes transiently of ECV304 cells. However, DNA fragmentation and DNA content analysis demonstrated that this metalloproteinase could not induce ECV304 cells apoptosis.     

A biochemical and pharmacological examination of Rhamphiophis oxyrhynchus (Rufous beaked snake) venom.

The venom of R. oxyrhynchus, a member of the psammophiine subfamily of the colubrid assemblage, was examined for biological activity using biochemical and pharmacological techniques. Venom displayed a high protein content, a complex electrophorectic profile and PLA2 activity but no detectable proteolytic or haematological activities. In the chick biventer cervicis nerve muscle preparation, venom (1-10 microg/ml) displayed postsynaptic neurotoxic activity as evidenced by inhibition of indirect (0.1 Hz, 0.2 ms, supramaximal V) twitches and responses to exogenous acetylcholine (1 mM) and carbachol (20 microM). This inhibitory effect was poorly reversible by washing. Venom (30-50 microg/ml) caused a rapid and readily reversible inhibition of direct (0.1 Hz, 2 ms, supramaximal V) twitches of the chick biventer cervicis nerve muscle preparation without morphological changes to the muscle fibers. Venom (30-100 microg/ml) inhibited electrically-evoked (0.2 Hz, 0.3 ms, 70-100 V) twitches of the prostatic segment of the rat vas deferens. This inhibitory effect was not significantly attenuated by 8-phenyltheophylline (8-PT; 20 microM), idazoxan (1 microM), a combination of ranitidine (0.2 microM) and thioperamide (10 microM) or capsazepine (10 microM). Venom (5 mg/kg) induced hypotension with subsequent cardiovascular collapse in the anaesthetised rat. The cardiovascular collapse was prevented by artificial respiration of the animals prior to venom administration. The biological activities demonstrated by R. oxyrhynchus venom may aid in prey envenomation strategies such as prey immobilisation. This study provides further evidence that colubrid venoms are comprised of multiple components which can display a variety of actions, some of which may be novel, therefore reinforcing the largely untapped potential of colubrid venoms.     

The purification and partial characterisation of two novel metalloproteinases from the venom of the West African carpet viper, Echis ocellatus.

Separation of previously uncharacterised Echis ocellatus venom by phenyl-Superose FPLC (Fast Liquid Protein Chromatography) yielded eight protein fractions. Three of these displayed high proteolytic activity when assayed by in vivo and in vitro assays (including enzyme linked immunosorbant assay), and were further separated using Superdex 75 and Mono-Q FPLC. This resulted in the purification of a non-haemorrhagic 24 kDa metalloproteinase (EoVMP1, pI 7.0), and a haemorrhagic 56 kDa metalloproteinase (EoVMP2, pI 5.5). Following tryptic digest, short amino acid sequences of EoVMP1 and EoVMP2 were obtained using Edman degradation. Both sequences displayed homology when aligned with existing snake venom metalloproteinases (SVMPs). The strong homology observed among previously well-characterised SVMPs suggests that principles governing the interaction of substrates and inhibitors are likely to be similar for EoVMP1, EoVMP2 and all members of the reprolysin family.     

Cardiovascular, haematological and neurological effects of the venom of the Papua New Guinean small-eyed snake (Micropechis ikaheka) and their neutralisation with CSL polyvalent and black snake antivenoms.

Cardiovascular and haematological effects of venom of the small-eyed Snake (Micropechis ikaheka) were examined in ventilated anaesthetised piglets. Neurotoxic effects were examined in chick biventer cervicis nerve-muscle preparations. Immunoreactivity of venom was tested against the monovalent antivenom components in a CSL Ltd Venom Detection Kit. Neutralisation was tested in vivo and in vitro with CSL Ltd polyvalent snake and Black Snake (Pseudechis australis) antivenoms. Venom in 0.1% bovine serum albumin in saline was infused into piglets in doses 1-2000 microg/kg. Pulmonary hypertension (P= 0.0007) and depression of cardiac output (P= 0.002) were observed up to 3 h after 150-160 microg/kg. The concentration of plasma free-haemoglobin increased more than 50-fold, indicating haemolysis. Neither coagulopathy nor thrombocytopenia occurred. Creatine phosphokinase and serum potassium levels did not increase suggesting absence of acute rhabdomyolysis. The venom caused post-synaptic neurotoxicty. Immunoreactivity of venom with Black Snake antivenom was observed at very high venom concentrations. Cardiovascular effects were absent and haemolysis was less after venom was pre-incubated at 37 degrees C for 30 min with polyvalent antivenom. Neutralisation by Black Snake antivenom was less effective. The neurotoxicity was neutralised by polyvalent or Black Snake antivenoms. Human envenomation may be treated with CSL Ltd polyvalent snake antivenom.     

Biochemistry and toxicology of toxins purified from the venom of the snake Bothrops asper

The isolation and study of individual snake venom components paves the way for a deeper understanding of the pathophysiology of envenomings – thus potentially contributing to improved therapeutic modalities in the clinical setting – and also opens possibilities for the discovery of novel toxins that might be useful as tools for dissecting cellular and molecular processes of biomedical importance. This review provides a summary of the different toxins that have been isolated and characterized from the venom of Bothrops asper, the snake species causing the majority of human envenomings in Central America. This venom contains proteins belonging to at least eight families: metalloproteinase, serine proteinase, C-type lectin-like, l-amino acid oxidase, disintegrin, DC-fragment, cystein-rich secretory protein, and phospholipase A₂. Some 25 venom proteins within these families have been isolated and characterized. Their main biochemical properties and toxic actions are described, including, in some cases, their possible relationships to the pathologic effects induced by B. asper venom.     

A portable device for the electrical extraction of scorpion venom

An attractive technique to extract scorpion venom is the use of a physiologically stimulating electrical signal across the muscles of the venom gland, resulting in the expression of venom from the aculeus. A Grass™ stimulator is typically used for this purpose, but is difficult to use in the field. The present communication describes a circuit which is battery-powered and simply constructed. Also described is the technique for its construction and housing. The circuit was successfully tested on two species of scorpion. The method for calculating the required values of passive circuit components is given to allow the adaptation and refinement of this circuit for producing different signals, as may be required for use in other species.     

A simply and sensitive fluorometric method for determination of gentamicin in liposomal suspensions

A new method for measuring gentamicin in liposomes fluorometrically is described. The assay is based on the reaction between the amino groups in the gentamicin molecule and o-phthaldialdehyde (OPA), under basic pH conditions; the product's fluorescence can be read directly on a simple fluorimeter. The effects of several factors (time of reaction, volume of the OPA reagent, and product stability) were investigated. The standard curve was linear in the concentration range of 0.5-4.0microg, showing an excellent determination coefficient of r(2)=0.99. Additionally, the influence of different liposomal lipids on gentamicin determination was tested. Liposomal lipids containing no free amino groups (PC, Chol, DOTAP) have no influence on the reaction when present in the reaction mixture. In contrast, amino groups containing lipid (SA) showed intense method interference. Therefore, a method of lipid extraction was adapted to remove undesired lipids. The described method was successfully utilised during 2 years of liposomal gentamicin experiments.     

Development of a rapid and sensitive enzyme-linked immunosorbent assay (ELISA) for measuring venom antigens after an experimental snake bite

, , and . Development of a rapid and sensititve enzyme-linked immunosorbent assay (ELISA) for measuring venom antigens after an experimental snake bite. Toxicon 26, 1157–1167, 1988.—We describe a new ELISA which allows the measurement of the concentration of venom antigens in whole blood. The assay can be performed in less than 20 min and requires a 200 μl sample of blood. It allows the accurate evaluation of concentrations of Vipera ammodytes venom in quantities smaller than 1 ng/ml of blood. Using this ELISA, we were able to follow in rabbits the kinetics of experimental envenomation with non-lethal doses of venom. This ELISA was also used to measure post mortem the level of venom antigens in various tissues such as liver, kidney, muscles and abdominal serosity of a rabbit. The method, which might be adapted to measure envenomation by other snake species, seems to be sufficiently rapid and sensitive to allow routine evaluation of the gravity of a snake bite in humans and to estimate the efficacy of immunotherapy.     

A simple kinetic method for rapid mechanistic analysis of reversible enzyme inhibitors

A simple diagnostic method for mechanistic analysis of reversible enzyme inhibitors is presented. The method involves simple experimentation to determine how the inhibition by a reversible inhibitor changes in response to the substrate concentration varied in the assay. Four types of inhibitors are categorized based on their kinetic characteristic: (1) competitive or mutually exclusive inhibitors that compete with the substrate for the enzyme; (2) noncompetitive inhibitors that are independent of the substrate for binding to the enzyme; (3) antagonistic inhibitors where the binding affinity of the inhibitor is partially reduced by the substrate binding to the enzyme; and (4) synergistic inhibitors where inhibitor binding is enhanced by substrate binding. An equation was derived for data fitting and subsequent determination of inhibitor binding mode. The method was evaluated in three model enzyme systems, i.e., PK, adenylate kinase, and LDH, with known inhibitors. The method was also used to characterize a large number of unknown Csp3 inhibitors identified from HTS of a compound library consisting of 120,000 distinct chemical entities, a field test that validated the utility of the method. Among 76 Csp3 inhibitors analyzed, 70 were found to be non-mutually exclusive inhibitors, suggesting the existence of an allosteric site(s) in Csp3 for effective inhibition. The implication of this observation is discussed.     

Isolation and pharmacological characterisation of papuantoxin-1, a postsynaptic neurotoxin from the venom of the Papuan black snake (Pseudechis papuanus)

The Papuan black snake (Pseudechis papuanus) is found throughout the southern coastal regions of Papua New Guinea and is thought to occur in the adjacent region of Iriyan Jaya. Neurotoxicity is a major symptom of envenomation by this species. This study describes the isolation of the first neurotoxin papuantoxin-1 from the venom of P. papuanus. Papuantoxin-1 (6738Da), which accounts for approximately 5% of the whole venom, was purified to homogeneity using successive steps of RP-HPLC. The toxin (0.3-1.0 microM) caused concentration dependent inhibition of indirect twitches (0.1 Hz, 0.2 ms and supramaximal V) and inhibited the responses to nicotinic agonists in the chick biventer cervicis nerve-muscle preparation, indicating a postsynaptic mode of action. However, papuantoxin-1 displayed no signs of myotoxicity. Papuantoxin-1 displayed pseudo-irreversible antagonism of cumulative concentration-response curves to carbachol at the skeletal muscle nicotinic receptors with an estimated pA2 value of 6.9+/-0.3. CSL black snake antivenom, which is raised against the venom of the Australian black snake Pseudechis australis, appears to be effective in reversing the effects of papuantoxin-1. Thus, black snake antivenom should be considered for the treatment of the neurotoxic effects following envenomation by the Papaun black snake.     

Development and validation of rapid and sensitive HPLC method for the quantitative determination of doxorubicin in human plasma

An isocratic reversed-phase high-performance liquid chromatographic method with ultraviolet detection at 254?nm has been developed for the determination of doxorubicin in human plasma. Plasma samples were extracted by a selective one-step liquid–liquid extraction using dichloromethane. Doxorubicin and the internal standard epirubicin were separated using a column packed with C₁₈ material, using a mobile phase consisting of water:acetonitrile (75:25v/v). The calibration graph for doxorubicin was linear in the range 0.2–10?μg/mL, with a correlation coefficient, R²?=?0.9986. Lower limit of quantitation was 0.2?μg/mL, using 1?mL plasma samples. The extraction recovery ranged from 98.5–101.1%, and the recovery rate was consistent for drug and internal standard examined at each level. The interday and intraday precision values ranged between 0.5–2%. Validation data showed that the assay for doxorubicin is sensitive, selective, accurate, and reproducible. The assay has been used in population pharmacokinetics study.