Inhibition of red cell Ca2+-dependent K+ channels by snake venoms

We have investigated the effects of several snake venoms on the Ca2+-dependent K+ channels of human red cells. A heat-resistant component of the venom of the snake Notechis scutatus irreversibly inhibited Ca2+-dependent K+ transport with a Ki value of 0.1-0.2 micrograms/ml. Metabolic changes of the cells modified the maximal effect of the venom. Binding of the venom required extracellular Ca2+ and was quick, but development of full inhibition required additional time. The effects of the venoms from Notechis scutatus and Leiurus quinquestriatus were additive, suggesting that both venoms act through different mechanisms. Venoms of the snakes Vipera russelli russelli and Oxyuranus scutellatus also inhibited Ca2+-dependent K+ transport with the same characteristics as the Notechis scutatus venom.     

Inhibition of Sendai virus by various snake venom.

Viperid, elapid and crotalid snake venoms were screened in vitro for antiviral activity against Sendai virus. The hemolysis of 10(8) human erythrocytes in 1 ml, caused by 70 HAU of Sendai virus, was abolished when the virions were pretreated with 10 ug of the viperid venom of Echis coloratus, and was considerably diminished when pretreated with 10 ug of the venom of Echis carinatus sochureki, the cobra venoms of Naja atra and Naja nigricollis nigricollis. These venoms did not affect the erythrocytes but inhibited the virions themselves irreversibly. All other examined snake venoms had low or no antiviral activity. There was no correlation between the proteolytic and the antiviral activity of the venoms.     

The "echis carinatus venom" prothrombin assay in coumarin treated patients. A comparison with one-stage and immunological assays.

Prothrombin (factor II) was assayed in a group of coumarin treated patients using the Echis carinatus venom as thromboplastin. The levels obtained were comparable to those observed using the classical one-stage method. A good correlation was in fact observed between the two methods. The levels observed by the Echis carinatus method were definitely lower than those obtained using two immunological methods indicating that Echis carinatus venom activated, in our system, only normal prothrombin. However, even the levels obtained immunologically were slightly decreased, regardless of the method used, as compared to pooled normal plasma. In congenital prothrombin deficiency (homozygotes and heterozygotes) the level obtained by the Echis carinatus method was comparable to that observed by the one-stage method. On the contrary, in a congenital dysprothrombinemia (prothrombin Padua) a normal level was observed whereas the one-stage and two-stage methods yielded constantly levels of about 50% of normal.     

Monoclonal antibody immunoaffinity chromatography of the nerve growth factor from snake venoms

1. Pure monoclonal antibodies to Vipera lebetina venom nerve growth factor have been isolated by affinity chromatography using CNBr-agarose bound antigen. 2. Nerve growth factors from ten snake venoms (Vipera lebetina, Vipera russellii, Vipera berus berus, Vipera ursini, Echis carinatus, Agkistrodon halys, Bungarus caeruleus, Naja naja oxiana, Naja naja, Naja naja atra) were purified using monoclonal antibodies against NGF linked to BrCN-activated agarose.     

Cytoskeletal rearrangements in human red blood cells induced by snake venoms: light microscopy of shapes and NMR studies of membrane function

RBCs (red blood cells) circulating through narrow blood capillaries withstand major deformation. The mechanical and chemical stresses commonly exerted on RBCs continue to attract interest for the study of membrane structure and function. Snake venoms are lethal biochemical 'cocktails' that often contain haemotoxins, metalloproteinases, myotoxins, neurotoxins, phosphodiesterases, phospholipases and proteases. We have monitored the effects of 4 snake venoms (Pseudechis guttatus, Oxyuranus scutellatus, Notechis scutatus and Naja kaouthia) on human RBCs using NMR spectroscopy, DIC (differential interference contrast) and confocal light microscopy. RBCs underwent reproducible stomatocytosis, with unusual geographical-like indentations, spherocytosis, followed by rapid lysis. Confocal micrographs using a fluorescent dye linked to phalloidin showed that the change in morphology was associated with the aggregation of actin in the cytoskeleton. (31)P NMR saturation transfer experiments recorded transport of the univalent anion HPA (hypophosphite) on a subsecond time scale, thereby reporting on the function of capnophorin or Band 3 linked to the cytoskeleton; anion-exchange activity was substantially reduced by venom treatment. We propose a molecular-cytological hypothesis for the shape and functional changes that is different from, or supplementary to, the more 'traditional' bilayer-couple hypothesis more often used to account for similar morphological changes invoked by other reagents.     

Evidence for existence of venom 5' nucleotidase in multiple forms through inhibition of concanavalin A

Pharmacologically active 5' nucleotidase is a ubiquitously distributed enzyme in snake venoms. In this study the effect of concanavalin A (Con-A) on different snake venoms 5' nucleotidase activity is tested in order to know the protein nature which will ultimately help in purification of the enzyme with high yield. Con-A inhibited Naja naja, Naja kauthia, Naja melanoleuca, Naja naja sputatrix, Agistrodon halys blomhoffii, Bothrops asper and Oxyranus scutellas venom 5' nucleotidase activity at different concentrations. This indicates the presence of glycopart in the protein, thus glycoprotein in nature. Vipera russellii, Vipera plaestenae, Agistrodon contratrix, Bitis orientis, Echis carinatus and Trimeresures malabaricus was not inhibited by Con-A, indicating absence of glycopart in the protein. This study for the first time shows existence of 5' nucleotidase in multimeric forms.     

Short term effects of animal venoms on the mitotic index of the duodenal mucosa of albino rats.

Short term administration of the venoms of the snakes Naja haje, Naja nigricollis, and Cerastes vipera and of the scorpion Leiurus quinquestriatus on the mitotic index of the duodenal mucosal cells of the white rat, Rattus rattus, has been studied. All the venoms increased the number of dividing cells of the duodenal mucosa significantly. Naja haje crude venom was fractionated into three fractions. Fraction I had no effect on the mitotic index whereas fractions II and III increased it significantly. Treatment of rats with Naja haje venom fractions II and III after blocking the histamine or the serotonin receptors did not affect the stimulatory action of the two venom fractions on the mitotic index, which it increased significantly. It was suggested that the venoms of Naja haje, Naja nigricollis, Cerastes vipera, and Leiurus quinquestriatus and Naja haje venom fractions possessed a mitogenic activity. Fraction II of Naja haje venom acted through both the muscarinic and adrenergic receptors while fraction III acted on the adrenergic ones.     

Biochemical and functional characterization of an L-amino acid oxidase isolated from Bothrops pirajai snake venom

In this work we describe the isolation of a new l-amino acid oxidase (LAAO) referred to as BpirLAAO-I from Bothrops pirajai snake venom, which was highly purified using a combination of molecular exclusion, affinity, and hydrophobic chromatography steps. BpirLAAO-I homodimeric acid glycoprotein (approximate Mr and pI of 130,000 and 4.9, respectively) displays high specificity toward hydrophobic/aromatic amino acids, while deglycosylation does not alter its enzymatic activity. The N-terminal LAAO sequence of its first 49 amino acids presented a high similarity between a amino acid sequence with other LAAOs from: Bothrops spp., Crotalus spp., Calloselasma rhodostoma, Agkistrodon spp., Trimeresurus spp., Pseudechis australis, Oxyuranus scutellatus, and Notechis scutatus. BpirLAAO-I induces time-dependent platelet aggregation, mouse paw edema, cytotoxic activity against Escherichia coli, Pseudomonas aeruginosa, Leishmania sp., and tumor cells, and also a typical fago (M13mp18) DNA fragmentation. Platelet aggregation, leishmanicidal and antitumoral activities were reduced by catalase. Thus, BpirLAAO-I is a multifunctional protein with promising biotechnological and medical applications.     

Proteolytic activities of cobra venoms based on inactivation of alpha 2-macroglobulin

The venoms of various cobra species showed a wide range of abilities to cleave hide powder azure, with Naja naja kaouthia and Ophiophagus hannah venoms showing the lowest activities and Naja nivea venom showing the greatest activity on this dye-linked substrate. The activities of the venoms on hide powder did not completely correlate with their ability to inactivate the alpha 2-macroglobulin of human serum. Incubation of 4-5 micrograms of Naja nigricollis venom per microliter of serum for 30 min caused loss of 95% of the alpha 2-macroglobulin activity of the serum. The inactivation was rapid, reaching 80% inactivation 5 min after mixing. This loss of alpha 2-macroglobulin activity was used to quantitate the weak proteolytic activity of N. nigricollis venom and a partially purified sample of the major fibrinogenolytic proteinase of the venom. The inactivation of alpha 2-macroglobulin was also used to compare the proteinase activities of venoms from seven species or subspecies of cobra. Based on alpha 2-macroglobulin inactivation, N. nigricollis had the highest proteinase activity among the tested venoms. The measurement of alpha 2-macroglobulin inactivation should provide a useful alternative to hide powder digestion for demonstration of weak proteolytic activities in venoms.     

Cytotoxic activity of various snake venoms on melanoma, B16F10 and chondrosarcoma

Elapid, crotalid and viperid venoms were screened in vitro and in vivo for cytotoxicity towards B16F10 melanoma and chondrosarcoma cell lines. The cytotoxic activity of elapid venoms was considerably higher than that of viperid or crotalid venoms. Elapid venoms disrupted the cell membrane within the first hour, leading to cell death. The strongest activity was found in the venom of Naja nigricollis. The venoms of some Viperidae and of all Crotalidae examined caused the cells to become rounded, without loss in their original volume, and to form aggregates. These changes were reversible when cells were changed to fresh medium. In vivo experiments with the venom of Naja nigricollis were in total agreement with the results achieved in vitro with melanoma cells and the venom exhibited similar cytotoxic activity on chondrosarcoma, inhibiting its development in vivo.     

A comparative study of the biological properties of Australian elapid venoms

1. The hemorrhagic, procoagulant, anticoagulant, protease, phosphodiesterase, alkaline phosphomonoesterase, L-amino acid oxidase, acetylcholinesterase, arginine ester hydrolase, phospholipase A, 5'-nucleotidase and hyaluronidase activities of 39 samples of venoms from 13 species (15 taxa) of Australian elapids were determined and the Sephadex G-75 gel filtration patterns for some of the venoms were also examined. 2. The results indicate that Australian elapid venoms can be divided into two groups: procoagulant Australian venoms (including N. scutatus, N. ater, O. scutellatus, O. microlepidotus, P. porphyriacus, T. carinatus, H. stephensii and P. textilis) and non-procoagulant Australian venoms (including A. superbus, P. colletti, P. australis, P. guttatus and A. antarcticus). 3. The non-procoagulant Australian venoms exhibited biological properties similar to other elapid venoms, while the procoagulant Australian venoms exhibited some properties characteristic of viperid venoms. 4. The data show that information on venom biological properties can be used for differentiation of many species of Australian elapids. 5. Particularly useful for this purpose are the hyaluronidase, alkaline phosphomonoesterase, acetylcholinesterase, and the procoagulant activities and the Sephadex G-75 gel filtration patterns of the venoms.     

Notechis 11'2, a non-toxic phospholipase A2 from the venom of Notechis scutatus scutatus.

Previously, we deduced the amino acid sequence of a novel phospholipase-A2-like protein (PLA2) from the nucleotide sequence of a cDNA isolated from a library prepared from the venom gland of the Australian elapid Notechis scutatus scutatus. The corresponding protein has now been identified, purified from the venom and named Notechis 11'2. Its complete amino acid sequence has been determined by automated Edman degradation of both the whole protein and peptides generated by Staphylococcus aureus protease digestion and chemical cleavage at a tryptophan residue. As predicted from its sequence which contains all the residues putatively required for PLA2 activity, Notechis 11'2 exhibits an esterase activity, preferentially against neutral phospholipids. However, despite its sequence homology with other highly toxic PLA2 present in the venom of Notechis scutatus scutatus, notechis 11'2 has no lethal activity. This observation further supports the view that the lethal activity of PLA2 from Notechis scutatus scutatus is not due to the esterasic activity only.     

Amino acid sequence of a presynaptic neurotoxin from the venom of Notechis scutatus scutatus (Australian tiger snake).

The complete amino acid sequence of notexin, a presynaptic neurotoxin from the venom of Notechis scutatus scutatus (Australian tiger snake), has been elucidated. The protein consists of a single chain of 119 amino acids cross-linked by seven disulfide bridges and has a formula weight of 13,578. The main fragmentation of the peptide chain was accomplished with a staphylococcal protease specific for glutamoyl bonds. A cyanogen bromide fragment and tryptic peptides were used to align the five major staphylococcal protease peptides. The sequence was determined by Edman degradation using the direct phenylthiohydantoin method and with carboxypeptidase A. Notexin is shown to be homologous to both porcine pancreatic phospholipase A and a phospholipase A from the venom of Naja melanoleuca.     

The biological properties of venoms from juvenile and adult taipan (Oxyuranus scutellatus) snakes.

1. The biological properties of four venom pooled samples from adult taipan (Oxyuranus scutellatus) snakes and one pooled venom sample from six juvenile taipan snakes (11 months old) were compared. 2. The intravenous LD50 (median lethal dose), procoagulant activity and enzymatic activities of the juvenile venom were not significantly different from those of the adult venoms. 3. The juvenile and adult venoms exhibited similar polyacrylamide gel electrophoretic (PAGE) and SDS-PAGE patterns, indicating that they possessed a similar protein composition. 4. The results suggest that there is no significant age-dependency in the biological properties of taipan venom.     

Novel natriuretic peptides from the venom of the inland taipan (Oxyuranus microlepidotus): isolation, chemical and biological characterisation

Three natriuretic-like peptides (TNP-a, TNP-b, and TNP-c) were isolated from the venom of Oxyuranus microlepidotus (inland taipan) and were also present in the venoms of Oxyuranus scutellatus canni (New Guinea taipan) and Oxyuranus scutellatus scutellatus (coastal taipan). They were isolated by HPLC, characterised by mass spectrometry and Edman analysis, and consist of 35-39 amino acid residues. These molecules differ from ANP/BNP through replacement of invariant residues within the 17-membered ring structure and by inclusion of proline residues in the C-terminal tail. TNP-c was equipotent to ANP in specific GC-A assays or aortic ring assays whereas TNP-a and TNP-b were either inactive (GC-A over-expressing cells and endothelium-denuded aortic rings) or weakly active (endothelium-intact aortic rings). TNP-a and TNP-b were also unable to competitively inhibit the binding of TNP-c in endothelium-denuded aortae (GC-A) or endothelium-intact aortae (NPR-C). Thus, these naturally occurring isoforms provide a new platform for further investigation of structure-function relationships of natriuretic peptides.     

Comparative proteomic analysis of the venom of the taipan snake, Oxyuranus scutellatus, from Papua New Guinea and Australia: role of neurotoxic and procoagulant effects in venom toxicity

The venom proteomes of populations of the highly venomous taipan snake, Oxyuranus scutellatus, from Australia and Papua New Guinea (PNG), were characterized by reverse-phase HPLC fractionation, followed by analysis of chromatographic fractions by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. Proteins belonging to the following seven protein families were identified in the two venoms: phospholipase A(2) (PLA(2)), Kunitz-type inhibitor, metalloproteinase (SVMP), three-finger toxin (3FTx), serine proteinase, cysteine-rich secretory proteins (CRISP), and coagulation factor V-like protein. In addition, C-type lectin/lectin-like protein and venom natriuretic peptide were identified in the venom of specimens from PNG. PLA(2)s comprised more than 65% of the venoms of these two populations. Antivenoms generated against the venoms of these populations showed a pattern of cross-neutralization, corroborating the immunological kinship of these venoms. Toxicity experiments performed in mice suggest that, at low venom doses, neurotoxicity leading to respiratory paralysis represents the predominant mechanism of prey immobilization and death. However, at high doses, such as those injected in natural bites, intravascular thrombosis due to the action of the prothrombin activator may constitute a potent and very rapid mechanism for killing prey.     

Snake venomics of African spitting cobras: toxin composition and assessment of congeneric cross-reactivity of the pan-African EchiTAb-Plus-ICP antivenom by antivenomics and neutralization approaches

Venomic analysis of the venoms of Naja nigricollis, N. katiensis, N. nubiae, N. mossambica, and N. pallida revealed similar compositional trends. The high content of cytotoxins and PLA(2)s may account for the extensive tissue necrosis characteristic of the envenomings by these species. The high abundance of a type I α-neurotoxin in N. nubiae may be responsible for the high lethal toxicity of this venom (in rodents). The ability of EchiTAb-Plus-ICP antivenom to immunodeplete and neutralize the venoms of African spitting cobras was assessed by antivenomics and neutralization tests. It partially immunodepleted 3FTx and PLA(2)s and completely immunodepleted SVMPs and CRISPs in all venoms. The antivenom neutralized the dermonecrotic and PLA(2) activities of all African Naja venoms, whereas lethality was eliminated in the venoms of N. nigricollis, N. mossambica, and N. pallida but not in those of N. nubiae and N. katiensis. The lack of neutralization of lethality of N. nubiae venom may be of medical relevance only in relatively populous areas of the Saharan region. The impaired activity of EchiTAb-Plus-ICP against N. katiensis may not represent a major concern. This species is sympatric with N. nigricollis in many regions of Africa, although very few bites have been attributed to it.     

Snake venoms as a source of compounds modulating sperm physiology: Secreted phospholipases A2 from Oxyuranus scutellatus scutellatus impact sperm motility,acrosome reaction and in vitro fertilization in mice

The goal of this study was to identify new compounds from venoms able to modulate sperm physiology and more particularly sperm motility. For this purpose, we screened the effects of 16 snake venoms cleared of molecules higher than 15 kDa on sperm motility. Venoms rich in neurotoxins like those from Oxyuranus scutellatus scutellatus or Daboia russelii, were highly potent inhibitors of sperm motility. In contrast, venoms rich in myotoxins like those from Echis carinatus, Bothrops alternatus and Macrovipera lebetina, were inactive. From the main pharmacologically-active fraction of the Taipan snake O. scutellatus s., a proteomic approach allowed us to identify 16 different proteins, among which OS1 and OS2, two secreted phospholipases A2 (sPLA₂). Purified OS1 and OS2 mimicked the inhibitory effect on sperm motility and were likely responsible for the inhibitory effect of the active fraction. OS1 and OS2 triggered sperm acrosome reaction and induced lipid rearrangements of the plasma membrane. The catalytic activity of OS2 was required to modulate sperm physiology since catalytically inactive mutants had no effect. Finally, sperm treated with OS2 were less competent than control sperm to initiate in vitro normal embryo development. This is the first report characterizing sPLA₂ toxins that modulate in vitro sperm physiology.     

Pulmonary fibrin microembolism with Echis carinatus venom in dogs: effects of a synthetic thrombin inhibitor

We produced pulmonary fibrin microembolism using an infusion of a prothrombin activator (Echis carinatus venom, 30 min, 0.5 NIH thrombin equivalent units/kg) in open-chest mongrel dogs. To determine the nonclotting effects of this venom on edemagenesis we infused an irreversible thrombin inhibitor, D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK, 57 nmol X kg-1 X min-1 for 120 min), alone (n = 5) or with venom (Echis + PPACK, n = 5). The control group (n = 5) was given 1 ml of 0.9% NaCl. A decline in left atrial pressure (means +/- SE, 5.3 +/- 0.4 to 4.0 +/- 0.5 mmHg, P less than 0.05) and cardiac index (149 +/- 10 to 82 +/- 13 ml X min-1 X kg-1, P less than 0.01) in association with a marked increase in pulmonary arterial pressure (14.5 +/- 0.6 to 26.6 +/- 2.5 mmHg, P less than 0.001) and pulmonary vascular resistance (64 +/- 5 to 304 +/- 42 mmHg X ml-1 X min-1 X kg-1, P less than 0.001) was observed after 20 min of venom infusion. During this interval, pulmonary artery wedge pressure increased (4 +/- 1 to 12 +/- 4 mmHg, P less than 0.01) in four of eight animals. Fibrinogen declined below measurable levels and fibrin microemboli were seen in many pulmonary arterioles. These changes were not observed in the Echis + PPACK, PPACK, or control groups. Leukopenia and thrombocytopenia were observed in the Echis and Echis + PPACK groups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Blood coagulation induced by the venom of Bothrops atrox. 1. Identification, purification, and properties of a prothrombin activator

In this paper, we show that the procoagulant action of Bothrops atrox venom is due in part to a protein component that activates prothrombin. The venom prothrombin activator was purified by ion-exchange chromatography and gel filtration. It was separated from a protease by affinity chromatography in a p-aminobenzamidine-CH-Sepharose column. It is a protein of about Mr 70,000, consisting of a single polypeptide chain. We have studied the kinetics of activation of prothrombin under different experimental conditions. The prothrombin activator from B. atrox venom is insensitive to reagents of serine and thiol proteases but is inactivated by ion chelators and by various divalent ions. These results suggest that it is a metalloenzyme. The prothrombin activator from B. atrox venom is inactive on the chromogenic substrates S-2337 and S-2238, and it is selective for prothrombin since it does not act on other blood coagulation factors such as fibrinogen and factor X. We have also studied the pattern of peptide cleavages produced in the human prothrombin molecule during the activation by the activator from B. atrox venom and compared it to that obtained with ecarin, a prothrombin activator from Echis carinatus venom. In the presence of thrombin inhibitors, e.g., hirudin, we found that the activators from B. atrox venom and ecarin act in a similar, or identical, manner by producing a thrombin intermediate, meizothrombin. In the absence of thrombin inhibitors, several peptides are generated, and alpha-thrombin is produced as a consequence of meizothrombin action.