A snake bite by the Burrowing Asp, Atractaspis engaddensis.

During routine milking of a group of Burrowing Asps Atractaspis engaddensis, one of the authors was bitten in the index finger by one fang, as is characteristic of bites by snakes of the genus. Local effects, oedema, erythema and numbness appeared within minutes, followed by systemic effects, including general weakness, sweating, pallor, fluctuations in the level of consciousness, vomiting and watery non-bloody diarrhoea. Gross oedema of the hand developed and extended up to the forearm. Two hours after admission to the hospital, blood pressure rose to 180/110, the ECG showed normal sinus rhythm and no signs of atrioventricular conduction block. An ECG obtained 24 h after the bite showed new T-wave inversions in leads V5 + 6, which gradually returned to baseline within several days. The local effects healed during the following weeks, but some discoloration and tenderness remained even 10 months after the bite. A maximal exercise (SPECT) study carried out five months after the bite was normal and a multigated radionuclear ventriculogram (MUGA) showed normal left-ventricular function. It may be assumed that the rise in blood pressure observed in this case reflects a systemic vasoconstrictive effect of the sarafotoxins, while the ST changes may have been caused by the direct effect of the toxins on the heart or indirectly by vasoconstriction of the coronary arteries. However, ischaemia secondary to a rise in blood pressure or to excitement could also explain the observed ECG-changes.

Venomous snakes of Israel: ecology and snakebite.

Ten species of venomous snakes belonging to three families occur in Israel and in Jordan, some of which may pose a serious threat to humans. Specific, local antivenins are available against only two of the species, while against others regional or European preparations are used. It is suggested that in addition to the monospecific anti-Vipera palaestinae, a polyspecific antivenin be prepared against the clinically most important venomous snakes of the region, namely, Echis coloratus, Pseudocerastes fieldi, Cerastes cerastes, Walterinnesia aegyptia, and Atractaspis engaddensis.

Resistance of the Egyptian mongoose to sarafotoxins.

The Egyptian mongoose (Herpestes ichneumon) is known for its resistance to viperid and elapid venoms. The current work demonstrates that it is also resistant to the venom of Atractaspis and its most toxic component, sarafotoxin-b. Intravenous administration of this toxin, at a dose of about 13 times LD100 for mice, resulted in disturbance in electrocardiograms in the mongoose, which returned to normal after several hours. Sarafotoxin-b failed to induce contraction of mongoose aortal preparations. Endothelin-1, which was demonstrated in tissue extracts of the mongoose by immunological methods, induced contraction of the isolated mongoose aorta. This contraction, however, was greatly reduced when endothelin-1 was applied on top of sarafotoxin-b. Binding studies revealed endothelin/sarafotoxin-specific binding sites in brain and cardiovascular preparations of the mongoose. It is suggested that some structural features of endothelin/sarafotoxin receptors in the mongoose enable them to differentiate between the two peptides.

The binding site of the nicotinic acetylcholine receptor in animal species resistant to alpha-bungarotoxin.

The ligand binding site of the nicotinic acetylcholine receptor (AChR) is located in the alpha-subunit, within a small fragment containing the tandem cysteines at positions 192 and 193. We have been analyzing the binding site domain of AChRs from several animal species exhibiting various degrees of resistance to alpha-bungarotoxin (alpha-BTX). Our earlier work on the snake and mongoose AChR, both of which do not bind alpha-BTX, suggested that amino acid substitutions at positions 187, 189, and 194 of the AChR alpha-subunit are important in determining the resistance of these AChRs to alpha-BTX. In the present study, we have examined the correlation between alpha-BTX binding and the structure of the binding site domain of AChR from the hedgehog, shrew, cat, and human. Fragments of the AChR alpha-subunit corresponding to residues 122-205 from these species were cloned, sequenced, and expressed in Escherichia coli. The hedgehog fragment does not bind alpha-BTX, in common with the snake and mongoose AChR, and the human fragment is a partial binder. The shrew and cat fragments bind alpha-BTX to a similar extent as the mouse fragment. The hedgehog and human AChRs have nonaromatic amino acid residues at positions 187 and 189 of the alpha-subunit, as is seen with the "toxin resistant" snake and mongoose, and in contrast with the "toxin binders", which have aromatic residues at these two positions.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological activities of [Thr2]sarafotoxin-b, a synthetic analogue of sarafotoxin-b.

The 21 amino acid sarafotoxins (SRTX) c and d/e as well as endothelin-3 (ET-3) are known to be less toxic and weaker pharmacologically than the other isopeptides SRTX-a, SRTX-b and ET-1. Since SRTX-c, SRTX-d/e and ET-3 possess a Thr instead of a Ser at position 2, we investigated the possibility that this mutation could be responsible for the observed biological differences. Here we show that the synthetic [Thr2]SRTX-b has indeed a lower vasoconstriction efficacy (approximately 35%) in the rabbit aorta, but it is nearly as potent as SRTX-b in toxicity tests and in influencing contraction of the rat uterus. Using monoclonal antibodies directed against the structurally related endothelin-1, we also show that the antigenicity of the analogue is comparable to that of SRTX-b, suggesting that the overall structure of the two peptides is similar, despite the substitution at position 2. We suggest that the Thr2 substitution contributes to the lower activity of the 'weak' peptides in some systems; however, additional substitutions found in the 'weak' peptides of the ET/SRTX family most probably contribute to their low pharmacological activity.

Sarafotoxins and endothelins: evolution, structure and function.

The venom of the burrowing asp Atractaspis engaddensis contains several 21 amino acid residue peptides known as sarafotoxins. The sarafotoxins are homologous to the mammalian endothelin family, and they have similar biological activities. This review covers recent advances in the study of the chemical and biological properties of the sarafotoxins and endothelins.

Cloning and sequence analysis of cDNAs encoding precursors of sarafotoxins. Evidence for an unusual "rosary-type" organization.

Sarafotoxins (SRTXs) are 21-amino acid peptides structurally and functionally similar to endothelins (ETs). To understand how SRTXs are overproduced in venom glands of the snakes Atractaspis engaddensis and hence used as toxins, we cloned cDNAs encoding SRTXs and elucidated their nucleotide sequences. We predict that SRTX precursors are large prepropolypeptide chains with an unusual "rosary-type" structure made of 12 successive similar stretches of 40 residues (39 in the first stretch). Each stretch begins with a "spacer" of 19 invariant residues (18 in the first stretch) immediately followed by the sequence of one SRTX isoform. Six different isoforms are identified within a single precursor molecule. Maturation of the precursor may require endopeptidases that cleave the Leu-Cys bond and the Trp-Arg/Lys bond invariably found at the SRTX N and C termini, respectively.

How the mongoose can fight the snake: the binding site of the mongoose acetylcholine receptor.

The ligand binding site of the nicotinic acetylcholine receptor (AcChoR) is within a short peptide from the alpha subunit that includes the tandem cysteine residues at positions 192 and 193. To elucidate the molecular basis of the binding properties of the AcChoR, we chose to study nonclassical muscle AcChoRs from animals that are resistant to alpha-neurotoxins. We have previously reported that the resistance of snake AcChoR to alpha-bungarotoxin (alpha-BTX) may be accounted for by several major substitutions in the ligand binding site of the receptor. In the present study, we have analyzed the binding site of AcChoR from the mongoose, which is also resistant to alpha-neurotoxins. It was shown that mongoose AcChoR does not bind alpha-BTX in vivo or in vitro. cDNA fragments of the alpha subunit of mongoose AcChoR corresponding to codons 122-205 and including the presumed ligand binding site were cloned, sequenced, and expressed in Escherichia coli. The expressed protein fragments of the mongoose, as well as of snake receptors, do not bind alpha-BTX. The mongoose fragment is highly homologous (greater than 90%) to the respective mouse fragment. Out of the seven amino acid differences between the mongoose and mouse in this region, five cluster in the presumed ligand binding site, close to cysteines 192 and 193. These changes are at positions 187 (Trp----Asn), 189 (Phe----Thr), 191 (Ser----Ala), 194 (Pro----Leu), and 197 (Pro----His). The mongoose like the snake AcChoR has a potential glycosylation site in the binding site domain. Sequence comparison between species suggests that substitutions at positions 187, 189, and 194 are important in determining the resistance of mongoose and snake AcChoR to alpha-BTX. In addition, it was shown that amino acid residues that had been reported to be necessary for acetylcholine binding are conserved in the toxin-resistant animals as well.

The secretion of Duvernoy's gland of Malpolon monspessulanus induces haemorrhage in the lungs of mice.

A toxic protein that induces death of mice with profuse bleeding from the nostrils was isolated from the secretion of Duvernoy's gland of Malpolon monspessulanus (Colubridae). The toxic protein, referred to as CM-b, showed mainly one band on SDS-PAGE corresponding to a mol. wt of 24,000. Its intravenous LD50 in mice was 1 microgram/g and i.v. injections of lethal or sublethal doses induced haemorrhage in the lungs. When injected into the skin of mice, however, the toxin was not haemorrhagic. CM-b showed no proteolytic or procoagulant activity. The nature of this and other components from Duvernoy's secretion of colubrids that cause similar effects remains to be established.

Inhibition of the proteolytic activity of hemorrhagin-e from Crotalus atrox venom by antihemorrhagins from homologous serum.

Antihemorrhagic proteins from Crotalus atrox serum were tested for their ability to inhibit the proteolytic activity of the hemorrhagic toxin-e from Crotalus atrox venom and of several other proteolytic enzymes: trypsin, collagenase and thermolysin. The antihemorrhagic proteins inhibited the proteolytic activity of hemorrhagin-e when tested on gelatin type I and collagen type IV, the proteolytic activity of trypsin on photofilm gelatin and the proteolytic activity of whole venom when tested on azocollagen and photofilm gelatin. The antihemorrhagins failed to inhibit the proteolytic activity of trypsin when tested on the specific synthetic substrate N-acetyl-DL-phenylalanine-beta-naphthyl ester (APNE), the activity of microbial collagenase on N-(3-[2-furyl]acryloyl)-Leu-Gly-Pro-Ala (FALGPA) or on azocollagen and the activity of thermolysin on N-(3-[2-furyl]acryloyl)-Gly-Leu amide (FAGLA). It is tentatively suggested that the antihemorrhagins from snake blood serum are proteinase inhibitors that underwent specialization towards the neutralization of the proteolytic activity of hemorrhagic toxins.

Activity of sarafotoxin/endothelin peptides in the heart and brain of lower vertebrates.

The effects of sarafotoxin-b (SRTX-b) and endothelin-1 (ET-1) were tested in the fish tilapia (Ore niloticus x O. aureus hybrids) and torpedo (Torpedo ocellata), the toad (Bufo viridis), the agama lizard (Agama stellio) and water snake (Natrix tessellata). In isolated heart preparations of the fish and agama, peptide doses of 0.05-0.5 micrograms/ml induced positive inotropic effects, reduction of the contraction rate and arrhythmia, leading to cardiac arrest. In the toad, a negative inotropic effect and a reduction of the contraction rate were observed, whereas the water snake was hardly affected by either SRTX-b or ET-1. In the agama, an i.v. injection of 15 micrograms of SRTX-b caused changes in the ECG, culminating in A-V block that led to cardiac arrest, while in the toad an injection of 45 micrograms induced only transient disturbances in the ECG. Binding studies with 125I-SRTX-b revealed specific binding sites for SRTX-b and ET-1 in the heart and brain preparations of fish (tilapia and torpedo) and agama, whereas no specific binding could be demonstrated in the toad or in the snake. These results suggest that most vertebrates tested are sensitive to SRTX/ET, while the snake may possess receptors that are of a different structure.

Endothelins and sarafotoxins: effects on motility, binding properties and phosphoinositide hydrolysis during the estrous cycle of the rat uterus.

The effects of four peptides of the endothelin/sarafotoxin (ET/SRTX) family on the motility of the rat uterus were examined during the different stages of the estrous cycle. ET-1, ET-3, SRTX-b and SRTX-c showed similar effects on the contraction of the uterus: a slight increase in the maximum tension of the spontaneous rhythmic contractions, a suppression of the relaxation phase of these contractions and an increase in their rate. All three effects were concentration dependent. Of the four peptides, ET-1 and SRTX-b showed the highest potency and efficacy, suggesting that among the various peptides of this family so far studied, ET-1 and SRTX-b are the two full agonists. The rank order of susceptibility of the different stages was, in most cases: proestrus greater than estrus greater than metestrus. Freshly excised diestrus uteri showed no spontaneous contractions and did not respond to any of the peptides. The binding potency of ET-1 and SRTX-b to uterine membranes was similar at the various estrous stages, but their maximal binding decreased gradually from proestrus to diestrus. All four peptides induced phosphoinositide (PI) hydrolysis in uterine slices at all four different stages, with ET-1 and SRTX-b again being more potent than ET-3 or SRTX-c. The maximal PI hydrolysis correlated with the increased rate of the rhythmic contractions. It is suggested that the reaction of the rat uterus to the ET/SRTX peptides depends on its hormonal status and that ET may act in concert with steroid hormones in the modulation of the estrous cycle.

Endothelin and sarafotoxin: influence on steroid-regulated motility of rat uterus.

The sarafotoxins (SRTX) and endothelins (ET) were shown to influence the motility of the isolated rat uterus by inducing an increase in the rate and in the maximum tension of the spontaneous rhythmic contractions and a suppression of the relaxation phase of these contractions. Ovariectomized rats, 24 weeks post-operation, show no spontaneous motility of their uteri and the SRTX/ET peptides induce only a slight tonic increase in the uterine tension. Treatment with 17 beta estradiol restores spontaneous motility and sensitivity to the SRTX/ET peptides in all three contraction modes. It is concluded that the influence of the SRTXs and ETs on uterine motility depends on the hormonal status of the animal.

Contractile effects and binding properties of endothelins/sarafotoxins in the guinea pig ileum.

Seven of the eight known isopeptides of the endothelin/sarafotoxin (ET/SRTX) family were tested on the isolated guinea pig ileum and found to cause a concentration-dependent increase in basal tone. The rate or the amplitude of the spontaneous rhythmic contractions of the ileal smooth muscle were essentially not affected by any of the peptides. The maximum contraction elicited by vasoactive intestinal contractor (VIC) was slightly stronger than that induced by endothelin-1 (ET-1) or sarafotoxin-b (SRTX-b), and significantly stronger than the maximal contractions elicited by sarafotoxin-a (SRTX-a), sarafotoxin-c (SRTX-c), or endothelin-3 (ET-3). Sarafotoxin-d (SRTX-d) caused, essentially, no contraction but a rather marked relaxation. The potencies of the various peptides to induce the increase in tension, in terms of EC50 values (cumulative effective concentrations that induce half-maximum response), ranged between 6 and 95 nM depending on the peptide. VIC, ET-1, SRTX-b and SRTX-a had similar potencies and were significantly more potent than SRTX-c and ET-3. A high concentration of SRTX-b elicited no additional response when applied to the organ bath after one of the other peptides had shown a maximal effect. Binding experiments with ileal membranes revealed similar binding properties for the various peptides. Competition with iodinated SRTX-b showed no meaningful differences between the various peptides. It is concluded that all the ET/SRTX peptides compete for the same receptor subtype in the ileum. In terms of efficacy, VIC can be considered as a full agonist of this receptor, SRTX-d is probably an antagonist, while all the other peptides behave as partial agonists.

Kinetic and cross-linking studies indicate different receptors for endothelins and sarafotoxins in the ileum and cerebellum.

Kinetics of ligand/receptor interactions using ET-1, ET-3 and SRTX-b were studied and cross-linking experiments carried out in guinea pig ileum and rat cerebellar preparations. Dissociation studies indicate that the two regions are characterized by different receptor subtypes and different modes of ligand binding. Autoradiographic patterns obtained following cross-linking of ET-1 and ET-3 to the different tissues support these conclusions.

Evolution of the sarafotoxin/endothelin superfamily of proteins.

Sixteen protein and nucleic acid sequences from the vasoconstrictor sarafotoxin/endothelin/endothelin-like superfamily of peptides were studied, and the evolutionary relationships between the sarafotoxin and endothelin gene families as well as the phylogenetic topology within each gene family and the three endothelin subfamilies was reconstructed. The endothelin gene family has diverged from an ancestral gene that has experienced an exon duplication event followed by two gene duplication events. The sarafotoxins' lineage diverged from the ancestral gene prior to the first endothelin gene duplication event. Analysis of the resulting phylogenetic trees revealed that in several lineages, the peptides have independently accumulated identical replacements in position 2, therefore supporting the hypothesis that residue 2 is crucial to their activity.

Purification and properties of an antivenom factor from the plasma of the South American rattlesnake (Crotalus durissus terrificus).

The lethal toxicity of Crotalus durissus terrificus (Crotalinae, Viperidae) can be attributed mainly to the presence of a neurotoxic protein, crotoxin, which also shows phospholipase A2 activity. It has been previously demonstrated that both lethal and phospholipase A2 activities of crotoxin can be neutralized by an alpha 1-globulin factor that is present in the homologous blood. Crotalus durissus terrificus plasma also renders some degree of protection to mice against the lethal toxicity of heterologous venoms from snakes of the genus Bothrops (Crotalinae, Viperidae), but not of the genus Micrurus (Elapinae, Elapidae). An anti-toxic factor was purified to homogeneity from C.d. terrificus plasma after three chromatographic steps (DEAE-Sephacel anion exchange, CM-Sepharose cation exchange and Pro-RPC reverse phase chromatography); it is named CNF for Crotalus neutralizing factor. The purification process was accompanied by polyacrylamide gel electrophoresis in the presence of SDS and by measurements of phospholipase A2 inhibition. After the first two purification steps, an 86-fold increase of the inhibitory activity of CNF was observed; however, the third step caused an apparent inactivation of the factor. The inactive CNF was shown to correspond to the previously active plasma material and to be homogeneous on electrophoresis, immunoelectrophoresis and partial amino-terminal sequence. The mol. wt of CNF was estimated as 23,600 by SDS-PAGE.

Antihemorrhagic factors from the blood serum of the western diamondback rattlesnake Crotalus atrox.

Several antihemorrhagic factors isolated from C. atrox serum are glycoproteins with mol. wt ranging from 65,000 to 80,000. The antihemorrhagic activity of these factors was stable at a pH range of 1.3-11.5 and at temperatures up to 85 degrees C, for 30 min. The isolated antihemorrhagins also neutralized the proteolytic activity of C. atrox venom, as tested with azocollagen and gelatin, and formed a complex with hemorrhagic toxin e isolated from the same venom. The neutralization capacity of the isolated antihemorrhagins was six times as high as the commercial polyvalent antivenom produced for clinical use.