Taipan Research Articles

Secretory phospholipase A 2 potentiates glutamate-induced rat striatal neuronal cell death in vivo

The secretory phospholipases A 2 (sPLA 2) OS 2 (10, 20 and 50 pmol) or OS 1, (50 pmol) purified from taipan snake Oxyuranus scutellatus scutellatus venom, and the excitatory amino acid glutamate (Glu) (2.5 and 5.0 μmol) were injected into the right striatum of male Wistar rats. Injection of 10 and 20 pmol OS 2 caused no neurological abnormalities or tissue damage. OS 2 (50 pmol) caused apathy and circling towards the injection side. Histology revealed an infarct at the injection site. Injection of 50 pmol OS 1 showed very little or no signs of neurotoxicity. Injection of 2.5 μmol Glu caused no tissue damage or neurological abnormality. After injection of 5.0 μmol Glu, the animals initially circled towards the side of injection, and gradually developed generalized clonic convulsions. These animals showed a well demarcated striatal infarct. When non-toxic concentrations of 20 pmol OS 2 and 2.5 μmol Glu were co-injected, a synergistic neurotoxicity was observed. Extensive histological damage occurred in the entire right hemisphere, and in several rats comprising part of the contralateral hemisphere. These animals were apathetic in the immediate hours following injection, with circling towards the side of injection in the following days. Thus, OS 2 greatly potentiates glutamate excitoxicity in vivo.

A pharmacological examination of venom from the Papuan taipan: ( Oxyuranus scutellatus canni)

The Papuan taipan ( Oxyuranus scutellatus canni) is the third most venomous terrestrial snake in the world, however, little is know about the pharmacology of the venom. In the chick biventer cervicis muscle, venom (10 μg/ml) abolished nerve-mediated twitches (time to 90% inhibition ( t 90) 44±5 min, n=9). This inhibition was unaffected by prior incubation of the venom with the phospholipase A inhibitor 4-bromophenacyl bromide (4-BPB; 0.72 mM) ( t 90 48±7 min, n=8). The mouse phrenic nerve diaphragm preparation displayed greater sensitivity to venom (10 μg/ml) ( t 90 25±1 min, n=6). In the chick biventer muscle, venom (10 μg/ml) significantly inhibited responses to acetylcholine (1 mM) and carbachol (20 μM), but not KCl (40 mM), indicating activity at post-synaptic nicotinic receptors. Venom (10 μg/ml) did not affect direct muscle stimulation. Venom (3–30 μg/ml) produced dose-dependant contractions of the guinea-pig ileum. Contractile responses were significantly inhibited by indomethacin (1 μM) or prior incubation of the venom with 4-BPB (0.72 mM) indicating involvement of a PLA component. In rat phenylephrine (0.3 μM) precontracted aortae, venom (3–100 μg/ml) produced endothelium-independent relaxation which was unaffected by prior incubation of venom (30 μg/ml) with 4-BPB (0.72 mM). In anaesthetised rats, 10 μg/kg (i.v.) venom produced rapid respiratory and cardiovascular collapse while 5 μg/kg (i.v.) venom produced only a small transient decrease in mean arterial blood pressure. Prior administration of 5 μg/kg (i.v.) venom enabled subsequent administration of 10 and 100 μg/kg (i.v.) venom without respiratory or cardiovascular collapse. Further work is required to identify specific toxins with the above pharmacological activity.

Snake Bite, Rhabdomyolysis, and Renal Failure

APPROXIMATELY 5 million snake bites, scorpion stings, and anaphylactic reactions to insect stings occur worldwide annually, causing over 100,000 human deaths each year.1 Snake bites are estimated to cause 30,000 deaths annually in Asia and 1,000 deaths each in Africa and South America. By contrast, in Australia and the United States, the number of deaths is minimal. The animal toxin can cause a spectrum of effects, from local reaction with pain and swelling to severe systemic response and vital organ involvement. Intravascular hemolysis, disseminated intravascular coagulation, rhabdomyolysis, and muscular paralysis are common and can lead to or contribute to devastating hemodynamic effects. Myoglobinuria and hemoglobinuria are often observed. In the tropics, animal toxins are an important cause of rhabdomyolysis. Insect stings (wasps, hornets, and bees) and snake bites are the most common cause of myoglobinuric acute renal failure (ARF). Examination of renal biopsy specimens using immunoperoxidase staining techniques has revealed myoglobin in the renal tubules of 60% of patients with various tropical diseases, yet clinical myoglobinuria is observed in only 9%. Snakes known to have toxins with myotoxicity include Notechis scutatus (Australian tiger snake), Oxyuranus scutellatus (Australian Taipan snake), Bungarus multicinctus (Taiwanese krait), Bungarus caeruleus (Indian krait), Crotalus durissus terrificus (South American rattlesnake), Micropechis ikeheka (small-eyed snake of New Guinea), Pseudechis australis (Mulga snake), Pseudechis colletti (Collett’s snake), Pseudechis affinis (Dugite snake), Pseudechis porphyriacus (red-bellied black snake), Pseudonaja textitis (common brown snake), Tropidechis carinatus (rough-scaled snake), Austrelaps superbus (copperhead), Cryptophis nigrescens (smalleyed snake), Vipera russelli pulchella (Sri Lankan Russell’s viper), and sea snakes. All myotoxic snake venoms contain phospholipase A2, which is responsible for the rhabdomyolysis. The venoms also have neurotoxin components. Some have presynaptic effects, while a few have predilection for postsynaptic action. Postsynaptic neurotoxins account for approximately 60% of the toxic protein of sea snakes (Enhydrina schistosa). Nevertheless, the clinical picture of poisoning is predominantly myotoxic rather than neurotoxic.2 Muscular pains and myoglobinuria are the most characteristic symptoms in human snake bite victims. Myalgia starts after the bite, reaches a peak a few days later, and usually lasts for 1 week. Muscular pains are associated with myoglobinuria, muscular paresis, and areflexia. Myoglobinuria lasts for approximately 1 week, while muscular paresis and areflexia can last as long as 3 weeks. ARF occurs in the majority of patients. Hyperkalemia, hyperuricemia, hypocalcemia, hyperphosphatemia, and a low ratio of serum urea nitrogen to serum creatinine concentrations are common laboratory findings. Serum creatine phosphokinase and lactate dehydrogenase levels are variable. Of clinical interest is the observation that hemodialysis in sea snake (Enhydrina schistosa) bite victims improves the muscular symptoms, including paresis. Although the clinical aspects of the Australian elapid and sea snake bites are well described, little is known about the sequential pathologic changes of the effects of myoglobinuria in the kidney. A recent renal pathologic study in mice at

P18 Haemostatic changes induced in 84 humans bitten by the Papuan taipan (Oxyuranus scutellatus canni) and recovery following antivenom

Haemophilia and Haemostasis Centre, Department of Haematology, Royal Free Hospital and School of Medicine, London, UK

A Novel Small Conductance Ca2+-activated K+ Channel Blocker from Oxyuranus scutellatusTaipan Venom: RE-EVALUATION OF TAICATOXIN AS A SELECTIVE Ca2+CHANNEL PROBE

Taicatoxin, isolated from the venom of the Australian taipan snake Oxyuranus scutellatus, has been previously regarded as a specific blocker of high threshold Ca2+ channels in heart. Here we show that taicatoxin (in contrast to a range of other Ca2+ channel blockers) interacts with apamin-sensitive, small conductance, Ca2+-activated potassium channels on both chromaffin cells and in the brain. Taicatoxin displays high affinity recognition of 125I-apamin acceptor-binding sites, present on rat synaptosomal membranes (Ki = 1.45 +/- 0.22 nM) and also specifically blocks affinity-labeling of a 33-kDa 125I-apamin-binding polypeptide on rat brain membranes. Taicatoxin (50 nM) completely blocks apamin-sensitive after-hyperpolarizing slow tail K+ currents generated in rat chromaffin cells (mean block 97 +/- 3%, n = 12) while only partially reducing total voltage-dependent Ca2+ currents (mean block 12 +/- 4%, n = 6). In view of these findings, the use of taicatoxin as a specific ligand for Ca2+ channels should now be reconsidered.

Severe envenomation by the taipan (Oxyuranus scutellatus)

Severe envenomation by the taipan <i>(Oxyuranus scutellatus)</i>

Severe envenomation by the taipan(Oxyuranus scutellatus)

Severe envenomation by the taipan(Oxyuranus scutellatus)

Electrocardiographic abnormalities in patients bitten by taipans ( Oxyuranus scutellatus canni) and other elapid snakes in Papua New Guinea

Envenoming by a number of species of snake may affect the myocardium or cause electrocardiographic changes; several different mechanisms have been proposed. In a prospective study of snake bite in Papua New Guinea, electrocardiographic changes were observed in 36 of 69 patients (52%) envenomed by the taipan ( Oxyuranus scutellatus), 2 of 6 (33%) envenomed by death adders ( Acanthophis sp.) and one envenomed by the brown snake ( Pseudonaja textilis). Septal T wave inversion and bradycardias, including atrioventricular block, were the commonest abnormalities. There was no haemodynamic deterioration. The cause of these changes is uncertain; only 2 of 24 patients (8·3%) with electrocardiographic changes had markedly elevated plasma concentrations of cardiac troponin T, a sensitive and specific marker of myocardial damage. This suggests that myocardial damage is uncommon following bites by these species. Electrocardiographic abnormalities are most likely to have been caused by a direct toxic effect of a venom component upon cardiac myocyte function; in taipan bites, taicatoxin, a calcium channel blocker, might be responsible.

Rational Design, Recombinant Preparation, and in Vitro and in Vivo Characterization of Human Prothrombin-derived Hirudin Antagonists

A mutant derivative of human prothrombin in which active site aspartate at position 419 is replaced by an asparagine (D419N-prothrombin) has been designed, expressed in recombinant Chinese hamster ovary cells, and purified to homogeneity. D419N-prothrombin was converted to the related molecules D419N-meizothrombin and D419N-thrombin by limited proteolysis by Echis carinatus and Oxyuranus scutellatus venom protease, respectively, and affinity-purified using an immobilized modified C-terminal hirudin-derived peptide. Neither D419N-thrombin nor D419N-meizothrombin exhibited thrombin activity. Titration resulted in no detection of the active site, but binding to the most specific thrombin inhibitor, hirudin, was conserved in both proteins. In vitro examinations showed that D419N-thrombin and D419N-meizothrombin bind to immobilized hirudin, neutralize hirudin in human blood plasma as well as in the purified system, and reactivate the thrombin-hirudin complex. Animal model studies confirmed that D419N-thrombin and D419N-meizothrombin act as hirudin antagonist in blood circulation without detectable effects on the coagulation system. Thus, both D419N-thrombin and D419N-meizothrombin combine for the first time hirudin-neutralizing properties with the advantages of recombinant production of human coagulation factors.

Effects on behaviour and EEG of single chain phospholipases A2 from snake and bee venoms injected into rat brain: search for a functional antagonism.

Three phospholipase A2 (PLA2s), OS1 and OS1 purified from the taipan snake venom Oxyuranus scutellatus scutellatus and bee venom PLA2 were injected to rats by the intracerebroventricular route. OS1 showed no sign of neurotoxicity at doses at which OS2 and bee venom PLA2 produced multiform dose-dependent behavioural effects including motor disturbances (stereotyped movements), compulsive scratching, convulsions and breathing difficulties. EEG recordings showed at the very time when the animal was motionless the induction of several episodes of a low frequency hippocampal theta rhythm, index of long-term changes in synaptic neuroplasticity. Spike-wave discharges were also produced but the occurrence was not systematic. These seizures were often accompanied with behavioural convulsions. Blockers of NMDA receptors and drugs modifying the GABAergic transmission could not abolish the neurotoxic effects of PLA2s except for diazepam (10 mg/kg intraperitoneally) that prevented only OS2-induced disturbances. Blockers of L-type Ca2+ channels and K+ channel openers were also without effect. The toxicity of OS2 and bee venom PLA2 is probably due to their initial specific binding to their neuronal receptor sites.

Failure of 3,4-diaminopyridine and edrophonium to produce significant clinical benefit in neurotoxicity following the bite of Papuan taipan ( Oxyuranus scutellatus canni)

Progressive systemic neurotoxicity is a common feature in patients envenomed following the bite of a Papuan taipan ( Oxyuranus scutellatus canni). Respiratory paralysis, which commonly results, accounts for considerable morbidity and mortality. Established neurotoxicity does not respond to antivenom. In this study, a combination of clinical and electrophysiological variables was used to assess the effect of edrophonium and 3,4-diaminopyridine in patients with significant neurotoxicity. Both drugs produced minor electrophysiological and clinical changes in envenomed patients. This effect was maximal when the 2 drugs were used in combination, but was insufficient to be of significant clinical benefit. Neither drug can be recommended for use in the management of Papuan taipan bite.

Electrophysiological findings in patients envenomed following the bite of a Papuan taipan ( Oxyuranus scutellatus canni)

Electrophysiological studies were done on patients with systemic neurotoxicity following the bite of a Papuan taipan ( Oxyuranus scutellatus canni). Evoked compound muscle action potentials decreased and increased in tandem with clinical deterioration and recovery. Nerve conduction velocities did not change in envenomed patients and were consistent with control studies. Repetitive nerve stimulation studies showed decrementai responses in envenomed patients with post-tetanic potentiation followed by post-tetanic exhaustion. The findings are consistent with studies in vitro which suggested that the major action of neurotoxins in Australian taipan venom is at the synapse. The observation that electrophysiological data correlate closely with the clinical condition of the patient has potential application in the assessment of interventions in the management of snake bite victims.

Snake bites by the Papuan taipan (Oxyuranus scutellatus canni): paralysis, hemostatic and electrocardiographic abnormalities, and effects of antivenom.

One hundred sixty-six patients with enzyme immunoassay-proven bites by taipans (Oxyuranus scutellatus canni) were studied in Port Moresby, Papua New Guinea. One hundred thirty-nine (84%) showed clinical evidence of envenoming: local signs were trivial, but most developed hemostatic disorders and neurotoxicity. The blood of 77% of the patients was incoagulable and 35% bled spontaneously, usually from the gums. Fifty-one per cent had microscopic hematuria. Neurotoxic signs (ptosis, ophthalmoplegia, bulbar paralysis, and peripheral muscular weakness) developed in 85%. Endotracheal intubation was required in 42% and mechanical ventilation in 37%. Electrocardiographic abnormalities (sinus bradycardia and septal T wave inversion) were found in 52% of a group of 69 unselected patients. Specific antivenom raised against Australian taipan venom was effective in stopping spontaneous systemic bleeding and restoring blood coagulability but, in most cases, it neither reversed nor prevented the evolution of paralysis even when given within a few hours of the bite. However, early antivenom treatment was associated statistically with decreased incidence and severity of neurotoxic signs. The low case fatality rate of 4.3% is attributable mainly to the use of mechanical ventilation, a technique rarely available in Papua New Guinea. Earlier use of increased doses of antivenoms of improved specificity might prove more effective.

The efficacy of antivenom in the treatment of bites by the Papuan taipan ( Oxyuranus scutellatus canni)

A prospective series of 156 patients systemically envenomed following the bite of a Papuan taipan ( Oxyuranus scutellatus canni) were studied. All patients were treated with appropriate antivenom and clinical course and outcome were compared. The proportion of patients requiring intubation was significantly smaller, and the time to resolution of neurotoxicity and discharge from hospital significantly shorter, in patients receiving antivenom no more than 4 h after the bite. No significant difference in outcome was demonstrated between patients receiving antivenom at various times after 4 h. No difference was demonstrated in the times to restoration of coagulability between the 2 groups. The only significant difference between a small number of patients given 2 vials of antivenom and patients given a single vial at the same time after envenoming was a marginally shorter duration of intubation in those who required it. The study suggests that, to achieve significant clinical benefit in Papuan taipan bite, antivenom must be given as early as possible.

Coagulopathy following bites by the Papuan taipan (Oxyuranus scutellatus canni).

The mechanisms of haemostatic failure were studied in 87 patients bitten by the Papuan taipan (Oxyuranus scutellatus canni). Eighty (92%) had evidence of a coagulopathy on laboratory testing; 36 (41.4%) developed spontaneous systemic bleeding, although this was rarely of clinical significance. Coagulation assays in 48 completely defibrinated patients showed marked reductions in factors V and VIII and reductions in factors II, IX, XI, XII and XIIIA. There was a reduction in plasminogen and alpha 2-antiplasmin levels and both total and cross-linked fibrin(ogen) degradation products (FDP) levels were elevated. The mean platelet count was initially decreased and fell further during admission. Similar but less severe changes were seen in patients who were mildly defibrinated. Following treatment with antivenom, fibrinogen levels rose rapidly and coagulability was restored within 6-12 h in 93% of patients. These abnormalities may be primarily attributable to the prothrombin activator present in taipan venom, but it is likely that other uncharacterized venom components contributed.

Neurotoxicity and haemostatic disturbances in patients envenomed by the Papuan black snake ( Pseudechis papuanus)

Among 335 patients presenting with snakebites in Central Province, Papua New Guinea, nine were proved by enzyme immunoassay to have been bitten by Papuan black snakes ( Pseudechis papuanus). Seven showed clinical evidence of envenoming. Early symptoms included vomiting and tender local lymph nodes. Five patients had neurotoxic signs and one required mechanical ventilation. Spontaneous systemic bleeding occurred in two patients. Coagulation studies in four patients showed thrombocytopenia, prolongation of prothrombin time, mild defibrination and depletion of other clotting factors with elevated fibrin(ogen) degradation products and other evidence of fibrinolysis. One patient developed mild renal dysfunction. There was no evidence of intravascular haemolysis or rhabdomyolysis. These clinical observations, which do not distinguish victims of P. papuanus from those of taipans ( Oxyuranus scutellatus canni), suggest that the venom contains neurotoxic, haemorrhagic and mild procoagulant activities. Only two other cases of proven envenoming by this species have been reported. There appears to have been a decline in the abundance of this species, and hence its medical importance, over the last 25 years.

The effect of bites by the Papua New Guinean taipan (Oxyuranus scutellatus canni): D. Lalloo, A. Trevett, M. Paul, A. Korinihona, J. Mapao, B. Danga-Christian, I. Laurenson, B. Mayo, A. Sawer, S. Naraqi, D. Theakston and D. Warrell (Department of Clinical Sciences, University of Papua New Guinea; Liverpool School of Tropical Medicine, U.K.; and Centre for Tropical Medicine, University of

The effect of bites by the Papua New Guinean taipan (Oxyuranus scutellatus canni): D. Lalloo, A. Trevett, M. Paul, A. Korinihona, J. Mapao, B. Danga-Christian, I. Laurenson, B. Mayo, A. Sawer, S. Naraqi, D. Theakston and D. Warrell (Department of Clinical Sciences, University of Papua New Guinea; Liverpool School of Tropical Medicine, U.K.; and Centre for Tropical Medicine, University of

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

1. 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. 2. The intravenous ld 50 (median lethal dose), procoagulant activity and enzymatic activities of the juvenile venom were not significantly different from those of the adult venoms. 3. 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. 4.The results suggest that there is no significant age-dependency in the biological properties of taipan venom.

Isolation and physiological characterization of taicatoxin, a complex toxin with specific effects on calcium channels

Taicatoxin is a new complex oligomeric toxin that was isolated from the venom of the Australian taipan snake Oxyuranus scutellatus scutellatus. It is composed of three different molecular entities: an α-neurotoxin-like peptide of mol. wt 8000, a neurotoxic phospholipase of mol. wt of 16,000 and a serine protease inhibitor of mol. wt 7000, linked by non-covalent bonds, at an approximate stoichiometry of 1:1:4. The most active form of the complex was isolated by ion exchange chromatography through DE-Cellulose followed by two steps of CM-Cellulose chromatography at pH 4.7 and pH 6.0, respectively. At this stage the complex migrates as a single component in β-alanine-acetate-urea gel electrophoresis and is very toxic to mice (1 or 2 μg of the complex protein kills a mouse of 20 g within 2 hr). It blocks the high threshold calcium channel current of excitable membranes in heart and does not affect the low threshold calcium channel current. The block occurs at a site that is accessible extracellularly but not intracellularly. The block is selective for calcium channels, reversible, does not affect single channel conductance but only changes channel gating, and is voltage dependent with higher affinity for inactivated channels. The phospholipase activity of the complex toxin can be separated by affinity-chromatography using a phospholipid analog (PC-Sepharose). The resulting complex contains only α-neurotoxin and protease inhibitor and is still capable of blocking calcium channels, although with less potency than the native oligomeric form. Sephadex G-50 gel filtration chromatography in the presence of high salt (1M NaCl) at alkaline pH (8.2), separates the α-neurotoxin-like peptide from the protease inhibitor, but at this stage the resulting peptides lose physiological activity towards the calcium channels. The amino acid sequence of the protease inhibitor was determined by automatic Edman degradation. The α-neurotoxin-like peptide and two isosubunits displaying phospholipase activity were sequenced at the N-terminal part of the molecule.

Comparative study of juvenile and adult taipan (Oxyuranus scutellatus) venoms: N.-H. Tan, A. Armugam, P. J. Mirtschin and P. Masci (Department of Biochemistry, University of Malaya, Kuala Lumpur, Malaysia; Venom Supplies, South Australia; Department of Medicine, University of Queensland, Australia).

Comparative study of juvenile and adult taipan (Oxyuranus scutellatus) venoms: N.-H. Tan, A. Armugam, P. J. Mirtschin and P. Masci (Department of Biochemistry, University of Malaya, Kuala Lumpur, Malaysia; Venom Supplies, South Australia; Department of Medicine, University of Queensland, Australia).