Chick Biventer Cervicis Nerve-muscle Preparation Research Articles

Isolation and characterization of rufoxin, a novel protein exhibiting neurotoxicity from venom of the psammophiine, Rhamphiophis oxyrhynchus (Rufous beaked snake)

Colubrid snake venoms potentially represent a vast source of novel biological actives and structural motifs owing to their diverse phylogeny. The present study describes the identification of rufoxin, a neurotoxin from the venom of Rhamphiophis oxyrhynchus (Rufous beaked snake) which is a member of the African colubrid lineage, the psammophiines. Rufoxin (1 μM) displayed reversible post-synaptic neurotoxic activity as evidenced by significant inhibition of indirect twitches and responses to exogenous nicotinic agonists in the chick biventer cervicis nerve–muscle preparation. Rufoxin (0.1–1.0 μM) also caused a rightward parallel shift of cumulative concentration–response curves to carbachol (CCh; 0.6–80 μM) without a significant depression of the maximum response, suggestive of classical competitive antagonism at the skeletal muscle nicotinic receptor. Rufoxin lacks NH 2-terminal sequence homology to previously identified snake venom toxins. This work indicates a wider distribution of neurotoxins across the advanced snake superfamily than previously described.

Oxylepitoxin-1, a reversible neurotoxin from the venom of the inland taipan ( Oxyuranus microlepidotus)

This study describes the characterization of oxylepitoxin-1 (MW 6789), the first postsynaptic neurotoxin isolated from the venom of the Inland taipan ( Oxyuranus microlepidotus), which is the most venomous snake in the world. Oxylepitoxin-1, purified using successive steps of size-exclusion and reverse phase-high performance liquid chromatography, produced concentration-dependent (0.3–1.0 μM) inhibition of nerve-mediated (0.1 Hz, 0.2 ms, supramaximal V) twitches of the chick biventer cervicis nerve-muscle preparation. Taipan antivenom (5 units/ml) prevented the neurotoxic activity of whole venom (10 μg/ml), but had no significant effect on oxylepitoxin-1 (1 μM). The toxin-induced inhibition of nerve-mediated twitches was significantly reversed upon washing the tissue at 5 min intervals. Oxylepitoxin-1 (30–300 nM) displayed competitive antagonism at the skeletal muscle nicotinic receptor with a pA 2 value of 7.16 ± 0.28 (i.e. approximately 10-fold more potent than tubocurarine). The venom had a high level of PLA 2 activity (765 ± 73 μmol/min/mg) while oxylepitoxin-1 displayed no PLA 2 activity. Partial N-terminal sequencing of oxylepitoxin-1 shows high sequence identity (i.e. 93%) to postsynaptic toxins isolated from the venom of the closely related coastal taipan ( Oxyuranus scutellatus scutellatus).

Isolation and pharmacological characterisation of hostoxin-1, a postsynaptic neurotoxin from the venom of the Stephen's banded snake ( Hoplocephalus stephensi)

Envenoming by the Stephen's banded snake ( Hoplocephalus stephensi) is not usually characterised by neurotoxicity. The present study describes the pharmacological characterisation of hostoxin-1 (MW 6660 Da), the first neurotoxin to be isolated from the venom of the Stephen's banded snake. Hostoxin-1 (0.3–1.0 μM) caused concentration-dependent inhibition of indirect twitches of the chick biventer cervicis nerve–muscle preparation. The neurotoxic activity of hostoxin-1 (0.3 μM) was irreversible by washing, but significantly reversed by the addition of CSL tiger snake antivenom (5 units/ml) added at t 90 (i.e. time at which twitches were inhibited by 90%). In addition, hostoxin-1 (0.3 μM) inhibited responses to exogenous acetylcholine and carbachol, but not KCl, indicating a postsynaptic mode of action. Hostoxin-1 (5–30 nM) displayed pseudo-irreversible antagonism at the skeletal muscle nicotinic receptor with a pA 2 value of 8.45 ± 0.32 (i.e. approximately 100-fold more potent than tubocurarine). H. stephensi venom displayed a high level of PLA 2 activity (specific activity 100.1 ± 4.4 μmol/min/mg). However, the activity of hostoxin-1 was negligible. Partial N-terminal sequencing of hostoxin-1 indicates that it has high sequence homology with other elapid short-chain neurotoxins.

IN VITRO NEUROTOXIC AND MYOTOXIC EFFECTS OF THE VENOM FROM THE BLACK WHIP SNAKE (DEMANSIA PAPUENSIS)

1. Black whip snakes belong to the family elapidae and are found throughout the northern coastal region of Australia. The black whip snake (Demansia papuensis) is considered to be potentially dangerous due to its size and phylogenetic distinctiveness. Previous liquid chromatography-mass spectrometry analysis of D. papuensis venom indicated a number of components within the molecular mass ranges compatible with neurotoxins. For the first time, this study examines the in vitro neurotoxic and myotoxic effects of the venom from D. papuensis. 2. Venom (10 microg/mL) caused significant inhibition of twitches elicited by stimulation (0.2 ms, 0.1 Hz, supramaximal V) of motor nerves in the chick biventer cervicis nerve-muscle preparation. This neurotoxic effect, which was postsynaptic in origin, was weak in comparison to that of most other Australian elapids. Prior addition (10 min) of polyvalent (PSAV) or tiger snake (TSAV) antivenom (5 units/mL) prevented venom-induced twitch inhibition. Addition of PSAV (5 units/mL) at t(50) failed to reverse the inhibitory effect but prevented further inhibition of nerve-mediated twitches. 3. The venom (20-50 microg/mL) is also myotoxic as indicated by a slowly developing contracture and inhibition of twitches elicited by direct stimulation (2 ms, 0.1 Hz, supramaximal V, in the presence of tubocurarine 10 micromol/L) of the chick biventer muscle. This activity was confirmed by histological examination of the muscle. 4. Fractionation and characterization of venom components is required to further investigate the reasons for the weak neurotoxic activity of D. papuensis venom.

Isolation and Pharmacological Characterization of Cannitoxin, a Presynaptic Neurotoxin from the Venom of the Papuan Taipan (Oxyuranus scutellatus canni)

The Papuan taipan (Oxyuranus scutellatus canni) is widely distributed throughout much of Papua New Guinea. Although neurotoxicity is a major symptom of envenomation, no neurotoxins have been isolated from this venom. Using a series of size exclusion chromatography steps, we report the isolation of cannitoxin, a presynaptic neurotoxin (44,848 Da) that represents approximately 16% of the whole venom. The toxin displayed high phospholipase A2 (PLA2 activity (330 +/- 5 micromol/min/mg) and caused concentration-dependent (11-66 nM) inhibition of indirect (0.2 ms; 0.1 Hz; supramaximal V) twitches of the chick biventer cervicis nerve-muscle preparation without effecting nicotinic receptor agonists. Prior addition of CSL Taipan antivenom (5 U/ml) or inhibition of phospholipase A2 activity by incubation with 4-bromophenacyl bromide prevented the inhibition of twitches. Cannitoxin is composed of three different subunits, alpha, beta, and gamma, with the possibility of two beta isomers. However, only the alpha subunit displayed in vitro neurotoxic activity of its own. Thus, cannitoxin is similar in structure and pharmacology to taipoxin, which has been isolated from the closely related Australian species O. scutellatus scutellatus (coastal taipan).

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 (6738 Da), 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 μM) 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 pA 2 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.

Phospholipase A2‐dependent effects of the venom from the new guinean small‐eyed snake Micropechis ikaheka

The New Guinean small-eyed snake (Micropechis ikaheka) is a cause of life-threatening envenoming. Previous studies on M. ikaheka venom have indicated the presence of neurotoxins as well as myotoxins. This study examined the in vitro myotoxic effects of M. ikaheka venom and the efficacy of a polyvalent antivenom in neutralizing these effects. Venom (50 microg/ml) produced a slowly developing contracture and inhibition of direct twitches of the chick biventer cervicis nerve-muscle preparation in the presence of tubocurarine (10 microM). Myotoxicity was confirmed by subsequent histological examination of tissues. This myotoxicity was prevented by the prior addition of polyvalent snake antivenom (30 U/ml). However, the addition of antivenom (30 U/ml) 1 h after venom administration failed to reverse or prevent the further inhibition of direct twitches. In addition, venom (1-10 microg/ml) produced concentration-dependent contractions of the guinea-pig isolated ileum. These effects were dependent on phospholipase A2 (PLA2) activity of the venom as evidenced by the ability of the PLA2 inhibitor 4-bromophenacyl bromide (4-BPB; 1.8 mM) to prevent this activity. This study indicates that M. ikaheka venom causes significant myotoxicity and that polyvalent snake antivenom may be a potential treatment for the myotoxic effects in patients envenomed by this species.

Pharmacological characterisation of a neurotoxin from the venom of Boiga dendrophila (Mangrove catsnake)

In this study, we have pharmacologically characterised boigatoxin-A, a three finger toxin isolated from the venom of the colubrid, Boiga dendrophila (Mangrove catsnake). In the chick biventer cervicis nerve-muscle preparation boigatoxin-A (1 μM) displayed poorly reversible postsynaptic blockade as evidenced by the inhibition of indirect (0.1 Hz, 0.2 ms, supramaximal V) twitches and responses to exogenous acetylcholine (1 mM) and carbachol (20 μM). Boigatoxin-A (0.3–0.5 μM) caused a concentration-dependent depression of the maximum response of cumulative concentration response curves to CCh (0.6–80 μM). Boigatoxin-A (1 μM) induced readily reversible inhibition of electrically evoked (0.2 Hz, 0.3 ms, 70–100 V) twitches of the prostatic segment of the rat vas deferens. This inhibition was not significantly attenuated by 8-phenyltheophylline (20 μM) or idazoxan (1 μM). Boigatoxin-A (1 μM) did not affect α,β-mATP (10 μM) or noradrenaline (25 μM) responses in unstimulated epididymal segments of the rat vas deferens. Our data suggests that this toxin has weak postsynaptic neurotoxicity in skeletal muscle and also prejunctional neurotoxic activity in the smooth muscle of the rat vas deferens to inhibit the release of neurotransmitter(s), but not via prejunctional purinergic or adrenergic receptors. This is the first report of such activity for a toxin isolated from snake venom and reinforces the largely untapped potential of colubrid venoms.

Neurotoxic effects of venoms from seven species of australasian black snakes (Pseudechis): Efficacy of black and tiger snake antivenoms

1. Pseudechis species (black snakes) are among the most widespread venomous snakes in Australia. Despite this, very little is known about the potency of their venoms or the efficacy of the antivenoms used to treat systemic envenomation by these snakes. The present study investigated the in vitro neurotoxicity of venoms from seven Australasian Pseudechis species and determined the efficacy of black and tiger snake antivenoms against this activity. 2. All venoms (10 microg/mL) significantly inhibited indirect twitches of the chick biventer cervicis nerve-muscle preparation and responses to exogenous acetylcholine (ACh; 1 mmol/L), but not to KCl (40 mmol/L), indicating activity at post-synaptic nicotinic receptors on the skeletal muscle. 3. Prior administration of either black or tiger snake antivenom (5 U/mL) prevented the inhibitory effects of all Pseudechis venoms. 4. Black snake antivenom (5 U/mL) added at t90 (i.e. the time-point at which the original twitch height was reduced by 90%) significantly reversed the effects of P. butleri (28+/-5%), P. guttatus (25+/-8%) and P. porphyriacus (28+/-10%) venoms. Tiger snake antivenom (5 U/mL) added at the t90 time-point significantly reversed the neurotoxic effects of P. guttatus (51+/-4%), P. papuanus (47+/-5%) and P. porphyriacus (20+/-7%) venoms. 5. We show, for the first time, the presence of neurotoxins in the venom of these related snake species and that this activity is differentially affected by either black snake or tiger snake antivenoms.

The efficacy of two antivenoms against the in vitro myotoxic effects of black snake ( Pseudechis) venoms in the chick biventer cervicis nerve-muscle preparation

Neurotoxicity is rarely seen following human systemic envenoming by Australasian black snakes (genus Pseudechis) with myotoxicity being the most prominent feature following bites by some species. This study investigated the in vitro myotoxicity of venoms from seven species of Australasian Pseudechis and determined the efficacy of CSL black and tiger snake antivenoms. All Pseudechis venoms (10 μg/ml) significantly inhibited direct twitches of the chick biventer cervicis nerve-muscle preparation ( p<0.05, one-way ANOVA). Prior addition of black snake antivenom (5 U/ml) prevented the inhibitory effects of all Pseudechis venoms ( p<0.05, one-way ANOVA), except Pseudechis butleri. Prior addition of tiger snake antivenom (5 U/ml) prevented the venom-induced reduction in direct twitches to Notechis scutatus venom and all Pseudechis venoms ( p<0.05, one-way ANOVA), with the exception of Pseudechis australis and Pseudechis colletti venoms. Black or tiger snake antivenom (5 U/ml) added 1 h after the addition of venom inhibited further reduction of direct twitches by N. scutatus and most Pseudechis venoms, but did not significantly restore twitch height. PLA 2 activity was found in all venoms with the following rank order: Pseudechis porphyriacus> P. australis> Pseudechis papuanus> P. butleri> Pseudechis guttatus≥ Pseudechis pailsii> P. colletti> N. scutatus. The results of the present study suggest that Australasian Pseudechis venoms possess variable myotoxic activity. The ability of black or tiger snake antivenom to prevent or inhibit further venom-induced effects varied across the genus.

Clinical and in vitro evidence for the efficacy of Australian red-back spider ( Latrodectus hasselti) antivenom in the treatment of envenomation by a Cupboard spider ( Steatoda grossa)

We report the case of a 22-year-old female who was bitten on the shoulder by a spider subsequently identified as a female Cupboard spider ( Steatoda grossa). She developed nausea, vomiting, and severe local and regional pain, similar to that seen in latrodectism. Symptoms were treated successfully with red-back spider antivenom (RBSAV). We also present in vitro data, which supports this clinical observation, and suggests that S. grossa venom is immunogenically reactive with both RBSAV and latrotoxin (LTx)-specific antibodies by Western blotting. Moreover, the effects of S. grossa venom on the isolated chick biventer cervicis nerve–muscle preparation are dose-dependent and similar to those seen with Latrodectus spp. venoms. S. grossa venom produced a sustained muscle contracture which could be prevented by pre-incubation of venom with RBSAV. Venom effects could also be reversed by the addition of antivenom after application of venom to the preparation. Although severe envenomation is uncommon following the bite of Steatoda spp. it may resemble latrodectism. These results indicate that RBSAV is likely to be effective in reversing symptoms of envenomation and should be considered in the treatment of patients with distressing or persisting symptoms.

Cross-reactivity of Sydney funnel-web spider antivenom: neutralization of the in vitro toxicity of other Australian funnel-web ( Atrax and Hadronyche) spider venoms

Australian funnel-web spiders are recognized as one of the most venomous spiders to humans world-wide. Funnel-web spider antivenom (FWS AV) reverses clinical effects of envenomation from the bite of Atrax robustus and a small number of related Hadronyche species. This study assessed the in vitro efficacy of FWS AV in neutralization of the effects of funnel-web spider venoms, collected from various locations along the eastern seaboard of Australia, in an isolated chick biventer cervicis nerve–muscle preparation. Venoms were separated by SDS-PAGE electrophoresis to compare protein composition and transblotted for Western blotting and incubation with FWS AV. SDS-PAGE of venoms revealed similar low and high molecular weight protein bands. Western blotting with FWS AV showed similar antivenom binding with protein bands in all the venoms tested. Male funnel-web spider venoms (7/7) and female venoms (5/10) produced muscle contracture and fasciculation when applied to the nerve–muscle preparation. Venom effects were reversed by subsequent application of FWS AV or prevented by pretreatment of the preparation with antivenom. FWS AV appears to reverse the in vitro toxicity of a number of funnel-web spider venoms from the eastern seaboard of Australia. FWS AV should be effective in the treatment of envenomation from most, if not all, species of Australian funnel-web spiders.

Red-back spider (Latrodectus hasselti) antivenom prevents the toxicity of widow spider venoms

Study Objectives: Widow spiders of the genus Latrodectus are found worldwide and produce similar clinical envenomation syndromes. In Australia, red-back spider antivenom (RBS-AV) is effective therapy for Latrodectus hasselti envenomation and it has been reported to reverse envenomation by other widow spiders. This study assessed the efficacy of RBS-AV in preventing in vitro and in vivo toxicity of widow spider venoms of North America and Europe. Methods: The binding of RBS-AV to α-latrotoxin and Latrodectus venoms (Latrodectus spp mactans, hesperus, lugubris, tredecimguttatus, hasselti ) was assayed using Western blotting. Prevention of in vitro toxicity to α-latrotoxin and the same venoms was tested by pretreating an isolated chick biventer cervicis nerve-muscle preparation with RBS-AV. Prevention of in vivo toxicity was determined by a lethality study in male Balb/c mice (2.5 to 5× median lethal dose [LD50]) or α-latrotoxin (10× LD50) preincubated with antivenom or without RBS-AV (control). Results: In Western blots, RBS-AV bound to α-latrotoxin and similar widow spider proteins in all venoms tested, indicating antigenic similarity with proteins found in RBS venom. Antivenom prevented the typical in vitro muscle contracture and loss of twitch tension seen with α-latrotoxin and the venoms tested. Control mice rapidly developed signs of envenomation, but mice treated with RBS-AV remained free of signs of envenomation. Conclusion: RBS-AV prevented both in vitro and in vivo toxicity from Latrodectus venoms and α-latrotoxin in mice. These data suggest that RBS-AV may be clinically effective in the treatment of envenomation resulting from the bite of other widow spiders. [Graudins A, Padula M, Broady K, Nicholson GM. Red-back spider (Latrodectus hasselti) antivenom prevents the toxicity of widow spider venoms. Ann Emerg Med. February 2001;37:154-160.]

A pharmacological examination of venoms from three species of death adder ( Acanthophis antarcticus, Acanthophis praelongus and Acanthophis pyrrhus)

The common ( A. antarcticus), northern ( A. praelongus) and desert ( A. pyrrhus) death adders are species belonging to the Acanthophis genus. The present study compared some pharmacological aspects of the venoms of these species and examined the in vitro efficacy of death adder antivenom. Neurotoxicity was determined by the time to produce 90% inhibition ( t 90) of indirect (0.1 Hz, 0.2 ms, supramaximal voltage) twitches in the chick biventer cervicis nerve-muscle (3–10 μg/ml) and mouse phrenic nerve-diaphragm (10 μg/ml) preparations. A. praelongus venom was significantly less neurotoxic than A. antarcticus venom but was not significantly different from A. pyrrhus venom. In the biventer muscle, all three venoms (3–10 μg/ml) abolished responses to exogenous ACh (1 mM) and carbachol (20 μM), but not KCl (40 mM), indicating activity at post-synaptic nicotinic receptors. All venoms (30 μg/ml) failed to produce significant inhibition of direct twitches (0.1 Hz, 2.0 ms, supramaximal voltage) in the chick biventer cervicis nerve-muscle preparation. However, A. praelongus (30 μg/ml) venom initiated a significant direct contracture of muscle, indicative of some myotoxic activity. The prior (10 min) administration of death adder antivenom (1 unit/ml), which is raised against A. antarcticus venom, markedly attenuated the twitch blockade produced by all venoms (10 μg/ml). Administration of antivenom (1.5 units/ml) at t 90 markedly reversed, over a period of 4 h, the inhibition of twitches produced by A. praelongus (3 μg/ml, 72±6% recovery) and A. pyrrhus (3 μg/ml, 51±9% recovery) but was less effective against A. antarcticus venom (3 μg/ml, 22±7% recovery). These results suggest that all three venoms contain postsynaptic neurotoxins. Death adder antivenom displayed differing efficacy against the in vitro neurotoxicity of the three venoms.

Mode of action of Duvernoy's gland extracts from the colubrid Dryadophis bifossatus in the chick biventer cervicis nerve-muscle preparation

Extracts and secretion of the Duvernoy's gland are venomous, inducing in several cases motor paralysis in experimental animals. The extracts of the Duvernoy's gland from the aglyphous colubrid Dryadophis bifossatus are very toxic, eliciting flaccid paralysis in pigeons, rabbits and Hylae (Brazil and Vellard, 1926). In the present study, the neuromuscular action of the extracts of Duvernoy's gland from this Colubridae was investigated in the chick biventer cervicis nerve-muscle preparation. The muscle was indirectly stimulated with supramaximal pulses, and by addition of acetylcholine or carbachol to the organ bath. Direct muscle stimulation was carried out in curarized preparations. The extracts induced an irreversible neuromuscular blockade and also inhibited irreversibly the contracture of the biventer cervicis produced by either acetylcholine or carbachol. The twitches elicited by direct muscle stimulation were not depressed. These results show that the neurotoxin(s) of the extracts interact with the end-plate cholinergic receptors.

Purification and partial amino acid sequences of a new presynaptic toxin and a cytotoxin from venom of pit veper Agkistrodon blomhoffii brevicaudus.

The technique of the reverse-phase performance liquid chromatography (RP-HPLC) was employed to separate and purify the toxic proteins from the venom of Agkistrodon blomhoffii brevicaudus collected in China. 3 toxic proteins marked as AgTx-1, AgTx-2 and AgTx-3 consisting of about 122 amino acid residues were screened. The toxicities (LD50) of the AgTx-1, AgTx-2 and AgTx-3 were 0.075, 0.51 and 6.6 mg per kg weight of mice respectively. Toxicological experiment in the chick biventer cervicis nerve-muscle preparation showed that the acetylcholine (Ach) sensitivity of the preparation was unchanged after the total failure of the indirect contraction caused by AgTx-1 and AgTx-2, suggesting that they were presynaptic blockers, namely beta-type of snake toxins. However, the amplitude of indirect contraction of the preparation was gradually reduced due to its incomplete relaxation caused by AgTx-3, indicating that it should belong to the category of cytotoxins. The partial amino acid sequences of 3 toxins have been established. It was found in ref. [1] that the sequences of the first 32 N-terminal amino acid residues of AgTx-1 and AgTx-2, as well as beta-agkistrodotoxin (beta-AgTx) reported previously were identical (the residue at the position 30 of beta-AgTx should be Trp). In view of the similarity in toxicities, and the amounts in the venom and other properties, it was concluded that AgTx-1 should be beta-AgTx and consequently was renamed beta 1-AgTx. AgTx-2 should be the isoform of beta 1-AgTx, and correspondingly named beta 2-AgTx.

The use of the chick biventer cervicis preparation to assess the protective activity of six international reference antivenoms on the neuromuscular effects of snake venoms in vitro

A. Barfaraz and A. L. Harvey. The use of the chick biventer cervicis preparation to assess the protective activity of six international reference antivenoms on the neuromuscular effects of snake venoms in vitro. Toxicon 32, 267–272, 1994.—The protective activity of some antivenoms on the neuromuscular activity in vitro of six snake venoms was established in order to test the feasibility of using a simple isolated nerve-muscle preparation to compare different antivenoms. Six venoms designated as International Reference Venoms by the World Health Organization (WHO) were used: Echis carinatus (Iran), Echis carinatus (Mali), Naja naja kaouthia, Notechis scutatus, Trimeresurus flavoviridis, and Vipera russelli (Thailand). The chick biventer cervicis nerve-muscle preparation was used to detect neurotoxic and myotoxic effects. The ratio of the amount of antivenom needed to neutralize a dose of venom (w/w) was calculated in order to quantify the potencies of the antivenoms. The antivenom to venom ratios were 1000 for Echis carinatus (Mali) and for Notechis scutatus; 100 for Naja naja kaouthia and Vipera russelli; and 10 for Echis carinatus (Iran) and Trimeresurus flavoviridis. It is concluded that in vitro assays using the chick biventer cervicis preparation could be used to compare the relative potencies of different antivenoms at neutralizing myotoxic and neurotoxic toxins in snake venoms.

Neuromuscular actions of sodium selenite on chick biventer cervicis nerve-muscle preparation

Sodium selenite was found to be toxic to chicks, with an LD 50 of 8.5 μg g , which was increased to 16.3 μg g by NaCN. The major symptoms of chicks, treated with selenite, were sedation and then dyspnea and paralysis. The cause of death by selenite was apparently due to the respiratory failure. The possible mechanism of toxicity was explored in the isolated chick biventer cervicis nerve-muscle preparation. Selenite initially increased the amplitude of the twitch, reversed the suppression of the twitch caused by d-tubocurarine, Mg 2+, Cd 2+ or Mn 2+ and significantly increased the quantal content and amplitude of endplate potentials. Subsequently, selenite depressed the amplitude of the twitch, blocked the axonal conduction and inhibited excitatory postsynaptic potentials. Both NH + 4 and K + enhanced the action of selenite in depressing the twitches. In addition, selenite induced a sustained contracture of the muscle, which was partially inhibited by removal of external Ca 2+ and markedly blocked by EGTA. Entry of Ca 2+ and release of the internal Ca 2+ were considered to be responsible for inducing contracture by selenite. Pretreatment with trypsin, glutathione (GSH) and cyanide profoundly inhibited the effects of selenite, indicating that the site of action of selenite was on the outer membrane and the binding of selenite to the sulfhydryl groups of membrane proteins was proposed to be an essential step for selenite-induced contracture and neuromuscular action. These findings suggest that neuromuscular blockade and tetanic spasm, produced by selenite in chicks, may play a role in causing respiratory failure in vivo.

Investigation of the Anti-Snake Venom Activity ofSchumanniophyton magnificum

Polar fractions of the methanolic extracts of the stem- and root-barks of SCHUMANNIOPHYTON MAGNIFICUM (Rubiaceae) reduced the effects of cobra venom cardiotoxin in the chick biventer cervicis nerve-muscle preparation IN VITRO. No activity was observed against a curaremimetic neurotoxin from cobra venom. Further fractionation of the extracts showed that the activity was not due to alkaloidal components, but the active substances could not be identified or isolated.

Effect of lignocaine on the responses produced by depolarizing agents at the chick neuromuscular junction.

The effect of lignocaine on the depolarization and contracture responses produced by acetylcholine (ACh) or tetraethylammonium (TEA) was studied in the isolated chick biventer cervicis (BVC) nerve-muscle preparation using the moving fluid electrode technique. Lignocaine (37 mumol X 1-1-1.8 mmol X 1-1) produced concentration-dependent contractures in the chick BVC muscle. The contractures produced by lignocaine were not accompanied by membrane depolarizations. ACh (5.5 mumol X 1-1-11.0 mmol X 1-1) and TEA (0.48-24.0 mmol X 1-1) produced concentration-dependent depolarizations and contractures in the chick BVC muscle. Lignocaine (0.93 mmol X 1-1) greatly reduced the responses produced by ACh, while it markedly increased the contracture responses produced by TEA. The depolarizations produced by TEA were reduced in lignocaine (0.93 mmol X 1-1). It was concluded that the local anaesthetic lignocaine produces contractures in the chick BVC muscle. Lignocaine potentiates the contractures produced by TEA, while it greatly reduces those produced by ACh.