Toxic activity and protein identification from the parotoid gland secretion of the common toad Bufo bufo.

Anuran toxins released from the skin glands are involved in defence against predators and microorganisms. Secretion from parotoid macroglands of bufonid toads is a rich source of bioactive compounds with the cytotoxic, cardiotoxic and hemolytic activity. Bufadienolides are considered the most toxic components of the toad poison, whereas the protein properties are largely unknown. In the present work, we analysed the cardio-, myo-, and neurotropic activity of extract and the selected proteins from Bufo bufo parotoids in in vitro physiological bioassays carried out on two standard model organisms: beetles and frogs. Our results demonstrate a strong cardioactivity of B. bufo gland extract. The toad poison stimulates (by 16%) the contractility of the insect heart and displays the cardioinhibitory effect on the frog heartbeat frequency (a 27% decrease), coupled with an irreversible cardiac arrest. The gland extract also exhibits significant myotropic properties (a 10% decrease in the muscle contraction force), whereas its neuroactivity remains low (a 4% decrease in the nerve conduction velocity). Among identified peptides present in the B. bufo parotoid extract are serine proteases, muscle creatine kinase, phospholipid hydroperoxide glutathione peroxidase, cytotoxic T-lymphocyte protein, etc. Some proteins contribute to the cardioinhibitory effect. Certain compounds display the paralytic (myo- and neurotropic) properties. As the toad gland extract exhibits a strong cardiotoxic activity, we conclude that the poison is a potent agent capable of slaying a predator. Our results also provide the guides for the use of toad poison-peptides in therapeutics and new drug development.

Purification and partial characterization of a novel neurotoxic protein from eggs of black widow spiders (Latrodectus tredecimguttatus).

Up to now, there have been a few reports on the toxic components purified from black widow spider (Latrodectus tredecimguttatus) eggs. In the present study, a novel neurotoxic protein was purified from the eggs by gel filtration combined with ion-exchange chromatography. Its molecular weight was 23.752 kDa determined by electrospray mass spectrometry. The protein could block the neuromuscular transmission in mouse-isolated phrenic nerve-hemidiaphragm preparations completely in a reversible manner and activate tetrodotoxin-sensitive sodium current in rat dorsal root ganglion cells. The N-terminal sequence of the protein was identified by the Edman degradation to be N-S-I-A-D-D-R-Y-R-W-P-G-Y-P-G-A-G-L-I-P-Y-I-I-D-S-. When the sequence was used to search against protein database with a sequence query in Mascot engine there was no matched sequence or protein whereas the Basic Local Alignment Search Tool (BLAST) analysis indicated that no significant similarity was found. These results demonstrated that the protein (named Latroeggtoxin-I) is a novel neurotoxic protein purified from the eggs of black widow spiders.

Marine algal pinnatoxins E and F cause neuromuscular block in an in vitro hemidiaphragm preparation.

Members of the cyclic imine group of toxins, gymnodimine and spirolides, have been found to be potent antagonists of both muscle type and neuronal nicotinic acetylcholine receptors. These toxins exhibit fast acting toxicity in vivo, causing death within minutes by respiratory depression. This toxicity is shared by the novel cyclic imine pinnatoxins E and F, produced by marine dinoflagellates and recently isolated from New Zealand shellfish. However, there is currently very little data available regarding the mechanism of action for any of the pinnatoxins, and no data at all on the novel pinnatoxins E and F. The aim of the current study was to investigate potential antagonism of nicotinic acetylcholine receptors by pinnatoxins E and F using two in vitro tissue preparations. Compound muscle action potentials elicited by stimulation of the phrenic nerve were recorded from the hemidiaphragm in order to test effects on muscle type heteromeric nicotinic receptors, while effects on α7 homomeric neuronal nicotinic receptors were investigated by recording gamma oscillations in response to tetanic stimulation of the CA1 region of the hippocampus. Both a crude extract containing a mixture of pinnatoxins E and F, as well as pure pinnatoxin F, had no effect on gamma oscillation spectral density or spike count at any concentrations. Conversely, at these same concentrations, both crude and pure pinnatoxin caused an almost complete abolition of nerve-evoked hemidiaphragm action potential responses, without any effect on electrically-evoked (direct) responses. This neuromuscular block could not be reversed by neostigmine. These results show that pinnatoxins E and F block neuromuscular transmission and suggest that observed in vivo muscle paralysis by pinnatoxin is due to selective antagonism of muscle type nicotinic acetylcholine receptors.

Neuromuscular activity of BaTX, a presynaptic basic PLA2 isolated from Bothrops alternatus snake venom.

We have previously isolated a Lys49 phospholipase A(2) homolog (BaTX) from Bothrops alternatus snake venom using a combination of molecular exclusion chromatography and reverse phase HPLC and shown its ability to cause neuromuscular blockade. In this work, we describe a one-step procedure for the purification of this toxin and provide further details of its neuromuscular activity. The toxin was purified by reverse phase HPLC and its purity and molecular mass were confirmed by SDS-PAGE, MALDI-TOF mass spectrometry, amino acid analysis and N-terminal sequencing. BaTX (0.007-1.4 microM) produced time-dependent, irreversible neuromuscular blockade in isolated mouse phrenic nerve-diaphragm and chick biventer cervicis preparations (time to 50% blockade with 0.35 microM toxin: 58+/-4 and 24+/-1 min, respectively; n=3-8; mean+/-S.E.) without significantly affecting the response to direct muscle stimulation. In chick preparations, contractures to exogenous acetylcholine (55 and 110 microM) or KCl (13.4 mM) were unaltered after complete blockade by all toxin concentrations. These results, which strongly suggested a presynaptic mechanism of action for this toxin, were reinforced by (1) the inability of BaTX to interfere with the carbachol-induced depolarization of the resting membrane, (2) a significant decrease in the frequency and amplitude of miniature end-plate potentials, and (3) a significant reduction (59+/-4%, n=12) in the quantal content of the end-plate potentials after a 60 min incubation with the toxin (1.4 microM). In addition, a decrease in the organ bath temperature from 37 degrees C to 24 degrees C and/or the replacement of calcium with strontium prevented the neuromuscular blockade, indicating a temperature-dependent effect possibly mediated by enzymatic activity.

Bipinnatins K-Q, minor cembrane-type diterpenes from the West Indian Gorgonian Pseudopterogorgia kallos: isolation, structure assignment, and evaluation of biological activities.

An extensive chemical study of the secondary metabolites found in the crude organic extract of the gorgonian octocoral Pseudopterogorgia kallos has led to the isolation of seven new cembranolides, bipinnatins K-Q ( 2- 8), and one known compound, bipinnatin E ( 9). The molecular structures of compounds 2- 8, many of which contain unusual structural features, were assigned mainly by 2D NMR spectroscopic methods and X-ray crystallographic analysis. The discovery of compounds 2- 8 may lend support to previously proposed mechanisms for the biosynthesis of recently isolated bioactive natural products from the same gorgonian specimen. The in vitro cytotoxicity of bipinnatins 4, 6, and 7 against the NCI tumor cells MCF breast cancer, NCI-H460 non-small cell lung cancer, and SF-268 CNS cancer was evaluated. However, only bipinnatin Q ( 6) displayed significant cytotoxic activity. Some of the compounds isolated proved to be inhibitors of acetylcholine receptors.

Functional characterization of a basic D49 phospholipase A2 (LmTX-I) from the venom of the snake Lachesis muta muta (bushmaster).

The whole venom of Lachesis muta muta is preponderantly neurotoxic but moderately myotoxic on the chick biventer cervicis preparation (BCp). We have now examined these toxic activities of a basic phospholipase A(2), LmTX-I, isolated from the whole venom. LmTX-I caused a significant concentration-dependent neuromuscular blockade in the BCp. The time to produce 50% neuromuscular blockade was 14.7+/-0.75 min (30 microg/ml), 23.6+/-0.9 min (10 microg/ml), 34+/-1.7 min (2.5 microg/ml) and 39.2+/-3.6 min (1 microg/ml), (n=5/concentration; p<0.05). Complete blockade with all tested concentrations was not accompanied by inhibition of the response to ACh. At the highest concentration, LmTX-I (30 microg/ml) significantly reduced contractures elicited by exogenous KCl (20mM), increased the release of creatine kinase (1542.5+/-183.9 IU/L vs 442.7+/-39.8 IU/L for controls after 120 min, p<0.05), and induced the appearance of degenerating muscle fibers ( approximately 15%). Quantification of myonecrosis indicated 14.8+/-0.8 and 2.0+/-0.4%, with 30 and 10 microg/mlvenom concentration, respectively, against 1.07+/-0.4% for control preparations. The findings indicate that the basic PLA(2) present on venom from L. m. muta (LmTX-I) possesses a dominant neurotoxic action on isolated chick nerve-muscle preparations, whereas myotoxicity was mainly observed at the highest concentration used (30 microg/ml). These effects of LmTX-I closely reproduce the effects of the whole venom of L. m. muta in chick neuromuscular preparations.

Identification of four additional myoinhibitory peptides (MIPs) from the ventral nerve cord of Manduca sexta.

Four new myoinhibitory peptides were isolated and identified from the ventral nerve cord of adult Manduca sexta. The new peptides are related to two previously identified myoinhibitory peptides also isolated from adult M. sexta, Mas-MIP I and Mas-MIP II. The sequences of the new peptides are APEKWAAFHGSWamide (Mas-MIP III), GWNDMSSAWamide (Mas-MIP IV), GWQDMSSAWamide (Mas-MIP V), and AWSALHGAWamide (Mas-MIP VI). Mas-MIPs III-VI were found to inhibit spontaneous peristalsis of the adult M. sexta anterior hindgut (ileum) in vitro.

Isolation of subunits, alpha, beta and gamma of the complex taipoxin from the venom of Australian taipan snake (Oxyuranus s. scutellatus): characterization of beta taipoxin as a potent mitogen.

The venom of Australian taipan snake (Oxyuranus s. scutellatus) is extremely potent due to the presence of taipoxin. The intact complex molecule of taipoxin having molecular weight 45.6 kDa is composed of alpha, beta and gamma subunits. This report describes the high pressure liquid chromatography (HPLC) separation of alpha, beta (beta-1 and beta-2) and gamma subunits from taipan crude venom. The fractions containing the taipoxin subunits were further purified to obtain homogeneous proteins. The toxicity in mice showed the alpha subunit as most toxic, the gamma subunit as moderately toxic and the beta-1 and beta-2 subunits were nontoxic. The proteins beta-1 and beta-2 were found to be mitogenic having neurotrophic activity on PC12 cells in culture similar to nerve growth factor. Immunologically, alpha, beta-1, beta-2 and gamma subunits were found to be different, showing cross reactivity, and beta-1 and beta-2 were found to be identical for biological properties and molecular weight. Further characterization of unexpected mitogenic activity of beta subunits is underway.

Neuromuscular blocking activity of methanolic extract of Piper sarmentosum leaves in the rat phrenic nerve-hemidiaphragm preparation.

Methanolic extract of Piper sarmentosum Roxb. (Piperaceae) leaves was studied for the neuromuscular blocking activity in rat phrenic nerve-hemidiaphragm preparations. The plant extract, at concentrations of 3.2, 4.0, 4.8 and 6.4 mg/ml, exhibited an initially transient increase in twitch tension which was followed by a marked dose-related neurally-evoked twitch depression. The neuromuscular blocking effect produced by the plant extract was compared with d-tubocurarine (dTC) and succinylcholine (SCh). The EC50 for neurally-evoked twitch depression of the extract, dTC and SCh was 4.07 mg/ml, 1.1 microM and 15 microM, respectively. The neurally-evoked twitch depression produced by the extract was partially antagonized by tetraethylammonium (TEA) but not by neostigmine (NS). These findings suggested that the plant extract possessed a marked neuromuscular blocking activity at the neuromuscular junction and a possible mechanism which was likely to inhibit neurotransmitter (acetylcholine) release at the presynaptic terminal.

Occurrence of paralytic shellfish poisons in Thai freshwater puffers.

Screening tests were carried out on the toxicity of freshwater puffers Tetraodon leiurus complex and Tetraodon suvatii collected from Udonthani province, north-eastern Thailand. Toxicity was highest in the liver and varied according to the location and season of fish catch. Fish which were reared in tap water for 3 months reduced the toxicity substantially. Partial purification was achieved by an ultrafiltration technique. Toxin components were consequently identified by high-performance liquid chromatography. It was found that toxins separated from the eggs, liver, skin and muscle of these puffers were composed of saxitoxin, neosaxitoxin and decarbamoylsaxitoxin.

Toxin production of Alexandrium minutum (Dinophyceae) from the Bay of Plenty, New Zealand.

Paralytic shellfish toxins of two clonal cultures of Alexandrium minutum isolated during the 1993 toxic shellfish events in the Bay of Plenty, New Zealand, were analyzed using high-performance liquid chromatography. Toxin composition profiles of both cultures showed neosaxitoxin (> 65 mole%) as the principal toxin, with saxitoxin and gonyautoxins (GTX1-4) as minor components. Neither C-toxins (C1-4) nor GTX5-6 were detectable in the two isolates. Bay of Plenty isolates of A. minutum have a unique toxin profile not found in any other isolates of this species that have been characterized. This weakens the hypothesis that A. minutum was recently introduced to New Zealand waters by ballast water or other long-distance transport mechanisms, and argues instead that the species was endemic to the area, but not noticed in the past. The average toxicity of the cultures was 8.8 and 11.0 pg saxitoxin equiv. cell-1 with acetic acid or HCl extraction, respectively. These are at the high end of the range of toxicity reported for A. minutum strains from around the world, and on a cell volume basis are comparable to the most toxic strains of the Alexandrium tamarense group. The toxin profile of A. minutum most closely matches that of mussels and to a lesser degree tuatua harvested from the Bay during the 1993 outbreak, but is quite different from the profile measured in scallops and pipi. Plausible mechanisms for bioconversion of the ingested algal toxins within the latter two shellfish species can be proposed, but it seems more likely that either other strains of A. minutum or other saxitoxin-producing dinoflagellates were ingested by those shellfish. This study established that A. minutum from the Bay of Plenty contains saxitoxins, has a unique toxin composition compared to all other isolates of this species, and was responsible for at least part of the PSP toxicity measured in shellfish during the 1993 outbreak.

Occurrence of paralytic toxin in Taiwanese crab Atergatopsis germaini.

Paralytic toxicity was detected by paralytic shellfish poison bioassay for all 17 specimens of the xanthid crab A. germaini collected from northern Taiwan in November 1993. The average toxicity of crab specimens was 3809 +/- 2591 mouse units (mean +/- S.D.). The toxin was partially purified from ethanolic extract of the crab by ultrafiltration and Bio-Gel P-2 column chromatography. Electrophoresis, TLC, HPLC, ultraviolet spectrum and GC-MS analyses indicated that the crab toxin was composed of gonyautoxin 3 (50%), neosaxitoxin and saxitoxin (7%), a novel paralytic shellfish poison-like toxin (40%) and tetrodotoxin (3%).

A lethal protein toxin (toxin-PC) from the Indian catfish (Plotosus canius, Hamilton) venom.

Isolation and purification of a lethal protein toxin from the Indian catfish Plotosus canius, Hamilton, venom is described. The purification procedure involved ammonium sulfate precipitate of crude venom followed by DEAE-ion exchange chromatography. The purified toxin (toxin-PC) was homogeneous on one-dimensional PAGE and PAS-negative, and had a molecular weight 15 Kd. Toxin-PC was lethal (LD50 225 micrograms/kg, intravenous, in mice) and cardiotoxic, having neuromuscular blocking activity. Toxin-PC produced cardiac arrest on isolated toad and guinea pig hearts. Prior administration of atropine and propanolol failed to counteract toxin activity on isolated heart preparations. On isolated chick biventer cervicis, toxin-PC produced total blockage of electrically-induced twitch response without affecting carbachol- and acetylcholine-induced contraction. The tension developed by the muscle was Ca++ ion-dependent. Neuromuscular blocking time was reduced when K+ ion concentration was increased in the medium. Antiserum raised against toxin-PC failed to antagonize lethal activity of toxin-PC in mice. Toxin-PC probably represents a major toxic component of catfish venom (P. canius), and was responsible for the pathophysiological changes.

Growth and toxin production of the toxic dinoflagellate Pyrodinium bahamense var. compressum in laboratory cultures.

Toxin production of a Malaysian isolate of the toxic red tide dinoflagellate Pyrodinium bahamense var. compressum was investigated at various stages of the batch culture growth cycle and under growth conditions affected by temperature, salinity, and light intensity variations. In all the experiments conducted, only 5 toxins were ever detected. Neosaxitoxin (NEO) and gonyautoxin V (GTX5) made up 80 mole percent or more of the cellular toxin content and saxitoxin (STX), GTX6 and decarbamoylsaxitoxin (dcSTX) made up the remainder. No gonyautoxins I-IV or C toxins were ever detected. In nutrient-replete batch cultures, toxin content rapidly peaked during early exponential phase and just as rapidly declined prior to the onset of plateau phase. Temperature had a marked effect on toxin content, which increased 3-fold as the temperature decreased from the optimum of 28 degrees C to 22 degrees C. Toxin content was constant at salinities of 24% or higher, but increased 3-fold at 20%. Toxin content decreased 2-fold and chlorophyll content increased 3-fold when light intensity was reduced from 90 to 15 microE m-2 s-1. This accompanied a 30% decrease in growth rate. Toxin composition (mole % individual toxin cell-1) remained constant throughout the course of the nutrient-replete culture and during growth at various salinities, but varied significantly with temperature and light intensity changes. At 22 degrees C, GTX5 was 25 mole % and NEO was 65 mole %, while at 34 degrees C, GTX5 increased to 55 mole % and NEO decreased proportionally to 40 mole %. When light intensity was reduced from 90 to 15 microE m-2 s-1, NEO decreased from 55 to 38 mole %, while GTX5 increased from 40 to 58 mole %. These data suggest that low light and high temperature both somehow enhance sulfo-transferase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and purification of a lethal scorpion toxin with neuromuscular blocking activity.

Toxin-L a lethal neuromuscular blocking agent was isolated from the venom of the scorpion, Lychas laevifrons (Pocock), by the CM-cellulose ion-exchange chromatography. It was a homogenous, thermolabile and low molecular weight protein. The toxin produced irreversible blockade of indirect stimulation induced twitch responses on innervated rat phrenic nerve-diaphragm and chick biventer cervicis preparation. The toxin did not produce any contractile response on toad rectus abdominis muscle preparation. On chronically denervated rat diaphragm, the toxin failed to alter the responses induced by direct stimulation, exogenous acetylcholine, potassium chloride and caffeine. Acetylcholine and carbachol induced contractions on isolated chick biventer cervicis remained unaltered by the toxin. Neostigmine failed to alter toxin induced neuromuscular blockade on innervated rat diaphragm. The toxin released a significant amount of acetylcholine from innervated rat diaphragm. It may be concluded that the toxin acts presynaptically through the release of acetylcholine, thereby producing neuromuscular blockade.

Postsynaptic block of a glutamatergic synapse by low molecular weight fractions of spider venom.

Fractions of low molecular weight (ca. 600-1000 dalton) isolated by high performance liquid chromatography (HPLC) and thin layer chromatography (TLC) from the venoms of the New-World spiders, Argiope trifasciata and Araneus gemma block transmission at glutamatergic nerve-muscle junctions in the locust, Schistocerca gregaria. These fractions are probably small peptides containing phenolic or indolic residues. Their effects on the neurally evoked twitch contraction, the junctional potential to glutamate ionophoresis and the voltage-clamped excitatory postsynaptic current of locust muscle indicate uncompetitive antagonism of the glutamate receptor channel complex through open channel block. In view of their slow reversibility of action they should make useful tools for future biochemical studies of locust glutamate receptors.