Presynaptic Activity of an Isolated Fraction from Rhinella schneideri Poison

Purpose: Rhinella schneideri is a toad found in many regions of the South America. The poison of the glands has cardiotoxic effect in animals and neuromuscular effects in mice and avian preparation. The purpose of this work was to identify the toxin responsible for the neuromuscular effect in avian and mice neuromuscular preparation. Methods: The methanolic extract from R. schneideri poison was fractioned by reversed phase HPLC. The purity and molecular mass were determined by LC/MS mass spectrometry. Chick biventer cervicis and mouse phrenic-nerve diaphragm were used as neuromuscular preparations to identify the toxin. Results: The purification resulted in 32 fractions, which 4 of them were active in neuromuscular preparation. The toxin of fraction 20 were chosen for better reproducibility of the whole extract activity and its molecular mass was 730.6 Da. The toxin produced facilitation of the muscle contraction followed by a complete neuromuscular blockade in chick biventer cervicis preparation in 90 min without interfering with the exogenous response to ACh and KCl. The quantal content was increased from 128 ± 13 (control) to 216 ± 44 (after 5 min and sustained until 60 min) in the presence of the toxin. Conclusion: In conclusion, our results demonstrated that the neuromuscular action of the poison of Rhinella schneideri is a multitoxin effect. More, the present work first isolated a 730.6 Da toxin that better represent the whole poison neuromuscular effect, to which is attributed a presynaptic action in avian and mouse neuromuscular preparation

Neurotoxic and convulsant effects induced by jack bean ureases on the mammalian nervous system

Ureases are microbial virulence factors either because of the enzymatic release of ammonia or due to many other non-enzymatic effects. Here we studied two neurotoxic urease isoforms, Canatoxin (CNTX) and Jack Bean Urease (JBU), produced by the plant Canavalia ensiformis, whose mechanisms of action remain elusive. The neurotoxins provoke convulsions in rodents (LD50 ∼2 mg/kg) and stimulate exocytosis in cell models, affecting intracellular calcium levels. Here, electrophysiological and brain imaging techniques were applied to elucidate their mode of action. While systemic administration of the toxins causes tonic-clonic seizures in rodents, JBU injected into rat hippocampus induced spike-wave discharges similar to absence-like seizures. JBU reduced the amplitude of compound action potential from mouse sciatic nerve in a tetrodotoxin-insensitive manner. Hippocampal slices from CNTX-injected animals or slices treated in vitro with JBU failed to induce long term potentiation upon tetanic stimulation. Rat cortical synaptosomes treated with JBU released L-glutamate. JBU increased the intracellular calcium levels and spontaneous firing rate in rat hippocampus neurons. MicroPET scans of CNTX-injected rats revealed increased Fluoro-deoxyglucose uptake in epileptogenesis-related areas like hippocampus and thalamus. Curiously, CNTX did not affect voltage-gated sodium, calcium or potassium channels currents, neither did it interfere on cholinergic receptors, suggesting an indirect mode of action that could be related to the ureases' membrane-disturbing properties. Understanding the neurotoxic mode of action of C. ensiformis ureases could help to unveil the so far underappreciated relevance of these toxins in diseases caused by urease-producing microorganisms, in which the human central nervous system is affected

In vitro and in vivo safety evaluation of Dipteryx alata Vogel extract

<p>Abstract</p> <p>Background</p> <p><it>Dipteryx alata </it>Vogel popularly known as "baru" is an important commercial leguminous tree species from the Brazilian Cerrado, which possess medicinal properties, besides its fruits consumption by animals and humans. The use of the "naturally occurring plants" as herbal remedies and foods mainly from leaves, seeds, flowers and roots of plants or extracts require precautions before ensuring these are safe and efficacious. The objective of this study was to evaluate the safety of <it>D. alata </it>barks extract.</p> <p>Methods</p> <p>Vegetal drugs of <it>D. alata </it>barks were submitted to quality control assays and further to the safety assays under 1) <it>in vitro </it>parameter by <it>Salmonella </it>(Ames) mutagenicity, and 2) <it>in vivo </it>parameter on the pregnancy of rats.</p> <p>Results</p> <p>The extract was non-mutagenic to any of the assessed strains TA97a, TA98, TA100 and TA102 even after metabolic activation (+S9). All <it>in vivo </it>parameters (reproductive ability evaluation, physical development of rat offsprings, and neurobehavioral development assays) showed no changes related to control group.</p> <p>Conclusion</p> <p><it>D. alata </it>barks extract is neither mutagenic by the Ames test nor toxic in the pregnancy of rats, with no physical-neurobehavioral consequences on the rat offsprings development.</p

The neuromuscular activity of paradoxin: a presynaptic neurotoxin from the venom of the inland taipan (Oxyuranus microlepidotus)

The inland taipan is the world's most venomous snake. However, little is known about the neuromuscular activity of the venom or paradoxin (PDX), a presynaptic neurotoxin from the venom. Venom (10microg/ml) and PDX (65nM) abolished indirect twitches of the chick biventer cervicis and mouse phrenic nerve diaphragm preparations. The time to 90% inhibition by PDX was significantly increased by replacing Ca(2+) (2.5mM) in the physiological solution with Sr(2+) (10mM). In the biventer cervicis muscle, venom (10microg/ml), but not PDX (65nM), significantly inhibited responses to ACh (1mM) and carbachol (20microM), but not KCl (40mM). In the mouse diaphragm (low Ca(2+); room temperature), the inhibitory effect of PDX (6.5nM) was delayed and a transient increase (746+/-64%; n=5) of contractions observed. In intracellular recording experiments using the mouse hemidiaphragm, PDX (6.5-65nM) significantly increased quantal content and miniature endplate potential frequency prior to blocking evoked release of acetylcholine. In extracellular recording experiments using the mouse triangularis sterni, PDX (2.2-65nM) significantly inhibited the voltage-dependent K(+), but not Na(+), waveform. In patch clamp experiments using B82 mouse fibroblasts stably transfected with rKv 1.2, PDX (22nM; n=3) had no significant effect on currents evoked by 10mV step depolarisations from -60 to +20mV. PDX exhibits all the pharmacology associated with beta-neurotoxins, and appears to be one of the most potent, if not the most potent beta-neurotoxin yet discovered

På jagt efter guldhornenes findested

Hunting for the find spots for the golden horns of GallehusIt is common knowledge that the golden horns were found at Gallehus in 1639 and 1734 and, consequently, so early that information on the find spots and finds circumstances is extremely sparse. In 1855, C.C. Rafn reached the conclusion that the horns were discovered in an undeveloped common area in the town (fig. 2), while in 1908, P. Lauridsen believed he had established the precise find spots, which were then marked with commemorative stones (figs. 3 and 4). With the discovery in 1951 of a report from 1734, it became clear that Lauridsen’s locations were incorrect and that the two finds spots are unlikely to have been more than 7 m apart, i.e. significantly less than the distance he concluded (fig. 1). This prompted Professor P.V. Glob to launch an investigation of the area in 1952, aimed at finding possible evidence that could explain these depositions. Glob continued his investigations in 1964, 1969 and 1971-72 (fig. 5). But he never managed to write a concluding report, and this article is an attempt to summarise his findings. The most important of these was the discovery of numerous pits, most of which were not very deep and had a very flat base. Many of them had apparently stood open for a shorter or longer period and they were therefore interpreted as clay pits (figs. 6-8). In 1969, some of these pits were found in an area corresponding to the find spot for one of the golden horns as specified in the 1734 source. Glob therefore believed he had found the actual find spot (figs. 10 and 12). The investigation in 1972 showed that these clay pits lay in the northeastern part of a larger more or less coherent complex of clay pits (fig. 7). Secure dating of these was not possible, but the fact that a house was built in 1832 over the southwesternmost corner of the complex testifies to a considerable age.Sven ThorsenNykøbing Falste

In Vitro Antiophidian Mechanisms of Hypericum brasiliense Choisy Standardized Extract: Quercetin-Dependent Neuroprotection

The neuroprotection induced by Hypericum brasiliense Choisy extract (HBE) and its main active polyphenol compound quercetin, against Crotalus durissus terrificus (Cdt) venom and crotoxin and crotamine, was enquired at both central and peripheral mammal nervous system. Cdt venom (10 μg/mL) or crotoxin (1 μg/mL) incubated at mouse phrenic nerve-diaphragm preparation (PND) induced an irreversible and complete neuromuscular blockade, respectively. Crotamine (1 μg/mL) only induced an increase of muscle strength at PND preparations. At mouse brain slices, Cdt venom (1, 5, and 10 μg/mL) decreased cell viability. HBE (100 μg/mL) inhibited significantly the facilitatory action of crotamine (1 μg/mL) and was partially active against the neuromuscular blockade of crotoxin (1 μg/mL) (data not shown). Quercetin (10 μg/mL) mimicked the neuromuscular protection of HBE (100 μg/mL), by inhibiting almost completely the neurotoxic effect induced by crotoxin (1 μg/mL) and crotamine (1 μg/mL). HBE (100 μg/mL) and quercetin (10 μg/mL) also increased cell viability in mice brain slices. Quercetin (10 μg/mL) was more effective than HBE (100 μg/mL) in counteracting the cell lysis induced by Cdt venom (1 and 10 μg/mL, resp.). These results and a further phytochemical and toxicological investigations could open new perspectives towards therapeutic use of Hypericum brasiliense standardized extract and quercetin, especially to counteract the neurotoxic effect induced by snake neurotoxic venoms

Inhibition of Kv2.1 Potassium Channels by MiDCA1, A Pre-Synaptically Active PLA2-Type Toxin from Micrurus dumerilii carinicauda Coral Snake Venom

MiDCA1, a phospholipase A2 (PLA2) neurotoxin isolated from Micrurus dumerilii carinicauda coral snake venom, inhibited a major component of voltage-activated potassium (Kv) currents (41 &plusmn; 3% inhibition with 1 &mu;M toxin) in mouse cultured dorsal root ganglion (DRG) neurons. In addition, the selective Kv2.1 channel blocker guangxitoxin (GxTx-1E) and MiDCA1 competitively inhibited the outward potassium current in DRG neurons. MiDCA1 (1 &micro;M) reversibly inhibited the Kv2.1 current by 55 &plusmn; 8.9% in a Xenopus oocyte heterologous system. The toxin showed selectivity for Kv2.1 channels over all the other Kv channels tested in this study. We propose that Kv2.1 channel blockade by MiDCA1 underlies the toxin&rsquo;s action on acetylcholine release at mammalian neuromuscular junctions

Pharmacological and structural characterization of a novel phospholipase A2 from Micrurus dumerilii carinicauda venom

We have isolated a new phospholipase A2 (MiDCA1) from the venom of the coral snake Micrurus dumerilii carinicauda. This toxin, which had a molecular mass of 15,552Da, shared high sequence homology with the PLA2 toxins MICNI A and B from Micrurus nigrocinctus venom (77.7% and 73.1%, respectively). In chick biventer cervicis preparations, MiDCA1 produced concentration- and time-dependent neuromuscular blockade that reached 100% after 120 min (2.4 microM, n = 6); contractures to exogenously applied carbachol (8 microM) and KCl (13 mM) were still seen after complete blockade. In mouse phrenic-nerve diaphragm preparations, MiDCA1 (2.4 microM; n = 6) caused triphasic changes followed by partial neuromuscular blockade. Intracellular recordings of end-plate potentials (EPPs) and miniature end-plate potentials (MEPPs) from mouse diaphragm preparations showed that MiDCA1 increased the quantal content by 386+/-12% after 10 min (n = 14; p<0.05) and caused a triphasic change in the frequency of MEPPs. MiDCA1 also decreased the resting membrane potential, an effect that was prevented by tetrodotoxin and/or low extracellular calcium, but not by d-tubocurarine. The toxin increased the amplitude of mouse sciatic-nerve compound action potentials by 30+/-9% (0.6 microM; p<0.05). Potassium currents elicited in freshly dissociated dorsal root ganglia neurones were blocked by 31+/-1% (n = 4; p<0.05) in the presence of 2.4 microM MiDCA1. These results show that MiDCA1 is a new presynaptic phospholipase A2 that produces neuromuscular blockade in vertebrate nerve-muscle preparations. The triphasic effects seen in mammalian preparations and the facilitatory response were probably caused mainly by the activation of sodium channels, complemented by the blockade of nerve terminal potassium channels. The inability of d-turocurarine to prevent the depolarization by MiDCA1 indicated that cholinergic nicotinic receptors were not involved in this phenomenon