Weak Neurotoxins Research Articles

Overlooked Risks of Biochars: Persistent Free Radicals trigger Neurotoxicity in Caenorhabditis elegans.

In recent years, biochars have gained increasing interest in mitigating climate changes and revitalizing contaminated or drained soil. Studies determining their impact on the ecosystem, especially on soil invertebrates, however, are still scarce and the neurotoxic potential of biochars has never been evaluated before. Using the model organism Caenorhabditis elegans we determined the neurotoxic effect of biochar produced from rice straw by pyrolysis at 500 °C at concentrations ranging from 0 to 2000 mg C·L-1. Biochar had a hormetic effect on locomotion behavior. Furthermore, high concentrations impaired defecation as well as the recognition and response to a chemical attractant. None of the potential toxic chemicals in the biochar had sufficient high concentrations to explain the detected neurotoxic effect. Using electron paramagnetic resonance (EPR) spectroscopy, we detected free radicals in the biochar. Detrimental reaction of free radicals with biotic macromolecules can induce oxidative stress responses and are a potential reason for the evaluated neurotoxic effect of biochar. Overall, we were able to prove that biochars have the potential to act as weak neurotoxins to soil organisms and effects of persistent free radicals should be investigated further.

Venomics of Naja sputatrix, the Javan spitting cobra: A short neurotoxin-driven venom needing improved antivenom neutralization

The venom proteome of Naja sputatrix (Javan spitting cobra) was elucidated through reverse-phase HPLC, nano-ESI-LCMS/MS and data mining. A total of 97 distinct protein forms belonging to 14 families were identified. The most abundant proteins are the three-finger toxins (3FTXs, 64.22%) and phospholipase A2 (PLA2, 31.24%), followed by nerve growth factors (1.82%), snake venom metalloproteinase (1.33%) and several proteins of lower abundance (<1%) including a variety of venom enzymes. At subproteome, the 3FTx is dominated by cytotoxins (48.08%), while short neurotoxins (7.89%) predominate over the long neurotoxins (0.48%) among other neurotoxins of lesser toxicity (muscarinic toxin-like proteins, 5.51% and weak neurotoxins, 2.26%). The major SNTX, CTX and PLA2 toxins were isolated with intravenous median lethal doses determined as 0.13, 1.06 and 0.50μg/g in mice, respectively. SABU, the Indonesia manufactured homologous tri-specific antivenom could neutralize the CTX and PLA2 fraction with moderate potency (potency=0.14–0.16mg toxin per ml antivenom). The SNTX, however, was very poorly neutralized with a potency level of 0.034mg/ml, indicating SNTX as the main limiting factor in antivenom neutralization. The finding helps elucidate the inferior efficacy of SABU reported in neutralizing N. sputatrix venom, and supports the call for antivenom improvement. Biological significanceThe Javan spitting cobra, Naja sputatrix is by itself a unique species and should not be confused as the equatorial and the Indochinese spitting cobras. The distinction among the spitting cobras was however unclear prior to the revision of cobra systematics in the mid-90's, and results of some earlier studies are now questionable as to which species was implicated back then. The current study successfully profiled the venom proteome of authenticated N. sputatrix, and showed that the venom is made up of approximately 64% three-finger toxins (including neurotoxins and cytotoxins) and 31% phospholipases A2 by total venom proteins. The findings verified that the paralyzing components in the venom i.e. neurotoxins are predominantly the short-chain subtype (SNTX) far exceeding the long-chain subtype (LNTX) which is more abundant in the venoms of monocled cobra and Indian common cobra. The neurotoxicity of N. sputatrix venom is hence almost exclusively SNTX-driven, and effective neutralization of the SNTX is the key to early reversal of paralysis. Unfortunately, as shown through a toxin-specific assay, the immunological neutralization of the SNTX using the Indonesian antivenom (SABU) was extremely weak, implying that SABU has limited therapeutic efficacy in treating N. sputatrix envenomation clinically. From the practical standpoint, actions need to be taken at all levels from laboratory to production and policy making to ensure that the shortcoming is overcome.

Proteomic analysis of Moroccan cobra Naja haje legionis venom using tandem mass spectrometry

The proteome of the venom of Naja haje legionis, the only medically important elapid species in Morocco, has been elucidated by using a combination of proteomic techniques that includes size exclusion chromatography, reverse-phase HPLC, Tricine/SDS-Page, tryptic digestion, Q-TOF tandem mass spectrometry and database search. The sequence analysis of venom fractions revealed a highly complex venom proteome which counts a total of 76 proteins identified from database that can be assigned into 9 proteins families. We report the identification of: cobra venom factor (CVF), l-amino-acid oxidases (LAAO), acetylcholinesterase (AChE), snake venom metalloproteinases (SVMP), cysteine rich secretory proteins (CRISP), venom nerve growth factor (vNGF), phospholipases A2 (PLA2), vespryns, kunitz-type inhibitor, short neurotoxins, long neurotoxins, weak neurotoxins, neurotoxin like proteins, muscarinic toxins, cardiotoxins and cytotoxins. Comparison of these proteins showed high sequence homology with proteins from other African and Asian cobras. Further works are needed to assess the contribution of individual toxins in venom toxicity. Naja haje legionis is one of the medically important snakes implicated in the pathogenesis of snake bite in Morocco. The absence of information about venom composition and clinical manifestations of envenomation by this cobra represents an obstacle for the management of this environmental disease in the country. The elucidation of Moroccan cobra venom composition will provide a reasonable guidance for clinician to understand the pathophysiological conditions associated with cobra envenomation and the elaboration of better management strategies.

225. Proteomic Analysis and Pharmacological Activities of the Venom of the Moroccan Cobra Naja haje legionis.

Background: In Morocco, envenomation by snake bites poses a serious problem to public health. The cobra Naja haje legionis is endemic of the country and one of the most dangerous species known. In this work, we report the proteomic and pharmacological characterizations of biologically active proteins from the venom of this species. Methods: The various proteins in the crude venom were fractionated and analyzed by a combination of chromatographic separations and proteomic analysis techniques such as gel filtration, RP-HPLC, 1D electrophoresis, in-gel digestion, tandem mass spectrometry and protein database search. The pharmacological properties of Naja haje legionis venom were assessed using in vitro preparations using rodents and chicks. Results: Our venomic strategy allowed the identification of 64 proteins and peptides from known database which can classified into 17 families according to their biological activities. We were able to identify cobra venom factor, L-aminoacid oxidases, acetylcholinesterase, metalloproteinase, disintegrin, cysteine-rich secretory proteins, nerve growth factor, phospholipases A2, vespryns, kunitz-type inhibitor, short neurotoxins, long neurotoxins, weak neurotoxins, neurotoxin like proteins, muscarinic toxins, cytotoxins and cardiotoxins. Pharmacological tests showed that Naja haje legionis venom contained neurotoxic activities inducing irreversible blockage of neuromuscular transmission in both rodent and chick nerve-muscle preparations. This venom also exhibit myotoxic and cardiotoxic activities. Conclusions: These results showed the potency and the complexity of the various proteins present in the venom of Naja haje legionis,which present a very similar pattern to other cobra venoms. The contribution of the different components in venom toxicity deserves further investigations.

Venomic analysis and evaluation of antivenom cross-reactivity of South American Micrurus species

Coral snakes from Micrurus genus are the main representatives of the Elapidae family in South America. However, biochemical and pharmacological features regarding their venom constituents remain poorly investigated. Here, venomic analyses were carried out aiming at a deeper understanding on the composition of M. frontalis, M. ibiboboca, and M. lemniscatus venoms. In the three venoms investigated, proteins ranging from 6 to 8 kDa (3FTx) and 12 to 14 kDa (PLA(2)) were found to be the most abundant. Also, the N-terminal sequences of four new proteins, purified from the M. lemniscatus venom, similar to 3FTx, PLA(2) and Kunitz-type protease inhibitor from other Micrurus and elapid venoms are reported. Cross-reactivity among different Micrurus venoms and homologous or heterologous antivenoms was carried out by means of 2D-electrophoresis and immunoblotting. As, expected, the heterologous anti-Elapid venom displayed the highest degree of cross-reactivity. Conversely, anti-M. corallinus reacted weakly against the tested venoms. In gel digestions, followed by mass spectrometry sequencing and similarity searching, revealed the most immunogenic protein families as similar to short and long neurotoxins, weak neurotoxins, PLA(2), β-bungarotoxin, venom protein E2, frontoxin III, LAO and C-type lectin. The implications of our results for the production of Micrurus antivenoms are discussed.

Biochemical characterization of the Micrurus pyrrhocryptus venom

Snake venom toxicity is the consequence of a combination of peptides and proteins whose identification and characterization are of great importance to understand envenomation and develop new clinical treatments. The Elapinae subfamily includes coral snakes whose bite causes mainly neurotoxic effects which disable muscle contraction and paralyse the heart as well as inhibit respiration. However, the structure–function relationship of venom toxins has been investigated only for a few species. We herein study biological aspects of the Micrurus pyrrhocryptus venom such as LD 50, hemorrhagic, necrotic, coagulant, myotoxic and hemolytic activity as well as the ability of venom components to compete with alpha-Bungarotoxin for the ligand-binding site of the nicotinic acetylcholine receptor. Besides, we report the determination of the molecular mass and N-terminal sequence of toxins including PLA2s, short, long and weak neurotoxins. The complete sequence of one of the short neurotoxins has also been obtained, this being the first sequence of an alpha-neurotoxin determined in the M. pyrrhocryptus venom and one of the few fully determined in members of the Micrurus genus.

Phospholipase A 2-interacting weak neurotoxins from venom of monocled cobra Naja kaouthia display cell-specific cytotoxicity

The molecular weights of two phospholipase A 2 (PLA 2)-interacting polypeptides (kaouthiotoxins (KTXs)—KTX-A and KTX-B) purified from the venom of monocled cobra Naja kaouthia, were estimated by mass spectrometry as 7722 and 7627 Da, respectively. Binary sequence alignment showed that both KTXs share substantial sequence homology with weak neurotoxins from cobra venom and they were devoid of any enzymatic activity. Their p I was determined at pH 8.1 showing basic nature of these proteins. KTXs displayed cell-specific cytotoxicity on mammalian and insect cells.

Behavioural Effects in Mice and Intoxication Symptomatology of Weak Neurotoxin from Cobra Naja kaouthia

Weak neurotoxins belong to the superfamily of three-finger toxins from snake venoms. In general, weak toxins have a low toxicity and, contrary to other three-finger toxins, their molecular targets are not well characterized: in vitro tests indicate that these may be nicotinic acetylcholine receptors. Here, we report the influence of intraperitoneal and intravenous injections of weak neurotoxin from Naja kaouthia venom on mouse behaviour. Dose-dependent suppression of orientation-exploration and locomotion activities as well as relatively weak neurotropic effects of weak neurotoxin were observed. The myorelaxation effect suggests a weak antagonistic activity against muscle-type nicotinic acetylcholine receptors. Neurotoxic effects of weak neurotoxin were related to its influence on peripheral nervous system. The symptomatology of the intoxication was shown to resemble that of muscarinic agonists. Our data suggest that, in addition to interaction with nicotinic acetylcholine receptors observed earlier in vitro, weak neurotoxin interacts in vivo with some other molecular targets. The results of behavioural experiments are in accord with the pharmacological profile of weak neurotoxin effects on haemodynamics in mice and rat indicating the involvement of both nicotinic and muscarinic acetylcholine receptors.

Novel genes encoding six kinds of three-finger toxins in Ophiophagus hannah (king cobra) and function characterization of two recombinant long-chain neurotoxins.

Three-finger toxins are a family of low-molecular-mass toxins (<10 kDa) having very similar three-dimensional structures. In the present study, 19 novel cDNAs coding three-finger toxins were cloned from the venom gland of Ophiophagus hannah (king cobra). Alignment analysis showed that the putative peptides could be divided into six kinds of three-finger toxins: LNTXs (long-chain neurotoxins), short-chain neurotoxins, cardiotoxins (CTXs), weak neurotoxins, muscarinic toxins and a toxin with a free SH group. Furthermore, a phylogenetic tree was established on the basis of the toxin cDNAs and the previously reported similar nucleotide sequences from the same source venom. It indicated that three-finger-toxin genes in O. hannah diverged early in the course of evolution by long- and short-type pathways. Two LNTXs, namely rLNTX1 (recombinant LNTX1) and rLNTX3, were expressed and showed cytolytic activity in addition to their neurotoxic function. By comparing the functional residues, we offer some possible explanations for the differences in their neurotoxic function. Moreover, a plausible elucidation of the additonal cytolytic activity was achieved by hydropathy-profile analysis. This, to our knowledge, is the first observation that recombinant long chain alpha-neurotoxins have a CTX-like cytolytic activity.

Weak neurotoxin from Naja kaouthia cobra venom affects haemodynamic regulation by acting on acetylcholine receptors

Recent in vitro studies of weak neurotoxins from snake venoms have demonstrated their ability to interact with both muscle-type and neuronal α7 nicotinic acetylcholine receptors (nAChR). However, the biological activity in vivo of weak neurotoxins remains largely unknown. We have studied the influence of weak neurotoxin (WTX) from the venom of cobra Naja kaouthia on arterial blood pressure (BP) and heart rate (HR) in rats and mice. It was found that intravenous injection of WTX induced a dose-dependent decrease in BP and an increase in HR in both species, the rats being more sensitive to WTX. Application of WTX following blockade of nAChRs or muscarinic acetylcholine receptors (mAChR) by hexamethonium or atropine, respectively, showed that both nAChRs and mAChRs are involved in the haemodynamic effects of WTX. Blockade of either nAChRs or mAChRs affected WTX action differently in rats and mice, thus reflecting interspecies differences in haemodynamic regulation.

On the neurotoxicity of glutaric, 3-hydroxyglutaric, and trans-glutaconic acids in glutaric acidemia type 1.

Glutaric acidemia type 1 (GA1) is an autosomal recessively inherited deficiency of glutaryl-CoA dehydrogenase. Accumulating metabolites, 3-hydroxyglutaric (3-OH-GA), glutaric (GA), and trans-glutaconic (TG) acids, have been proposed to be involved in the development of the striatal degeneration seen in children with GA1 via an excitotoxic mechanism. We have studied the extent to which 3-OH-GA, GA, and TG are neurotoxic and whether neurotoxicity is caused by an excitotoxic mechanism in which 3-OH-GA, GA, or TG overactivates N-methyl-D-aspartate (NMDA) receptors. In cultured mouse neocortical neurons, all three compounds were weakly neurotoxic, possibly through activation of NMDA receptors. However, further studies in the rat cortical wedge preparation and with NMDA receptors expressed in Xenopus oocytes could not confirm an interaction of the compounds with NMDA receptors. It is concluded that the metabolites 3-OH-GA, GA, and TG are only weak neurotoxins and that the neurodegenerative cascade destroying the striatum in patients with GA1 involves mainly mechanisms other than excitoxicity.

Structurally conserved α-neurotoxin genes encode functionally diverse proteins in the venom of Naja sputatrix

The structure and organization of the genes encoding the long-chain neurotoxins and four other isoforms of weak neurotoxins in the venom of Naja sputatrix are reported. The genes contained three exons interrupted by two introns, a structure similar to other members of the three-finger toxin family. The proteins encoded by these genes, however, show varied affinity towards nicotinic acetylcholine receptors. Phylogenetic analysis of these genes showed that the weak neurotoxin gene is confined to a distinct group. We also observe that specific mutations of the gene provide the diversity in function in these toxins while maintaining a common structural scaffold. This forms the first report where the molecular basis of evolution of postsynaptic neurotoxins from an ancestral gene can be demonstrated using the same species of snake.

First tryptophan-containing weak neurotoxin from cobra venom

With the purpose of studying structure–function relationships among weak neurotoxins (called so because of their low toxicity), we have isolated a toxin (WTX) from the venom of cobra Naja kaouthia using a combination of gel-filtration and ion-exchange chromatography. The amino acid sequence of the isolated toxin was determined by means of Edman degradation and MALDI mass spectrometry, the primary structure obtained being confirmed by 1H-NMR in the course of spatial structure analysis. The WTX sequence differs slightly from that of the toxin CM-9a isolated earlier from the same venom (Joubert and Taljaard, Hoppe-Seyler's Z. Physiol. Chem., 361 (1980) 425). The differences include an extra residue (Trp36) between Ser35 and Arg37 as well as interchanging of two residues (Tyr52 and Lys50) in the C-terminal part of the toxin molecule. These changes improve the alignment that can be made with other weak neurotoxin sequences. An extended sequence comparison reveals that WTX is the first case of a tryptophan-containing weak neurotoxin isolated from cobra venom. WTX was found to compete with radioiodinated α-bungarotoxin for binding to the membrane-bound nicotinic acetylcholine receptor from Torpedo californica.