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The cobra (genus Naja ( N. )) is one of the most common venomous snakes. Due to its frequency and deadly complications of muscle paralysis, local necrosis, and chronic musculoskeletal disability, it should not be ignored. The pathology of devastating tissue destruction, even though specific antivenoms exist, is not fully clear. Here, we attempted to dig in envenomed tissues to study the clinical toxicology of cobra venom. Four cases of N. atra snake envenomation, in which the subjects developed advanced tissue injury, were involved in this study. We used enzyme-ligand sandwich immunoassay (ELISA) to assay the whole venom, cytotoxin A3 and short-chain neurotoxin (sNTX) in blood, bullae, wound discharge, and debrided tissue. We found that persistently high concentrations of venom and toxins, especially cytotoxin A3, were detected in bullae, wound discharge fluid and necrotic tissue of these patients even after large doses of specific antivenom treatment, and wide excision and advanced debridement could largely remove these toxins, lessen the size of necrosis, and promote wound healing. We also found that the point-of-care apparatus, ICT-Cobra kit, might be used to promptly monitor the wound condition and as one of the indicators of surgical intervention in cases of cobra envenomation in Taiwan.

Development of a rapid, on-site detection tool for snakebite is highly sought after, owing to its clinically and forensically relevant medicolegal significance. Polyvalent antivenom therapy in the management of such envenomation cases is finite due to its poor venom neutralization capabilities as well as diagnostic ramifications manifested as untoward immunological reactions. For precise molecular diagnosis of elapid venoms of the big four snakes, we have developed a lateral flow kit using a monoclonal antibody (AB1; IgG1 - κ chain; Kd: 31 nM) generated against recombinant cytotoxin-7 (rCTX-7; 7.7 kDa) protein of the elapid venom. The monoclonal antibody specifically detected the venoms of Naja naja (p < 0.0001) and Bungarus caeruleus (p<0.0001), without showing any immunoreactivity against the viperidae snakes in big four venomous snakes. The kit developed attained the limit of quantitation of 170 pg/μL and 2.1 ng/μL in spiked buffer samples and 28.7 ng/μL and 110 ng/μL in spiked serum samples for detection of N. naja and B. caeruleus venoms, respectively. This kit holds enormous potential in identification of elapid venom of the big four snakes for effective prognosis of an envenomation; as per the existing medical guidelines., Competing Interests: The authors have declared that no competing interests exist.

Three cytotoxin-like proteins from the venom of Ophiophagus hannah were isolated by a combination of ion exchange chromatography and reverse phase HPLC. Amino acid sequence analysis revealed that these proteins all consisted of 63 amino acids and shared approximate 50% and 56% sequence identity with Naja naja atra cardiotoxins and cardiotoxin-like basic proteins (CLBPs), respectively. CD spectra revealed that their secondary structure was dominated with beta-sheet as those noted with cardiotoxins and CLBPs. O. hannah cytotoxin-like protein exhibited a cell-lytic activity on SK-N-SH cells, but its activity was more weak than that noted for N. naja atra cardiotoxin 3. Alternatively, apoptotic cell death was induced by the addition of N. naja atra CLBP. Based on the sequence information with the toxin molecules, the functional residues and regions related to the differential activity with O. hannah cytotoxin-like protein, cardiotoxin and CLBP are discussed.

A study has been made of the cytotoxic activity of basic phospholipase A2 of venom from Naja nigricollis on different types of cells and of the participation of esterase activity in this cytotoxic activity. It was previously shown that the cytotoxicity observed is not due to a contaminant, since the cytotoxic action vanished after immunoprecipitation of basic phospholipase A2 by specific monoclonal antibodies. All eukaryotic cells tested were sensitive to the cytotoxic action of basic phospholipase A2. In contrast, Escherichia coli K-12 wild strain was resistant. Participation of cell membranes in the sensitive or resistant character of cells to the phospholipase A2 attack was investigated using E. coli K-12 membrane mutants. Some membrane mutants were sensitive and the sensitivity or resistance to basic phospholipase A2 was found to be related to a single mutation in the locus envA. The requirement for esterase activity of phospholipase A2 in cytotoxic attack has been shown to be dependent on the cell line tested. Indeed, when the esterase activity of basic phospholipase A2 was eliminated by treatment with p-bromophenacyl bromide, the enzyme retained a cytotoxic potency inducing necrosis of certain tumoral cells grown in vitro, but not other cells such as erythrocytes, for which concomitant esterase activity was also necessary. In vivo studies of toxicity showed that the loss of either cytotoxic potency or esterase activity eliminates the lethal character of basic phospholipase A2. This leads us to propose that in vivo toxicity of phospholipase A2 depends on simultaneous expression of esterase activity and a non-enzymatic property, manifested by in vitro cytotoxic potency.

This paper describes how small angle neutron scattering (SANS) can be used to confirm that gel filtration results are free of dimerization effects. After characterization by analytical gel filtration, concentrated solutions (in heavy water, D2O) of a cobra neurotoxin, a cytotoxin, and a cytotoxin analog are studied by SANS. Small differences in shape are shown to be discernible by means of least-square fits to ellipsoidal models. The parallel axis theorem is then invoked to assess dimerization levels statistically. The results are briefly discussed on the basis of function in relation to structure.

A cytotoxin-like basic protein has been isolated from the venom of the nominate race of cobra (Naja naja naja from Pakistan) by a single step of high-performance liquid chromatography. The primary structure was determined and consists of 62 amino acid residues in a single polypeptide chain. It is highly similar to that of the cytotoxin-like basic proteins isolated from other Naja species, but differs in two of the SS-loop structures from that of cytotoxins.

Interaction between cytotoxin of the Central Asia cobra venom and dimiristoylphosphatidylcholine bilayer depending on its phase state was studied by ESR with spin label. A conclusion can be drawn that the efficiency of cytotoxin effect on the membranes depends on their phase state. Cytotoxin molecules are incorporated into myophile region of the bilayer, only if the latter is in the liquid crystal state. The interaction between cytotoxins and lipids of the bilayer in a gel state is in the main conditioned by electrostatic forces.

Two kinds of distance matrices have been formed from minimum mutational distances and absolute hydrophobicity differences obtained by comparison of aligned homologous sequences of 56 toxins from venom of snakes belonging to 7 genera. Phylogenetic trees were constructed from these distance matrices, employing the unweighted pair-group method using arithmetic averages (UPGMA). The Pearson product-moment correlation coefficient has been used to estimate the agreement between the original distance matrix and that obtained directly from the dendrogram. For all these procedures the set of computer programs PHYTREE (written in BASIC for micro-computer, and available from the author) has been used.

The review will critically assess the information available on the conformation of homologous neurotoxins and cytotoxins isolated from Elapid snakes. Particular attention will be given to the dynamics of the molecules in solution because there is the possibility that defined intramolecular rearrangements are involved at the sites of action. Such properties will be then reconciled with the known X-ray crystallographic and sequence data in order to derive likely structure-activity relationships.

A less-cytotoxic polypeptide, designated as LCBP, was isolated from the venom of Naja naja by gel filtration on Sephadex G-50 followed by CM-cellulose chromatography. The cytotoxicity toward Yoshida sarcoma cells and lethal toxicity toward mice of LCBP were both one order of magnitude lower than that of cytotoxins and that of toxin A, respectively. LCBP is a single polypeptide consisting of 61 amino acid residues with four intramolecular disulfide linkages, and the amino acid sequence is the same as that of cardiotoxin-like basic polypeptide (CLBP) isolated from the venom of Naja naja atra. This is the first time that the same polypeptides were isolated from different cobra venoms.

It is suggested, on the basis of the structural information available from the literature, that the molecules of cobramine B and homologous cytotoxins, in contrast to snake venom neurotoxins, are amphiphilic in the sense that they are composed of a predominantly hydrophobic multi-stranded beta-sheet and other regions sharply hydrophilic. It is possible that the direct lytic activity of snake venom cytotoxins is due, at least in part, to their amphiphathy.