Naja annulifera Snake: New insights into the venom components and pathogenesis of envenomation.

BACKGROUND: Naja annulifera is a medically important venomous snake occurring in some of the countries in Sub-Saharan Africa. Accidental bites result in severe coagulation disturbances, systemic inflammation and heart damage, as reported in dogs, and death, by respiratory arrest, in humans. Despite the medical importance of N. annulifera, little is known about its venom composition and the pathogenesis of envenomation. In this paper, the toxic, inflammatory and immunogenic properties of N. annulifera venom were analyzed. METHODOLOGY/PRINCIPAL FINDINGS: Venom proteomic analysis identified 79 different proteins, including Three Finger Toxins, Cysteine Rich Secretory Proteins, Metalloproteinases, Phospholipases A2 (PLA2), Hyaluronidase, L-amino-acid oxidase, Cobra Venom Factor and Serine Proteinase. The presence of PLA2, hyaluronidase, fibrinogenolytic and anticoagulant activities was detected using functional assays. The venom was cytotoxic to human keratinocytes. In an experimental murine model of envenomation, it was found that the venom induced local changes, such as swelling, which was controlled by anti-inflammatory drugs. Moreover, the venom caused death, which was preceded by systemic inflammation and pulmonary hemorrhage. The venom was shown to be immunogenic, inducing a strong humoral immune response, with the production of antibodies able to recognize venom components with high molecular weight and to neutralize its lethal activity. CONCLUSIONS/SIGNIFICANCE: The results obtained in this study demonstrate that N. annulifera venom contains toxins able to induce local and systemic inflammation, which can contribute to lung damage and death. Moreover, the venom is immunogenic, an important feature that must be considered during the production of a therapeutic anti-N. annulifera antivenom.

Rapid ligand fishing for identification of acetylcholinesterase-binding peptides in snake venom reveals new properties of dendrotoxins.

Acetylcholinesterase (AChE) from Electrophorus electricus (eel) was immobilized on the surface of amino-modified paramagnetic beads to serve as a model for the development, validation and application of a new affinity-based ligand-fishing assay for the discovery of bioactive peptides from complex protein mixtures such as venoms. Nano liquid chromatography-mass spectrometry (nanoLC-MS) was used for the analysis of trapped peptides. Using enzyme-functionalized beads, the ligand-fishing assay was evaluated and optimized using a peptide reference mixture composed of one acetylcholinesterase binder (fasciculin-II) and five non-binders (mambalgin-1, angiotensin-II, bradykinin, cardiotoxin and α-bungarotoxin). As proof of concept, snake venom samples spiked with fasciculin-II demonstrated assay selectivity and sensitivity, fishing the peptide binder from complex venom solutions at concentrations as low as 1.0 µg/mL. As negative controls for method validation, venoms of four different snake species, not known to harbor AChE binding peptides, were screened and no AChE binders were detected. The applicability of the ligand fishing assay was subsequently demonstrated with venom from the black mamba, Jameson's mamba and western green mamba (Dendroaspis spp.), which have previously been reported to contain the AChE binding fasciculins. Unknown peptides (i.e. not fasciculins) with affinity to AChE were recovered from all mamba venoms tested. Tryptic digestion followed by nano-LC-MS analysis of the material recovered from black mamba venom identified the peptide with highest AChE-binding affinity as dendrotoxin-I, a pre-synaptic neurotoxin previously not known to interact with AChE. Co-incubation of AChE with various dendrotoxins in vitro revealed reduced inactivation of AChE activity over time, thus demonstrating that these toxins stabilize AChE.

Non-neurotoxic activity of Malayan krait (Bungarus candidus) venom from Thailand

Background: Envenoming by kraits (genus Bungarus) is a medically significant issue in South Asia and Southeast Asia. Malayan krait (Bungarus candidus) venom is known to contain highly potent neurotoxins. In recent years, there have been reports on the non-neurotoxic activities of krait venom that include myotoxicity and nephrotoxicity. However, research on such non-neurotoxicity activities of Malayan krait venom is extremely limited. Thus, the aim of the present study was to determine the myotoxic, cytotoxic and nephrotoxic activities of B. candidus venoms from northeastern (BC-NE) and southern (BC-S) Thailand in experimentally envenomed rats. Methods: Rats were administered Malayan krait (BC-NE or BC-S) venom (50 g/kg, i.m.) or 0.9% NaCl solution (50 L, i.m.) into the right hind limb. The animals were sacrificed 3, 6 and 24 h after venom administration. The right gastrocnemius muscle and both kidneys were collected for histopathological analysis. Blood samples were also taken for determination of creatine kinase (CK) and lactate dehydrogenase (LDH) levels. The human embryonic kidney cell line (HEK-293) was used in a cell proliferation assay to determine cytotoxic activity. Results: Administration of BC-NE or BC-S venom (50 g/kg, i.m.) caused time-dependent myotoxicity, characterized by an elevation of CK and LDH levels. Histopathological examination of skeletal muscle displayed marked muscle necrosis and myofiber disintegration 24 h following venom administration. Both Malayan krait venoms also induced extensive renal tubular injury with glomerular and interstitial congestion in rats. BC-NE and BC-S venoms (1000.2 g/ mL) caused concentration-dependent cytotoxicity on the HEK-293 cell line. However, BC-NE venom (IC50 =8 ± 1 g/mL; at 24 h incubation; n = 4) was found to be significantly more cytotoxic than BC-S venom (IC50 =15 ± 2 g/mL; at 24 h incubation; n = 4). In addition, the PLA2 activity of BC-NE venom was significantly higher than that of BC-S venom...

In-Depth Glyco-Peptidomics Approach Reveals Unexpected Diversity of Glycosylated Peptides and Atypical Post-Translational Modifications in Dendroaspis angusticeps Snake Venom.

Animal venoms represent a valuable source of bioactive peptides that can be derived into useful pharmacological tools, or even innovative drugs. In this way, the venom of Dendroaspis angusticeps (DA), the Eastern Green Mamba, has been intensively studied during recent years. It mainly contains hundreds of large toxins from 6 to 9 kDa, each displaying several disulfide bridges. These toxins are the main target of venom-based studies due to their valuable activities obtained by selectively targeting membrane receptors, such as ion channels or G-protein coupled receptors. This study aims to demonstrate that the knowledge of venom composition is still limited and that animal venoms contain unexpected diversity and surprises. A previous study has shown that Dendroaspis angusticeps venom contains not only a cocktail of classical toxins, but also small glycosylated peptides. Following this work, a deep exploration of DA glycopeptidome by a dual nano liquid chromatography coupled to electrospray ionization mass spectrometry (nanoLC-ESI-MS) and Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) analyses was initiated. This study reveals unsuspected structural diversity of compounds such as 221 glycopeptides, displaying different glycan structures. Sequence alignments underline structural similarities with natriuretic peptides already characterized in Elapidae venoms. Finally, the presence of an S-cysteinylation and hydroxylation of proline on four glycopeptides, never described to date in snake venoms, is also revealed by proteomics and affined by nuclear magnetic resonance (NMR) experiments.

Australian elapid snake envenomation in cats: Clinical priorities and approach.

Practical relevance: No fewer than 140 species of terrestrial snakes reside in Australia, 92 of which possess venom glands. With the exception of the brown tree snake, the venom-producing snakes belong to the family Elapidae. The venom of a number of elapid species is more toxic than that of the Indian cobra and eastern diamondback rattle snake, which has earned Australia its reputation for being home to the world's most venomous snakes. Clinical challenges: The diagnosis of elapid snake envenomation is not always easy. Identification of Australian snakes is not straightforward and there are no pathognomonic clinical signs. In cats, diagnosis of envenomation is confounded by the fact that, in most cases, there is a delay in seeking veterinary attention, probably because snake encounters are not usually witnessed by owners, and also because of the tendency of cats to hide and seek seclusion when unwell. Although the administration of antivenom is associated with improved outcomes, the snake venom detection kit and antivenom are expensive and so their use may be precluded if there are financial constraints. Evidence base: In providing comprehensive guidance on the diagnosis and treatment of Australian elapid snake envenomation in cats, the authors of this review draw on the published veterinary, medical and toxicology literature, as well as their professional experience as specialists in medicine, and emergency medicine and critical care.

Proteomics and antivenomics of Papuan black snake (Pseudechis papuanus) venom with analysis of its toxicological profile and the preclinical efficacy of Australian antivenoms.

The Papuan black snake (Pseudechis papuanus Serpentes: Elapidae) is endemic to Papua New Guinea, Indonesian Papua and Australia's Torres Strait Islands. We have investigated the biological activity and proteomic composition of its venom. The P. papuanus venom proteome is dominated by a variety (n≥18) of PLA2s, which together account for ~90% of the venom proteins, and a set of low relative abundance proteins, including a short-neurotoxic 3FTx (3.1%), 3-4 PIII-SVMPs (2.8%), 3 cysteine-rich secretory proteins (CRISP; 2.3%) 1-3 l-amino acid oxidase (LAAO) molecules (1.6%). Probing of a P. papuanus cDNA library with specific primers resulted in the elucidation of the full-length nucleotide sequences of six new toxins, including vespryn and NGF not found in the venom proteome, and a calglandulin protein involved in toxin expression with the venom glands. Intravenous injection of P. papuanus venom in mice induced lethality, intravascular haemolysis, pulmonary congestion and oedema, and anticoagulation after intravenous injection, and these effects are mainly due to the action of PLA2s. This study also evaluated the in vivo preclinical efficacy of Australian black snake and polyvalent Seqirus antivenoms. These antivenoms were effective in neutralising the lethal, PLA2 and anticoagulant activities of P. papuanus venom in mice. On the other hand, all of the Seqirus antivenoms tested using an antivenomic approach exhibited strong immunorecognition of all the venom components. These preclinical results suggest that Australian Seqirus1 antivenoms may provide paraspecific protection against P. papuanus venom in humans. SIGNIFICANCE PARAGRAPH: The toxicological profile and proteomic composition of the venom of the Papuan black snake, Pseudechis papuanus, a large diurnal snake endemic to the southern coast of New Guinea and a handful of close offshore islands, were investigated. Intravenous injection of P. papuanus venom in mice induced intravascular hemolysis, pulmonary congestion and edema, anticoagulation, and death. These activities could be assigned to the set of PLA2 molecules, which dominate the P. papuanus venom proteome. This study also showed that Australian Seqirus black snake or polyvalent antivenoms were effective in neutralising the lethal, PLA2 and anticoagulant activities of the venom. These preclinical results support the continued recommendation of these Seqirus antivenoms in the clinical management of P. papuanus envenoming in Australia, Papua New Guinea or Indonesian Papua Province.

Exploring the venom of the forest cobra snake: Toxicovenomics and antivenom profiling of Naja melanoleuca.

A toxicovenomic analysis of the venom of the forest cobra, N. melanoleuca, was performed, revealing the presence of a total of 52 proteins by proteomics analysis. The most abundant proteins belong to the three-finger toxins (3FTx) (57.1wt%), which includes post-synaptically acting α-neurotoxins. Phospholipases A2 (PLA2) were the second most abundant group of proteins (12.9wt%), followed by metalloproteinases (SVMPs) (9.7wt%), cysteine-rich secretory proteins (CRISPs) (7.6wt%), and Kunitz-type serine proteinase inhibitors (3.8wt%). A number of additional protein families comprised each <3wt% of venom proteins. A toxicity screening of the fractions, using the mouse lethality test, identified toxicity in RP-HPLC peaks 3, 4, 5 and 8, all of them containing α-neurotoxins of the 3FTx family, whereas the rest of the fractions did not show toxicity at a dose of 0.53mg/kg. Three polyspecific antivenoms manufactured in South Africa and India were tested for their immunoreactivity against crude venom and fractions of N. melanoleuca. Overall, antivenoms immunorecognized all fractions in the venom, the South African antivenom showing a higher titer against the neurotoxin-containing fractions. This toxicovenomic study identified the 3FTx group of α-neurotoxins in the venom of N. melanoleuca as the relevant targets to be neutralized. BIOLOGICAL SIGNIFICANCE: A toxicovenomic analysis of the venom of the forest cobra, also known as black cobra, Naja melanoleuca, was performed. Envenomings by this elapid species are characterized by a progressive descending paralysis which starts with palpebral ptosis and, in severe cases, ends up with respiratory arrest and death. A total of 52 different proteins were identified in this venom. The most abundant protein family was the three-finger toxin (3FTx) family, which comprises almost 57.1wt% of the venom, followed by phospholipases A2 (PLA2) (12.9wt%). In addition, several other protein families were identified in a much lower percentage in the venom. A toxicity screening of the fractions, using the mouse lethality assay, identified four peaks as those having toxicity higher than that of the crude venom. These fractions predominantly contain α-neurotoxins of the 3FTx family. This toxicovenomic characterization agrees with the clinical and experimental manifestations of envenomings by this species, in which a strong neurotoxic effect predominates. Therefore, our findings suggest that immunotherapy against envenomings by N. melanoleuca should be directed towards the neutralization of 3FTxs; this has implications for the improvement of current antivenoms and for the development of novel antivenoms based on biotechnological approaches. A screening of the immunoreactivity of three antivenoms being distributed in sub-Saharan Africa revealed that they immunoreact with the fractions containing α-neurotoxins, although with different antibody titers.

Top-down venomics of the East African green mamba, Dendroaspis angusticeps, and the black mamba, Dendroaspis polylepis, highlight the complexity of their toxin arsenals.

We report the characterization, by combination of high-resolution on-line molecular mass and disulfide bond profiling and top-down MS/MS analysis, of the venom proteomes of two congeneric African snake species of medical importance, Dendroaspis angusticeps (green mamba) and D. polylepis (black mamba). Each of these mamba venoms comprised more than two-hundred polypeptides belonging to just a few toxin families. Both venom proteomes are overwhelmingly composed of post-synaptically-acting short- and long-chain neurotoxins that potently inhibit muscle- and neuronal-type nicotinic acetylcholine receptors; muscarinic cardiotoxins; and dendrotoxins, that block some of the Kv1, n-class of K+ channels. However, the identity of the major proteins and their relative abundances exhibit marked interspecific variation. In addition, the greater resolution of the top-down venomic analytical approach revealed previously undetected protein species, isoforms and proteoforms, including the identification and precise location of modified lysine residues in a number of proteins in both venoms, but particularly in green mamba toxins. This comparative top-down venomic analysis unveiled the untapped complexity of Dendroaspis venoms and lays the foundations for rationalizing the notably different potency of green and black mamba lethal arsenals at locus resolution. SIGNIFICANCE PARAGRAPH: We report the characterization, by combination of high-resolution on-line molecular mass and disulfide bond profiling and top-down MS/MS analysis, of the venom proteomes of two congeneric African snake species of medical importance, Dendroaspis angusticeps (green mamba) and D. polylepis (black mamba). Each of these mamba venoms comprised more than two-hundred polypeptides belonging to just a few toxin families. Both venom proteomes are overwhelmingly composed of post-synaptically-acting short- and long-chain neurotoxins that potently inhibit muscle- and neuronal-type nicotinic acetylcholine receptors; muscarinic cardiotoxins; and dendrotoxins, that block some of the Kv1, n-class of K+ channels. However, the identity of the major proteins and their relative abundances exhibit marked interspecific variation. In addition, the greater resolution of the top-down venomic analytical approach revealed previously undetected protein species, isoforms and proteoforms, including the identification and precise location of modified lysine residues in a number of proteins in both venoms, but particularly in green mamba toxins. This comparative top-down venomic analysis unveiled the untapped complexity of Dendroaspis venoms and lays the foundations for rationalizing the notably different potency of green and black mamba lethal arsenals at locus resolution.

Unveiling the elusive and exotic: Venomics of the Malayan blue coral snake (Calliophis bivirgata flaviceps).

The venom proteome of the Malayan blue coral snake, Calliophis bivirgata flaviceps from west Malaysia was investigated by 1D-SDS-PAGE and shotgun-LCMS/MS. A total of 23 proteins belonging to 11 protein families were detected from the venom proteome. For the toxin proteins, the venom consists mainly of phospholipase A2 (41.1%), cytotoxin (22.6%), SVMPs (18.7%) and vespryns (14.6%). However, in contrast to the venoms of New World coral snakes and most elapids, there was no post-synaptic α-neurotoxin detected. The proteome also revealed a relatively high level of phosphodiesterase (1.3%), which may be associated with the reported high level of adenosine in the venom. Also detected were 5'-nucleotidase (0.3%), hyaluronidase (0.1%) and cysteine-type endopeptide inhibitor (0.6%). Enzymatic studies confirmed the presence of phospholipase A2, phosphodiesterase, 5'-nucleotidase and acetylcholinesterase activities but not l-amino acid oxidase activity. The venom exhibited moderate cytotoxic activity against CRL-2648 fibroblast cell lines (IC50=62.14±0.87 µg/mL) and myotoxicity in mice, presumably due to the action of its cytotoxin or its synergistic action with phospholipase A2. Interestingly, the venom lethality could be cross-neutralized by a neurotoxic bivalent antivenom from Taiwan. Together, the findings provide insights into the composition and functions of the venom of this exotic oriental elapid snake. BIOLOGICAL SIGNIFICANCE: While venoms of the New World coral snake have been extensively studied, literature pertaining to the Old World or Asiatic coral snake venoms remains lacking. This could be partly due to the inaccessibility to the venom of this rare species and infrequent cases of envenomation reported. This study identified and profiled the venom proteome of the Malayan blue coral snake (C. b. flaviceps) through SDS-PAGE and a high-resolution nano-LCMS/MS method, detailing the types and abundance of proteins found in the venom. The biological and toxic activities of the venom were also investigated, offering functional correlation to the venom proteome studied. Of note, the venom contains a unique toxin profile predominated with phospholipase A2 and cytotoxin with no detectable post-synaptic neurotoxin. The venom is moderately lethal to mice and the fatal effect could be cross-neutralized by a heterologous elapid bivalent antivenom from Taiwan. The findings enrich snake toxin databases and provide insights into the composition and pathogenesis of the venom of this exotic species.

Modulated mechanism of phosphatidylserine on the catalytic activity of Naja naja atra phospholipase A2 and Notechis scutatus scutatus notexin.

Phosphatidylserine (PS) externalization is a hallmark for apoptotic death of cells. Previous studies showed that Naja naja atra phospholipase A2 (NnaPLA2) and Notechis scutatus scutatus notexin induced apoptosis of human cancer cells. However, NnaPLA2 and notexin did not markedly disrupt the integrity of cellular membrane as evidenced by membrane permeability of propidium iodide. These findings reflected that the ability of NnaPLA2 and notexin to hydrolyze membrane phospholipids may be affected by PS externalization. To address that question, this study investigated the membrane-interacted mode and catalytic activity of NnaPLA2 and notexin toward outer leaflet (phosphatidylcholine/sphingomyelin/cholesterol, PC/SM/Chol) and inner leaflet (phosphatidylserine/phosphatidylethanolamine/cholesterol, PS/PE/Chol) of plasma membrane-mimicking vesicles. PS incorporation promoted enzymatic activity of NnaPLA2 and notexin on PC and PC/SM vesicles, but suppressed NnaPLA2 and notexin activity on PC/SM/Chol and PE/Chol vesicles. PS incorporation increased the membrane fluidity of PC vesicles but reduced membrane fluidity of PC/SM, PC/SM/Chol and PE/Chol vesicles. PS increased the phospholipid order of all the tested vesicles. Moreover, PS incorporation did not greatly alter the binding affinity of notexin and NnaPLA2 with phospholipid vesicles. Acrylamide quenching studies and trinitrophenylation of Lys residues revealed that membrane-bound mode of notexin and NnaPLA2 varied with the targeted membrane compositions. The fine structure of catalytic site in NnaPLA2 and notexin in all the tested vesicles showed different changes. Collectively, the present data suggest that membrane-inserted PS modulates PLA2 interfacial activity via its effects on membrane structure and membrane-bound mode of NnaPLA2 and notexin, and membrane compositions determine the effect of PS on PLA2 activity.

Histopathological analysis and in situ localisation of Australian tiger snake venom in two clinically envenomed domestic animals.

OBJECTIVE: To assess histopathological changes in clinically envenomed tiger snake patients and identify tissue specific localisation of venom toxins using immunohistochemistry. SAMPLES: One feline and one canine patient admitted to the Murdoch Pet Emergency Centre (MPEC), Murdoch University with tiger snake (Notechis sp.) envenoming. Both patients died as a result of envenomation. Non-envenomed tissue was also collected and used for comparison. METHODOLOGY: Biopsy samples (heart, lung, kidney andskeletal muscle tissue) were retrieved 1-2 h post death and processed for histopathological examination using Haemotoxylin and Eosin, Martius Scarlet Blue and Periodic Acid Schiff staining. Tissues were examined by light microscopy and tissue sections subjected to immunohistochemical staining using in-house generated monoclonal and polyclonal antibodies against Notechis venoms. RESULTS: Venom-induced pathological changes were observed in the lungs, kidneys and muscle tissue of both patients. Evidence, not previously noted, of procoagulant venom effects were apparent, with formed thrombi in the heart, lungs (small fibrillar aggregates and larger, discrete thrombi) and kidneys. Immunohistochemical assays revealed venom present in the pulmonary tissue, in and around the glomerular capsule and surrounding tubules in renal tissue and scattered throughout the Gastrocnemius muscle tissue. CONCLUSION: This work has shown pathological evidence of procoagulant venom activity supporting previous suggestions that an initial thrombotic state occurs in envenomed patients. We have shown that venom toxins are able to be localised to specific tissues, in this case, venom was detected in the lung, kidney and muscle tissues of clinically envenomed animals. Future work will examine specific toxin localisation using monoclonal antibodies and identify if antivenom molecules are able to reach their target tissues.

Non-lethal polypeptide components in cobra venom.

Snakes from several genera (mostly from Naja genus) belonging to the Elapidae family are usually named cobras. The effect of cobra bites is mainly neurotoxic. This is explained by the presence of highly potent alpha-neurotoxin in their venoms. The other two highly toxic components of cobra venoms are cytotoxins and phospholipases A(2). These three types of toxins constitute a major part of cobra venom. They have attracted the attention of researchers for many years and have been very well studied and thoroughly described. However cobra venoms contain also many other less abundant components which possess very low toxicity or even are not toxic at all. These components, mostly proteins, belong to different structural and functional types, and the reason for their presence in the venom is not always evident. Some of them are known for many years (e.g., nerve growth factor and cobra venom factor); others (e.g., cysteine rich secretory proteins, CRISPs) were discovered only recently. There are non-lethal proteins with unique biological activities that can be used as biochemical tools, while others may be regarded as potential leads for drug design. This review is the first attempt to systemize the available data on non-lethal components of cobra venom.

Atrial natriuretic peptide and related peptides.

In recent years, biomarkers have been recognized as important tools for diagnosis, risk stratification, and therapeutic decision-making in cardiovascular diseases. Currently, the clinical potential of several natriuretic peptides is under scientific investigation. The well-known counter-regulatory hormones are atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), dendroaspis natriuretic peptide (DNP) and urodilatin, which play an important role in the homeostasis of body fluid volume. ANP and BNP have already been demonstrated to have diagnostic usefulness in a great number of studies, which have progressed from bench to bedside. This article summarizes existing data on ANP and related peptides in cardiovascular and other disorders, and outlines the potential clinical usefulness of these markers.

New method for characterizing highly disulfide-bridged peptides in complex mixtures: application to toxin identification from crude venoms.

Animal venoms are highly complex mixtures that can contain many disulfide-bridged toxins. This work presents an LC-MALDI approach allowing (1) a rapid classification of toxins according to their number of disulfide bonds and (2) a rapid top-down sequencing of the toxins using a new MALDI matrix enhancing in-source decay (ISD). The crude venom is separated twice by LC: the fractions of the first separation are spotted on the MALDI matrix alpha-cyano-4-hydroxycinnamic acid (CHCA) and the others using 1,5-diaminonaphthalene (1,5-DAN). CHCA spots are more convenient for obtaining a precise mass fingerprint of a large number of peptides; however, the analysis of 1,5-DAN spots allows the number of disulfide bridges to be counted owing to their partial in-plume reduction by this particular matrix. Subsequently, the disulfide bonds of all peptides present in the crude venom were reduced by an excess of tris(carboxyethyl)phosphine before the LC separation and were subjected to the same analysis in CHCA and 1,5-DAN. Toxins were sequenced using a TOF/TOF analysis of metastable fragments from CHCA spots and ISD fragmentation from 1,5-DAN spots. Novel conotoxin sequences were found using this approach. The use of 1,5-DAN for ISD top-down sequencing is also illustrated for higher molecular weight toxins such as snake cardiotoxins and neurotoxins (>6500 Da), where sequence coverage >70% is obtained from the c-ion series.

Quantitation of Indian krait (Bungarus caeruleus) venom in human specimens of forensic origin by indirect competitive inhibition enzyme-linked immunosorbent assay.

An indirect competitive inhibition enzyme-linked immunosorbent assay was reported to detect krait venom in human specimens of forensic origin. Polyclonal anti-krait venom antibodies were characterized by indirect antibody capture assay. The calibration plot was constructed based on linear regression analysis (y = 72.85 - 12.29x, r(2) = 0.98) with concentration ranges from 0.013 to 1000 ng/well of krait venom with a limit of detection of 0.2 ng/mL in the assay system. The IC50 (inhibitory concentration at 50% displacement) value of krait venom was observed to be 70 ng. Spiking studies indicated recoveries of 95-100% and 94-100% when various concentrations of krait venom were spiked to rat tissues (skin, liver, and kidneys) and pooled human serum, respectively. Polyclonal anti-krait venom antibodies showed no cross-reactivity with cobra and viper venom when tested in the assay system. The coefficient of variation of various concentrations of working range in intra-assay (n = 6) was <5%, whereas in interassay (n = 6) it was observed to be < or 7%. Further, the method was used to quantitate krait venom in human autopsy and biopsy specimens of forensic origin. Concentration of krait venom was found to be in the range of 4-172 ng/100 mg skin or skin scrapings and 64-378 ng/mL blood or serum. The methodology may find application in forensic laboratories to assess the cause of death in the cases of krait-bite victims.

Purification and characterization of Ophiophagus hannah cytotoxin-like proteins.

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.

Use of egg yolk antibody (IgY) as an immunoanalytical tool in the detection of Indian cobra (Naja naja naja) venom in biological samples of forensic origin.

An immunoglobulin Y (IgY) based indirect double antibody sandwich enzyme linked immunosorbent assay (ELISA) was developed for the detection of Indian cobra (Naja naja naja) venom in the biological samples of forensic origin. Polyclonal antibodies were raised and purified from chick egg yolk and rabbit serum. The cobra venom was sandwiched between immobilized affinity purified IgY and the rabbit IgG. The detection concentration of cobra venom was in the range of 0.1 to 300ng. The calibration plot was based on linear regression analysis (y=0.2581x+0.4375, r(2)=0.9886). The limit of detection of the assay was found to be 0.1ng. The coefficient of variation (CV) of different concentrations of working range in inter (n=6) and intra-assay (n=6) was observed to be less than 10%. The recovery of venom was found to be in the range of 80-99%, when different concentrations (0.002, 0.1, 0.2, 1, and 2microg) of cobra venom were spiked to pooled normal human serum (ml(-1)). No cross reactivity was observed with krait and viper venom in the immunoassay system in the concentration range of 0.1-1000ng. The method was initially, validated by analyzing specimens (autopsy) of experimental rats injected with cobra venom (1.2mgkg(-1) body mass). Further, human specimens (autopsy and biopsy) of snake bite victims of forensic origin were also analyzed. The methodology developed may find diagnostic application in forensic laboratories.

Enzyme immunoassays in brown snake (Pseudonaja spp.) envenoming: detecting venom, antivenom and venom-antivenom complexes.

Although a commercial snake venom detection kit (SVDK) is available to distinguish between the five major snake groups in Australia, there is no assay for quantifying venom or antivenom concentrations in envenomed patients. Serum samples were obtained from patients with brown snake (Pseudonaja spp.) envenoming before and after the administration of antivenom and patients with suspected brown snake bites but no evidence of envenoming. Enzyme immunoassays (EIAs) were developed for free venom, free antivenom and the venom-antivenom complex. Standard samples measured in duplicate had a coefficient of variation of less than 10%. The EIA for venom was able to detect brown snake venom down to concentrations of 3 ng/mL. A high baseline absorbance was measured in some patients that did not change with the addition of excess antivenom to the samples. In these patients, the baseline absorbance was subtracted from all measurements to calculate the true venom concentration. The EIA for brown snake antivenom had a limit of detection of 20 microg/mL, but 50 microg/mL was used as a cut-off based on assays in patients who had not received antivenom. The EIA for venom-antivenom complexes was unable to detect these at the low venom concentrations that occurred in patients. Quantification of venom and antivenom will help to determine the dose of antivenom required to bind venom and to establish appropriate end points for antivenom treatment.

Cobra venom contains a pool of cysteine-rich secretory proteins.

A large family of cysteine-rich secretory proteins (CRISPs) includes proteins of different origin, the function of the majority of CRISPs being unknown. For CRISPs isolated from snake venom, two types of activities were found: two proteins blocked cyclic nucleotide-gated ion channels, several others blocked potassium-stimulated smooth muscle contraction. Thus, snake CRISPs represent potentially valuable tools for studies of ion channels, which makes promising a search for new CRISPs. Here we report on the isolation of several novel CRISPs from the venoms of Asian cobra Naja kaouthia and African cobra Naja haje using a combination of different types of liquid chromatography. Four CRISP variants were identified in N. kaouthia venom and three proteins, one of them acidic, were found in N. haje venom. Acidic CRISP was found in a reptilian venom for the first time. Our data suggest that each cobra venom contains a pool of different CRISPs.

Identification of PLA(2) and alpha-neurotoxin proteins in the venom of Pseudonaja affinis (dugite).

The Western brown snake Pseudonaja affinis (dugite), common to the Perth area of Western Australia, possesses one of the most lethal venoms in the world. Little is known, however, about the toxic protein constituents of the venom, other than those causing coagulopathic and procoagulant effects. The current study was therefore undertaken in order to identify other protein constituents and activities present. Crude venom induced a contraction in rat tracheal preparations through phospholipase A(2) (PLA(2)) activity, as shown by the complete and partial inhibition of contraction by PLA(2) inhibitors 4-bromophenacyl bromide and quinacrine. Further, a reduced degree of smooth muscle contraction in the presence of the leukotriene receptor antagonist SKF104353 suggested that this effect was mediated by leukotriene metabolites. The venom-induced contraction did not reoccur upon a second administration of the venom, despite the muscle retaining its contractile function and appearing histologically undamaged. Chromatographic separation of the protein constituents of the venom showed that PLA(2) activity was associated with all protein fractions. A low-molecular-weight component of the venom was further investigated through N-terminal sequencing and found to possess high identity to the short-chain alpha-neurotoxin family of toxins. Venom activity on cultured rat cardiac myocytes and cultured cortical neurons was also examined. The crude venom was found to temporarily inhibit the beating of the cardiac myocytes, after which the beating resumed erratically. Cortical neurons, however, were irreversibly affected, showing concentration-dependent cell death.