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Bungarus (krait) envenomings are well-known for their life-threatening neurotoxic effects. However, their impact on coagulation remains largely unexplored experimentally or clinically. This study, examined the effect of begins to examine venoms from four Bungarus species-B. caeruleus, B. candidus, B. fasciatus, and B. flaviceps on human platelet poor plasma coagulation parameters using thromboelastography and coagulation inhibition assays. B. flaviceps completely inhibited clotting, while B. caeruleus only delayed clot formation. In contrast, B. candidus and B. fasciatus did not affect clotting. Subsequent examinations into the anticoagulant biochemical mechanisms demonstrated divergent pathophysiological pathways. B. caeruleus venom anticoagulant effects were prevented by the addition of an excess of phospholipids, with anticoagulation thereby the result of phospholipid depletion. In contrast B. flaviceps anticoagulation was not affected by the addition of an excess of phospholipids. Further investigations demonstrated that B. flaviceps mediates its anticoagulant toxicity through the inactivation of coagulation enzymes. The anticoagulant effects of both B. flaviceps and B. caeruleus were nullified by varespladib, a phospholipase A 2 (PLA 2 ) inhibitor, revealing the toxin class involved. These results uncover previously unrecognized and unexplored anticoagulant effects of Bungarus venoms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Assam, a Northeastern State of India, is inhabited by several venomous snake species causing substantial morbidity and mortality. The data on the epidemiology of snakebites and their management is underreported in this region. Hence, a secondary health-based retrospective study was carried out at Demow Model Hospital, Sivasagar, Assam, to evaluate the clinical and epidemiological profile of snakebite cases reported in this rural hospital and their management. Snakebites occurring between April 2018 to August 2022 were reviewed based on socio-demographic details of the patient, clinical symptoms, and treatment using a standard questionnaire. Out of the 1011 registered snakebite cases, 139 patients (13.7%) counted for venomous bites, among which 92 patients (66.19%) accounted for viper bites (green pit viper and Salazar's pit viper), and 30 patients (21.5%) were bitten by elapid snakes (Indian monocled Cobra, banded krait, and greater/lesser black krait). A maximum number of snakebite cases (80.5%) were reported from the interior rural villages and documented from July to September (51.3%). Elapid snake envenomed patients, except one, were successfully treated with commercial antivenom, neostigmine, and glycopyrrolate. Because commercial polyvalent antivenom against "Big Four" venomous snakes of India showed poor neutralization of pit-vipers envenomation; therefore, pit-viper bite patients were treated with repurposed drugs magnesium sulfate and glycerin compression dressing. Adverse serum reactions were reported only in 3 (11.1%) cases. The preventive measures and facilities adopted at the Demow Model Hospital significantly reduce snakebite death and morbidity; therefore, they can be s practised across various states in India as a prototype., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Cobra (Naja spp.) envenoming is a life-threatening medical emergency, and a correct diagnosis is crucial to initiating timely and appropriate antivenom treatment. However, snakebite diagnostics remain unavailable in Southeast Asia. This study, therefore, developed an immunodetection assay with a potential diagnostic application for cobra envenoming. The cytotoxin of Naja kaouthia (Thai Monocled Cobra) (Nk-CTX) was purified from its venom to produce CTX-specific antibodies in rabbits and chickens. A double-antibody sandwich enzyme-linked immunosorbent assay was developed using the purified anti-Nk-CTX antibodies (immunoglobulin G and immunoglobulin Y), and its selectivity, specificity, and sensitivity for the venoms of five major cobra species in Southeast Asia (N. kaouthia, Naja sumatrana, Naja sputatrix, Naja siamensis, and Naja philippinensis) were studied. The results showed the immunoassay discriminates cobra venoms from other species commonly implicated in snakebites in Southeast Asia, i.e., the Malayan Krait, Many-banded Krait, King Cobra, Eastern Russell's Viper, Malayan Pit Viper and White-lipped Pit Viper. The immunoassay has a high sensitivity for the five cobra venoms, with detection limits (LoD) ranging from 0.6 to 2.6 ng/ml. Together, the findings suggest the potential diagnostic application of the cytotoxin immunoassay for cobra envenoming. The immunoassay was found to exhibit high immunoreactivity toward ten Asiatic cobra venoms (absorbance > 1.5), in contrast to African cobra venoms with low immunoreactivity (absorbance < 0.9). Considering the varying CTX antigenicity between Asiatic and African cobras, the immunoassay for African cobras should utilize antibodies produced specifically from the cytotoxins of African cobra venoms., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

In the Southern Asian countries, snakebite takes a substantial toll in terms of human life, inflicts acute morbidity and long term disability both physical and psychological, and therefore represents a neglected socio-economic problem and severe health issue that requires immediate medical attention. The 'Big Four' venomous snakes, viz. Daboia russelii, Naja naja, Bungarus caeruleus and Echis carinatus, are prominent, medically important species and are the most dangerous snakes of this region; therefore, the commercial polyvalent antivenom (PAV) contains antibodies against the venoms of these snakes. However, envenomations by species other than the 'Big Four' snakes are grossly neglected, and PAV is only partially effective in neutralizing the venom of these snakes. Many issues confounding effective treatment of snakebite are discussed in this review, and these hurdles preventing successful treatment of snakebite must be addressed. However, in South Asian countries, the pre-hospital treatment and appropriate first aid are equally important to mitigate the problem of snakebite and therefore, these issues are also highlighted here. Further, this review suggests a roadmap and guidelines for the prevention of snakebite and improvement of hospital management of snakebite in these Southern Asian countries., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Ceylon krait (Bungarus ceylonicus) of the family Elapidae is a highly venomous endemic species inhabiting in the wet zone and some parts of the intermediate climatic zones of Sri Lanka. Clinical records of its bites are rare and limited to five case reports in the literature. It is of interest to note that there are several non-venomous snakes in Sri Lanka having similar morphological appearance to kraits causing identification difficulties which lead to unnecessary and unindicated administration of antivenom. We report two paediatric cases of proven Ceylon krait bites and three adult patients with similar looking non-venomous snakebites. These children were 1½ and 13 years old and developed neuroparalysis without progressing to respiratory failure and recovered. Both the children were administered Indian polyvalent antivenom which has not developed against endemic Ceylon krait venom. The two adult patients also received antivenom due to the misidentification without clinical and laboratory evidence of envenoming., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Snakebites are common in India and the most common neurotoxic snakebites in India are due to Common krait (Bungarus caeruleus) and cobra (Naja naja). Severe envenomation may mimic brain death or a locked-in state with flaccid paralysis in a descending manner and total ophthalmoplegia. Usually, patients who receive timely antivenom and ventilator support recover completely without any sequalae. We are reporting two cases of krait bite with an unusually long period of flaccid paralysis, which required prolong ventilation. While case 1 required 10 days of mechanical ventilation followed by 5 days of non-invasive ventilation, case 2 required 11 days of mechanical ventilation followed by 5 days of non-invasive ventilation. Both the cases had delayed recovery and residual weakness at 3-month follow up. These case reports suggest that krait bite may cause prolong neuromuscular weakness in children, which has implications for both acute and chronic management., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

In South Asia, the "Big-4" venomous snakes Naja naja, Bungarus caeruleus, Daboia russelii, and Echis carinatus are so-called because they are the most medically important snakes in the region. Antivenom is the only effective treatment option for snakebite envenoming but antivenom is not produced domestically in Pakistan making the country reliant on polyvalent products imported from India and Saudi Arabia. The present study investigated the toxin composition and activity of the venoms of Pakistani specimens by means of proteomic and physio/pharmacological experiments. To evaluate the composition of venoms, 1D/2D-PAGE of crude venoms and RP-HPLC followed by SDS-PAGE were performed. Enzymatic, hemolytic, coagulant and platelet aggregating activities of crude venoms were assayed and were concordant with expectations based on the abundance of protein species in each. Neutralization assays were performed using Bharat polyvalent antivenom (BPAV), a product raised against venoms from Big-4 specimens from southern India. BPAV exhibited cross-reactivity against the Pakistani venoms, however, neutralization of clinically relevant activities was variable and rarely complete. Cumulatively, the presented data not only highlight geographical variations present in the venoms of the Big-4 snakes of South Asia, but also demonstrate the neutralization potential of Indian polyvalent against the venom of Pakistani specimens. Given the partial neutralization observed, it is clear that whilst BPAV is a life-saving product in Pakistan, in future it is hoped that a region-specific product might be manufactured domestically, using venoms of local snakes in the immunising mixture., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Snake venoms are mixtures of biologically-active proteins and peptides, and several studies have described the characteristics of some of these toxins. However, complete proteomic profiling of the venoms of many snake species has not yet been done. The Indian cobra (Naja naja) and common krait (Bungarus caeruleus) are elapid snake species that are among the 'Big Four' responsible for the majority of human snake envenomation cases in India. As understanding the composition and complexity of venoms is necessary for successful treatment of envenomation in humans, we utilized three different proteomic profiling approaches to characterize these venoms: i) one-dimensional SDS-PAGE coupled with in-gel tryptic digestion and electrospray tandem mass spectrometry (ESI-LC-MS/MS) of individual protein bands; ii) in-solution tryptic digestion of crude venoms coupled with ESI-LC-MS/MS; and iii) separation by gel-filtration chromatography coupled with tryptic digestion and ESI-LC-MS/MS of separated fractions. From the generated data, 81 and 46 different proteins were identified from N. naja and B. caeruleus venoms, respectively, belonging to fifteen different protein families. Venoms from both species were found to contain a variety of phospholipases A 2 and three-finger toxins, whereas relatively higher numbers of snake venom metalloproteinases were found in N. naja compared to B. caeruleus venom. The analyses also identified less represented venom proteins including L-amino acid oxidases, cysteine-rich secretory proteins, 5'-nucleotidases and venom nerve growth factors. Further, Kunitz-type serine protease inhibitors, cobra venom factors, phosphodiesterases, vespryns and aminopeptidases were identified in the N. naja venom, while acetylcholinesterases and hyaluronidases were found in the B. caeruleus venom. We further analyzed protein coverage (Lys/Arg rich and poor regions as well as potential glycosylation sites) using in-house software. These studies expand our understanding of the proteomes of the venoms of these two medically-important species., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Kraits belong to Elapideae and are widely distributed in East and South-East Asian countries. Krait venom possesses neurotoxins, membrane toxins, cardiotoxins, three finger toxins, metalloproteinases, cholinesterases, L-amino acid oxidases and serine proteases. The therapeutic potential of krait venom in pathophysiological conditions such as microbial and parasitic infections, cancer, arthritis, inflammation and blood coagulation disorder is discussed in this review. More intensive new research ventures are required to establish the therapeutic potential of krait venom in complex and emerging diseases., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The Ceylon krait (Bungarus ceylonicus) is a deadly venomous elapid snake endemic to Sri Lanka. Its habitat is mainly in the wet zone of the island. Despite its frequent encounters in and near human dwellings, reports of envenoming are rare and limited to four in the literature. The first and last fatal reports envenoming by B. ceylonicus was in 1993. After over two decades, we report two confirmed cases of B. ceylonicus bites-one a dry bite and the other with signs and symptoms of moderate envenoming. The envenoming occurred at night while the victim was asleep, causing tightness in the chest and dyspnoea on waking up, followed by neuromuscular paralysis that did not cause respiratory failure and complete recovery was observed three days following the bite., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A heat stable protein BF-F47 was purified from the crude venom of Bungarus fasciatus by CM cellulose ion exchange chromatography and HPLC. Osteoarthritis (OA) was developed in male albino Wistar rats by collagenase injection. BF-F47 treatment significantly restored urinary hydroxyproline and glucosamine in OA rats. Serum acid phosphatase, alkaline phosphatase, creatinine and serum molecular markers TNF-α, IL-1β, IL-17, cytokine induced neutrophil chemoattractant-1, matrix metalloproteinase-1, cathepsin-K, osteocalcin and PGE2 were also significantly altered. BF-F47 showed partial restoration of osteoarthritis joints. Thus, BF-F47 induced anti-osteoarthritic activity in Wistar rats acted through molecular markers of arthritis and inflammation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Snakebite is a global health problem affecting millions of people. According to WHO, India has the highest mortality and/or morbidity due to snakebite. In spite of commendable research on Indian BIG FOUR venomous species; Naja naja and Bungarus caeruleus (elapid); Daboia russelii and Echis carinatus (viperid), no significant progress has been achieved in terms of diagnosis and management of biting species with appropriate anti-snake venom. Major hurdle is identification of offending species. Present study aims at differentiation of Indian BIG FOUR snake venoms based on their distinguish action on rodent blood coagulation. Assessment of coagulation alterations by elapid venoms showed negligible effect on re-calcification time, prothrombin time, activated partial thromboplastin time and factors assay (I, II, V, VIII and X) both in vitro and in vivo. However, viperid venoms demonstrated significant anticoagulant status due to their remarkable fibrinogen degradation potentials as supported by fibrinogenolytic activity, fibrinogen zymography and rotational thromboelastometry. Though results provide hint on probable alterations of Indian BIG FOUR snake venoms on blood coagulation, the study however needs validation from human victim's samples to ascertain its reliability for identification of biting snake species., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Kraits are venomous snakes of genus Bungarus from family Elapidae. Krait venoms are generally neurotoxic, but toxicity strongly depends on the particular species and regional origin of snakes. We analyzed the proteomes of Vietnamese Bungarus multicinctus and Bungarus fasciatus venoms both qualitatively and quantitatively. It should be noted that no proteomic data for B. multicinctus venom existed so far. We have found that in this venom, almost half (45%) of the proteins by weight was represented by β-bungarotoxins, followed by three finger toxins (28%) and phospholipases A2 (16%), other proteins being present at the level of 1-3%. In B. fasciatus venom, phospholipase A2 was the main component (71%), followed by oxidase of l-amino acids (8%), acetylcholinesterase (5%) and metalloproteinases (4%). Unexpectedly, extremely low amount of three finger toxins (1%) was found in this venom. Interestingly, the presence of complement depleting factor was observed in both venoms. Although our data showed the presence of the same toxin families in Vietnamese krait venoms as those found earlier in the venoms of kraits from other geographic regions, their relative ratio is completely different. This concerns especially B. fasciatus venom with predominant content of phospholipases A2 and very low amount of three finger toxins., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

BF-CT1, a 13 kDa protein isolated from Bungarus fasciatus snake venom through CM cellulose ion exchange chromatography at 0.02 M NaCl salt gradient showed cytotoxicity in in vitro and in vivo experimental models. In in vivo Ehrlich ascites carcinoma (EAC) induced BALB/c mice model, BF-CT1 treatment reduced EAC cell count significantly through apoptotic cell death pathway as evidenced by FACS analysis, increased caspase 3, 9 activity and altered pro, antiapoptotic protein expression. BF-CT1 treatment caused cell shrinkage, chromatin condensation and induced apoptosis through increased caspase 3, caspase 9 activity, PARP cleavage and down regulation of heat shock proteins in U937 leukemic cell line. Cytosolic cytochrome C production was increased after BF-CT1 treatment upon U937 cell line. BF-CT1 treated U937 cell showed cell cycle arrest at sub G1 phase through cyclin D and CDK down regulation with up regulation of p15 and p16. It also down regulated PI3K/AKT pathway and MAPkinase pathway and promoted apoptosis and regulated cell proliferation in U937 cells. BF-CT1 prevented angiogenesis in in vitro U937 cell line through decreased VEGF and TGF-β1 production., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

L-amino acid oxidases (LAAOs) are widely distributed in snake venoms, which contribute to the toxicity of venoms. However, LAAO from Bungarus fasciatus (B. fasciatus) snake venom has not been isolated previously. In the present study, LAAO from B. fasciatus snake venom was purified by SP-Sepharose HP anion exchange chromatography followed by Heparin-Sepharose FF affinity chromatography procedure and the purified enzyme was named BF-LAAO. BF-LAAO presented an estimated molecular weight of 55kDa in SDS-PAGE and an apparent molecular weight of 70kDa in size-exclusion chromatography suggesting that BF-LAAO is a monomeric protein. Kinetics studies showed that BF-LAAO was very active against L-Tyr, L-Asp, L-Phe, L-Glu, L-Trp, L-His, L-Gln, L-Ile, L-Met, L-Leu and moderately active against L-Lys, L-Arg, L-Ala and L-Asn. BF-LAAO exhibited a cytotoxic effect on A549 cells and caused up to 41.2% apoptosis of A549 cells following 12h incubation period. In the mouse peritoneum, BF-LAAO provoked a marked increase in the number of neutrophils, lymphocytes and macrophages following injection. It also induced rabbit platelet aggregation in a dose-dependent manner. At 3h following injection, BF-LAAO elicited severe inflammation in the gastrocnemius muscles of mice, but failed to induce significant organ damage. In conclusion, the cytotoxic and proinflammatory activities of BF-LAAO could be the main cause of the local inflammation, which helps us to understand the pathogenesis of snakebite.

Five-month-old white leghorn chickens were immunized with 50 microg of Common Cobra (Naja naja) and 30 microg of Krait venoms (Bungarus caeruleus) to generate antivenom antibodies against the venom antigen. Chickens received booster doses of increasing concentrations of venom at 14 days time intervals to raise the antivenom level in egg yolk. The antivenom from immunized chicken egg yolk was extracted by polyethylene glycol (PEG) and ammonium sulphate precipitation method which was further purified by DEAE cellulose ion exchange column chromatography. A high molecular weight protein of 180 kDa was detected by electrophoretic analysis which shows the purity of antivenom generated in chicken. Antibodies generated were specific and sensitive to the venom antigen. Various pharmacological activities of Cobra and Krait venoms were carried out by both in-vivo and in-vitro methods. The neutralization of lethality, hemorrhagic, edema, PLA(2) and procoagulant activity was evaluated in assays involving pre-incubation of venom and antivenom prior to testing. The antivenom was effective in neutralizing the toxic and enzymatic activities of venom. The LD(50) of venom for 18 g of mice was found to be 10 microg for Cobra and 3 microg for Krait venoms. The median effective dose (ED(50)) of anti-Cobra venom was 4.48 mg/5LD(50) and 1.0 ml neutralized 0.127 mg of Cobra venom and the median effective dose (ED(50)) of anti-Krait venom was 3.18 mg/5LD(50) and 1.0 ml neutralized 0.051 mg of Krait venom. The results indicate that antivenom generated in chicken could be used for therapeutic purposes in case of snakebite envenomation.

beta-Bungarotoxin (beta-Bgt), a presynaptic phospholipase A(2) (PLA(2)) neurotoxin isolated from the venom of Bungarus multicinctus, consists of A chain and B chain. The goal of the present study is to explore the functional contribution of the two subunits to the toxicity of beta-Bgt. beta-Bgt was found to induce apoptotic death of SK-N-SH cells via elevating intracellular Ca(2+) and intracellular ROS production. Moreover, an activation of p38 MAPK was associated with the cytotoxicity of beta-Bgt. SB202190 (p38 MAPK inhibitor), N-acetylcysteine (antioxidant reagent), 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA) (Ca(2+) chelator) and the inhibitors of Ca(2+) release from intracellular depots (ruthenium red and 2-aminoethoxydiphenyl borate) effectively attenuated the cytotoxicity of beta-Bgt. In sharp contrast to the inability of A chain, B chain was able to induce cytotoxic effects on SK-N-SH cells as beta-Bgt did. Abolishment of PLA(2) activity did not significantly alter the cytotoxic activity of beta-Bgt. MK801 (an NMDA receptor antagonist), antibodies against NMDA receptor and 4-aminopyridine (a potassium channel blocker) markedly reduced the cytotoxic effects of beta-Bgt, B chain and catalytically inactivated beta-Bgt. Moreover, antibodies against NMDA receptor blocked the binding of rhodamine-labeled beta-Bgt to SK-N-SH cells. Taken together, our data indicate that B chain is a functional subunit responsible for the cytotoxicity of beta-Bgt, and suggest that the cytotoxicity of beta-Bgt is mediated by NMDA receptor and potassium conductance.

The structural organization of the genes encoding B2, B4, B5 and B6 chains of beta-bungarotoxins are reported in this study. These genes shared virtually identical overall organization with three exons interrupted by two introns in similar positions. On the contrary, intron 1 of these genes had a similar size, a notable variation with the size of intron 2 was observed. It was found that two regions at the second intron of B1 and B2 chains were absent in that of B4, B5 and B6 chains. RT-PCR analyses indicated that Bungarus multicinctus venom gland, heart, liver and muscle expressed the RNA transcripts showing sequence similarity with the intronic segment being deleted in B4, B5 and B6 chain genes. This reflects that the ancestral gene of the intronic segment might insert in multiple loci of B. multicinctus genome. Comparative analyses of B chain genes showed that the protein-coding regions of the exons are more diverse than introns, except for in the signal peptide domain. These results suggest that intron insertions or deletions occur with the evolution of B chains, and that accelerated evolution may diversify the protein-coding sequence of B chain genes same as snake phospholipase A2, neurotoxin and cardiotoxin genes.

Using B1 chain of beta-bungarotoxin (beta-Bgt) as bait in yeast two-hybrid screen, we found that KChIP3 was a binding protein of B1 chain. Thus, protein-protein interaction between beta-Bgt and KChIP3 is investigated in the present study. Pull-down assay showed that recombinant KChIP3 proteins were associated with beta-Bgt as well as B1 chain, whereas the inability of KChIPs 1, 2 and 4 to bind with beta-Bgt was observed. Although Ca2+ was not a crucial factor essential for the binding of KChIP3 with beta-Bgt and B1 chain, their interaction could be enhanced by the addition of Ca2+. Alternatively, the association of A1 chain of beta-Bgt with KChIP3 was marginally detected. The dissociation constant of beta-Bgt with KChIP3 were 12.2 and 6.08 microM in the absence and presence of 2mM Ca2+, respectively. Moreover, native KChIP3 from rat brain was to be isolated by beta-Bgt-Sepharose. These observations indicate that KChIP3 is a binding protein of beta-Bgt. In view of the multiple functions of KChIP3 in neuronal cells, the interaction of KChIP3 with beta-Bgt may represent an event for the manifestation of the biological activities of beta-Bgt.

Candoxin (PDB #1JGK), a three-finger neurotoxin from Bungarus candidus venom, inhibits post-synaptic neuromuscular and neuronal alpha7nACh-receptors, and induces delayed cell-death throughout the glial population. When applied to cultured human glial cell lines, candoxin (CDX) induced cell death in a concentration (EC(50) approximately 1muM) and time dependent manner. Results of TUNEL-histochemistry further confirm CDX-induced brain (hippocampus, frontal cortex, and temporal regions) damage when administered intracerebroventricularly (i.c.v) in adult mice. In this study, we explored differential gene expression profiles following exposure of human glial (Hs 683) cell lines to CDX at various time intervals using Affymetrix-GeneChips. By means of MAS and GeneSpring analyses, 105 genes whose expression was significantly (P<0.01) altered by at least 3-fold were selected. Results of the genome analysis reveal that the potential role of CDX at molecular level involves the regulation of genes in signal transduction, ubiquitin-inflammation, mitochondrial-dysfunction, and damage-response pathways. In addition, using QRT-PCR and rationally designed specific CDX-binding peptide (P-NT.II), we identified the genes-IL7R, IL13RA2, IL-1beta, TNFRSF12A, GADD45A, CD44 and IFI44-that might play an important role in CDX-induced glial inflammation, DNA-damage and degeneration. These findings reveal new insight into the molecular mechanisms of glial-driven neurodegeneration after exposure to neurotoxins.

In the present study, Xenopus nerve-muscle cultures were used to explore the functional roles of A chain (a phospholipase A(2) subunit) and B chain (a non-phospholipase A(2) subunit) of Bungarus multicinctus beta-bungarotoxin. It was found that beta-bungarotoxin induced an increment of the frequency of spontaneous synaptic currents (SSCs) in the nerve-muscle cultures. Modification of beta-bungarotoxin with pyridoxal-5'-phosphate or substitution of Ca(2+) with Ba(2+) in buffer abolished the phospholipase A(2) activity of beta-bungarotoxin and the facilitatory phase of SSC as well. Antibodies that were directed specifically against A chain or B chain effectively inhibited phospholipase A(2) activity, and as a consequence the SSC frequency was not greatly different from the control rate. These results suggest that both A and B chains are indispensable parts of beta-bungarotoxin for inducing the facilitation of SSC frequency with Xenopus nerve-muscle cultures.

The presence of phospholipase A(2) (PLA(2)) in the venom of Malayan krait (Bungarus candidus) and its structure were studied. The PLA(2) cDNAs from the venom gland of B. candidus (Indonesia origin) were amplified by the polymerase chain reactions (PCR) and cloned. The primers used were based on the cDNA sequences of several homologous B. multicinctus venom PLA(2)s. In addition to the A-chains of beta-bungarotoxins, a novel B. candidus PLA(2) was cloned and its full amino acid sequence deduced. Having totally 125 amino acid residues, the PLA(2) contains a pancreatic loop and is 61% identical to the acidic PLA(2) of king cobra venom. However, the enzyme was not detected from the venom sample. Its structural relationships to other elapid venom PLA(2)s were analyzed with a phylogenetic tree and discussed.

The major lethal toxin in the venom of Bungarus flaviceps has been isolated by ion-exchange chromatography, absorption chromatography and RP-HPLC with a 14-fold purification and an overall yield of 16.5% of the lethal toxicity contained in crude venom. Its sublethal dose (LD(50)) determined in mice weighing 18-20 g was 0.25 (0.19-0.32) microg per mouse. The lethal toxin was pure according to disc- and SDS-PAGE as well as gel HPLC. Its apparent molecular weight determined by SDS-PAGE was 29 kDa. It is a basic protein consisting of two polypeptide chains having apparent molecular weights of 17 and 8 kDa, respectively. The toxin has PLA activity but is free of ACE activity.

This paper presents the nucleotide sequences of the cDNAs encoding two long chain alpha-neurotoxins from Laticauda semifasciata venom gland. The deduced amino acid sequences are Ls-III and its iso-neurotoxin.

A specific activator of blood coagulation factor X was purified from the venom of Bungarus fasciatus by gel filtration and by ion-exchange chromatography on a Mono-Q column (FPLC). It consisted of a single polypeptide chain, with a mol. wt of 70,000 in reducing and non-reducing conditions. The enzyme had an amidolytic activity towards the chromogenic substrates S-2266 and S-2302 but it did not hydrolyse S-2238, S2251 or S-2222, which are specific substrates for thrombin, plasmin and factor Xa, respectively. The enzyme activated factor X in vitro and the effect was Ca2+ dependent with a Hill coefficient of 7.9. As with physiological activators, the venom activator cleaves the heavy chain of factor X, producing the activated factor Xa alpha. The purified factor X activator from B. fasciatus venom did not activate prothrombin, nor did it cleave or clot purified fibrinogen. The amidolytic activity and the factor X activation activity of the factor X activator from B. fasciatus venom were readily inhibited by serine protease inhibitors such as diisopropyl fluorophosphate (DFP), phenylmethanesulfonyl fluoride (PMSF), benzamidine and by soybean trypsin inhibitor but not by EDTA. These observations suggest that the factor X activator from B. fasciatus venom is a serine protease. It therefore differs from those of activators obtained from Vipera russelli and Bothrops atrox venoms, which are metalloproteinases.