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2.
Blood Coagul Fibrinolysis ; 30(8): 379-384, 2019 Dec.
Article in English | MEDLINE | ID: mdl-31415248

ABSTRACT

BACKGROUND: A phenomena of interest is the in vitro anticoagulant effects of neurotoxins found in elapid venoms that kill by paralysis. These enzymes include phospholipase A2 (PLA2), and it has recently been demonstrated that carbon monoxide inhibits the PLA2-dependent neurotoxin contained in Mojave rattlesnake type A venom. The purpose of this investigation was to assess if the anticoagulant activity of elapid venoms containing PLA2 and/or three finger toxins could be inhibited by carbon monoxide. METHODS: Venoms collected from Bungarus multicinctus, Micrurus fulvius, and five Naja species were exposed to carbon monoxide via carbon monoxide releasing molecule-2 prior to placement into human plasma. Coagulation kinetics were assessed via thrombelastography. RESULTS: Compared with plasma without venom addition, all venoms had significant anticoagulant effects, with a 160-fold range of concentrations having similar anticoagulant effects in a species-specific manner. Carbon monoxide significantly inhibited the anticoagulant effect of all venoms tested, but inhibition was not complete in all cases. CONCLUSION: Given that individual neurotoxin activity often depends on intact activity that includes anticoagulant action, it may be possible that carbon monoxide inhibits neurotoxicity. Future investigation is justified to assess such carbon monoxide mediated inhibition with purified neurotoxins in vitro and in vivo.


Subject(s)
Anticoagulants , Carbon Monoxide/pharmacology , Snake Venoms/pharmacology , Animals , Blood Coagulation/drug effects , Blood Specimen Collection , Bungarotoxins/antagonists & inhibitors , Bungarotoxins/chemistry , Bungarotoxins/pharmacology , Bungarus , Coral Snakes , Elapid Venoms/antagonists & inhibitors , Elapid Venoms/chemistry , Elapid Venoms/pharmacology , Elapidae , Humans , Neurotoxins/antagonists & inhibitors , Proteome/analysis , Snake Venoms/antagonists & inhibitors , Snake Venoms/chemistry , Thrombelastography
3.
J Proteomics ; 193: 243-254, 2019 02 20.
Article in English | MEDLINE | ID: mdl-30385415

ABSTRACT

The proteome of the Pakistani B. sindanus venom was investigated with reverse-phase HPLC and nano-ESI-LCMS/MS analysis. At least 36 distinct proteins belonging to 8 toxin protein families were identified. Three-finger toxin (3FTx), phospholipase A2 (including ß-bungarotoxin A-chains) and Kunitz-type serine protease inhibitor (KSPI) were the most abundant, constituting ~95% of total venom proteins. The other toxin proteins of low abundance are snake venom metalloproteinase (SVMP), L-amino acid oxidase (LAAO), acetylcholinesterase (AChE), vespryn and cysteine-rich secretory protein (CRiSP). The venom was highly lethal to mice with LD50 values of 0.04 µg/g (intravenous) and 0.15 µg/g (subcutaneous). The 3FTx proteins are diverse, comprising kappa-neurotoxins, neurotoxin-like protein, non-conventional toxins and muscarinic toxin-like proteins. Kappa-neurotoxins and ß-bungarotoxins represent the major toxins that mediate neurotoxicity in B. sindanus envenoming. Alpha-bungarotoxin, commonly present in the Southeast Asian krait venoms, was undetected. The Indian VINS Polyvalent Antivenom (VPAV) was immunoreactive toward the venom, and it moderately cross-neutralized the venom lethality (potency = 0.25 mg/ml). VPAV was able to reverse the neurotoxicity and prevent death in experimentally envenomed mice, but the recovery time was long. The unique toxin composition of B. sindanus venom may be considered in the formulation of a more effective pan-regional, polyspecific antivenom. BIOLOGICAL SIGNIFICANCE: Bungarus sindanus, an endemic krait species distributed mainly in the Sindh Province of Pakistan is a cause of snake envenomation. Its specific antivenom is, however, lacking. The proteomic study of its venom revealed a substantial presence of κ-bungarotoxins and ß-bungarotoxins. The toxin profile corroborates the potent neurotoxicity and lethality of the venom tested in vivo. The heterologous Indian VINS polyvalent antivenom (VPAV) cross-reacted with B. sindanus venom and cross-neutralized the venom neurotoxicity and lethality in mice, albeit the efficacy was moderate. The findings imply that B. sindanus and the phylogenetically related B. caeruleus of India share certain venom epitopes. Research should be advanced to improve the efficacy spectrum of a pan-regional polyspecific antivenom.


Subject(s)
Antivenins , Bungarotoxins , Bungarus/metabolism , Proteome , Animals , Antivenins/chemistry , Antivenins/pharmacology , Bungarotoxins/antagonists & inhibitors , Bungarotoxins/metabolism , Bungarotoxins/toxicity , Cross Reactions , Mice , Pakistan , Proteome/antagonists & inhibitors , Proteome/metabolism , Proteome/toxicity
4.
J Med Case Rep ; 11(1): 330, 2017 Nov 24.
Article in English | MEDLINE | ID: mdl-29169382

ABSTRACT

BACKGROUND: Ceylon krait (Bungarus ceylonicus) is a venomous elapid snake endemic to Sri Lanka. It inhabits shaded home gardens and forests in the wet zone of Sri Lanka and might creep into houses in the night. Despite frequent encounters with humans, reports of envenoming are very rare. CASE PRESENTATION: We report a case of a 26-year-old Sri Lankan Sinhalese man with confirmed Ceylon krait envenoming presenting with bilateral partial ptosis, ophthalmoplegia, facial muscle weakness, and dysphagia. Single fiber electromyography and repetitive nerve stimulation confirmed neuromuscular paralysis. He was administered polyvalent anti-venom serum immediately following admission without a prompt clinical response. Complete recovery was observed 3 days following the bite. CONCLUSIONS: Because of the rarity of envenoming, precise and detailed information on the clinical manifestations following envenoming is lacking. However, Ceylon krait bite can be potentially fatal; so, treating physicians should be aware of species identification, habitat, and biting habits and clinical presentation of envenoming of Ceylon krait. This case report adds knowledge to the existing limited literature available on Ceylon krait envenoming; a rare but potentially fatal clinical entity.


Subject(s)
Antivenins/therapeutic use , Bungarotoxins/antagonists & inhibitors , Bungarus , Facial Paralysis/physiopathology , Immunologic Factors/therapeutic use , Snake Bites/drug therapy , Adult , Animals , Electromyography , Facial Paralysis/drug therapy , Facial Paralysis/immunology , Humans , Male , Snake Bites/immunology , Snake Bites/physiopathology , Species Specificity , Sri Lanka , Time Factors , Treatment Outcome
5.
Toxicon ; 127: 44-48, 2017 Mar 01.
Article in English | MEDLINE | ID: mdl-28062164

ABSTRACT

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.


Subject(s)
Bungarotoxins/antagonists & inhibitors , Bungarus , Snake Bites/drug therapy , Adult , Animals , Antivenins/therapeutic use , Female , Humans , Male , Middle Aged , Paralysis/etiology , Snake Bites/pathology , Sri Lanka
7.
PLoS One ; 9(8): e105404, 2014.
Article in English | MEDLINE | ID: mdl-25144237

ABSTRACT

Antibody-based technology is the main method for diagnosis and treatment of snake bite envenoming currently. However, the development of an antibody, polyclonal or monoclonal, is a complicated and costly procedure. Aptamers are single stranded oligonucleotides that recognize specific targets such as proteins and have shown great potential over the years as diagnostic and therapeutic agents. In contrast to antibodies, aptamers can be selected in vitro without immunization of animals, and synthesized chemically with extreme accuracy, low cost and high degree of purity. In this study we firstly report on the identification of DNA aptamers that bind to ß-bungarotoxin (ß-BuTx), a neurotoxin from the venom of Bungarus multicinctus. A plate-SELEX method was used for the selection of ß-BuTx specific aptamers. After 10 rounds of selection, four aptamer candidates were obtained, with the dissociation constant ranged from 65.9 nM to 995 nM measured by fluorescence spectroscopy. Competitive binding assays using both the fluorescently labeled and unlabeled aptamers revealed that the four aptamers bound to the same binding site of ß-BuTx. The best binder, ßB-1, bound specifically to ß-BuTx, but not to BSA, casein or α-Bungarotoxin. Moreover, electrophoretic mobility shift assay and enzyme-linked aptamer assay demonstrated that ßB-1 could discriminate B. multicinctus venom from other snake venoms tested. The results suggest that aptamer ßB-1 can serve as a useful tool for the design and development of drugs and diagnostic tests for ß-BuTx poisoning and B. multicinctus bites.


Subject(s)
Aptamers, Nucleotide/pharmacology , Bungarotoxins/antagonists & inhibitors , Bungarus , Animals , Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/metabolism , Base Sequence , Binding Sites , Bungarotoxins/metabolism , Bungarus/metabolism , SELEX Aptamer Technique , Snake Venoms/antagonists & inhibitors , Snake Venoms/metabolism
8.
Bioorg Khim ; 37(5): 609-15, 2011.
Article in Russian | MEDLINE | ID: mdl-22332355

ABSTRACT

Lynx1 expresses in the central nervous system and plays important role in a regulation of nicotinic acetylcholine receptors. Successful milligram-quantitive expression of ws-Lynx1 was achieved only in the case of its production in the form of cytoplasm inclusion bodies. Different conditions of ws-Lynx1 refolding for yield optimization were performed. The obtained recombinant protein was characterized by means of mass spectrometry and CD spectroscopy. The binding experiments on the nAChRs from Torpedo californica membranes revealed that ws-Lynxl is biologically active and blocks muscle nAChR with IC50-20-30 microM.


Subject(s)
GPI-Linked Proteins/biosynthesis , Neurotransmitter Agents/biosynthesis , Receptors, Nicotinic/metabolism , Recombinant Proteins/biosynthesis , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Animals , Base Sequence , Bungarotoxins/antagonists & inhibitors , Electric Organ/chemistry , Electric Organ/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , GPI-Linked Proteins/chemistry , GPI-Linked Proteins/pharmacology , Gene Expression , Genetic Vectors , Humans , Molecular Sequence Data , Neurotransmitter Agents/chemistry , Neurotransmitter Agents/pharmacology , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/pharmacology , Solubility , Torpedo
9.
Anal Chem ; 81(7): 2708-14, 2009 Apr 01.
Article in English | MEDLINE | ID: mdl-19278248

ABSTRACT

The gymnodimines and spirolides are phycotoxins classified into a heterogeneous group of marine biocompounds called cyclic imines. Although there is no clear evidence of their toxicity to humans, gymnodimines and spirolides are highly toxic to rodents and constitute a source of false positives in lipophilic toxin detection by the mouse bioassay. Using nicotinic acetylcholine receptor-enriched membranes of Torpedo, and fluorescent alpha-bungarotoxin, we developed a fluorescence polarization assay to detect and quantify gymnodimine-A and 13-desmethyl C spirolide. The presence of these cyclic imines in solution inhibited the interaction of fluorescent-labeled alpha-bungarotoxin with nicotinic acetylcholine receptors in a concentration-dependent manner. The sensitivity of the assay is in the order of nanomolar concentrations of gymnodimine and 13-desmethyl C spirolide. Okadaic acid, yessotoxin, and brevetoxin-2, three lipophilic marine toxins, did not interfere with this assay. A suitable extraction method in shellfish was also developed. The gymnodimine-A and 13-desmethyl C spirolide recovery rates of mussel matrix extraction with acetone/chloroform were 63.6% +/- 3.5% and 87.4% +/- 5.3%, respectively. In summary, this inhibition assay is capable of gymnodimine-A and 13-desmethyl C spirolide detection in mussel extracts with enough sensitivity and specificity to quantify these toxins in the range of 50-2000 microg/kg and 70-700 microg/kg of shellfish meat, respectively.


Subject(s)
Heterocyclic Compounds, 3-Ring/analysis , Heterocyclic Compounds, 3-Ring/toxicity , Hydrocarbons, Cyclic/analysis , Hydrocarbons, Cyclic/toxicity , Imines/analysis , Imines/toxicity , Lactones/analysis , Lactones/toxicity , Animals , Bungarotoxins/antagonists & inhibitors , Bungarotoxins/metabolism , Electric Organ/metabolism , Fluorescence Polarization , Heterocyclic Compounds, 3-Ring/metabolism , Hydrocarbons, Cyclic/metabolism , Imines/metabolism , Lactones/metabolism , Receptors, Nicotinic/metabolism , Shellfish , Time Factors , Torpedo/metabolism
10.
Biosens Bioelectron ; 24(7): 2289-93, 2009 Mar 15.
Article in English | MEDLINE | ID: mdl-19162466

ABSTRACT

We report a liquid crystal (LC)-based sensor for real-time and label-free identification of phospholipase-like toxins. Beta-bungarotoxin exhibits Ca(2+)-dependent phospholipase A(2) activity whereas alpha-bungarotoxin and myotoxin II do not exhibit any phospholipase activity. The sensor can selectively identify beta-bungarotoxin, when it hydrolyzes a phospholipid monolayer self-assembled at aqueous-LC interface, through orientational responses of LCs. As a result, optical signals that reflect the spatial and temporal distribution of phospholipids during the hydrolysis can therefore be generated in a real-time manner. The sensor is very sensitive and requires less than 5pg of beta-bungarotoxin for the detection. When phospholipase A(2) inhibitors are introduced together with beta-bungarotoxin, no orientational response of LCs can be observed. In addition, the regeneration of the sensor can be done without affecting the sensing performance. This work demonstrates a simple and cost-effective LC-based sensor for identifying phospholipase-like toxins and for screening compound libraries to find potential toxin inhibitors.


Subject(s)
Biosensing Techniques/instrumentation , Bungarotoxins/analysis , Liquid Crystals/chemistry , Phospholipases/analysis , Phospholipids/chemistry , Refractometry/instrumentation , Bungarotoxins/antagonists & inhibitors , Bungarotoxins/chemistry , Computer Systems , Equipment Design , Equipment Failure Analysis , Phospholipases/chemistry , Reproducibility of Results , Sensitivity and Specificity
11.
J Cancer Res Clin Oncol ; 132(10): 653-63, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16835749

ABSTRACT

Identification of the mechanisms leading to malignant transformation of respiratory cells may prove useful in the prevention and treatment of tobacco-related lung cancer. Nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) can induce tumors both locally and systemically. In addition to the genotoxic effect, they have been shown to affect lung cells due to ligating the nicotinic acetylcholine receptors (nAChRs) expressed on the plasma membrane. In this study, we sought to establish the role for nAChRs in malignant transformation caused by NNK and NNN. We used the BEP2D cells that represent a suitable model for studying the various stages of human bronchial carcinogenesis. We found that these cells express alpha1, alpha3, alpha5, alpha7, alpha9, alpha10, beta1, beta2, and beta4 nAChR subunits that can form high-affinity binding sites for NNK and NNN. Exposure of BEP2D cells to either NNK or NNN in both cases increased their proliferative potential which could be abolished in the presence of nAChR antagonists alpha-bungarotoxin, which worked most effectively against NNK, or mecamylamine, which was most efficient against NNN. The BEP2D cells stimulated with the nitrosamines showed multifold increases of the transcription of the PCNA and Bcl-2 genes by both real-time polymerase chain reaction and in-cell western assays. To gain a mechanistic insight into NNK- and NNN-initiated signaling, we investigated the expression of genes encoding the signal transduction effectors GATA-3, nuclear factor-kappaB, and STAT-1. Experimental results indicated that stimulation of nAChRs with NNK led to activation of all three signal transduction effectors under consideration, whereas NNN predominantly activated GATA-3 and STAT-1. The GATA-3 protein-binding activity induced by NNK and NNN correlated with elevated gene expression. The obtained results support the novel concept of receptor-mediated action of NNK and NNN placing cellular nAChRs in the center of the pathophysiologic loop, and suggest that an nAChR antagonist may serve as a chemopreventive agent.


Subject(s)
Anticarcinogenic Agents/pharmacology , Lung/drug effects , Nicotinic Antagonists/pharmacology , Nitrosamines/antagonists & inhibitors , Binding Sites , Bungarotoxins/antagonists & inhibitors , Cell Proliferation/drug effects , Cells, Cultured , GATA3 Transcription Factor/genetics , Humans , Lung/cytology , NF-kappa B/genetics , Nitrosamines/toxicity , Proliferating Cell Nuclear Antigen/genetics , Protein Subunits/metabolism , Proto-Oncogene Proteins c-bcl-2/genetics , Receptors, Nicotinic/metabolism , STAT1 Transcription Factor/genetics , Nicotiana/chemistry , Transcription, Genetic , Up-Regulation
12.
Biochemistry ; 43(39): 12700-8, 2004 Oct 05.
Article in English | MEDLINE | ID: mdl-15449960

ABSTRACT

The alpha-conotoxins MI and GI display stronger affinities for the alphagamma agonist site on the Torpedo californica electrocyte nicotinic acetylcholine receptor (ACHR) than for the alphadelta agonist site, while alpha-conotoxin SI binds with the same affinity to both sites. Prior studies reported that the arginine at position 9 on GI and the tyrosine at position 111 on the receptor gamma subunit were responsible for the stronger alphagamma affinities of GI and MI, respectively. This study was undertaken to determine if the alpha-conotoxin midchain cationic residues interact with Torpedo gammaY111. The findings show that lysine 10 on MI is responsible for the alphagamma selectivity of MI and confirm the previously reported importance of R9 on GI and on the SI analogue, SIP9R. The results also show that gammaY111 contributes substantially to the selective alphagamma high affinity of all three peptides. Double-mutant cycle analyses reveal that, in the alphagamma site, K10 on MI and R9 on SIP9R interact with the aromatic ring of gammaY111 to stabilize the high-affinity complex, while in contrast, R9 on GI does not. The substitution of Y for R at position 113 on the delta subunit converts the alphadelta site into a high-affinity site for MI, GI, and SIP9R through the interacting of deltaY113 with K10 on MI and with R9 on both GI and SIP9R. The overall data show that the residues in the two sites with which MI interacts, other than at gamma111/delta113, are either the same or similar enough to exert equivalent effects on MI, indicating that MI binds in the same orientation at the alphagamma and alphadelta sites. Similar findings show that SIP9R probably also binds in the same orientation at the wild-type alphagamma and alphadelta sites. The finding that R9 on GI interacts closely with deltaR113Y but not with gammaY111 means that GI binds in different orientations at the alphagamma and alphadelta sites. This report also discusses the molecular basis of the difference in the MI high-affinity sites on Torpedo and embryonic mouse muscle ACHRs.


Subject(s)
Conotoxins/metabolism , Protein Subunits/genetics , Protein Subunits/metabolism , Receptors, Nicotinic/genetics , Receptors, Nicotinic/metabolism , Tyrosine/metabolism , Animals , Binding, Competitive/genetics , Bungarotoxins/antagonists & inhibitors , Bungarotoxins/metabolism , Cell Line , Humans , Mice , Mutagenesis, Site-Directed , Protein Binding/genetics , Protein Subunits/biosynthesis , Protein Subunits/chemistry , Radioligand Assay , Receptors, Nicotinic/biosynthesis , Receptors, Nicotinic/chemistry , Thermodynamics , Torpedo , Transfection , Tyrosine/genetics
13.
Toxicol Appl Pharmacol ; 189(1): 45-55, 2003 May 15.
Article in English | MEDLINE | ID: mdl-12758059

ABSTRACT

We demonstrated that beta-bungarotoxin (beta-BuTX), a snake presynaptic neurotoxin, exhibited a potent cytotoxic effect on cultured cerebellar granule neurons. The mechanism of action of beta-BuTX and the cytoprotective agents against beta-BuTX were studied. The neuronal death of cerebellar granule neurons induced by beta-BuTX was manifested with apoptosis and necrosis processes as revealed by neurite fragmentation, morphological alterations, and staining apoptotic bodies with the fluorescent dye Hoechst 33258. By means of microspectrofluorimetry and fura-2, we measured intracellular Ca2+ concentration, [Ca2+]i and found that [Ca2+]i was increased markedly prior to the morphological changes and cytotoxicity. The downstream pathway of the increased [Ca2+]i was investigated: there was increased production of free radicals, decreased mitochondrial membrane potential, and depleted cellular ATP content. MK801 and suramin effectively suppressed these detrimental effects of beta-BuTX. Furthermore, the [3H]MK801 binding was reduced by unlabeled MK801, beta-BuTX, and suramin. Thus, activation of N-methyl-D-aspartate (NMDA) receptors appeared to play a crucial role in the cytotoxic effects following betaBuTX exposure. In conclusion, the novel finding of this study was that a polypeptide beta-BuTX exerted a potent cytotoxic effect through sequential events, including activating NMDA receptors followed by increasing [Ca2+]i, ROS production, and impaired mitochondrial energy metabolism. Suramin, clinically used as a trypanocidal agent, was an effective antagonist against beta-BuTX. Data suggest that suramin might have value to detect the possible pathway of certain neuropathological disorders.


Subject(s)
Bungarotoxins/antagonists & inhibitors , Bungarotoxins/toxicity , Neurons/drug effects , Neurons/metabolism , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Receptors, N-Methyl-D-Aspartate/metabolism , Suramin/pharmacology , Animals , Cell Survival/drug effects , Cell Survival/physiology , Cells, Cultured , Cerebellum/cytology , Cerebellum/drug effects , Cerebellum/metabolism , Dose-Response Relationship, Drug , Neurons/cytology , Rats , Rats, Wistar
14.
Naunyn Schmiedebergs Arch Pharmacol ; 367(5): 451-61, 2003 May.
Article in English | MEDLINE | ID: mdl-12700884

ABSTRACT

The aim of this study was to elucidate the mechanism underlying the neurotoxic effect of beta-bungarotoxin (beta-BuTX) on cultured cerebellar granular neurons (CGN). Beta-BuTX had a potent time- and concentration-dependent neurotoxic effect on mature CGN. Beta-BuTX appeared to destroy initially the neurites and then caused neuronal death by both apoptotic and necrotic processes. Inspection using Nomarski optics showed that these neurons displayed morphological features of necrotic cells, including cell swelling, loss of membrane integrity and eventual dissolution of the cell. Staining with the fluorescent dye Hoechst 33258 showed that beta-BuTX-treated neuron bodies stained more densely with smaller apoptotic bodies. Using microspectrofluorimetry and fura-2 to measure cytosolic [Ca(2+)] ([Ca(2+)](i)), beta-BuTX markedly increased [Ca(2+)](i). BAPTA-AM, EGTA, MK 801 and diltiazem not only attenuated the beta-BuTX-mediated rise in [Ca(2+)](i) but also attenuated beta-BuTX-mediated neurotoxicity. In addition, these Ca(2+) inhibitors prevented the beta-BuTX-induced generation of reactive nitrogen species. The NO synthase inhibitor N(G)-methyl- l-arginine) also exhibited neuroprotection. This is the first report showing that beta-BuTX-induced CGN death is mediated, at least in part, by excessive generation of NO triggered by [Ca(2+)](i) overloading. Activation of NMDA receptors and L-type calcium channels is apparently involved in the increase in [Ca(2+)](i) induced by this neurotoxin. This potent neurotoxin will be a useful tool for studying neurotoxic processes and using this model system will allow us to find neuroprotective agents.


Subject(s)
Bungarotoxins/toxicity , Calcium/metabolism , Cell Death/drug effects , Egtazic Acid/analogs & derivatives , Neurons/drug effects , Nitric Oxide/biosynthesis , Animals , Bungarotoxins/antagonists & inhibitors , Bungarotoxins/metabolism , Calcium/adverse effects , Calcium/chemistry , Calcium Channel Blockers/pharmacology , Calcium Channels, L-Type/physiology , Cells, Cultured , Cerebellum/cytology , Diltiazem/pharmacology , Dizocilpine Maleate/pharmacology , Dose-Response Relationship, Drug , Egtazic Acid/pharmacology , Neurons/ultrastructure , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/pharmacology , Rats , Rats, Wistar , Receptors, N-Methyl-D-Aspartate/antagonists & inhibitors , Receptors, N-Methyl-D-Aspartate/drug effects , Receptors, N-Methyl-D-Aspartate/physiology , Snakes
15.
Biochem Pharmacol ; 65(1): 131-42, 2003 Jan 01.
Article in English | MEDLINE | ID: mdl-12473387

ABSTRACT

The aim of this study was to elucidate the mechanism of the neurotoxic effect of beta-bungarotoxin (beta-BuTX, a snake presynaptic neurotoxin isolated from the venom of Bungarus multicinctus) on cultured cerebellar granule neurons. beta-BuTX exerted a potent, time-dependent, neurotoxic effect on mature granule neurons. Mature neurons, with an abundance of neurite outgrowths, were obtained after 7-8 days in culture. By means of microspectrofluorimetry and fura-2, we measured the intracellular Ca(2+) concentration ([Ca(2+)](i)) and found it to be increased markedly. BAPTA-AM [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tertrakis(acetoxymethyl ester)], EGTA, MK801 (dizocilpine maleate), and diltiazem prevented not only the elevation of [Ca(2+)](i), but also the beta-BuTX-induced neurotoxic effect. The signaling pathway involved in the elevation of [Ca(2+)](i) in beta-BuTX-induced neurotoxicity was studied. The results obtained indicated that beta-BuTX initially increased the production of reactive oxygen species and subsequently reduced mitochondrial membrane potential and depleted ATP. All of these events in the signaling pathway were blocked by MK801, diltiazem, EGTA, and BAPTA-AM. These findings suggest that the neurotoxic effect of beta-BuTX is mediated, at least in part, by a cascade of events that include the direct or indirect activation of N-methyl-D-aspartate (NMDA) receptors and L-type calcium channels that, in turn, lead to Ca(2+) influx, oxidative stress, mitochondrial dysfunction, and ATP depletion. Therefore, we suggest that this polypeptide neurotoxin, as a result of its high potency and irreversible properties, is a useful tool to elucidate the mechanisms of neurodegenerative diseases.


Subject(s)
Bungarotoxins/pharmacology , Calcium Channel Blockers/pharmacology , Calcium Channels, L-Type/metabolism , Neurons/drug effects , Receptors, N-Methyl-D-Aspartate/metabolism , Adenosine Triphosphate/metabolism , Animals , Bungarotoxins/antagonists & inhibitors , Calcium/metabolism , Cell Death/drug effects , Cell Survival/drug effects , Diltiazem/pharmacology , Dizocilpine Maleate/pharmacology , Drug Interactions , Excitatory Amino Acid Antagonists/pharmacology , Magnesium Chloride/pharmacology , Membrane Potentials/drug effects , Membrane Potentials/physiology , Mitochondria/drug effects , Mitochondria/physiology , Neurons/pathology , Rats , Rats, Wistar , Reactive Oxygen Species/metabolism , Receptors, N-Methyl-D-Aspartate/drug effects
16.
Toxicology ; 183(1-3): 235-42, 2003 Feb 01.
Article in English | MEDLINE | ID: mdl-12504354

ABSTRACT

In an attempt to elucidate the mechanism by which excessive fluoride damages the central nervous system, the effects of exposure of PC12 cells to different concentrations of fluoride for 48 h on nicotinic acetylcholine receptors (nAChRs) were characterized here. Significant reductions in the number of binding sites for both [3H]epibatidine and [125I]alpha-bungarotoxin, as well as a significant decrease in the B(max) value for the high-affinity of epibatidine binding site were observed in PC12 cells subjected to high levels of fluoride. On the protein level, the alpha 3 and alpha 7 subunits of nAChRs were also significantly decreased in the cells exposed to high concentrations of fluoride. In contrast, such exposure had no significant effect on the level of the beta 2 subunit. These findings suggest that selective decreases in the number of nAChRs may play an important role in the mechanism(s) by which fluoride causes dysfunction of the central nervous system.


Subject(s)
Receptors, Nicotinic/metabolism , Sodium Fluoride/pharmacology , Animals , Binding Sites , Binding, Competitive , Blotting, Western , Bridged Bicyclo Compounds, Heterocyclic/antagonists & inhibitors , Bridged Bicyclo Compounds, Heterocyclic/metabolism , Bungarotoxins/antagonists & inhibitors , Bungarotoxins/metabolism , Cell Membrane/metabolism , Kinetics , Nicotinic Agonists/metabolism , PC12 Cells , Pyridines/antagonists & inhibitors , Pyridines/metabolism , Rats , Receptors, Nicotinic/biosynthesis , Sodium Fluoride/toxicity
17.
Biochemistry ; 41(32): 10194-9, 2002 Aug 13.
Article in English | MEDLINE | ID: mdl-12162733

ABSTRACT

We previously produced synthetic peptides mimicking the snake neurotoxin binding site of the nicotinic receptor. These peptide mimotopes bind the snake neurotoxin alpha-bungarotoxin with higher affinity than peptides reproducing native receptor sequences and inhibit toxin binding to nicotinic receptors in vitro; yet their efficiency in vivo is low. Here we synthesized one of the peptide mimotopes in a tetrabranched MAP form. The MAP peptide binds alpha-bungarotoxin in solution and inhibits its binding to the receptor with a K(A) and an IC(50) similar to the monomeric peptide. Nonetheless, it is at least 100 times more active in vivo. The MAP completely neutralizes toxin lethality when injected in mice at a dose compatible with its use as a synthetic antidote in humans. The in vivo efficacy of the tetrameric peptide cannot be ascribed to a kinetic and thermodynamic effect and is probably related to different pharmacokinetic behavior of the tetrameric molecule, with respect to the monomer. Our findings bring new perspectives to the therapeutic use of multimeric peptides.


Subject(s)
Antidotes/chemical synthesis , Antidotes/pharmacology , Bungarotoxins/antagonists & inhibitors , Peptide Fragments/chemical synthesis , Peptide Fragments/pharmacology , Receptors, Nicotinic/metabolism , Animals , Antidotes/administration & dosage , Antidotes/metabolism , Binding Sites , Binding, Competitive , Bungarotoxins/toxicity , Epitopes/administration & dosage , Epitopes/chemistry , Epitopes/metabolism , Epitopes/pharmacology , Injections, Subcutaneous , Lethal Dose 50 , Ligands , Mice , Molecular Mimicry , Peptide Fragments/administration & dosage , Peptide Fragments/metabolism
18.
Postgrad Med J ; 78(919): 276-80, 2002 May.
Article in English | MEDLINE | ID: mdl-12151569

ABSTRACT

Common krait (Bungarus caeruleus) is the deadliest snake found commonly in the dry zone of Sri Lanka. In Anuradhapura, 210 farmers bitten by the common krait over a three year period were investigated prospectively from 1 January 1996. The sex ratio was equal, 110 (52%) patients were in the age group 10-30 years. One hundred and one (48%) patients were severely envenomed and needed mechanical ventilation from 12 hours to 29 days (mode two days). The bite occurred at night while the victims were asleep on the floor. In 99 (47%) situations killed specimens were available for identification. The cardinal symptom was abdominal pain developing within hours of the bite. Alteration in the level of consciousness was observed in 150 (71%) patients: drowsy in 91 (43%), semiconscious in 24 (11%), and deep coma in 35 (17%). Autonomic disturbances included transient hypertension, tachycardia, lacrimation, sweating, and salivation. These manifested in 139 (66%) patients with moderate to severe envenomation. One hundred and forty nine (71%) had hypokalaemia and 105 (50%) metabolic acidosis, anterograde memory loss in 84 (40%), and delayed neuropathy in 38 (22%) patients. Polyvalent antivenom had no significant benefit (t = 0.5) in reversing respiratory paralysis and preventing delayed neurological complications. Sixteen (7.6%) patients died and a submucosal haemorrhage in the stomach was seen at necropsy in three cases. Mortality could be minimised with early and free access to mechanical ventilation.


Subject(s)
Bungarus , Snake Bites/complications , Adolescent , Adult , Age Distribution , Aged , Animals , Antivenins/therapeutic use , Autonomic Nervous System Diseases/etiology , Bungarotoxins/antagonists & inhibitors , Child , Consciousness , Critical Care/methods , Female , Humans , Incidence , Male , Middle Aged , Prognosis , Prospective Studies , Respiratory Paralysis/etiology , Seasons , Sex Distribution , Snake Bites/epidemiology , Snake Bites/therapy , Social Class , Sri Lanka/epidemiology
19.
Toxicon ; 39(7): 1045-51, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11223094

ABSTRACT

Employing a combinatorial phage-peptide library, we previously identified the peptide MRYYESSLKSYPD (designated, library-peptide) that binds the snake toxin alpha-bungarotoxin (alpha-BTX) with a moderate binding constant of 10(-6)M (Balass et al., 1997. Proc. Natl. Acad. Sci. USA 94, 6054-6058). Under the experimental conditions employed, we found that the library-peptide did not protect mice from alpha-BTX lethality when injected concomitantly with the toxin. In order to improve the affinity of the peptide to alpha-BTX, we designed and synthesized the peptide CRYYESSLKSYCD (Met1 and Pro12 were replaced by cysteines), which following oxidation creates a single disulfide bond and forms a cyclic structure. The design of the cyclic peptide was based on our previous NMR analysis of the library-peptide/alpha-BTX complex (Scherf et al., 1997. Proc. Natl. Acad. Sci. USA 94, 6059-6064). The cyclic peptide binds alpha-BTX with affinity two orders of magnitude higher than that of the linear library selected peptide. Whereas the library peptide was ineffective, the cyclic peptide conferred protection from alpha-BTX lethality in mice, even when given 1h after the toxin injection. The cyclic peptide conferred complete protection from alpha-BTX lethality in mice when administered 40min prior to toxin injection. However, experiments with the whole venom of the snake Bungarus multicinctus showed that protection could be achieved only when the cyclic peptide was administered concomitantly with the venom.


Subject(s)
Bungarotoxins/antagonists & inhibitors , Bungarotoxins/toxicity , Peptides, Cyclic/pharmacology , Animals , Chromatography, High Pressure Liquid , Cyclization , Cysteine/chemistry , In Vitro Techniques , Oxidation-Reduction , Peptides, Cyclic/chemical synthesis , Protein Binding , Receptors, Cholinergic/metabolism , Torpedo
20.
Biochemistry ; 38(16): 4912-21, 1999 Apr 20.
Article in English | MEDLINE | ID: mdl-10213592

ABSTRACT

We have constructed a series of cysteine-substitution mutants in order to identify residues in the mouse muscle nicotinic acetylcholine receptor (AChR) that are involved in alpha-bungarotoxin (alpha-Bgtx) binding. Following transient expression in HEK 293-derived TSA-201 cells, covalent modification of the introduced cysteines with thiol-specific reagents reveals that alpha subunit residues W187, V188, F189, Y190, and P194 are solvent accessible and are in a position to contribute to the alpha-Bgtx binding site in native receptors. These results with the intact receptor are consistent with NMR studies of an alpha-Bgtx/receptor-dodecapeptide complex [Basus, V., Song., G., and Hawrot, E. (1993) Biochemistry 32, 12290-12298]. We pursued a more detailed analysis of the F189C mutant as this site varies substantially between AChRs that bind Bgtx and certain neuronal AChRs that do not. Treatment of intact cells expressing F189C with either bromoacetylcholine (BrACh) or [2-(trimethylammonium)ethyl] methane-thiosulfonate (MTSET), both methylammonium-containing thiol-modifying reagents with agonist properties, results in a marked decrease ( approximately 55-70%) in the number of alpha-Bgtx binding sites, as measured under saturating conditions. The decrease in sites appears to affect both alpha/gamma and alpha/delta sites to the same extent, as shown for alphaW187C and alphaF189C which were the two mutants examined on this issue. In contrast to the results obtained with MTSET and BrACh, modification with reagents that lack the alkylammonium entity, such as methylmethanethiosulfonate (MMTS), the negatively charged 2-sulfonatoethyl methane-thiosulfonate (MTSES), or the positively charged aminoethyl methylthiosulfonate (MTSEA), has little or no effect on the maximal binding of alpha-Bgtx to the alphaW187C, alphaV188C, or alphaF189C mutant receptors. The striking alkylammonium dependency suggests that an interaction of the tethered modifying group with the negative subsite within the agonist binding domain is primarily responsible for the observed blockade of toxin binding.


Subject(s)
Bungarotoxins/metabolism , Cysteine/genetics , Nicotinic Agonists/chemistry , Receptors, Nicotinic/chemistry , Acetylcholine/analogs & derivatives , Acetylcholine/chemistry , Animals , Bungarotoxins/antagonists & inhibitors , Bungarotoxins/genetics , Humans , Indicators and Reagents , Mesylates/chemistry , Mice , Mutagenesis, Site-Directed , Nicotinic Agonists/metabolism , Nicotinic Antagonists/chemistry , Nicotinic Antagonists/metabolism , Oxidation-Reduction , Peptide Fragments/genetics , Phenylalanine/genetics , Protein Binding/genetics , Receptors, Nicotinic/genetics , Receptors, Nicotinic/metabolism , Torpedo , Tryptophan/genetics , Valine/genetics
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