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Sample records for potent ichthyotoxic neurotoxins

  1. Brevetoxins, like ciguatoxins, are potent ichthyotoxic neurotoxins that accumulate in fish✩

    PubMed Central

    Naar, Jerome P.; Flewelling, Leanne J.; Lenzi, Allison; Abbott, Jay P.; Granholm, April; Jacocks, Henry M.; Gannon, Damon; Henry, Michael; Pierce, Richard; Baden, Daniel G.; Wolny, Jennifer; Landsberg, Jan H.

    2009-01-01

    Brevetoxins and ciguatoxins are closely related potent marine neurotoxins. Although ciguatoxins accumulate in fish to levels that are dangerous for human consumption, live fish have not been considered as potential sources of brevetoxin exposure in humans. Here we show that, analogous to ciguatoxins, brevetoxins can accumulate in live fish by dietary transfer. We experimentally identify two pathways leading to brevetoxin-contaminated omnivorous and planktivorous fish. Fish fed with toxic shellfish and Karenia brevis cultures remained healthy and accumulated high brevetoxin levels in their tissues (up to 2675 ng g−1 in viscera and 1540 ng g−1 in muscle). Repeated collections of fish from St. Joseph Bay in the Florida panhandle reveal that accumulation of brevetoxins in healthy fish occurs in the wild. We observed that levels of brevetoxins in the muscle of fish at all trophic levels rise significantly, but not to dangerous levels, during a K. brevis bloom. Concentrations were highest in fish liver and stomach contents, and increased during and immediately following the bloom. The persistence of brevetoxins in the fish food web was followed for 1 year after the K. brevis bloom. PMID:17675204

  2. Tetrodotoxin, an Extremely Potent Marine Neurotoxin: Distribution, Toxicity, Origin and Therapeutical Uses.

    PubMed

    Lago, Jorge; Rodríguez, Laura P; Blanco, Lucía; Vieites, Juan Manuel; Cabado, Ana G

    2015-10-01

    Tetrodotoxin (TTX) is a potent neurotoxin responsible for many human intoxications and fatalities each year. The origin of TTX is unknown, but in the pufferfish, it seems to be produced by endosymbiotic bacteria that often seem to be passed down the food chain. The ingestion of contaminated pufferfish, considered the most delicious fish in Japan, is the usual route of toxicity. This neurotoxin, reported as a threat to human health in Asian countries, has spread to the Pacific and Mediterranean, due to the increase of temperature waters worldwide. TTX, for which there is no known antidote, inhibits sodium channel producing heart failure in many cases and consequently death. In Japan, a regulatory limit of 2 mg eq TTX/kg was established, although the restaurant preparation of "fugu" is strictly controlled by law and only chefs qualified are allowed to prepare the fish. Due to its paralysis effect, this neurotoxin could be used in the medical field as an analgesic to treat some cancer pains. PMID:26492253

  3. Tetrodotoxin, an Extremely Potent Marine Neurotoxin: Distribution, Toxicity, Origin and Therapeutical Uses

    PubMed Central

    Lago, Jorge; Rodríguez, Laura P.; Blanco, Lucía; Vieites, Juan Manuel; Cabado, Ana G.

    2015-01-01

    Tetrodotoxin (TTX) is a potent neurotoxin responsible for many human intoxications and fatalities each year. The origin of TTX is unknown, but in the pufferfish, it seems to be produced by endosymbiotic bacteria that often seem to be passed down the food chain. The ingestion of contaminated pufferfish, considered the most delicious fish in Japan, is the usual route of toxicity. This neurotoxin, reported as a threat to human health in Asian countries, has spread to the Pacific and Mediterranean, due to the increase of temperature waters worldwide. TTX, for which there is no known antidote, inhibits sodium channel producing heart failure in many cases and consequently death. In Japan, a regulatory limit of 2 mg eq TTX/kg was established, although the restaurant preparation of “fugu” is strictly controlled by law and only chefs qualified are allowed to prepare the fish. Due to its paralysis effect, this neurotoxin could be used in the medical field as an analgesic to treat some cancer pains. PMID:26492253

  4. Basis Tetrapeptides as Potent Intracellular Inhibitors of type A Botulinum Neurotoxin Protease Activity

    SciTech Connect

    Hale, M.; Swaminathan, S.; Oyler, G.; Ahmed, S. A.

    2011-01-21

    Botulinum neurotoxins (BoNT) are the most potent of all toxins that cause flaccid muscle paralysis leading to death. They are also potential biothreat agents. A systematic investigation of various short peptide inhibitors of the BoNT protease domain with a 17-residue peptide substrate led to arginine-arginine-glycine-cysteine having a basic tetrapeptide structure as the most potent inhibitor. When assayed in the presence of dithiothreitol (DTT), the inhibitory effect was drastically reduced. Replacing the terminal cysteine with one hydrophobic residue eliminated the DTT effect but with two hydrophobic residues made the pentapeptide a poor inhibitor. Replacing the first arginine with cysteine or adding an additional cysteine at the N terminus did not improve inhibition. When assessed using mouse brain lysates, the tetrapeptides also inhibited BoNT/A cleavage of the endogenous SNAP-25. The peptides penetrated the neuronal cell lines, N2A and BE(2)-M17, without adversely affecting metabolic functions as measured by ATP production and P-38 phosphorylation. Biological activity of the peptides persisted within cultured chick motor neurons and rat and mouse cerebellar neurons for more than 40 h and inhibited BoNT/A protease action inside the neurons in a dose- and time-dependent fashion. Our results define a tetrapeptide as the smallest peptide inhibitor in the backdrop of a large substrate protein of 200+ amino acids having multiple interaction regions with its cognate enzyme. The inhibitors should also be valuable candidates for drug development.

  5. Crystallographic characterization of functional sites of crotoxin and ammodytoxin, potent β-neurotoxins from Viperidae venom.

    PubMed

    Faure, Grazyna; Saul, Frederick

    2012-09-15

    This review will focus on a description of the three-dimensional structures of two β-neurotoxins, the monomeric PLA(2) ammodytoxin from Vipera ammodytes ammodytes, and heterodimeric crotoxin from Crotalus durissus terrificus, and a detailed structural analysis of their multiple functional sites. We have recently determined at high resolution the crystal structures of two natural isoforms of ammodytoxin (AtxA and AtxC) (Saul et al., 2010) which exhibit different toxicity profiles and different anticoagulant properties. Comparative structural analysis of these two PLA(2) isoforms, which differ only by two amino acid residues, allowed us to detect local conformational changes and delineate the role of critical residues in the anticoagulant and neurotoxic functions of these PLA(2) (Saul et al., 2010). We have also determined, at 1.35Å resolution, the crystal structure of heterodimeric crotoxin (Faure et al., 2011). The three-dimensional structure of crotoxin revealed details of the binding interface between its acidic (CA) and basic (CB) subunits and allowed us to identify key residues involved in the stability and toxicity of this potent heterodimeric β-neurotoxin (Faure et al., 2011). The precise spatial orientation of the three covalently linked polypeptide chains in the mature CA subunit complexed with CB helps us to understand the role played by critical residues of the CA subunit in the increased toxicity of the crotoxin complex. Since the CA subunit is a natural inhibitor of the catalytic and anticoagulant activities of CB, identification of the CA-CB binding interface describes residues involved in this inhibition. We propose future research directions based on knowledge of the recently reported 3D structures of crotoxin and ammodytoxin.

  6. Potent tetravalent replicon vaccines against botulinum neurotoxins using DNA-based Semliki Forest virus replicon vectors.

    PubMed

    Yu, Yun-Zhou; Guo, Jin-Peng; An, Huai-Jie; Zhang, Shu-Ming; Wang, Shuang; Yu, Wei-Yuan; Sun, Zhi-Wei

    2013-05-01

    Human botulism is commonly associated with botulinum neurotoxin (BoNT) serotypes A, B, E and F. This suggests that the greatest need is for a tetravalent vaccine that provides protection against all four of these serotypes. In current study, we investigated the feasibility of generating several tetravalent vaccines that protected mice against the four serotypes. Firstly, monovalent replicon vaccine against BoNT induced better antibody response and protection than that of corresponding conventional DNA vaccine. Secondly, dual-expression DNA replicon pSCARSE/FHc or replicon particle VRP-E/FHc vaccine was well resistant to the challenge of BoNT/E and BoNT/F mixture as a combination vaccine composed of two monovalent replicon vaccines. Finally, the dual-expression DNA replicon or replicon particle tetravalent vaccine could simultaneously and effectively neutralize and protect the four BoNT serotypes. Protection correlated directly with serum ELISA titers and neutralization antibody levels to BoNTs. Therefore, replicon-based DNA or particle might be effective vector to develop BoNT vaccines, which might be more desirable for use in clinical application than the conventional DNA vaccines. Our studies demonstrate the utility of combining dual-expression DNA replicon or replicon particle vaccines into multi-agent formulations as potent tetravalent vaccines for eliciting protective responses to four serotypes of BoNTs.

  7. A Potent Peptidomimetic Inhibitor of Botulinum Neurotoxin Serotype a Has a Very Different Conformation Than SNAP-25 Substrate

    SciTech Connect

    Zuniga, J.E.; Schmidt, J.J.; Fenn, T.; Burnett, J.C.; Arac, D.; Gussio, R.; Stafford, R.G.; Badie, S.S.; Bavari, S.; Brunger, A.T.

    2009-05-28

    Botulinum neurotoxin serotype A is the most lethal of all known toxins. Here, we report the crystal structure, along with SAR data, of the zinc metalloprotease domain of BoNT/A bound to a potent peptidomimetic inhibitor (Ki = 41 nM) that resembles the local sequence of the SNAP-25 substrate. Surprisingly, the inhibitor adopts a helical conformation around the cleavage site, in contrast to the extended conformation of the native substrate. The backbone of the inhibitor's P1 residue displaces the putative catalytic water molecule and concomitantly interacts with the 'proton shuttle' E224. This mechanism of inhibition is aided by residue contacts in the conserved S1' pocket of the substrate binding cleft, and the induction of new hydrophobic pockets, which are not present in the apo form, especially for the P2' residue of the inhibitor. Our inhibitor is specific for BoNT/A as it does not inhibit other BoNT serotypes or thermolysin.

  8. Molecular evolution of α-latrotoxin, the exceptionally potent vertebrate neurotoxin in black widow spider venom.

    PubMed

    Garb, Jessica E; Hayashi, Cheryl Y

    2013-05-01

    Black widow spiders (members of the genus Latrodectus) are widely feared because of their potent neurotoxic venom. α-Latrotoxin is the vertebrate-specific toxin responsible for the dramatic effects of black widow envenomation. The evolution of this toxin is enigmatic because only two α-latrotoxin sequences are known. In this study, ~4 kb α-latrotoxin sequences and their homologs were characterized from a diversity of Latrodectus species, and representatives of Steatoda and Parasteatoda, establishing the wide distribution of latrotoxins across the mega-diverse spider family Theridiidae. Across black widow species, α-latrotoxin shows ≥ 94% nucleotide identity and variability consistent with purifying selection. Multiple codon and branch-specific estimates of the nonsynonymous/synonymous substitution rate ratio also suggest a long history of purifying selection has acted on α-latrotoxin across Latrodectus and Steatoda. However, α-latrotoxin is highly divergent in amino acid sequence between these genera, with 68.7% of protein differences involving non-conservative substitutions, evidence for positive selection on its physiochemical properties and particular codons, and an elevated rate of nonsynonymous substitutions along α-latrotoxin's Latrodectus branch. Such variation likely explains the efficacy of red-back spider, L. hasselti, antivenom in treating bites from other Latrodectus species, and the weaker neurotoxic symptoms associated with Steatoda and Parasteatoda bites. Long-term purifying selection on α-latrotoxin indicates its functional importance in black widow venom, even though vertebrates are a small fraction of their diet. The greater differences between Latrodectus and Steatoda α-latrotoxin, and their relationships to invertebrate-specific latrotoxins, suggest a shift in α-latrotoxin toward increased vertebrate toxicity coincident with the evolution of widow spiders.

  9. 3-Aminopropanal, formed during cerebral ischaemia, is a potent lysosomotropic neurotoxin.

    PubMed Central

    Li, Wei; Yuan, Xi-Ming; Ivanova, Svetlana; Tracey, Kevin J; Eaton, John W; Brunk, Ulf T

    2003-01-01

    Cytotoxic polyamine-derived amino aldehydes, formed during cerebral ischaemia, damage adjacent tissue (the so-called 'penumbra') not subject to the initial ischaemic insult. One such product is 3-aminopropanal (3-AP), a potent cytotoxin that accumulates in ischaemic brain, although the precise mechanisms responsible for its formation are still unclear. More relevant to the present investigations, the mechanisms by which such a small aldehydic compound might be cytotoxic are also not known, but we hypothesized that 3-AP, having the structure of a weak lysosomotropic base, might concentrate within lysosomes, making these organelles a probable focus of initial toxicity. Indeed, 3-AP leads to lysosomal rupture of D384 glioma cells, a process which clearly precedes caspase activation and apoptotic cell death. Immunohistochemistry reveals that 3-AP concentrates in the lysosomal compartment and prevention of this accumulation by the lysosomotropic base ammonia, NH(3), protects against 3-AP cytotoxicity by increasing lysosomal pH. A thiol compound, N-(2-mercaptopropionyl)glycine, reacts with and neutralizes 3-AP and significantly inhibits cytoxocity. Both amino and aldehyde functions of 3-AP are necessary for toxicity: the amino group confers lysosomotropism and the aldehyde is important for additional, presently unknown, reactions. We conclude that 3-AP exerts its toxic effects by accumulating intralysosomally, causing rupture of these organelles and releasing lysosomal enzymes which initiate caspase activation and apoptosis (or necrosis if the lysosomal rupture is extensive). These results may have implications for the development of new therapeutics designed to lessen secondary damage arising from focal cerebral ischaemia. PMID:12513695

  10. Interactions of a potent cyclic peptide inhibitor with the light chain of botulinum neurotoxin A: Insights from X-ray crystallography.

    PubMed

    Kumaran, Desigan; Adler, Michael; Levit, Matthew; Krebs, Michael; Sweeney, Richard; Swaminathan, Subramanyam

    2015-11-15

    The seven antigenically distinct serotypes (A-G) of botulinum neurotoxin (BoNT) are responsible for the deadly disease botulism. BoNT serotype A (BoNT/A) exerts its lethal action by cleaving the SNARE protein SNAP-25, leading to inhibition of neurotransmitter release, flaccid paralysis and autonomic dysfunction. BoNTs are dichain proteins consisting of a ∼ 100 kDa heavy chain and a ∼ 50 kDa light chain; the former is responsible for neurospecific binding, internalization and translocation, and the latter for cleavage of neuronal SNARE proteins. Because of their extreme toxicity and history of weaponization, the BoNTs are regarded as potential biowarfare/bioterrorism agents. No post-symptomatic therapeutic interventions are available for BoNT intoxication other than intensive care; therefore it is imperative to develop specific antidotes against this neurotoxin. To this end, a cyclic peptide inhibitor (CPI-1) was evaluated in a FRET assay for its ability to inhibit BoNT/A light chain (Balc). CPI was found to be highly potent, exhibiting a Ki of 12.3 nM with full-length Balc448 and 39.2 nM using a truncated crystallizable form of the light chain (Balc424). Cocrystallization studies revealed that in the Balc424-CPI-1 complex, the inhibitor adopts a helical conformation, occupies a high percentage of the active site cavity and interacts in an amphipathic manner with critical active site residues. The data suggest that CPI-1 prevents SNAP-25 from accessing the Balc active site by blocking both the substrate binding path at the surface and the Zn(2+) binding region involved in catalysis. This differs from linear peptide inhibitors described to date which block only the latter. PMID:26522088

  11. Therapeutic effectiveness of botulinum neurotoxin A: potent blockade of autonomic transmission by targeted cleavage of only the pertinent SNAP-25.

    PubMed

    Lawrence, Gary W; Ovsepian, Saak V; Wang, Jiafu; Aoki, K Roger; Dolly, J Oliver

    2013-07-01

    In search of a basis for the impressive potency of an endoprotease that cleaves SNAP-25, botulinum neurotoxin type A (BoNT/A), in treating numerous diseases due to hyper-active autonomic nerves, truncation of its target and inhibition of neurotransmission were studied in rat sympathetic neurons. Tetrodotoxin-sensitive spontaneous cholinergic neurotransmission was blocked >80% by 1 pM BoNT/A despite cleaving <20% of the SNAP-25. A maximum cleavage of ∼60% SNAP-25 could be achieved with >1 nM BoNT/A, despite an absence of non-cleavable SNAP-25 in the detergent-solubilised neurons. In contrast, BoNT/E (100 nM) truncated nearly all the SNAP-25 in the intact cells, but was unable to block neurotransmission at low concentrations like BoNT/A. Chimeras created by inserting the acceptor-binding HC domain of BoNT/A into BoNT/E still cleaved all the SNAP-25, indicating ubiquitous expression of BoNT/A acceptors. Accordingly, SV2 and SNAP-25 were found to be co-expressed and broadly co-localised in neurons, but absent from non-neuronal cells. On the other hand, partial cleavage by the BoNT/A protease persisted upon replacing its HC with counterparts from BoNT/E or BoNT/B. Moreover, limited cleavage of SNAP-25 was conferred onto the protease from BoNT/E when fused to the N-terminus of BoNT/A. Thus, the BoNT/A protease is uniquely well-adapted for selectively inactivating the SNAP-25 directly involved in neurotransmission; this together with the toxin's acceptor and its target being localised on the peri-somatic boutons likely contribute to its exceptional therapeutic utility in the clinic.

  12. Longer-acting and highly potent chimaeric inhibitors of excessive exocytosis created with domains from botulinum neurotoxin A and B.

    PubMed

    Wang, Jiafu; Zurawski, Tomas H; Bodeker, MacDara O; Meng, Jianghui; Boddul, Sanjay; Aoki, K Roger; Dolly, J Oliver

    2012-05-15

    Various human neurogenic hyper-excitability disorders are successfully treated with type A or B BoNT (botulinum neurotoxin). The BoNT/A complex is widely used because of its longer-lasting benefits; also, autonomic side-effects are more often reported for BoNT/B. To establish if this distinct effect of BoNT/B could be exploited therapeutically, BoNT/A was modified so that it would bind the more abundant BoNT/B acceptor in rodents while retaining its desirable persistent action. The advantageous protease and translocation domain of BoNT/A were recombinantly combined with the acceptor-binding moiety of type B [H(C)/B (C-terminal half of BoNT/B heavy chain)], creating the chimaera AB. This purified protein bound the BoNT/B acceptor, displayed enhanced capability relative to type A for intraneuronally delivering its protease, cleaved SNAP-25 (synaptosome-associated protein of 25 kDa) and induced a more prolonged neuromuscular paralysis than BoNT/A in mice. The BA chimaera, generated by substituting H(C)/A (C-terminal half of BoNT/A heavy chain) into BoNT/B, exhibited an extremely high specific activity, delivered the BoNT/B protease via the BoNT/A acceptor into neurons, or fibroblast-like synoviocytes that lack SNAP-25, cleaving the requisite isoforms of VAMP (vesicle-associated membrane protein). Both chimaeras inhibited neurotransmission in murine bladder smooth muscle. BA has the unique ability to reduce exocytosis from non-neuronal cells expressing the BoNT/A-acceptor and utilising VAMP, but not SNAP-25, in exocytosis.

  13. Alpha neurotoxins.

    PubMed

    Barber, Carmel M; Isbister, Geoffrey K; Hodgson, Wayne C

    2013-05-01

    α-Neurotoxins have been isolated from hydrophid, elapid and, more recently, colubrid snake venoms. Also referred to as postsynaptic neurotoxins or 'curare mimetic' neurotoxins, they play an important role in the capture and/or killing of prey by binding to the nicotinic acetylcholine receptor on the skeletal muscle disrupting neurotransmission. They are also thought to cause respiratory paralysis in envenomed humans. This review will discuss the historical background into the discovery, isolation, structure and mechanism of action of the α-neurotoxins, including targets and cellular outcomes, and then will examine the potential uses of α-neurotoxins as pharmacological tools and/or as drug leads.

  14. Second Generation Steroidal 4-Aminoquinolines Are Potent, Dual-Target Inhibitors of the Botulinum Neurotoxin Serotype A Metalloprotease and P. falciparum Malaria

    PubMed Central

    2015-01-01

    Significantly more potent second generation 4-amino-7-chloroquinoline (4,7-ACQ) based inhibitors of the botulinum neurotoxin serotype A (BoNT/A) light chain were synthesized. Introducing an amino group at the C(3) position of the cholate component markedly increased potency (IC50 values for such derivatives ranged from 0.81 to 2.27 μM). Two additional subclasses were prepared: bis(steroidal)-4,7-ACQ derivatives and bis(4,7-ACQ)cholate derivatives; both classes provided inhibitors with nanomolar-range potencies (e.g., the Ki of compound 67 is 0.10 μM). During BoNT/A challenge using primary neurons, select derivatives protected SNAP-25 by up to 89%. Docking simulations were performed to rationalize the compounds’ in vitro potencies. In addition to specific residue contacts, coordination of the enzyme’s catalytic zinc and expulsion of the enzyme’s catalytic water were a consistent theme. With respect to antimalarial activity, the compounds provided better IC90 activities against chloroquine resistant (CQR) malaria than CQ, and seven compounds were more active than mefloquine against CQR strain W2. PMID:24742203

  15. The insecticidal neurotoxin Aps III is an atypical knottin peptide that potently blocks insect voltage-gated sodium channels

    PubMed Central

    Bende, Niraj S.; Kang, Eunji; Herzig, Volker; Bosmans, Frank; Nicholson, Graham M.; Mobli, Mehdi; King, Glenn F.

    2013-01-01

    One of the most potent insecticidal venom peptides described to date is Aps III from the venom of the trapdoor spider Apomastus schlingeri. Aps III is highly neurotoxic to lepidopteran crop pests, making it a promising candidate for bioinsecticide development. However, its disulfide-connectivity, three-dimensional structure, and mode of action have not been determined. Here we show that recombinant Aps III (rAps III) is an atypical knottin peptide; three of the disulfide bridges form a classical inhibitor cystine knot motif while the fourth disulfide acts as a molecular staple that restricts the flexibility of an unusually large β hairpin loop that often houses the pharmacophore in this class of toxins. We demonstrate that the irreversible paralysis induced in insects by rAps III results from a potent block of insect voltage-gated sodium channels. Channel block by rAps III is voltage-independent insofar as it occurs without significant alteration in the voltage-dependence of channel activation or steady-state inactivation. Thus, rAps III appears to be a pore blocker that plugs the outer vestibule of insect voltage-gated sodium channels. This mechanism of action contrasts strikingly with virtually all other sodium channel modulators isolated from spider venoms that act as gating modifiers by interacting with one or more of the four voltage-sensing domains of the channel. PMID:23473802

  16. The insecticidal neurotoxin Aps III is an atypical knottin peptide that potently blocks insect voltage-gated sodium channels.

    PubMed

    Bende, Niraj S; Kang, Eunji; Herzig, Volker; Bosmans, Frank; Nicholson, Graham M; Mobli, Mehdi; King, Glenn F

    2013-05-15

    One of the most potent insecticidal venom peptides described to date is Aps III from the venom of the trapdoor spider Apomastus schlingeri. Aps III is highly neurotoxic to lepidopteran crop pests, making it a promising candidate for bioinsecticide development. However, its disulfide-connectivity, three-dimensional structure, and mode of action have not been determined. Here we show that recombinant Aps III (rAps III) is an atypical knottin peptide; three of the disulfide bridges form a classical inhibitor cystine knot motif while the fourth disulfide acts as a molecular staple that restricts the flexibility of an unusually large β hairpin loop that often houses the pharmacophore in this class of toxins. We demonstrate that the irreversible paralysis induced in insects by rAps III results from a potent block of insect voltage-gated sodium channels. Channel block by rAps III is voltage-independent insofar as it occurs without significant alteration in the voltage-dependence of channel activation or steady-state inactivation. Thus, rAps III appears to be a pore blocker that plugs the outer vestibule of insect voltage-gated sodium channels. This mechanism of action contrasts strikingly with virtually all other sodium channel modulators isolated from spider venoms that act as gating modifiers by interacting with one or more of the four voltage-sensing domains of the channel.

  17. A single-domain llama antibody potently inhibits the enzymatic activity of botulinum neurotoxin by binding to the non-catalytic alpha-exosite binding region.

    PubMed

    Dong, Jianbo; Thompson, Aaron A; Fan, Yongfeng; Lou, Jianlong; Conrad, Fraser; Ho, Mengfei; Pires-Alves, Melissa; Wilson, Brenda A; Stevens, Raymond C; Marks, James D

    2010-04-01

    Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K(d)) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K(d) for BoNT/A Lc of 1.47 x 10(-)(10) M and an IC(50) (50% inhibitory concentration) of 4.7 x 10(-)(10) M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 A resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.

  18. A Single-Domain Llama Antibody Potently Inhibits the Enzymatic Activity of Botulinum Neurotoxin by Binding to the Non-Catalytic [alpha]-Exosite Binding Region

    SciTech Connect

    Dong, Jianbo; Thompson, Aaron A.; Fan, Yongfeng; Lou, Jianlong; Conrad, Fraser; Ho, Mengfei; Pires-Alves, Melissa; Wilson, Brenda A.; Stevens, Raymond C.; Marks, James D.

    2010-08-13

    Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K{sub d}) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K{sub d} for BoNT/A Lc of 1.47 x 10{sup -10} M and an IC{sub 50} (50% inhibitory concentration) of 4.7 x 10{sup -10} M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 {angstrom} resolution. The structure reveals that the Aa1 VHH binds in the {alpha}-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc {alpha}-exosite as a target for inhibitor development.

  19. A branched peptide mimotope of the nicotinic receptor binding site is a potent synthetic antidote against the snake neurotoxin alpha-bungarotoxin.

    PubMed

    Bracci, Luisa; Lozzi, Luisa; Pini, Alessandro; Lelli, Barbara; Falciani, Chiara; Niccolai, Neri; Bernini, Andrea; Spreafico, Adriano; Soldani, Patrizia; Neri, Paolo

    2002-08-13

    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.

  20. Interactions of a potent cyclic peptide inhibitor with the light chain of botulinum neurotoxin A: insights from x-ray crystallography

    DOE PAGESBeta

    Kumaran, D.; Adler, M.; Levit, M.; Krebs, M.; Sweeney, R.; Swaminathan, S.

    2015-10-17

    The seven antigenically distinct serotypes (A to G) of botulinum neurotoxin (BoNT) are responsible for the deadly disease botulism. BoNT serotype A (BoNT/A) exerts its lethal action by cleaving the SNARE protein SNAP-25, leading to inhibition of neurotransmitter release, flaccid paralysis and autonomic dysfunction. BoNTs are dichain proteins: the heavy chain is responsible for neurospecific binding, internalization and translocation, and the light chain is responsible for substrate cleavage. Because of their extreme toxicity and prior history of weaponization, the BoNTs are considered to be potential bioterrorism agents. No post-symptomatic therapeutic interventions are available for BoNT intoxication other than critical care;more » therefore it is imperative to develop specific antidotes against this neurotoxin. To this end, a cyclic peptide inhibitor (CPI-1) was synthesized and found to inhibit BoNT/A light chain (Balc) with high affinity. When tested in a cell-free Förster resonance excitation transfer (FRET) assay, CPI-1 was found to have a Ki of 13.9 nM using full-length Balc448 and 42.1 nM using a truncated crystallizable form of light chain (Balc424). Co-crystallization of CPI-1 with Balc424 revealed that in the Balc-CPI-1 complex, the inhibitor adopts a helical conformation, occupies a high percentage of the active site cavity and interacts in an amphipathic manner with critical active site residues. The data suggest that CPI-1 prevents SNAP-25 from accessing the Balc active site by blocking both the substrate binding path at the surface and the Zn2+ binding region involved in catalysis. This is in contrast to linear peptide inhibitors described to date which block only the latter« less

  1. Interactions of a potent cyclic peptide inhibitor with the light chain of botulinum neurotoxin A: insights from x-ray crystallography

    SciTech Connect

    Kumaran, D.; Adler, M.; Levit, M.; Krebs, M.; Sweeney, R.; Swaminathan, S.

    2015-10-17

    The seven antigenically distinct serotypes (A to G) of botulinum neurotoxin (BoNT) are responsible for the deadly disease botulism. BoNT serotype A (BoNT/A) exerts its lethal action by cleaving the SNARE protein SNAP-25, leading to inhibition of neurotransmitter release, flaccid paralysis and autonomic dysfunction. BoNTs are dichain proteins: the heavy chain is responsible for neurospecific binding, internalization and translocation, and the light chain is responsible for substrate cleavage. Because of their extreme toxicity and prior history of weaponization, the BoNTs are considered to be potential bioterrorism agents. No post-symptomatic therapeutic interventions are available for BoNT intoxication other than critical care; therefore it is imperative to develop specific antidotes against this neurotoxin. To this end, a cyclic peptide inhibitor (CPI-1) was synthesized and found to inhibit BoNT/A light chain (Balc) with high affinity. When tested in a cell-free Förster resonance excitation transfer (FRET) assay, CPI-1 was found to have a Ki of 13.9 nM using full-length Balc448 and 42.1 nM using a truncated crystallizable form of light chain (Balc424). Co-crystallization of CPI-1 with Balc424 revealed that in the Balc-CPI-1 complex, the inhibitor adopts a helical conformation, occupies a high percentage of the active site cavity and interacts in an amphipathic manner with critical active site residues. The data suggest that CPI-1 prevents SNAP-25 from accessing the Balc active site by blocking both the substrate binding path at the surface and the Zn2+ binding region involved in catalysis. This is in contrast to linear peptide inhibitors described to date which block only the latter

  2. Comparison of oral toxicological properties of botulinum neurotoxin

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Botulinum neurotoxins (BoNTs) are among the most potent biological toxins for humans. Of the seven known serotypes (A-G) of BoNT, serotypes A, B and E cause most of the foodborne intoxications in humans. BoNTs in nature are associated with non-toxic accessory proteins known as neurotoxin-associated ...

  3. Spatial and temporal patterns of surface water quality and ichthyotoxicity in urban and rural river basins in Texas.

    PubMed

    Vanlandeghem, Matthew M; Meyer, Matthew D; Cox, Stephen B; Sharma, Bibek; Patiño, Reynaldo

    2012-12-15

    The Double Mountain Fork Brazos River (Texas, USA) consists of North (NF) and South Forks (SF). The NF receives urban runoff and twice-reclaimed wastewater effluent, whereas the SF flows through primarily rural areas. The objective of this study was to determine and compare associations between standard water quality variables and ichthyotoxicity at a landscape scale that included urban (NF) and rural (SF) sites. Five NF and three SF sites were sampled quarterly from March 2008 to March 2009 for specific conductance, salinity, hardness, pH, temperature, and turbidity; and a zebrafish (Danio rerio) embryo bioassay was used to determine ichthyotoxicity. Metal and nutrient concentrations at all sites were also measured in addition to standard water quality variables in spring 2009. Principal component analyses identified hardness, specific conductance, and salinity as the water variables that best differentiate the urban NF (higher levels) from rural SF habitat. Nutrient levels were also higher in the NF, but no landscape scale patterns in metal concentrations were observed. Ichthyotoxicity was generally higher in NF water especially in winter, and multiple regression analyses suggested a positive association between water hardness and ichthyotoxicity. To test for the potential influence of the toxic golden alga (Prymnesium parvum) on overall ichthyotoxicity, a cofactor known to enhance golden alga toxin activity was used in the bioassays. Golden alga ichthyotoxicity was detected in the NF but not the SF, suggesting golden alga may have contributed to overall ichthyotoxicity in the urban but not in the rural system. In conclusion, the physicochemistry of the urban-influenced NF water was conducive to the expression of ichthyotoxicity and also point to water hardness as a novel factor influencing golden alga ichthyotoxicity in surface waters.

  4. Spatial and temporal patterns of surface water quality and ichthyotoxicity in urban and rural river basins in Texas

    USGS Publications Warehouse

    VanLandeghem, Matthew M.; Meyer, Matthew D.; Cox, Stephen B.; Sharma, Bibek; Patino, Reynaldo

    2012-01-01

    The Double Mountain Fork Brazos River (Texas, USA) consists of North (NF) and South Forks (SF). The NF receives urban runoff and twice-reclaimed wastewater effluent, whereas the SF flows through primarily rural areas. The objective of this study was to determine and compare associations between standard water quality variables and ichthyotoxicity at a landscape scale that included urban (NF) and rural (SF) sites. Five NF and three SF sites were sampled quarterly from March 2008 to March 2009 for specific conductance, salinity, hardness, pH, temperature, and turbidity; and a zebrafish (Danio rerio) embryo bioassay was used to determine ichthyotoxicity. Metal and nutrient concentrations at all sites were also measured in addition to standard water quality variables in spring 2009. Principal component analyses identified hardness, specific conductance, and salinity as the water variables that best differentiate the urban NF (higher levels) from rural SF habitat. Nutrient levels were also higher in the NF, but no landscape scale patterns in metal concentrations were observed. Ichthyotoxicity was generally higher in NF water especially in winter, and multiple regression analyses suggested a positive association between water hardness and ichthyotoxicity. To test for the potential influence of the toxic golden alga (Prymnesium parvum) on overall ichthyotoxicity, a cofactor known to enhance golden alga toxin activity was used in the bioassays. Golden alga ichthyotoxicity was detected in the NF but not the SF, suggesting golden alga may have contributed to overall ichthyotoxicity in the urban but not in the rural system. In conclusion, the physicochemistry of the urban-influenced NF water was conducive to the expression of ichthyotoxicity and also point to water hardness as a novel factor influencing golden alga ichthyotoxicity in surface waters.

  5. Prymnesium parvum revisited: relationship between allelopathy, ichthyotoxicity, and chemical profiles in 5 strains.

    PubMed

    Blossom, Hannah E; Rasmussen, Silas A; Andersen, Nikolaj G; Larsen, Thomas O; Nielsen, Kristian F; Hansen, Per J

    2014-12-01

    Bioassay-guided discovery of ichthyotoxic algal compounds using in vivo fish assays is labor intensive, costly, and highly regulated. Since the mode of action of most known algal-mediated fish-killing toxins is damage to the cell membranes in the gills, various types of cell-based bioassays are often used for bioassay guided purification of new ichthyotoxins. Here we tested the hypothesis that allelopathy is related to ichthyotoxicity and thus that a microalgal bioassay can be used as a proxy for ichthyotoxicity by comparing the toxicity of five strains of Prymnesium parvum toward rainbow trout (Oncorhynchus mykiss, 10 g) and the microalga Teleaulax acuta. No relationship between median effective concentrations (EC50s) on fish and median lethal concentrations (LC50s) on algae was observed in the 5 strains showing that a microalgal bioassay cannot be used as a proxy for ichthyotoxicity. Fish were more sensitive to P. parvum with EC50s ranging from 6×10(3) to 40×10(3) cells ml(-1), compared to the test alga where LC50s ranged from 30×10(3) to nearly non-toxic at 500×10(3) cells ml(-1). In addition, the cellular concentrations of two recently suggested ichthyotoxins produced by P. parvum, the "golden algae toxins", GAT 512 and a novel GAT 510, did not show any relationship to either ichthyotoxicity or allelopathy, and are not the biologically relevant toxins, but are simply lipids found in algal chloroplasts. Finally, we demonstrate that the recently suggested ichthyotoxin, oleamide, could not be detected in any of the five P. parvum strains above the limit of detection, nor was it found in a (13)C-labeled strain. Instead we document that oleamide can easily be extracted from plastic materials, which may have been the source of oleamide reported previously. PMID:25456230

  6. Zebrafish Sensitivity to Botulinum Neurotoxins

    PubMed Central

    Chatla, Kamalakar; Gaunt, Patricia S.; Petrie-Hanson, Lora; Ford, Lorelei; Hanson, Larry A.

    2016-01-01

    Botulinum neurotoxins (BoNT) are the most potent known toxins. The mouse LD50 assay is the gold standard for testing BoNT potency, but is not sensitive enough to detect the extremely low levels of neurotoxin that may be present in the serum of sensitive animal species that are showing the effects of BoNT toxicity, such as channel catfish affected by visceral toxicosis of catfish. Since zebrafish are an important animal model for diverse biomedical and basic research, they are readily available and have defined genetic lines that facilitate reproducibility. This makes them attractive for use as an alternative bioassay organism. The utility of zebrafish as a bioassay model organism for BoNT was investigated. The 96 h median immobilizing doses of BoNT/A, BoNT/C, BoNT/E, and BoNT/F for adult male Tübingen strain zebrafish (0.32 g mean weight) at 25 °C were 16.31, 124.6, 4.7, and 0.61 picograms (pg)/fish, respectively. These findings support the use of the zebrafish-based bioassays for evaluating the presence of BoNT/A, BoNT/E, and BoNT/F. Evaluating the basis of the relatively high resistance of zebrafish to BoNT/C and the extreme sensitivity to BoNT/F may reveal unique functional patterns to the action of these neurotoxins. PMID:27153088

  7. Zebrafish Sensitivity to Botulinum Neurotoxins.

    PubMed

    Chatla, Kamalakar; Gaunt, Patricia S; Petrie-Hanson, Lora; Ford, Lorelei; Hanson, Larry A

    2016-01-01

    Botulinum neurotoxins (BoNT) are the most potent known toxins. The mouse LD50 assay is the gold standard for testing BoNT potency, but is not sensitive enough to detect the extremely low levels of neurotoxin that may be present in the serum of sensitive animal species that are showing the effects of BoNT toxicity, such as channel catfish affected by visceral toxicosis of catfish. Since zebrafish are an important animal model for diverse biomedical and basic research, they are readily available and have defined genetic lines that facilitate reproducibility. This makes them attractive for use as an alternative bioassay organism. The utility of zebrafish as a bioassay model organism for BoNT was investigated. The 96 h median immobilizing doses of BoNT/A, BoNT/C, BoNT/E, and BoNT/F for adult male Tübingen strain zebrafish (0.32 g mean weight) at 25 °C were 16.31, 124.6, 4.7, and 0.61 picograms (pg)/fish, respectively. These findings support the use of the zebrafish-based bioassays for evaluating the presence of BoNT/A, BoNT/E, and BoNT/F. Evaluating the basis of the relatively high resistance of zebrafish to BoNT/C and the extreme sensitivity to BoNT/F may reveal unique functional patterns to the action of these neurotoxins. PMID:27153088

  8. [Neurotoxins from snake venom].

    PubMed

    Larréché, S; Mion, G; Clapson, P; Debien, B; Wybrecht, D; Goyffon, M

    2008-04-01

    Many snakes are able to quickly immobilize prey, thanks to their venom neurotoxins. Most of these snakes belong to families Elapidae or Hydrophidae but neurotoxins were also isolated from families Viperidae and Colubridae. Ophidian neurotoxins can be classified into several categories: neurotoxins which inhibit synaptic transmission (postsynaptic and presynaptic neurotoxins) and neurotoxins which facilitate it excessively (dendrotoxin and fasciculin). Their toxicity is dose-dependent, and venom effects are extremely fast. The clinical feature is a potentially fatal neurological syndrome, the so called cobraic syndrome. Because death by respiratory arrest may occur quickly with cobraic syndrome, immunotherapy is a true emergency, because toxins irreversible fixing makes immunotherapy effect uncertain after a few hours passed.

  9. On Botulinum Neurotoxin Variability

    PubMed Central

    2015-01-01

    ABSTRACT  The rapidly growing number of botulinum neurotoxin sequences poses the problem of the possible evolutionary significance of the variability of these superpotent neurotoxins for toxin-producing Clostridium species. To progress in the understanding of this remarkable phenomenon, we suggest that researchers should (i) abandon an anthropocentric view of these neurotoxins as human botulism-causing agents or as human therapeutics, (ii) begin to investigate in depth the role of botulinum neurotoxins in animal botulism in the wilderness, and (iii) devote large efforts to next-generation sequencing of soil samples to identify novel botulinum neurotoxins. In order to compare the fitness of the different toxins, we suggest that assays of all the steps from toxin production to animal death should be performed. PMID:25564463

  10. DNA vaccines targeting heavy chain C-terminal fragments of Clostridium botulinum neurotoxin serotypes A, B, and E induce potent humoral and cellular immunity and provide protection from lethal toxin challenge.

    PubMed

    Scott, Veronica L; Villarreal, Daniel O; Hutnick, Natalie A; Walters, Jewell N; Ragwan, Edwin; Bdeir, Khalil; Yan, Jian; Sardesai, Niranjan Y; Finnefrock, Adam C; Casimiro, Danilo R; Weiner, David B

    2015-01-01

    Botulinum neurotoxins (BoNTs) are deadly, toxic proteins produced by the bacterium Clostridium botulinum that can cause significant diseases in humans. The use of the toxic substances as potential bioweapons has raised concerns by the Centers for Disease Control and Prevention and the United States Military. Currently, there is no licensed vaccine to prevent botulinum intoxication. Here we present an immunogenicity study to evaluate the efficacy of novel monovalent vaccines and a trivalent cocktail DNA vaccine targeting the heavy chain C-terminal fragments of Clostridium botulinum neurotoxin serotypes A, B, and E. These synthetic DNA vaccines induced robust humoral and polyfunctional CD4(+) T-cell responses which fully protected animals against lethal challenge after just 2 immunizations. In addition, naïve animals administered immunized sera mixed with the lethal neurotoxin were 100% protected against intoxication. The data demonstrate the protective efficacy induced by a combinative synthetic DNA vaccine approach. This study has importance for the development of vaccines that provide protective immunity against C. botulinum neurotoxins and other toxins.

  11. DNA vaccines targeting heavy chain C-terminal fragments of Clostridium botulinum neurotoxin serotypes A, B, and E induce potent humoral and cellular immunity and provide protection from lethal toxin challenge

    PubMed Central

    Scott, Veronica L; Villarreal, Daniel O; Hutnick, Natalie A; Walters, Jewell N; Ragwan, Edwin; Bdeir, Khalil; Yan, Jian; Sardesai, Niranjan Y; Finnefrock, Adam C; Casimiro, Danilo R; Weiner, David B

    2015-01-01

    Botulinum neurotoxins (BoNTs) are deadly, toxic proteins produced by the bacterium Clostridium botulinum that can cause significant diseases in humans. The use of the toxic substances as potential bioweapons has raised concerns by the Centers for Disease Control and Prevention and the United States Military. Currently, there is no licensed vaccine to prevent botulinum intoxication. Here we present an immunogenicity study to evaluate the efficacy of novel monovalent vaccines and a trivalent cocktail DNA vaccine targeting the heavy chain C-terminal fragments of Clostridium botulinum neurotoxin serotypes A, B, and E. These synthetic DNA vaccines induced robust humoral and polyfunctional CD4+ T-cell responses which fully protected animals against lethal challenge after just 2 immunizations. In addition, naïve animals administered immunized sera mixed with the lethal neurotoxin were 100% protected against intoxication. The data demonstrate the protective efficacy induced by a combinative synthetic DNA vaccine approach. This study has importance for the development of vaccines that provide protective immunity against C. botulinum neurotoxins and other toxins. PMID:26158319

  12. Botulinum Neurotoxin Injections

    MedlinePlus

    ... years without side effects from long-term use. Immunity Because botulinum neurotoxin is a biological product, it ... for the body to create antibodies and develop immunity to the effects of the toxin. Measures are ...

  13. Sublethal concentrations of ichthyotoxic alga Prymnesium parvum affect rainbow trout susceptibility to viral haemorrhagic septicaemia virus.

    PubMed

    Andersen, Nikolaj Gedsted; Lorenzen, Ellen; Snogdal Boutrup, Torsten; Hansen, Per Juel; Lorenzen, Niels

    2016-01-13

    Ichthyotoxic algal blooms are normally considered a threat to maricultured fish only when blooms reach lethal cell concentrations. The degree to which sublethal algal concentrations challenge the health of the fish during blooms is practically unknown. In this study, we analysed whether sublethal concentrations of the ichthyotoxic alga Prymnesium parvum affect the susceptibility of rainbow trout Oncorhynchus mykiss to viral haemorrhagic septicaemia virus (VHSV). During exposure to sublethal algal concentrations, the fish increased production of mucus on their gills. When fish were exposed to the algae for 12 h prior to the addition of virus, a marginal decrease in the susceptibility to VHSV was observed compared to fish exposed to VHSV without algae. If virus and algae were added simultaneously, inclusion of the algae increased mortality by 50% compared to fish exposed to virus only, depending on the experimental setup. We concluded that depending on the local exposure conditions, sublethal concentrations of P. parvum could affect susceptibility of fish to infectious agents such as VHSV. PMID:26758652

  14. Botulinum Neurotoxin Research at the Western Regional Research Center

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Botulinum neurotoxins (BoNTs) are some of the most potent toxins to humans. The most common route of intoxication is through ingestion of contaminated food or drink. In addition, these toxins are likely targets for use in intentional adulteration of food or animal feeds and are thus classified as Se...

  15. Botulinum neurotoxin: a deadly protease with applications to human medicine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Botulinum neurotoxins (BoNTs) are some of the most potent biological toxins to humans. They are synthesized by the gram-positive, spore-forming bacterium Clostridium botulinum. BoNT is secreted from the bacterium as a ~150 kDa polypeptide which is cleaved by bacterial or host proteases into a ~50 kD...

  16. Timbós: ichthyotoxic plants used by Brazilian Indians.

    PubMed

    Teixeira, J R; Lapa, A J; Souccar, C; Valle, J R

    1984-05-01

    The pharmacology of serjanosides, active principles isolated from the fish-poison plant Serjania lethalis St. Hil, a Sapindaceae, was investigated by comparing their actions in fishes and mammals with those of rotenone and certain saponins. The ichthyocid activity of the serjanosides was 2.5 times greater than that of the crude plant extract, approximately 10 times lower than the activity of rotenone, but from 10 to 50 times greater than the activity of the other saponins. When injected in mammals, the serjanosides induced deep prostration, dyspnea, cyanosis, ectopic heart beats, cardiovascular failure and respiratory arrest. These effects, leading to death that was not prevented by artificial respiration, indicated several mechanisms for the toxic action of the serjanosides. In vitro studies with these substances have shown that membrane depolarization and muscle contracture were probably due to unspecific surface actions. Rotenone, under the same experimental conditions induced hypotension, bradycardia and respiratory arrest. Death was prevented by artificial respiration. Ectopic foci, membrane depolarization, contractures and neuromuscular block were not observed after rotenone. Apparently, death from rotenone poisoning was a consequence of respiratory failure of central origin. The serjanosides are rather potent fish poison saponins. Mammals, however, are apparently insensitive to the same specific action since other toxic effects induced by those substances in rats and mice were also observed by employing saponins devoid of fish-killing activity.

  17. Botulinum neurotoxins: mechanism of action.

    PubMed

    Tighe, Ann P; Schiavo, Giampietro

    2013-06-01

    Botulinum neurotoxins are used clinically for conditions characterized by hyperexcitability of peripheral nerve terminals and hypersecretory syndromes. These neurotoxins are synthesized as precursor proteins with low activity, but their effects are mediated by the active form of the neurotoxin through a multistep mechanism. Following a high-affinity interaction with a protein receptor and polysialogangliosides on the synaptic membrane, botulinum neurotoxins enter the neuron and causes a sustained inhibition of synaptic transmission. The active neurotoxin is part of a high-molecular-weight complex that protects the neurotoxin from proteolytic degradation. Although complexing proteins do not affect diffusion of therapeutic neurotoxin, they may lead to the development of neutralizing antibodies that block responsiveness to it. Nerve terminal intoxication is reversible and its duration varies for different BoNT serotypes. Although it was previously assumed that botulinum neurotoxins exert effects only on the peripheral synapses, such as the neuromuscular junction, there is now substantial evidence that these neurotoxins affect neurotransmission at distal central nervous system sites as well.

  18. Coupling the Torpedo microplate-receptor binding assay with mass spectrometry to detect cyclic imine neurotoxins.

    PubMed

    Aráoz, Rómulo; Ramos, Suzanne; Pelissier, Franck; Guérineau, Vincent; Benoit, Evelyne; Vilariño, Natalia; Botana, Luis M; Zakarian, Armen; Molgó, Jordi

    2012-12-01

    Cyclic imine neurotoxins constitute an emergent family of neurotoxins of dinoflagellate origin that are potent antagonists of nicotinic acetylcholine receptors. We developed a target-directed functional method based on the mechanism of action of competitive agonists/antagonists of nicotinic acetylcholine receptors for the detection of marine cyclic imine neurotoxins. The key step for method development was the immobilization of Torpedo electrocyte membranes rich in nicotinic acetylcholine receptors on the surface of microplate wells and the use of biotinylated-α-bungarotoxin as tracer. Cyclic imine neurotoxins competitively inhibit biotinylated-α-bungarotoxin binding to Torpedo-nicotinic acetylcholine receptors in a concentration-dependent manner. The microplate-receptor binding assay allowed rapid detection of nanomolar concentrations of cyclic imine neurotoxins directly in shellfish samples. Although highly sensitive and specific for the detection of neurotoxins targeting nicotinic acetylcholine receptors as a class, the receptor binding assay cannot identify a given analyte. To address the low selectivity of the microplate-receptor binding assay, the cyclic imine neurotoxins tightly bound to the coated Torpedo nicotinic receptor were eluted with methanol, and the chemical nature of the eluted ligands was identified by mass spectrometry. The immobilization of Torpedo electrocyte membranes on the surface of microplate wells proved to be a high-throughput format for the survey of neurotoxins targeting nicotinic acetylcholine receptors directly in shellfish matrixes with high sensitivity and reproducibility.

  19. Toxicity of four potentially ichthyotoxic marine phytoflagellates determined by four different test methods.

    PubMed

    Meldahl, A S; Edvardsen, B; Fonnum, F

    1994-07-01

    The toxicity of the marine phytoflagellates Prymnesium parvum. Prymnesium patelliferum, Chrysochromulina polylepis, and Chrysochromulina leadbeateri isolated from ichthyotoxic blooms in Norwegian coastal waters was compared using four different test methods developed for the detection of toxins produced by these species. The test methods were (1) lethality to the crustacean Artemia salina exposed to living algae, (2) hemolytic activity (lysis of human erythrocytes) by crude algal lipid extracts, and inhibition of the uptake of the neurotransmitters L-glutamate and gamma-aminobutyric acid (GABA) into (3) synaptosomes and (4) synaptic vesicles of rat brain by crude algal lipid extracts. All test methods indicated different levels of toxicity among the algal species. Prymnesium parvum, P. patelliferum, and C. polylepis were toxic as determined by all four test methods. The cultured strain of C. leadbeateri, although isolated from a toxic algal bloom, appeared nontoxic by the methods used here, and served as a negative control. The hemolytic activity of P. parvum extract was about nine times higher than that of P. patelliferum extract, whereas the activity was only two to three times higher using the other three methods. Chrysochromulina polylepis had higher toxic activity than P. patelliferum except for less inhibitory effect on synaptosomes. The inhibition of synaptosomal and vesicular neurotransmitter uptake systems by extracts of P. parvum, P. patelliferum, and C. polylepis appeared to be due to similar mechanisms of action, since similar inhibition ratios between the uptake of L-glutamate and GABA were obtained in both synaptosomes and synaptic vesicles. We suggest that P. parvum, P. patelliferum, and C. polylepis produce a set of similar toxins and that the relative amounts of each toxin vary among the three species. PMID:7912738

  20. Origin of neurotoxins from defensins.

    PubMed

    Zhu, Li-Mei; Gao, Bin; Zhu, Shun-Yi

    2015-06-25

    There are at least three conserved protein folds shared by ion channel-targeted neurotoxins and antimicrobial defensins, including cysteine-stabilized α-helix and β-sheet fold (CSαβ), inhibitor cystine knot fold (ICK) and β-defensin fold (BDF). Based on a combined data of sequences, structures and functions, it has been proposed that these neurotoxins could originate from related ancient antimicrobial defensins by neofunctionalization. This provides an ideal system to study how a novel function emerged from a conserved structural scaffold during evolution. The elucidation of functional novelty of proteins not only has great significance in evolutionary biology but also will be helpful in guiding rational molecular design. This review describes recent progresses in origin of neurotoxins, focusing on the three conserved protein scaffolds.

  1. Botulinum neurotoxin A: a review.

    PubMed

    Berry, M G; Stanek, Jan J

    2012-10-01

    Despite its ubiquity in cosmetic circles and broad general awareness, a literature search of botulinum neurotoxin in JPRAS and BJPS yielded a mere 4 articles germane to cosmesis. A pair each detailing its application in masseteric hypertrophy(1,2) and the use of cryoanalgesia.(3,4) Given that botulinum neurotoxin A is the most commonly used cosmetic treatment, with American figures being most accurate,(5) a review of the background, development and scientific evidence would be perhaps useful, if not overdue, as Plastic Surgeons increasingly incorporate non-surgical interventions into their practices as part of a comprehensive facial rejuvenation strategy.

  2. Botulinum neurotoxin A: a review.

    PubMed

    Berry, M G; Stanek, Jan J

    2012-10-01

    Despite its ubiquity in cosmetic circles and broad general awareness, a literature search of botulinum neurotoxin in JPRAS and BJPS yielded a mere 4 articles germane to cosmesis. A pair each detailing its application in masseteric hypertrophy(1,2) and the use of cryoanalgesia.(3,4) Given that botulinum neurotoxin A is the most commonly used cosmetic treatment, with American figures being most accurate,(5) a review of the background, development and scientific evidence would be perhaps useful, if not overdue, as Plastic Surgeons increasingly incorporate non-surgical interventions into their practices as part of a comprehensive facial rejuvenation strategy. PMID:22552262

  3. Ligand-binding assays for cyanobacterial neurotoxins targeting cholinergic receptors.

    PubMed

    Aráoz, Rómulo; Vilariño, Natalia; Botana, Luis M; Molgó, Jordi

    2010-07-01

    Toxic cyanobacterial blooms are a threat to public health because of the capacity of some cyanobacterial species to produce potent hepatotoxins and neurotoxins. Cyanobacterial neurotoxins are involved in the rapid death of wild and domestic animals by targeting voltage gated sodium channels and cholinergic synapses, including the neuromuscular junction. Anatoxin-a and its methylene homologue homoanatoxin-a are potent agonists of nicotinic acetylcholine receptors. Since the structural determination of anatoxin-a, several mass spectrometry-based methods have been developed for detection of anatoxin-a and, later, homoanatoxin-a. Mass spectrometry-based techniques provide accuracy, precision, selectivity, sensitivity, reproducibility, adequate limit of detection, and structural and quantitative information for analyses of cyanobacterial anatoxins from cultured and environmental cyanobacterial samples. However, these physicochemical techniques will only detect known toxins for which toxin standards are commercially available, and they require highly specialized laboratory personnel and expensive equipment. Receptor-based assays are functional methods that are based on the mechanism of action of a class of toxins and are thus, suitable tools for survey of freshwater reservoirs for cyanobacterial anatoxins. The competition between cyanobacterial anatoxins and a labelled ligand for binding to nicotinic acetylcholine receptors is measured radioactively or non-radioactively providing high-throughput screening formats for routine detection of this class of neurotoxins. The mouse bioassay is the method of choice for marine toxin monitoring, but has to be replaced by fully validated functional methods. In this paper we review the ligand-binding assays developed for detection of cyanobacterial and algal neurotoxins targeting the nicotinic acetylcholine receptors and for high-throughput screening of novel nicotinic agents.

  4. Rapid microfluidic assay for the detection of botulinum neurotoxin in animal sera

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The potent botulinum neurotoxins (BoNTs) represent a threat to public health and safety. Botulism is a disease caused by BoNT intoxication that results in muscle paralysis that can be fatal. Sensitive assays capable of detecting BoNTs from different substrates and settings are essential to limit f...

  5. Use of monoclonal antibodies in the sensitive detection and neutralization of botulinum neurotoxin serotype B

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Botulinum neurotoxins (BoNT) are some of nature’s most potent toxins. Due to potential food contamination and bioterrorism concerns, the development of detection reagents, therapeutics and countermeasures are of urgent interest. Recently, we have developed sensitive electrochemiluminescent (ECL) i...

  6. Structure of a bimodular botulinum neurotoxin complex provides insights into its oral toxicity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Botulinum neurotoxins (BoNTs) are highly potent oral poisons produced by Clostridium botulinum. BoNTs are secreted along with several auxiliary proteins forming progenitor toxin complexes (PTC). Here, we report the structure of a ~760 kDa 14-subunit PTC using a combination of X-ray crystallography a...

  7. Maculotoxin: a neurotoxin from the venom glands of the octopus Hapalochlaena maculosa identified as tetrodotoxin.

    PubMed

    Sheumack, D D; Howden, M E; Spence, I; Quinn, R J

    1978-01-13

    Maculotoxin, a potent neurotoxin isolated from the posterior salivary glands of the blue-ringed octopus. Hapalochlaena maculosa, has now been identified as tetrodotoxin. This is the first reported case in which tetrodotoxin has been found to occur in a venom. PMID:619451

  8. DOMOIC ACID AS A DEVELOPMENTAL NEUROTOXIN

    PubMed Central

    Costa, Lucio G.; Giordano, Gennaro; Faustman, Elaine M.

    2010-01-01

    Domoic acid (DomA) is an excitatory aminoacid which can accumulate in shellfish and finfish under certain environmental conditions. DomA is a potent neurotoxin. In humans and in non-human primates, oral exposure to a few mg/kg DomA elicits gastrointestinal effects, while slightly higher doses cause neurological symptoms, seizures, memory impairment, and limbic system degeneration. In rodents, which appear to be less sensitive than humans or non-human primates, oral doses cause behavioral abnormalities (e.g. hindlimb scratching), followed by seizures and hippocampal degeneration. Similar effects are also seen in other species (from sea-lions to zebrafish), indicating that DomA exerts similar neurotoxic effects across species. The neurotoxicity of DomA is ascribed to its ability to interact and activate the AMPA/KA receptors, a subfamily of receptors for the neuroexcitatory neurotransmitter glutamate. Studies exploring the neurotoxic effects of DomA on the developing nervous system indicate that DomA elicits similar behavioral, biochemical and morphological effects as in adult animals. However, most importantly, developmental neurotoxicity is seen at doses of DomA that are one to two orders of magnitude lower than those exerting neurotoxicity in adults. This difference may be due to toxicokinetic and/or toxicodynamic differences. Estimated safe doses may be exceeded in adults by high consumption of shellfish contaminated with DomA at the current limit of 20 ug/g. Given the potential higher susceptibility of the young to DomA neurotoxicity, additional studies investigating exposure to, and effects of this neurotoxin during brain development are warranted. PMID:20471419

  9. Botulinum neurotoxin: the ugly duckling.

    PubMed

    Koussoulakos, Stauros

    2009-01-01

    This review presents a brief account of the most significant biological effects and clinical applications of botulinum neurotoxins, in a way comprehensive even for casual readers who are not familiar with the subject. The most toxic known substances in botulinum neurotoxins are polypeptides naturally synthesized by bacteria of the genus Clostridium. These polypeptides inhibit acetylcholine release at neuromuscular junctions, thus causing muscle paralysis involving both somatic and autonomic innervation. There is substantial evidence that this muscle-paralyzing feature of botulinum neurotoxins is useful for their beneficial influence on more than 50 pathological conditions such as spastic paralysis, cerebral palsy, focal dystonia, essential tremor, headache, incontinence and a variety of cosmetic interventions. Injection of adequate quantities of botulinum toxins in spastic muscles is considered as a highly hopeful procedure for the treatment of people who suffer from dystonia, cerebral palsy or have experienced a stroke. So far, numerous and reliable studies have established the safety and efficacy of botulinum neurotoxins and advocate wider clinical therapeutic and cosmetic applications. PMID:19365125

  10. A Novel Inhibitor Prevents the Peripheral Neuroparalysis of Botulinum Neurotoxins

    PubMed Central

    Azarnia Tehran, Domenico; Zanetti, Giulia; Leka, Oneda; Lista, Florigio; Fillo, Silvia; Binz, Thomas; Shone, Clifford C.; Rossetto, Ornella; Montecucco, Cesare; Paradisi, Cristina; Mattarei, Andrea; Pirazzini, Marco

    2015-01-01

    Botulinum neurotoxins (BoNTs) form a large class of potent and deadly neurotoxins. Given their growing number, it is of paramount importance to discover novel inhibitors targeting common steps of their intoxication process. Recently, EGA was shown to inhibit the action of bacterial toxins and viruses exhibiting a pH-dependent translocation step in mammalian cells, by interfering with their entry route. As BoNTs act in the cytosol of nerve terminals, the entry into an appropriate compartment wherefrom they translocate the catalytic moiety is essential for toxicity. Herein we propose an optimized procedure to synthesize EGA and we show that, in vitro, it prevents the neurotoxicity of different BoNT serotypes by interfering with their trafficking. Furthermore, in mice, EGA mitigates botulism symptoms induced by BoNT/A and significantly decreases the lethality of BoNT/B and BoNT/D. This opens the possibility of using EGA as a lead compound to develop novel inhibitors of botulinum neurotoxins. PMID:26670952

  11. A Novel Inhibitor Prevents the Peripheral Neuroparalysis of Botulinum Neurotoxins.

    PubMed

    Azarnia Tehran, Domenico; Zanetti, Giulia; Leka, Oneda; Lista, Florigio; Fillo, Silvia; Binz, Thomas; Shone, Clifford C; Rossetto, Ornella; Montecucco, Cesare; Paradisi, Cristina; Mattarei, Andrea; Pirazzini, Marco

    2015-01-01

    Botulinum neurotoxins (BoNTs) form a large class of potent and deadly neurotoxins. Given their growing number, it is of paramount importance to discover novel inhibitors targeting common steps of their intoxication process. Recently, EGA was shown to inhibit the action of bacterial toxins and viruses exhibiting a pH-dependent translocation step in mammalian cells, by interfering with their entry route. As BoNTs act in the cytosol of nerve terminals, the entry into an appropriate compartment wherefrom they translocate the catalytic moiety is essential for toxicity. Herein we propose an optimized procedure to synthesize EGA and we show that, in vitro, it prevents the neurotoxicity of different BoNT serotypes by interfering with their trafficking. Furthermore, in mice, EGA mitigates botulism symptoms induced by BoNT/A and significantly decreases the lethality of BoNT/B and BoNT/D. This opens the possibility of using EGA as a lead compound to develop novel inhibitors of botulinum neurotoxins. PMID:26670952

  12. High-Throughput Screening Uncovers Novel Botulinum Neurotoxin Inhibitor Chemotypes.

    PubMed

    Bompiani, Kristin M; Caglič, Dejan; Krutein, Michelle C; Benoni, Galit; Hrones, Morgan; Lairson, Luke L; Bian, Haiyan; Smith, Garry R; Dickerson, Tobin J

    2016-08-01

    Botulism is caused by potent and specific bacterial neurotoxins that infect host neurons and block neurotransmitter release. Treatment for botulism is limited to administration of an antitoxin within a short time window, before the toxin enters neurons. Alternatively, current botulism drug development targets the toxin light chain, which is a zinc-dependent metalloprotease that is delivered into neurons and mediates long-term pathology. Several groups have identified inhibitory small molecules, peptides, or aptamers, although no molecule has advanced to the clinic due to a lack of efficacy in advanced models. Here we used a homogeneous high-throughput enzyme assay to screen three libraries of drug-like small molecules for new chemotypes that modulate recombinant botulinum neurotoxin light chain activity. High-throughput screening of 97088 compounds identified numerous small molecules that activate or inhibit metalloprotease activity. We describe four major classes of inhibitory compounds identified, detail their structure-activity relationships, and assess their relative inhibitory potency. A previously unreported chemotype in any context of enzyme inhibition is described with potent submicromolar inhibition (Ki = 200-300 nM). Additional detailed kinetic analyses and cellular cytotoxicity assays indicate the best compound from this series is a competitive inhibitor with cytotoxicity values around 4-5 μM. Given the potency and drug-like character of these lead compounds, further studies, including cellular activity assays and DMPK analysis, are justified. PMID:27314875

  13. Three enzymatically active neurotoxins of Clostridium botulinum strain Af84: BoNT/A2, /F4, and /F5.

    PubMed

    Kalb, Suzanne R; Baudys, Jakub; Smith, Theresa J; Smith, Leonard A; Barr, John R

    2014-04-01

    Botulinum neurotoxins (BoNTs) are produced by various species of clostridia and are potent neurotoxins which cause the disease botulism, by cleaving proteins needed for successful nerve transmission. There are currently seven confirmed serotypes of BoNTs, labeled A-G, and toxin-producing clostridia typically only produce one serotype of BoNT. There are a few strains (bivalent strains) which are known to produce more than one serotype of BoNT, producing either both BoNT/A and /B, BoNT/A and /F, or BoNT/B and /F, designated as Ab, Ba, Af, or Bf. Recently, it was reported that Clostridium botulinum strain Af84 has three neurotoxin gene clusters: bont/A2, bont/F4, and bont/F5. This was the first report of a clostridial organism containing more than two neurotoxin gene clusters. Using a mass spectrometry based proteomics approach, we report here that all three neurotoxins, BoNT/A2, /F4, and /F5, are produced by C. botulinum Af84. Label free MS(E) quantification of the three toxins indicated that toxin composition is 88% BoNT/A2, 1% BoNT/F4, and 11% BoNT/F5. The enzymatic activity of all three neurotoxins was assessed by examining the enzymatic activity of the neurotoxins upon peptide substrates, which mimic the toxins' natural targets, and monitoring cleavage of the substrates by mass spectrometry. We determined that all three neurotoxins are enzymatically active. This is the first report of three enzymatically active neurotoxins produced in a single strain of Clostridium botulinum.

  14. Detection of botulinum neurotoxin serotypes A and B using a chemiluminescent versus electrochemiluminescent immunoassay in food and serum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Botulinum neurotoxins (BoNTs) are some of the most potent biological toxins. High-affinity monoclonal antibodies (mAbs) have been developed for the detection of BoNT serotypes A and B using a chemiluminescent capture enzyme-linked immunosorbent assay (ELISA). In an effort to improve toxin detection ...

  15. Detection of botulinum neurotoxin serotypes A and B using chemiluminescence and electrochemiluninescene immunoassays in food and serum matrices

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Botulinum neurotoxins (BoNT) are some of the most potent biological toxins with serotypes A and B being most prevalent in foodborne contaminations. BoNTs are also likely targets for use in intentional adulteration of food or animal feeds and are thus classified as Select Agents. In our laboratories,...

  16. Rapid and selective detection of botulinum neurotoxin serotypes-A and –B with a single immunochromatographic test strip

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Seven, antigenically distinct botulinum neurotoxins (BoNT) are produced by the bacterium Clostridium botulinum and classified into serotypes designated A-G. In animals these potent toxin acts to inhibit acetylcholine release, resulting in paralysis and death. BoNT/A and /B, together represent >80% o...

  17. Update on the botulinum neurotoxins.

    PubMed

    Carruthers, A; Carruthers, J

    2001-12-01

    The botulinum neurotoxins (BTX) are an exciting group of therapeutic agents with dramatically expanding clinical indications. The US FDA has approved BOTOX (BTX-A, Allergan) and Myobloc (BTX-B, Elan Pharmaceuticals) for the treatment of cervical dystonia. TPP Canada has also approved BOTOX for the treatment of glabellar frown lines. The US FDA is expected to approve this new indication before the end of 2002. These changes will dramatically expand the marketing of BTXs. Concerns about risks and side-effects diminish as clinical experience increases with this "most poisonous of poisons". In particular, the incidence of secondary resistance to the toxin's effect has been dramatically diminished with the reduction of the non-toxic protein in current batches of BOTOX. PMID:11813096

  18. Presynaptic neurotoxins: an expanding array of natural and modified molecules.

    PubMed

    Davletov, Bazbek; Ferrari, Enrico; Ushkaryov, Yuri

    2012-01-01

    The process of neurotransmitter release from nerve terminals is a target for a wide array of presynaptic toxins produced by various species, from humble bacteria to arthropods to vertebrate animals. Unlike other toxins, most presynaptic neurotoxins do not kill cells but simply inhibit or activate synaptic transmission. In this review, we describe two types of presynaptic neurotoxins: clostridial toxins and latrotoxins, which are, respectively, the most potent blockers and stimulators of neurotransmitter release. These toxins have been instrumental in defining presynaptic functions and are now widely used in research and medicine. Here, we would like to analyse the diversity of these toxins and demonstrate how the knowledge of their structures and mechanisms of action can help us to design better tools for research and medical applications. We will look at natural and synthetic variations of these exquisite molecular machines, highlighting recent advances in our understanding of presynaptic toxins and questions that remain to be answered. If we can decipher how a given biomolecule is modified by nature to target different species, we will be able to design new variants that carry only desired characteristics to achieve specific therapeutic, agricultural or research goals. Indeed, a number of research groups have already initiated a quest to harness the power of natural toxins with the aim of making them more specifically targeted and safer for future research and medical applications.

  19. Neurotoxin formation from pilot-scale incineration of synthetic ester turbine lubricants with a triaryl phosphate additive.

    PubMed

    Rubey, W A; Striebich, R C; Bush, J; Centers, P W; Wright, R L

    1996-01-01

    The high-temperature combustion of synthetic ester turbine engine lubricants has been performed by diluting the lubricant 5, 15, or 25% in diesel fuel and burning the mixture in a pilot-scale boiler facility. The effluent gas from this combustion system was carefully monitored for the formation of a potent neurotoxin, trimethylolpropane phosphate (TMPP). Although TMPP was not detected in the gaseous effluent, elevated levels of the neurotoxin were found in scrapings from the inside of the boiler system. Because of the extreme toxicity of this compound, significant dermal exposure could be a potential risk to incinerator operation and maintenance personnel. PMID:8783815

  20. Neurotoxin formation from pilot-scale incineration of synthetic ester turbine lubricants with a triaryl phosphate additive.

    PubMed

    Rubey, W A; Striebich, R C; Bush, J; Centers, P W; Wright, R L

    1996-01-01

    The high-temperature combustion of synthetic ester turbine engine lubricants has been performed by diluting the lubricant 5, 15, or 25% in diesel fuel and burning the mixture in a pilot-scale boiler facility. The effluent gas from this combustion system was carefully monitored for the formation of a potent neurotoxin, trimethylolpropane phosphate (TMPP). Although TMPP was not detected in the gaseous effluent, elevated levels of the neurotoxin were found in scrapings from the inside of the boiler system. Because of the extreme toxicity of this compound, significant dermal exposure could be a potential risk to incinerator operation and maintenance personnel.

  1. Structure- and Substrate- Based Inhibitor Design for Clostridium botulinum Neurotoxin Serotype A*

    SciTech Connect

    Kumaran,D.; Rawat, R.; Ludivico, M.; Ahmed, S.; Swaminathan, S.

    2008-01-01

    The seven antigenically distinct serotypes of Clostridium botulinum neurotoxins cleave specific soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex proteins and block the release of neurotransmitters that cause flaccid paralysis and are considered potential bioweapons. Botulinum neurotoxin type A is the most potent among the clostridial neurotoxins, and to date there is no post-exposure therapeutic intervention available. To develop inhibitors leading to drug design, it is imperative that critical interactions between the enzyme and the substrate near the active site are known. Although enzyme-substrate interactions at exosites away from the active site are mapped in detail for botulinum neurotoxin type A, information about the active site interactions is lacking. Here, we present the crystal structures of botulinum neurotoxin type A catalytic domain in complex with four inhibitory substrate analog tetrapeptides, viz. RRGC, RRGL, RRGI, and RRGM at resolutions of 1.6-1.8 Angstroms . These structures show for the first time the interactions between the substrate and enzyme at the active site and delineate residues important for substrate stabilization and catalytic activity. We show that OH of Tyr366 and NH2 of Arg363 are hydrogen-bonded to carbonyl oxygens of P1 and P1' of the substrate analog and position it for catalytic activity. Most importantly, the nucleophilic water is replaced by the amino group of the N-terminal residue of the tetrapeptide. Furthermore, the S1' site is formed by Phe194, Thr215, Thr220, Asp370, and Arg363. The Ki of the best inhibitory tetrapeptide is 157 nm.

  2. Substrate Binding Mode and its Implication on Drug Design for Botulinum Neurotoxin A

    SciTech Connect

    Kumaran, D.; Rawat, R; Ahmed, A; Swaminathan, S

    2008-01-01

    The seven antigenically distinct serotypes of Clostridium botulinum neurotoxins, the causative agents of botulism, block the neurotransmitter release by specifically cleaving one of the three SNARE proteins and induce flaccid paralysis. The Centers for Disease Control and Prevention (CDC) has declared them as Category A biowarfare agents. The most potent among them, botulinum neurotoxin type A (BoNT/A), cleaves its substrate synaptosome-associated protein of 25 kDa (SNAP-25). An efficient drug for botulism can be developed only with the knowledge of interactions between the substrate and enzyme at the active site. Here, we report the crystal structures of the catalytic domain of BoNT/A with its uncleavable SNAP-25 peptide 197QRATKM202 and its variant 197RRATKM202 to 1.5 A and 1.6 A, respectively. This is the first time the structure of an uncleavable substrate bound to an active botulinum neurotoxin is reported and it has helped in unequivocally defining S1 to S5? sites. These substrate peptides make interactions with the enzyme predominantly by the residues from 160, 200, 250 and 370 loops. Most notably, the amino nitrogen and carbonyl oxygen of P1 residue (Gln197) chelate the zinc ion and replace the nucleophilic water. The P1?-Arg198, occupies the S1? site formed by Arg363, Thr220, Asp370, Thr215, Ile161, Phe163 and Phe194. The S2? subsite is formed by Arg363, Asn368 and Asp370, while S3? subsite is formed by Tyr251, Leu256, Val258, Tyr366, Phe369 and Asn388. P4?-Lys201 makes hydrogen bond with Gln162. P5?-Met202 binds in the hydrophobic pocket formed by the residues from the 250 and 200 loop. Knowledge of interactions between the enzyme and substrate peptide from these complex structures should form the basis for design of potent inhibitors for this neurotoxin.

  3. Neurotoxins from Australo-Papuan elapids: a biochemical and pharmacological perspective.

    PubMed

    Kuruppu, Sanjaya; Smith, A Ian; Isbister, Geoffrey K; Hodgson, Wayne C

    2008-01-01

    Most of the medically important snakes in Papua New Guinea and Australia belong to the family Elapidae and are referred to as "Australo-Papuan" elapids. Neurotoxicity is often a life-threatening symptom of envenoming by these snakes; therefore, much attention has been paid to the isolation and detailed pharmacological and biochemical characterization of the presynaptic (beta) and postsynaptic (alpha) neurotoxins from these elapid venoms. These studies have highlighted the potential for these toxins to be used as highly potent and selective probes for biomedical research and, perhaps, the potential for their use as lead compounds for the development of pharmaceutical agents. Historically, the potency of neurotoxins/crude venoms has been determined using murine LD50 (lethal dose) assays. However, a different rank order of potency often results when crude venoms/toxins are ranked based on their in vitro pharmacological parameters (e.g., t90 values). The lack of neurotoxicity following envenoming by brown snakes, despite the presence of a potent neurotoxin in their venom, has puzzled clinical toxinologists for years. This paradox also appears to include envenoming by the Stephen's banded snake. Lastly, the in vitro studies examining the effectiveness of antivenoms as well as the potential for alternative compounds to reverse/prevent neurotoxicity are discussed. This review presents for the first time a detailed comparative analysis of the pharmacology and biochemistry of neurotoxins isolated from the Australo-Papuan elapids, placing emphasis on the time taken for onset of action, receptor binding parameters, reversibility, and the methods for determining potency.

  4. Synthesis and Biological Evaluation of Botulinum Neurotoxin A Protease Inhibitors

    PubMed Central

    Li, Bing; Pai, Ramdas; Cardinale, Steven C.; Butler, Michelle M.; Peet, Norton P.; Moir, Donald T.; Bavari, Sina; Bowlin, Terry L.

    2010-01-01

    NSC 240898 was previously identified as a botulinum neurotoxin A light chain (BoNT/A LC) endopeptidase inhibitor by screening the National Cancer Institute Open Repository diversity set. Two types of analogs have been synthesized and shown to inhibit BoNT/A LC in a FRET-based enzyme assay, with confirmation in an HPLC-based assay. These two series of compounds have also been evaluated for inhibition of anthrax lethal factor (LF), an unrelated metalloprotease, to examine enzyme specificity of the BoNT/A LC inhibition. The most potent inhibitor against BoNT/A LC in these two series is compound 12 (IC50 = 2.5 µM, FRET assay), which is 4.4-fold more potent than the lead structure, and 11.2-fold more selective for BoNT/A LC versus the anthrax LF metalloproteinase. Structure-activity relationship studies have revealed structural features important to potency and enzyme specificity. PMID:20155918

  5. Insights into the different catalytic activities of Clostridium neurotoxins.

    PubMed

    Chen, Sheng; Karalewitz, Andrew P A; Barbieri, Joseph T

    2012-05-01

    The clostridial neurotoxins are among the most potent protein toxins for humans and are responsible for botulism, a flaccid paralysis elicited by the botulinum toxins (BoNT), and spastic paralysis elicited by tetanus toxin (TeNT). Seven serotypes of botulinum neurotoxins (A-G) and tetanus toxin showed different toxicities and cleave their substrates with different efficiencies. However, the molecular basis of their different catalytic activities with respect to their substrates is not clear. BoNT/B light chain (LC/B) and TeNT light chain (LC/T) cleave vesicle-associated membrane protein 2 (VAMP2) at the same scissile bond but possess different catalytic activities and substrate requirements, which make them the best candidates for studying the mechanisms of their different catalytic activities. The recognition of five major P sites of VAMP2 (P7, P6, P1, P1', and P2') and fine alignment of sites P2 and P3 and sites P2 and P4 by LC/B and LC/T, respectively, contributed to their substrate recognition and catalysis. Significantly, we found that the S1 pocket mutation LC/T(K(168)E) increased the rate of native VAMP2 cleavage so that it approached the rate of LC/B, which explains the molecular basis for the lower k(cat) that LC/T possesses for VAMP2 cleavage relative to that of LC/B. This analysis explains the molecular basis underlying the VAMP2 recognition and cleavage by LC/B and LC/T and provides insight that may extend the pharmacologic utility of these neurological reagents.

  6. The long journey of botulinum neurotoxins into the synapse.

    PubMed

    Rummel, Andreas

    2015-12-01

    Botulinum neurotoxins (BoNT) cause the disease botulism, a flaccid paralysis of the muscle. They are also very effective, widely used medicines applied locally in sub-nanogram quantities. BoNTs are released together with several non-toxic, associated proteins as progenitor toxin complexes (PCT) by Clostridium botulinum to become highly potent oral poisons ingested via contaminated food. They block the neurotransmission in susceptible animals and humans already in nanogram quantities due to their specific ability to enter motoneurons and to cleave only selected neuronal proteins involved in neuroexocytosis. BoNTs have developed a sophisticated strategy to passage the gastrointestinal tract and to be absorbed in the intestine of the host to finally attack neurons. A non-toxic non-hemagglutinin (NTNHA) forms a binary complex with BoNT to protect it from gastrointestinal degradation. This binary M-PTC is one component of the bi-modular 14-subunit ∼760 kDa large progenitor toxin complex. The other component is the structurally and functionally independent dodecameric hemagglutinin (HA) complex which facilitates the absorption on the intestinal epithelium by glycan binding. Subsequent to its transcytosis the HA complex disrupts the tight junction of the intestinal barrier from the basolateral side by binding to E-cadherin. Now, the L-PTC can also enter the circulation by paracellular routes in much larger quantities. From here, the dissociated BoNTs reach the neuromuscular junction and accumulate via interaction with polysialo gangliosides, complex glycolipids, on motoneurons at the neuromuscular junction. Subsequently, additional specific binding to luminal segments of synaptic vesicles proteins like SV2 and synaptotagmin leads to their uptake. Finally, the neurotoxins shut down the synaptic vesicle cycle, which they had exploited before to enter their target cells, via specific cleavage of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE

  7. Emerging Opportunities for Serotypes of Botulinum Neurotoxins

    PubMed Central

    Peng Chen, Zhongxing; Morris, J. Glenn; Rodriguez, Ramon L.; Shukla, Aparna Wagle; Tapia-Núñez, John; Okun, Michael S.

    2012-01-01

    Background: Two decades ago, botulinum neurotoxin (BoNT) type A was introduced to the commercial market. Subsequently, the toxin was approved by the FDA to address several neurological syndromes, involving muscle, nerve, and gland hyperactivity. These syndromes have typically been associated with abnormalities in cholinergic transmission. Despite the multiplicity of botulinal serotypes (designated as types A through G), therapeutic preparations are currently only available for BoNT types A and B. However, other BoNT serotypes are under study for possible clinical use and new clinical indications; Objective: To review the current research on botulinum neurotoxin serotypes A-G, and to analyze potential applications within basic science and clinical settings; Conclusions: The increasing understanding of botulinal neurotoxin pathophysiology, including the neurotoxin’s effects on specific neuronal populations, will help us in tailoring treatments for specific diagnoses, symptoms and patients. Scientists and clinicians should be aware of the full range of available data involving neurotoxin subtypes A-G. PMID:23202312

  8. Fetal exposure to environmental neurotoxins in Taiwan.

    PubMed

    Jiang, Chuen-Bin; Hsi, Hsing-Cheng; Fan, Chun-Hua; Chien, Ling-Chu

    2014-01-01

    Mercury (Hg), lead (Pb), cadmium (Cd), and arsenic (As) are recognized neurotoxins in children that particularly affect neurodevelopment and intellectual performance. Based on the hypothesis that the fetal basis of adult disease is fetal toxic exposure that results in adverse outcomes in adulthood, we explored the concentrations of key neurotoxins (i.e., Hg, Pb, Cd, and As) in meconium to identify the risk factors associated with these concentrations. From January 2007 to December 2009, 545 mother-infant pairs were recruited. The geometric mean concentrations of Pb and As in the meconium of babies of foreign-born mothers (22.9 and 38.1 µg/kg dry weight, respectively) were significantly greater than those of babies of Taiwan-born mothers (17.5 and 33.0 µg/kg dry weight, respectively). Maternal age (≥30 y), maternal education, use of traditional Chinese herbs during pregnancy, and fish cutlet consumption (≥3 meals/wk) were risk factors associated with concentrations of key prenatal neurotoxins. The Taiwan government should focus more attention on providing intervention programs for immigrant mothers to help protect the health of unborn babies. Further investigation on how multiple neurotoxins influence prenatal neurodevelopment is warranted.

  9. Neurotoxins and fillers for skin rejuvenation.

    PubMed

    Mayor, Jessica; Grunebaum, Lisa

    2014-02-01

    The ephemeral effects of neurotoxins and fillers are well described for facial remodeling and rejuvenation. Less is known about their long-term effects on skin rejuvenation and neocollagenesis. This article aims to review current available science and literature to support the use of these cosmetic procedures as lasting antiaging treatments.

  10. Structure and function of tetanus and botulinum neurotoxins.

    PubMed

    Montecucco, C; Schiavo, G

    1995-11-01

    Tetanus and botulinum neurotoxins are produced by Clostridia and cause the neuroparalytic syndromes of tetanus and botulism. Tetanus neurotoxin acts mainly at the CNS synapse, while the seven botulinum neurotoxins act peripherally. Clostridial neurotoxins share a similar mechanism of cell intoxication: they block the release of neurotransmitters. They are composed of two disulfide-linked polypeptide chains. The larger subunit is responsible for neurospecific binding and cell penetration. Reduction releases the smaller chain in the neuronal cytosol, where it displays its zinc-endopeptidase activity specific for protein components of the neuroexocytosis apparatus. Tetanus neurotoxin and botulinum neurotoxins B, D, F and G recognize specifically VAMP/ synaptobrevin. This integral protein of the synaptic vesicle membrane is cleaved at single peptide bonds, which differ for each neurotoxin. Botulinum A, and E neurotoxins recognize and cleave specifically SNAP-25, a protein of the presynaptic membrane, at two different sites within the carboxyl-terminus. Botulinum neurotoxin type C cleaves syntaxin, another protein of the nerve plasmalemma. These results indicate that VAMP, SNAP-25 and syntaxin play a central role in neuroexocytosis. These three proteins are conserved from yeast to humans and are essential in a variety of docking and fusion events in every cell. Tetanus and botulinum neurotoxins form a new group of zinc-endopeptidases with characteristic sequence, mode of zinc coordination, mechanism of activation and target recognition. They will be of great value in the unravelling of the mechanisms of exocytosis and endocytosis, as they are in the clinical treatment of dystonias.

  11. Neurologic uses of botulinum neurotoxin type A

    PubMed Central

    Ney, John P; Joseph, Kevin R

    2007-01-01

    This article reviews the current and most neurologic uses of botulinum neurotoxin type A (BoNT-A), beginning with relevant historical data, neurochemical mechanism at the neuromuscular junction. Current commercial preparations of BoNT-A are reviewed, as are immunologic issues relating to secondary failure of BoNT-A therapy. Clinical uses are summarized with an emphasis on controlled clinical trials (as appropriate), including facial movement disorders, focal neck and limb dystonias, spasticity, hypersecretory syndromes, and pain. PMID:19300614

  12. Role of tetanus neurotoxin insensitive vesicle-associated membrane protein (TI-VAMP) in vesicular transport mediating neurite outgrowth.

    PubMed

    Martinez-Arca, S; Alberts, P; Zahraoui, A; Louvard, D; Galli, T

    2000-05-15

    How vesicular transport participates in neurite outgrowth is still poorly understood. Neurite outgrowth is not sensitive to tetanus neurotoxin thus does not involve synaptobrevin-mediated vesicular transport to the plasma membrane of neurons. Tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) is a vesicle-SNARE (soluble N-ethylmaleimide-sensitive fusion protein [NSF] attachment protein [SNAP] receptor), involved in transport to the apical plasma membrane in epithelial cells, a tetanus neurotoxin-resistant pathway. Here we show that TI-VAMP is essential for vesicular transport-mediating neurite outgrowth in staurosporine-differentiated PC12 cells. The NH(2)-terminal domain, which precedes the SNARE motif of TI-VAMP, inhibits the association of TI-VAMP with synaptosome-associated protein of 25 kD (SNAP25). Expression of this domain inhibits neurite outgrowth as potently as Botulinum neurotoxin E, which cleaves SNAP25. In contrast, expression of the NH(2)-terminal deletion mutant of TI-VAMP increases SNARE complex formation and strongly stimulates neurite outgrowth. These results provide the first functional evidence for the role of TI-VAMP in neurite outgrowth and point to its NH(2)-terminal domain as a key regulator in this process.

  13. Role of Tetanus Neurotoxin Insensitive Vesicle-Associated Membrane Protein (Ti-Vamp) in Vesicular Transport Mediating Neurite Outgrowth

    PubMed Central

    Martinez-Arca, Sonia; Alberts, Philipp; Zahraoui, Ahmed; Louvard, Daniel; Galli, Thierry

    2000-01-01

    How vesicular transport participates in neurite outgrowth is still poorly understood. Neurite outgrowth is not sensitive to tetanus neurotoxin thus does not involve synaptobrevin-mediated vesicular transport to the plasma membrane of neurons. Tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) is a vesicle-SNARE (soluble N-ethylmaleimide-sensitive fusion protein [NSF] attachment protein [SNAP] receptor), involved in transport to the apical plasma membrane in epithelial cells, a tetanus neurotoxin-resistant pathway. Here we show that TI-VAMP is essential for vesicular transport-mediating neurite outgrowth in staurosporine-differentiated PC12 cells. The NH2-terminal domain, which precedes the SNARE motif of TI-VAMP, inhibits the association of TI-VAMP with synaptosome-associated protein of 25 kD (SNAP25). Expression of this domain inhibits neurite outgrowth as potently as Botulinum neurotoxin E, which cleaves SNAP25. In contrast, expression of the NH2-terminal deletion mutant of TI-VAMP increases SNARE complex formation and strongly stimulates neurite outgrowth. These results provide the first functional evidence for the role of TI-VAMP in neurite outgrowth and point to its NH2-terminal domain as a key regulator in this process. PMID:10811829

  14. Unique Ganglioside Recognition Strategies for Clostridial Neurotoxins

    SciTech Connect

    Benson, Marc A.; Fu, Zhuji; Kim, Jung-Ja P.; Baldwin, Michael R.

    2012-03-15

    Botulinum neurotoxins (BoNTs) and tetanus neurotoxin are the causative agents of the paralytic diseases botulism and tetanus, respectively. The potency of the clostridial neurotoxins (CNTs) relies primarily on their highly specific binding to nerve terminals and cleavage of SNARE proteins. Although individual CNTs utilize distinct proteins for entry, they share common ganglioside co-receptors. Here, we report the crystal structure of the BoNT/F receptor-binding domain in complex with the sugar moiety of ganglioside GD1a. GD1a binds in a shallow groove formed by the conserved peptide motif E ... H ... SXWY ... G, with additional stabilizing interactions provided by two arginine residues. Comparative analysis of BoNT/F with other CNTs revealed several differences in the interactions of each toxin with ganglioside. Notably, exchange of BoNT/F His-1241 with the corresponding lysine residue of BoNT/E resulted in increased affinity for GD1a and conferred the ability to bind ganglioside GM1a. Conversely, BoNT/E was not able to bind GM1a, demonstrating a discrete mechanism of ganglioside recognition. These findings provide a structural basis for ganglioside binding among the CNTs and show that individual toxins utilize unique ganglioside recognition strategies.

  15. The zinc-dependent protease activity of the botulinum neurotoxins.

    PubMed

    Lebeda, Frank J; Cer, Regina Z; Mudunuri, Uma; Stephens, Robert; Singh, Bal Ram; Adler, Michael

    2010-05-01

    The botulinum neurotoxins (BoNT, serotypes A-G) are some of the most toxic proteins known and are the causative agents of botulism. Following exposure, the neurotoxin binds and enters peripheral cholinergic nerve endings and specifically and selectively cleaves one or more SNARE proteins to produce flaccid paralysis. This review centers on the kinetics of the Zn-dependent proteolytic activities of these neurotoxins, and briefly describes inhibitors, activators and factors underlying persistence of toxin action. Some of the structural, enzymatic and inhibitor data that are discussed here are available at the botulinum neurotoxin resource, BotDB (http://botdb.abcc.ncifcrf.gov).

  16. Computer-aided identification, synthesis, and biological evaluation of novel inhibitors for botulinum neurotoxin serotype A

    SciTech Connect

    Teng, Y. G.; Berger, W. T.; Nesbitt, N. M.; Kumar, K.; Balius, T. E.; Rizzo, R. C.; Tonge, P. J.; Ojima, I.; Swaminathan, S.

    2015-07-27

    Botulinum neurotoxins (BoNTs) are among the most potent biological toxin known to humans, and are classified as Category A bioterrorism agents by the Centers for Disease Control and prevention (CDC). There are seven known BoNT serotypes (A-G) which have been thus far identified in literature. BoNTs have been shown to block neurotransmitter release by cleaving proteins of the soluble NSF attachment protein receptor (SNARE) complex. Disruption of the SNARE complex precludes motor neuron failure which ultimately results in flaccid paralysis in humans and animals. Currently, there are no effective therapeutic treatments against the neurotoxin light chain (LC) after translocation into the cytosols of motor neurons. In this work, high-throughput virtual screening was employed to screen a library of commercially available compounds from ZINC database against BoNT/A-LC. Among the hit compounds from the in-silico screening, two lead compounds were identified and found to have potent inhibitory activity against BoNT/A-LC in vitro, as well as in Neuro-2a cells. A few analogues of the lead compounds were synthesized and their potency examined. One of these analogues showed an enhanced activity than the lead compounds

  17. Computer-aided identification, synthesis, and biological evaluation of novel inhibitors for botulinum neurotoxin serotype A

    DOE PAGESBeta

    Teng, Y. G.; Berger, W. T.; Nesbitt, N. M.; Kumar, K.; Balius, T. E.; Rizzo, R. C.; Tonge, P. J.; Ojima, I.; Swaminathan, S.

    2015-07-27

    Botulinum neurotoxins (BoNTs) are among the most potent biological toxin known to humans, and are classified as Category A bioterrorism agents by the Centers for Disease Control and prevention (CDC). There are seven known BoNT serotypes (A-G) which have been thus far identified in literature. BoNTs have been shown to block neurotransmitter release by cleaving proteins of the soluble NSF attachment protein receptor (SNARE) complex. Disruption of the SNARE complex precludes motor neuron failure which ultimately results in flaccid paralysis in humans and animals. Currently, there are no effective therapeutic treatments against the neurotoxin light chain (LC) after translocation intomore » the cytosols of motor neurons. In this work, high-throughput virtual screening was employed to screen a library of commercially available compounds from ZINC database against BoNT/A-LC. Among the hit compounds from the in-silico screening, two lead compounds were identified and found to have potent inhibitory activity against BoNT/A-LC in vitro, as well as in Neuro-2a cells. A few analogues of the lead compounds were synthesized and their potency examined. One of these analogues showed an enhanced activity than the lead compounds« less

  18. Therapeutic applications of botulinum neurotoxins in head and neck disorders

    PubMed Central

    Alshadwi, Ahmad; Nadershah, Mohammed; Osborn, Timothy

    2014-01-01

    Objective The aim of this article is to review the mechanism of action, physiological effects, and therapeutic applications of botulinum neurotoxins in the head and neck area. Study design An extensive literature search was performed using keywords. The resulting articles were analyzed for relevance in four areas: overview on botulinum neurotoxins, the role of botulinum neurotoxins in the management of salivary secretory disorders, the role of botulinum neurotoxins in the management of facial pain, and the role of botulinum neurotoxins in head and neck movement disorders. Institutional review board approval was not needed due the nature of the study. Results Botulinum neurotoxin therapy was demonstrated to be a valuable alternative to conventional medical therapy for many conditions affecting the head and neck area in terms of morbidly, mortality, and patient satisfaction with treatment outcomes. Conclusion Botulinum neurotoxin therapy provides viable alternatives to traditional treatment modalities for some conditions affecting the head and neck region that have neurological components. This therapy can overcome some of the morbidities associated with conventional therapy. More research is needed to determine the ideal doses of botulinum neurotoxin to treat different diseases affecting the head and neck regions. PMID:25544809

  19. The ichthyotoxic alga Chattonella marina induces Na{sup +}, K{sup +}-ATPase, and CFTR proteins expression in fish gill chloride cells in vivo

    SciTech Connect

    Tang, Janet Y.M.; Wong, Chris K.C.; Au, Doris W.T. . E-mail: bhdwtau@cityu.edu.hk

    2007-02-02

    Our previous studies demonstrated that the ichthyotoxic Chattonella marina stimulated proliferation of branchial chloride cell (CC) and induced osmotic distress akin to hyperactive elimination of ions in fish (Rhabdosargus sarba). To ascertain the in vivo effects of C. marina on key CC ion transporters, the localization and expression of Na{sup +}, K{sup +}-ATPase (NKA) and cystic fibrosis transmembrane conductance regulator (CFTR) proteins in response to C. marina exposure were investigated, using a quantitative immunocytochemical approach. The polarized distributions of NKA ({alpha} subunit) and CFTR proteins in branchial CCs of R. sarba remained unchanged under C. marina exposure. However, significant inductions of these two ion-transporters were detected in CCs of fish after 6 h exposure. By real-time PCR, no significant changes in gill NKA and CFTR mRNA expressions were detected, suggesting a post-transcriptional pathway is likely involved in regulating the ion transporters abundance. This study is the first to demonstrate the in vivo effects of harmful algal toxin on NKA and CFTR protein expressions in gill transepithelial cells. Taken together, an augmentation of branchial CCs together with hyper-stimulation of NKA and CFTR in CCs attribute to the rapid development of osmotic distress in C. marina susceptible fish.

  20. Substrate recognition of VAMP-2 by botulinum neurotoxin B and tetanus neurotoxin.

    PubMed

    Chen, Sheng; Hall, Cherisse; Barbieri, Joseph T

    2008-07-25

    Botulinum neurotoxin (BoNT; serotypes A-G) and tetanus neurotoxin elicit flaccid and spastic paralysis, respectively. These neurotoxins are zinc proteases that cleave SNARE proteins to inhibit synaptic vesicle fusion to the plasma membrane. Although BoNT/B and tetanus neurotoxin (TeNT) cleave VAMP-2 at the same scissile bond, their mechanism(s) of VAMP-2 recognition is not clear. Mapping experiments showed that residues 60-87 of VAMP-2 were sufficient for efficient cleavage by BoNT/B and that residues 40-87 of VAMP-2 were sufficient for efficient TeNT cleavage. Alanine-scanning mutagenesis and kinetic analysis identified three regions within VAMP-2 that were recognized by BoNT/B and TeNT: residues adjacent to the site of scissile bond cleavage (cleavage region) and residues located within N-terminal and C-terminal regions relative to the cleavage region. Analysis of residues within the cleavage region showed that mutations at the P7, P4, P2, and P1' residues of VAMP-2 had the greatest inhibition of LC/B cleavage (> or =32-fold), whereas mutations at P7, P4, P1', and P2' residues of VAMP-2 had the greatest inhibition of LC/TeNT cleavage (> or =64-fold). Residues within the cleavage region influenced catalysis, whereas residues N-terminal and C-terminal to the cleavage region influenced binding affinity. Thus, BoNT/B and TeNT possess similar organization but have unique residues to recognize and cleave VAMP-2. These studies provide new insights into how the clostridial neurotoxins recognize their substrates.

  1. Substrate Recognition of VAMP-2 by Botulinum Neurotoxin B and Tetanus Neurotoxin*

    PubMed Central

    Chen, Sheng; Hall, Cherisse; Barbieri, Joseph T.

    2008-01-01

    Botulinum neurotoxin (BoNT; serotypes A-G) and tetanus neurotoxin elicit flaccid and spastic paralysis, respectively. These neurotoxins are zinc proteases that cleave SNARE proteins to inhibit synaptic vesicle fusion to the plasma membrane. Although BoNT/B and tetanus neurotoxin (TeNT) cleave VAMP-2 at the same scissile bond, their mechanism(s) of VAMP-2 recognition is not clear. Mapping experiments showed that residues 60-87 of VAMP-2 were sufficient for efficient cleavage by BoNT/B and that residues 40-87 of VAMP-2 were sufficient for efficient TeNT cleavage. Alanine-scanning mutagenesis and kinetic analysis identified three regions within VAMP-2 that were recognized by BoNT/B and TeNT: residues adjacent to the site of scissile bond cleavage (cleavage region) and residues located within N-terminal and C-terminal regions relative to the cleavage region. Analysis of residues within the cleavage region showed that mutations at the P7, P4, P2, and P1′ residues of VAMP-2 had the greatest inhibition of LC/B cleavage (≥32- fold), whereas mutations at P7, P4, P1′, and P2′ residues of VAMP-2 had the greatest inhibition of LC/TeNT cleavage (≥64-fold). Residues within the cleavage region influenced catalysis, whereas residues N-terminal and C-terminal to the cleavage region influenced binding affinity. Thus, BoNT/B and TeNT possess similar organization but have unique residues to recognize and cleave VAMP-2. These studies provide new insights into how the clostridial neurotoxins recognize their substrates. PMID:18511417

  2. Transforming the domain structure of botulinum neurotoxins into novel therapeutics.

    PubMed

    Chaddock, John

    2013-01-01

    Botulinum neurotoxins are comprised of multiple identifiable protein domains. Recent advances in understanding the relationships between domain structure and neurotoxin function have provided a number of opportunities to engineer innovative therapeutic proteins that utilise the neurotoxins and neurotoxin domains. For example, recent insights into the properties of the catalytic, translocation and binding domains open up opportunities to develop botulinum neurotoxins with enhanced properties of selectivity, potency and duration of action. In parallel, the broad scope for utilisation of the individual domains is becoming clearer as significant advancements are made to exploit the unique biology of the catalytic and translocation domains. These opportunities and the status of their development will be reviewed in this chapter.

  3. Botulinum neurotoxins: genetic, structural and mechanistic insights.

    PubMed

    Rossetto, Ornella; Pirazzini, Marco; Montecucco, Cesare

    2014-08-01

    Botulinum neurotoxins (BoNTs) are produced by anaerobic bacteria of the genus Clostridium and cause a persistent paralysis of peripheral nerve terminals, which is known as botulism. Neurotoxigenic clostridia belong to six phylogenetically distinct groups and produce more than 40 different BoNT types, which inactivate neurotransmitter release owing to their metalloprotease activity. In this Review, we discuss recent studies that have improved our understanding of the genetics and structure of BoNT complexes. We also describe recent insights into the mechanisms of BoNT entry into the general circulation, neuronal binding, membrane translocation and neuroparalysis.

  4. Development of recombinant vaccines for botulinum neurotoxin.

    PubMed

    Smith, L A

    1998-11-01

    Synthetic genes encoding non-toxic, carboxyl-terminal regions (approximately 50 kDa) of botulinum neurotoxin (BoNT) serotypes A and B (referred to as fragment C or HC) were constructed and cloned into the methylotropic yeast, Pichia pastoris. Genes specifying BoNTA(HC) and BoNTB(HC) were expressed as both intracellular and secreted products. Recombinants, expressed intracellularly, yielded products with the expected molecular weight as judged by SDS PAGE and Western blot (immunoblot) analysis, while secreted products were larger due to glycosylation. Gene products were used to vaccinate mice and evaluated for their ability to elicit protective antibody titers in vivo. Mice given three intramuscular vaccinations with yeast supernatant containing glycosylated BoNTA(HC) were protected against an intraperitoneal challenge of 10(6) 50% mouse lethal doses (MLD50) of serotype A neurotoxin, a result not duplicated by its BoNTB(HC) counterpart. Vaccinating mice with cytoplasmically produced BoNTA(HC) and BoNTB(HC) protected animals from a challenge of 10(6) MLD50 of serotype A and B toxins, respectively. Because of the glycosylation encountered with secreted BoNT(HC), our efforts focused on the production and purification of products from intracellular expression. PMID:9792170

  5. Temporal characteristics of botulinum neurotoxin therapy

    PubMed Central

    Lebeda, Frank J; Cer, Regina Z; Stephens, Robert M; Mudunuri, Uma

    2010-01-01

    Botulinum neurotoxin is a pharmaceutical treatment used for an increasing number of neurological and non-neurological indications, symptoms and diseases. Despite the wealth of clinical reports that involve the timing of the therapeutic effects of this toxin, few studies have attempted to integrate these data into unified models. Secondary reactions have also been examined including the development of adverse events, resistance to repeated applications, and nerve terminal sprouting. Our primary intent for conducting this review was to gather relevant pharmacodynamic data from suitable biomedical literature regarding botulinum neurotoxins via the use of automated data-mining techniques. We envision that mathematical models will ultimately be of value to those who are healthcare decision makers and providers, as well as clinical and basic researchers. Furthermore, we hypothesize that the combination of this computer-intensive approach with mathematical modeling will predict the percentage of patients who will favorably or adversely respond to this treatment and thus will eventually assist in developing the increasingly important area of personalized medicine. PMID:20021324

  6. Evolution of neurotoxins: from research modalities to clinical realities.

    PubMed

    Kostrzewa, Richard M

    2009-01-01

    In the 1950s, the discovery of anti-nerve growth factor, an immunotoxin stunting sympathetic neural development, signaled the advent of neurotoxins as research modalities. Other selective neurotoxins were discovered in rapid succession. In the 1960s, 6-hydroxydopamine and 6-hydroxydopa were shown to destroy noradrenergic and dopaminergic nerves. Excitotoxins (glutamate, aspartate, and analogs) were discovered in the 1970s. DSP-4 [N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine] proved to be selective for noradrenergic destruction, while 5,6- and 5,7-dihydroxytryptamines were relatively selective for serotonin neurons. Additional neurotoxins were discovered, but it was MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) that predominated neurotoxicity research in the 1980s. Eventually, Clostridium botulinum neurotoxin (BoNT), discovered as a "poisonous" principle in the late 1800s, resurfaced in purified and standardized forms as a clinically useful drug. Neurotoxins represent chemical tools, useful not only for discerning neuronal mechanisms and animal modeling of neurological disorders, but also for their use in medicine and potential as treatments for medical disorders. This unit reviews the early discovery of neurotoxins, describes categories of neurotoxins, and finally characterizes their usefulness--first as research tools, and eventually as clinical therapeutic agents.

  7. Re-engineering clostridial neurotoxins for the treatment of chronic pain: current status and future prospects.

    PubMed

    Pickett, Andy

    2010-06-01

    Clostridial neurotoxins from the botulinum neurotoxin (BoNT) family are protein complexes, derived from the bacterium Clostridium botulinum, which potently inhibit acetylcholine release and result in a reversible blockade of the neuromuscular junction. This feature led to the clinical development of BoNT-A for a number of neuromuscular disorders. BoNT-A toxins are commercially available as three different preparations: Dysport/Azzalure, Botox/Vistabel, and Xeomin/Bocouture. Although BoNT-A preparations have not yet been approved for the treatment of pain, a substantial body of preclinical and clinical evidence shows that BoNT-A is effective in treating a number of different types of pain. It is thought to exert an analgesic effect both via muscle-relaxant properties and also directly, via inhibition of nociceptive neuropeptides. This review explores the mechanistic basis of this analgesic effect, summarizing current knowledge of the structure-function relationship of BoNT and discussing effects on both motor and pain neurons. For a complete picture of the analgesic properties of BoNT-A, clinical evidence of efficacy in myofascial pain and neuropathic pain is considered in tandem with a mechanistic rationale for activity. Patients experiencing chronic pain are clear candidates for treatment with a modified clostridial endopeptidase that would provide enduring inhibition of neurotransmitter release. A strong preclinical evidence base underpins the concept that re-engineering of BoNT could be used to enhance the analgesic potential of this neurotoxin, and it is hoped that the first clinical studies examining re-engineered BoNT-A will confirm this potential. PMID:20462283

  8. Re-engineering clostridial neurotoxins for the treatment of chronic pain: current status and future prospects.

    PubMed

    Pickett, Andy

    2010-06-01

    Clostridial neurotoxins from the botulinum neurotoxin (BoNT) family are protein complexes, derived from the bacterium Clostridium botulinum, which potently inhibit acetylcholine release and result in a reversible blockade of the neuromuscular junction. This feature led to the clinical development of BoNT-A for a number of neuromuscular disorders. BoNT-A toxins are commercially available as three different preparations: Dysport/Azzalure, Botox/Vistabel, and Xeomin/Bocouture. Although BoNT-A preparations have not yet been approved for the treatment of pain, a substantial body of preclinical and clinical evidence shows that BoNT-A is effective in treating a number of different types of pain. It is thought to exert an analgesic effect both via muscle-relaxant properties and also directly, via inhibition of nociceptive neuropeptides. This review explores the mechanistic basis of this analgesic effect, summarizing current knowledge of the structure-function relationship of BoNT and discussing effects on both motor and pain neurons. For a complete picture of the analgesic properties of BoNT-A, clinical evidence of efficacy in myofascial pain and neuropathic pain is considered in tandem with a mechanistic rationale for activity. Patients experiencing chronic pain are clear candidates for treatment with a modified clostridial endopeptidase that would provide enduring inhibition of neurotransmitter release. A strong preclinical evidence base underpins the concept that re-engineering of BoNT could be used to enhance the analgesic potential of this neurotoxin, and it is hoped that the first clinical studies examining re-engineered BoNT-A will confirm this potential.

  9. Ichthyotoxic Karlodinium veneficum (Ballantine) J Larsen in the Upper Swan River Estuary (Western Australia): Ecological conditions leading to a fish kill

    PubMed Central

    Adolf, Jason E.; Bachvaroff, Tsvetan R.; Deeds, Jonathan R.; Place, Allen R

    2015-01-01

    Ichthyotoxic Karlodinium veneficum has become a persistent problem in the eutrophic Swan River Estuary (SRE) near Perth, Western Australia. Karlotoxin (KmTx) concentrations and K. veneficum were sampled from March to July 2005, spanning a bloom confirmed by microscopy and genetics (ITS sequence), and a fish kill coincident with end of the bloom. The objective of this study was to investigate K. veneficum cell and toxin dynamics, and water quality conditions, leading up to the bloom and fish kill in this estuarine system. Abundance of K. veneficum increased as diatom abundance decreased over a 3-month period (Jan-Mar) preceding the bloom. Low freshwater flow to the SRE characterized the bloom initiation period, while elevated seasonal flows altered water quality and preceded the end of the bloom and fish kill. The bloom of K. veneficum was localized over a bottom layer of hypoxic water in a stratified water column. Low nitrate levels, DIN:DIP (mol) near unity, and particulate C:N:P of K. veneficum-rich water samples were consistent with nitrogen limitation of phytoplankton. A KmTx 2 congener was present in the concentration range 0–1052 ng KmTx mL−1, levels that were sufficient to kill larval fish in the laboratory within 4 h. A KmTx cell quota of 2.8 pg KmTx cell−1 was estimated for the bloom, which is moderately high for the species. Gill histopathology of fish from this fish kill showed signs of damage similar to those caused by KmTx in the lab. Results from this study suggest that conditions in the SRE, including elevated K. veneficum abundance and KmTx cell quotas, as well as hypoxia in the upper SRE, likely contribute to seasonal fish kills observed in this system. PMID:27642270

  10. A 1-D simulation analysis of the development and maintenance of the 2001 red tide of the ichthyotoxic dinoflagellate Karenia brevis on the West Florida shelf

    NASA Astrophysics Data System (ADS)

    Lenes, J. M.; Darrow, B. P.; Walsh, J. J.; Jolliff, J. K.; Chen, F. R.; Weisberg, R. H.; Zheng, L.

    2012-06-01

    A one-dimensional (1-D) ecological model, HABSIM, examined the initiation and maintenance of the 2001 red tide on the West Florida shelf (WFS). Phytoplankton competition among toxic dinoflagellates (Karenia brevis), nitrogen fixing cyanophytes (Trichodesmium erythraeum), large siliceous phytoplankton (diatoms), and small non-siliceous phytoplankton (microflagellates) explored the sequence of events required to support the observed red tide from August to December 2001. The ecological model contained 24 state variables within five submodels: circulation, atmospheric (iron deposition), bio-optics, pelagic (phytoplankton, nutrients, bacteria, zooplankton, and fish), and benthic (nutrient regeneration). The 2001 model results reaffirmed that diazotrophs are the basis for initiation of red tides of K. brevis on the WFS. A combination of selective grazing pressure, iron fertilization, low molar nitrogen to phosphorus ratios, and eventual silica limitation of fast-growing diatoms set the stage for dominance of nitrogen fixers. "New" nitrogen was made available for subsequent blooms of K. brevis through the release of ammonium and urea during nitrogen fixation, as well as during cell lysis, by the Trichodesmium population. Once K. brevis biomass reached ichthyotoxic levels, rapid decay of subsequent fish kills supplied additional organic nutrients for utilization by these opportunistic toxic algae. Both nutrient vectors represented organic non-siliceous sources of nitrogen and phosphorus, further exacerbating silica limitation of the diatom population. The model reproduced this spring transition from a simple estuarine-driven, diatom-based food chain to a complex summer-fall system of Trichodesmium and toxic dinoflagellates. While the model was able to replicate the initiation and maintenance of the 2001 red tide, bloom termination was not captured by this 1-D form on the WFS. Here, horizontal advection and perhaps cell lysis loss terms might play a significant role, to be

  11. Molecular assembly of botulinum neurotoxin progenitor complexes

    PubMed Central

    Benefield, Desirée A.; Dessain, Scott K.; Shine, Nancy; Ohi, Melanie D.; Lacy, D. Borden

    2013-01-01

    Botulinum neurotoxin (BoNT) is produced by Clostridium botulinum and associates with nontoxic neurotoxin-associated proteins to form high-molecular weight progenitor complexes (PCs). The PCs are required for the oral toxicity of BoNT in the context of food-borne botulism and are thought to protect BoNT from destruction in the gastrointestinal tract and aid in absorption from the gut lumen. The PC can differ in size and protein content depending on the C. botulinum strain. The oral toxicity of the BoNT PC increases as the size of the PC increases, but the molecular architecture of these large complexes and how they contribute to BoNT toxicity have not been elucidated. We have generated 2D images of PCs from strains producing BoNT serotypes A1, B, and E using negative stain electron microscopy and single-particle averaging. The BoNT/A1 and BoNT/B PCs were observed as ovoid-shaped bodies with three appendages, whereas the BoNT/E PC was observed as an ovoid body. Both the BoNT/A1 and BoNT/B PCs showed significant flexibility, and the BoNT/B PC was documented as a heterogeneous population of assembly/disassembly intermediates. We have also determined 3D structures for each serotype using the random conical tilt approach. Crystal structures of the individual proteins were placed into the BoNT/A1 and BoNT/B PC electron density maps to generate unique detailed models of the BoNT PCs. The structures highlight an effective platform that can be engineered for the development of mucosal vaccines and the intestinal absorption of oral biologics. PMID:23509303

  12. New Elements To Consider When Modeling the Hazards Associated with Botulinum Neurotoxin in Food

    PubMed Central

    Mura, Ivan; Malakar, Pradeep K.; Walshaw, John; Peck, Michael W.; Barker, G. C.

    2015-01-01

    Botulinum neurotoxins (BoNTs) produced by the anaerobic bacterium Clostridium botulinum are the most potent biological substances known to mankind. BoNTs are the agents responsible for botulism, a rare condition affecting the neuromuscular junction and causing a spectrum of diseases ranging from mild cranial nerve palsies to acute respiratory failure and death. BoNTs are a potential biowarfare threat and a public health hazard, since outbreaks of foodborne botulism are caused by the ingestion of preformed BoNTs in food. Currently, mathematical models relating to the hazards associated with C. botulinum, which are largely empirical, make major contributions to botulinum risk assessment. Evaluated using statistical techniques, these models simulate the response of the bacterium to environmental conditions. Though empirical models have been successfully incorporated into risk assessments to support food safety decision making, this process includes significant uncertainties so that relevant decision making is frequently conservative and inflexible. Progression involves encoding into the models cellular processes at a molecular level, especially the details of the genetic and molecular machinery. This addition drives the connection between biological mechanisms and botulism risk assessment and hazard management strategies. This review brings together elements currently described in the literature that will be useful in building quantitative models of C. botulinum neurotoxin production. Subsequently, it outlines how the established form of modeling could be extended to include these new elements. Ultimately, this can offer further contributions to risk assessments to support food safety decision making. PMID:26350137

  13. Mechanism of substrate recognition by the novel Botulinum Neurotoxin subtype F5

    PubMed Central

    Guo, Jiubiao; Chan, Edward Wai Chi; Chen, Sheng

    2016-01-01

    Botulinum Neurotoxins (BoNTs) are the causative agents of botulism, which act by potently inhibiting the neurotransmitter release in motor neurons. Seven serotypes of BoNTs designated as BoNT/A-G have been identified. Recently, two novel types of Botulinum neurotoxins, which cleave a novel scissile bond, L54-E55, of VAMP-2 have been reported including BoNT/F subtype F5 and serotype H. However, little has been known on how these BoNTs recognize their substrates. The present study addressed for the first time the unique substrate recognition mechanism of LC/F5. Our data indicated that the optimal peptide required for efficient LC/F5 substrate cleavage is VAMP-2 (20–65). Interestingly, the overall mode of substrate recognition adopted by LC/F5 was similar to LC/F1, except that its recognition sites were shifted one helix toward the N-terminus of VAMP-2 when compared to that of LC/F1. The composition of LC/F5 pockets were found to have changed accordingly to facilitate specific recognition of these new sites of VAMP-2, including the P2′, P1′, P2, P3, B3, B2 and B1 sites. The study provides direct evidence of the evolutionary adaption of BoNT to recognize its substrate which is useful for effective antitoxin and inhibitor development. PMID:26794648

  14. Botulinum neurotoxin type-A enters a non-recycling pool of synaptic vesicles

    PubMed Central

    Harper, Callista B.; Papadopulos, Andreas; Martin, Sally; Matthews, Daniel R.; Morgan, Garry P.; Nguyen, Tam H.; Wang, Tong; Nair, Deepak; Choquet, Daniel; Meunier, Frederic A.

    2016-01-01

    Neuronal communication relies on synaptic vesicles undergoing regulated exocytosis and recycling for multiple rounds of fusion. Whether all synaptic vesicles have identical protein content has been challenged, suggesting that their recycling ability may differ greatly. Botulinum neurotoxin type-A (BoNT/A) is a highly potent neurotoxin that is internalized in synaptic vesicles at motor nerve terminals and induces flaccid paralysis. Recently, BoNT/A was also shown to undergo retrograde transport, suggesting it might enter a specific pool of synaptic vesicles with a retrograde trafficking fate. Using high-resolution microscopy techniques including electron microscopy and single molecule imaging, we found that the BoNT/A binding domain is internalized within a subset of vesicles that only partially co-localize with cholera toxin B-subunit and have markedly reduced VAMP2 immunoreactivity. Synaptic vesicles loaded with pHrodo-BoNT/A-Hc exhibited a significantly reduced ability to fuse with the plasma membrane in mouse hippocampal nerve terminals when compared with pHrodo-dextran-containing synaptic vesicles and pHrodo-labeled anti-GFP nanobodies bound to VAMP2-pHluorin or vGlut-pHluorin. Similar results were also obtained at the amphibian neuromuscular junction. These results reveal that BoNT/A is internalized in a subpopulation of synaptic vesicles that are not destined to recycle, highlighting the existence of significant molecular and functional heterogeneity between synaptic vesicles. PMID:26805017

  15. Algal chloroplast produced camelid VHH antitoxins are capable of neutralizing botulinum neurotoxin

    PubMed Central

    Barrera, Daniel J.; Rosenberg, Julian N.; Chiu, Joanna G.; Chang, Yung-Nien; Debatis, Michelle; Ngoi, Soo-Mun; Chang, John T.; Shoemaker, Charles B.; Oyler, George A.; Mayfield, Stephen P.

    2015-01-01

    We have produced three antitoxins consisting of the variable domains of camelid heavy chain-only antibodies (VHH) by expressing the genes in the chloroplast of green algae. These antitoxins accumulate as soluble proteins capable of binding and neutralizing botulinum neurotoxin. Furthermore, they accumulate at up to 5% total soluble protein, sufficient expression to easily produce these antitoxins at scale from algae. The genes for the three different antitoxins were transformed into Chlamydomonas reinhardtii chloroplasts and their products purified from algae lysates and assayed for in vitro biological activity using toxin protection assays. The produced antibody domains bind to botulinum neurotoxin serotype A (BoNT/A) with similar affinities as camelid antibodies produced in Escherichia coli, and they are similarly able to protect primary rat neurons from intoxication by BoNT/A. Furthermore, the camelid antibodies were produced in algae without the use of solubilization tags commonly employed in E. coli. These camelid antibody domains are potent antigen binding proteins and the heterodimer fusion protein containing two VHH domains was capable of neutralizing BoNT/A at near equimolar concentrations with the toxin. Intact antibody domains were detected in the gastrointestinal (GI) tract of mice treated orally with antitoxin producing microalgae. These findings support the use of orally delivered antitoxins produced in green algae as a novel treatment for botulism. PMID:25229405

  16. Algal chloroplast produced camelid VH H antitoxins are capable of neutralizing botulinum neurotoxin.

    PubMed

    Barrera, Daniel J; Rosenberg, Julian N; Chiu, Joanna G; Chang, Yung-Nien; Debatis, Michelle; Ngoi, Soo-Mun; Chang, John T; Shoemaker, Charles B; Oyler, George A; Mayfield, Stephen P

    2015-01-01

    We have produced three antitoxins consisting of the variable domains of camelid heavy chain-only antibodies (VH H) by expressing the genes in the chloroplast of green algae. These antitoxins accumulate as soluble proteins capable of binding and neutralizing botulinum neurotoxin. Furthermore, they accumulate at up to 5% total soluble protein, sufficient expression to easily produce these antitoxins at scale from algae. The genes for the three different antitoxins were transformed into Chlamydomonas reinhardtii chloroplasts and their products purified from algae lysates and assayed for in vitro biological activity using toxin protection assays. The produced antibody domains bind to botulinum neurotoxin serotype A (BoNT/A) with similar affinities as camelid antibodies produced in Escherichia coli, and they are similarly able to protect primary rat neurons from intoxication by BoNT/A. Furthermore, the camelid antibodies were produced in algae without the use of solubilization tags commonly employed in E. coli. These camelid antibody domains are potent antigen-binding proteins and the heterodimer fusion protein containing two VH H domains was capable of neutralizing BoNT/A at near equimolar concentrations with the toxin. Intact antibody domains were detected in the gastrointestinal (GI) tract of mice treated orally with antitoxin-producing microalgae. These findings support the use of orally delivered antitoxins produced in green algae as a novel treatment for botulism.

  17. A Simple, Rapid and Sensitive FRET Assay for Botulinum Neurotoxin Serotype B Detection

    PubMed Central

    Li, Xuechen; Chen, Sheng

    2014-01-01

    Botulinum neurotoxins (BoNTs), the most potent naturally-occurring neurotoxins known to humans, comprise seven distinct serotypes (BoNT/A-G), each of which exhibits unique substrate specificity. Many methods have been developed for BoNT detection, in particular for BoNT/A, with various complexity and sensitivity, while substrate based FRET assay is considered as the most widely used approach due to its simplicity and sensitivity. In this study, we designed a vesicle-associated membrane protein 2 (VAMP2) based FRET assay based on the understanding of the VAMP2 and light chain/B (LC/B) interactions in our previous studies. The current design constituted the shortest peptide, VAMP2 (63–85), with FRET dyes (EDAN and Dabcyl) labelled at position 76 and 85, respectively, which showed minimal effect on VAMP2 substrate catalysis by LC/B and therefore enhanced the sensitivity of the assay. The FRET peptide, designated as FVP-B, was specific to LC/B, with a detection sensitivity as low as ∼20 pM in 2 h. Importantly, FVP-B showed the potential to be scaled up and used in high throughput screening of LC/B inhibitor. The currently developed FRET assay is one of the most economic and rapid FRET assays for LC/B detection. PMID:25437190

  18. Exploration of endogenous substrate cleavage by various forms of botulinum neurotoxins.

    PubMed

    Guo, Jiubiao; Wang, Jinglin; Chan, Edward Waichi; Chen, Sheng

    2015-06-15

    Botulinum neurotoxins are the most potent protein neurotoxin known to human. The dual roles of BoNTs as both the causative agent of human botulism and a widely used protein-based therapeutic agent for treatment of numerous neuromuscular disorders/cosmetic uses make it an extremely hot topic of research. Biochemical characterization of these toxins was mainly confined to the recombinant light chains and substrate and little is known about their efficiency on the cleavage of endogenous substrates. In the present study, we showed that BoNTs exhibited variable activities on their endogenous substrates and that their efficiency to cleave recombinant and endogenous substrate was not consistent, presumably due to the differential recognition of their respective substrates in the natural SNARE complex format. Through testing the combinatorial effects of different BoNTs on cleavage of endogenous substrates, we showed that the combinations of LC/A and LC/B, as well as LC/A and LC/F, could enhance the activity of each individual BoNT. This finding may shed light on the future development of new BoNT serotypes for clinical application, and formulation of combinatorial uses of different BoNTs to minimize the development of immuno-resistance by using a lower amount of individual type. PMID:25912942

  19. Algal chloroplast produced camelid VH H antitoxins are capable of neutralizing botulinum neurotoxin.

    PubMed

    Barrera, Daniel J; Rosenberg, Julian N; Chiu, Joanna G; Chang, Yung-Nien; Debatis, Michelle; Ngoi, Soo-Mun; Chang, John T; Shoemaker, Charles B; Oyler, George A; Mayfield, Stephen P

    2015-01-01

    We have produced three antitoxins consisting of the variable domains of camelid heavy chain-only antibodies (VH H) by expressing the genes in the chloroplast of green algae. These antitoxins accumulate as soluble proteins capable of binding and neutralizing botulinum neurotoxin. Furthermore, they accumulate at up to 5% total soluble protein, sufficient expression to easily produce these antitoxins at scale from algae. The genes for the three different antitoxins were transformed into Chlamydomonas reinhardtii chloroplasts and their products purified from algae lysates and assayed for in vitro biological activity using toxin protection assays. The produced antibody domains bind to botulinum neurotoxin serotype A (BoNT/A) with similar affinities as camelid antibodies produced in Escherichia coli, and they are similarly able to protect primary rat neurons from intoxication by BoNT/A. Furthermore, the camelid antibodies were produced in algae without the use of solubilization tags commonly employed in E. coli. These camelid antibody domains are potent antigen-binding proteins and the heterodimer fusion protein containing two VH H domains was capable of neutralizing BoNT/A at near equimolar concentrations with the toxin. Intact antibody domains were detected in the gastrointestinal (GI) tract of mice treated orally with antitoxin-producing microalgae. These findings support the use of orally delivered antitoxins produced in green algae as a novel treatment for botulism. PMID:25229405

  20. Identification of a Unique Ganglioside Binding Loop within Botulinum Neurotoxins C and D-SA

    SciTech Connect

    Karalewitz, Andrew P.-A.; Kroken, Abby R.; Fu, Zhuji; Baldwin, Michael R.; Kim, Jung-Ja P.; Barbieri, Joseph T.

    2010-09-22

    The botulinum neurotoxins (BoNTs) are the most potent protein toxins for humans. There are seven serotypes of BoNTs (A-G) based on a lack of cross antiserum neutralization. BoNTs utilize gangliosides as components of the host receptors for binding and entry into neurons. Members of BoNT/C and BoNT/D serotypes include mosaic toxins that are organized in D/C and C/D toxins. One D/C mosaic toxin, BoNT/D-South Africa (BoNT/D-SA), was not fully neutralized by immunization with BoNT serotype C or D, which stimulated this study. Here the crystal structures of the receptor binding domains of BoNT/C, BoNT/D, and BoNT/D-SA are presented. Biochemical and cell binding studies show that BoNT/C and BoNT/D-SA possess unique mechanisms for ganglioside binding. These studies provide new information about how the BoNTs can enter host cells as well as a basis for understanding the immunological diversity of these neurotoxins.

  1. Identification of a unique ganglioside binding loop within botulinum neurotoxins C and D-SA .

    PubMed

    Karalewitz, Andrew P-A; Kroken, Abby R; Fu, Zhuji; Baldwin, Michael R; Kim, Jung-Ja P; Barbieri, Joseph T

    2010-09-21

    The botulinum neurotoxins (BoNTs) are the most potent protein toxins for humans. There are seven serotypes of BoNTs (A-G) based on a lack of cross antiserum neutralization. BoNTs utilize gangliosides as components of the host receptors for binding and entry into neurons. Members of BoNT/C and BoNT/D serotypes include mosaic toxins that are organized in D/C and C/D toxins. One D/C mosaic toxin, BoNT/D-South Africa (BoNT/D-SA), was not fully neutralized by immunization with BoNT serotype C or D, which stimulated this study. Here the crystal structures of the receptor binding domains of BoNT/C, BoNT/D, and BoNT/D-SA are presented. Biochemical and cell binding studies show that BoNT/C and BoNT/D-SA possess unique mechanisms for ganglioside binding. These studies provide new information about how the BoNTs can enter host cells as well as a basis for understanding the immunological diversity of these neurotoxins.

  2. New Elements To Consider When Modeling the Hazards Associated with Botulinum Neurotoxin in Food.

    PubMed

    Ihekwaba, Adaoha E C; Mura, Ivan; Malakar, Pradeep K; Walshaw, John; Peck, Michael W; Barker, G C

    2016-01-01

    Botulinum neurotoxins (BoNTs) produced by the anaerobic bacterium Clostridium botulinum are the most potent biological substances known to mankind. BoNTs are the agents responsible for botulism, a rare condition affecting the neuromuscular junction and causing a spectrum of diseases ranging from mild cranial nerve palsies to acute respiratory failure and death. BoNTs are a potential biowarfare threat and a public health hazard, since outbreaks of foodborne botulism are caused by the ingestion of preformed BoNTs in food. Currently, mathematical models relating to the hazards associated with C. botulinum, which are largely empirical, make major contributions to botulinum risk assessment. Evaluated using statistical techniques, these models simulate the response of the bacterium to environmental conditions. Though empirical models have been successfully incorporated into risk assessments to support food safety decision making, this process includes significant uncertainties so that relevant decision making is frequently conservative and inflexible. Progression involves encoding into the models cellular processes at a molecular level, especially the details of the genetic and molecular machinery. This addition drives the connection between biological mechanisms and botulism risk assessment and hazard management strategies. This review brings together elements currently described in the literature that will be useful in building quantitative models of C. botulinum neurotoxin production. Subsequently, it outlines how the established form of modeling could be extended to include these new elements. Ultimately, this can offer further contributions to risk assessments to support food safety decision making. PMID:26350137

  3. Mechanism of substrate recognition by the novel Botulinum Neurotoxin subtype F5.

    PubMed

    Guo, Jiubiao; Chan, Edward Wai Chi; Chen, Sheng

    2016-01-01

    Botulinum Neurotoxins (BoNTs) are the causative agents of botulism, which act by potently inhibiting the neurotransmitter release in motor neurons. Seven serotypes of BoNTs designated as BoNT/A-G have been identified. Recently, two novel types of Botulinum neurotoxins, which cleave a novel scissile bond, L(54)-E(55), of VAMP-2 have been reported including BoNT/F subtype F5 and serotype H. However, little has been known on how these BoNTs recognize their substrates. The present study addressed for the first time the unique substrate recognition mechanism of LC/F5. Our data indicated that the optimal peptide required for efficient LC/F5 substrate cleavage is VAMP-2 (20-65). Interestingly, the overall mode of substrate recognition adopted by LC/F5 was similar to LC/F1, except that its recognition sites were shifted one helix toward the N-terminus of VAMP-2 when compared to that of LC/F1. The composition of LC/F5 pockets were found to have changed accordingly to facilitate specific recognition of these new sites of VAMP-2, including the P2', P1', P2, P3, B3, B2 and B1 sites. The study provides direct evidence of the evolutionary adaption of BoNT to recognize its substrate which is useful for effective antitoxin and inhibitor development. PMID:26794648

  4. CRYSTAL STRUCTURE OF CLOSTRIDIUM BOTULINUM NEUROTOXIN SEROTYPE B.

    SciTech Connect

    SWAMINATHAN,S.; ESWARAMOORTHY,S.

    2001-11-19

    The toxigenic strains of Clostridium botulinum produce seven serologically distinct types of neurotoxins labeled A - G (EC 3.4.24.69), while Clostridium tetani produces tetanus neurotoxin (EC 3.4.24.68). Botulinum and tetanus neurotoxins (BoNTs and TeNT) are produced as single inactive chains of molecular mass of approximately 150 kDa. Most of these neurotoxins are released after being cleaved into two chains, a heavy chain (HI) of 100 kDa and a light chain (L) of 50 kDa held together by an interchain disulfide bond, by tissue proteinases. BoNT/E is released as a single chain but cleaved by host proteinases [1]. Clostvidium botulinum neurotoxins are extremely poisonous proteins with their LD{sub 50} for humans in the range of 0.1 - 1 ng kg{sup -1} [2]. Botulinum neurotoxins are responsible for neuroparalytic syndromes of botulism characterized by serious neurological disorders and flaccid paralysis. BoNTs block the release of acetylcholine at the neuromuscular junction causing flaccid paralysis while TeNT blocks the release of neurotransmitters like glycine and {gamma}-aminobutyric acid (GABA) in the inhibitory interneurons of the spinal cord resulting in spastic paralysis. In spite of different clinical symptoms, their aetiological agents intoxicate neuronal cells in the same way and these toxins have similar structural organization [3].

  5. Cyanobacterial blooms and biomagnification of the neurotoxin BMAA in South Florida coastal waters

    NASA Astrophysics Data System (ADS)

    Brand, L.; Mash, D.

    2008-12-01

    Blooms of cyanobacteria have developed in Florida Bay, Biscayne Bay and other coastal waters of South Florida. It has recently been shown that virtually all cyanobacteria produce the potent neurotoxin, beta-N- methylamino-L-alanine (BMAA). Studies in Guam indicate that BMAA can biomagnify up the food chain from cyanobacteria to human food and humans. Recent studies in Guam and on human brains in North America suggest an association between BMAA and neurodegenerative diseases such as Alzheimer's, Parkinson's, and Amyotrophic Lateral Sclerosis (ALS). A variety of organisms from South Florida coastal waters are being analyzed for BMAA content to determine if BMAA is biomagnifying in these food chains and if it is a potential human health hazard. Some have extremely high concentrations of BMAA.

  6. Ichthyotoxic Brominated Diphenyl Ethers from a Mixed Assemblage of a Red Alga and Cyanobacterium: Structure Clarification and Biological Properties

    PubMed Central

    Suyama, Takashi L.; Cao, Zhengyu; Murray, Thomas F.; Gerwick, William H.

    2009-01-01

    Primary fractions from the extract of a tropical red alga mixed with filamentous cyanobacteria, collected from Papua New Guinea, were active in a neurotoxicity assay. Bioassay guided isolation led to two natural products (1, 2) with relatively potent calcium ion influx properties. The more prevalent of the neurotoxic compounds (1) was characterized by extensive NMR, mass spectrometry, and X-ray crystallography, and shown to be identical to a polybrominated diphenyl ether metabolite present in the literature, but reported with different NMR properties. To clarify this anomalous result, we synthesized a candidate isomeric polybrominated diphenyl ether (3), but this clearly had different NMR shifts than the reported compound. We conclude that the original isolate of 3,4,5-tribromo-2-(2,4-dibromophenoxy)phenol was contaminated with a minor compound, giving rise to the observed anomalous NMR shifts. The second and less abundant natural product (2) isolated in this study was a more highly brominated species. All three compounds showed a low micromolar ability to increase intracellular calcium ion concentrations in mouse neocortical neurons as well as toxicity to zebrafish. Because polybrominated diphenyl ethers have both natural as well as anthropomorphic origins, and accumulate in marine organisms at higher trophic level (mammals, fish, birds), these neurotoxic properties are of environmental significance and concern. PMID:19638282

  7. Atrazine selects for ichthyotoxic Prymnesium parvum, a possible explanation for golden algae blooms in lakes of Texas, USA.

    PubMed

    Yates, Brian S; Rogers, William J

    2011-11-01

    Prymnesium parvum Carter is a mixotrophic haptophyte which, under certain environmental conditions, produces potent toxins responsible for fish kills around the world since the 1930s. Many P. parvum blooms have occurred in catchments where crop agriculture is a dominant land use; however, the effects of herbicides on bloom dynamics have not yet been investigated. Aquatic microbial communities containing P. parvum were subjected to two separate experiments involving the addition of either atrazine or glyphosate at varying concentrations. After 14, 21, and 28 days at 10 μg/l atrazine we observed that the relative abundance of P. parvum was significantly higher compared to the control. After 28 days, the relative abundance of P. parvum was approximately 53% higher in 10 μg/l atrazine compared to the control. Glyphosate exhibited no statistically-significant effect on the relative abundance of P. parvum. Inadequate characterization of the microbial community and uncertainty due to ecological and allelopathic effects of P. parvum made it difficult to establish strong relationships between herbicide sensitivity and nutritional mode. Large volumes of mobile and persistent herbicides with high toxicity to phytoplankton are used in cotton defoliation in Texas prior to the typical P. parvum pre-bloom period. These results have important implications for management, such as whether reduction in herbicide runoff could decrease the frequency and duration of P. parvum blooms in the future.

  8. Botulinum Neurotoxin Type A in Neurology: Update

    PubMed Central

    Orsini, Marco; Leite, Marco Antonio Araujo; Chung, Tae Mo; Bocca, Wladimir; de Souza, Jano Alves; de Souza, Olivia Gameiro; Moreira, Rayele Priscila; Bastos, Victor Hugo; Teixeira, Silmar; Oliveira, Acary Bulle; Moraes, Bruno da Silva; Matta, André Palma; Jacinto, Luis Jorge

    2015-01-01

    This paper reviews the current and most neurological (central nervous system, CNS) uses of the botulinum neurotoxin type A. The effect of these toxins at neuromuscular junction lends themselves to neurological diseases of muscle overactivity, particularly abnormalities of muscle control. There are seven serotypes of the toxin, each with a specific activity at the molecular level. Currently, serotypes A (in two preparations) and B are available for clinical purpose, and they have proved to be safe and effective for the treatment of dystonia, spasticity, headache, and other CNS disorders in which muscle hyperactivity gives rise to symptoms. Although initially thought to inhibit acetylcholine release only at the neuromuscular junction, botulinum toxins are now recognized to inhibit acetylcholine release at autonomic cholinergic nerve terminals, as well as peripheral release of neuro-transmitters involved in pain regulation. Its effects are transient and nondestructive, and largely limited to the area in which it is administered. These effects are also graded according to the dose, allowing individualized treatment of patients and disorders. It may also prove to be useful in the control of autonomic dysfunction and sialorrhea. In over 20 years of use in humans, botulinum toxin has accumulated a considerable safety record, and in many cases represents relief for thousands of patients unaided by other therapy. PMID:26487928

  9. Engineered botulinum neurotoxins as new therapeutics.

    PubMed

    Masuyer, Geoffrey; Chaddock, John A; Foster, Keith A; Acharya, K Ravi

    2014-01-01

    Botulinum neurotoxins (BoNTs) cause flaccid paralysis by inhibiting neurotransmission at cholinergic nerve terminals. Each BoNT consists of three domains that are essential for toxicity: the binding domain, the translocation domain, and the catalytic light-chain domain. BoNT modular architecture is associated with a multistep mechanism that culminates in the intracellular proteolysis of SNARE (soluble N-ethylmaleimide-sensitive-fusion-protein attachment protein receptor) proteins, which prevents synaptic vesicle exocytosis. As the most toxic proteins known, BoNTs have been extensively studied and are used as pharmaceutical agents to treat an increasing variety of disorders. This review summarizes the level of sophistication reached in BoNT engineering and highlights the diversity of approaches taken to utilize the modularity of the toxin. Improved efficiency and applicability have been achieved by direct mutagenesis and interserotype domain rearrangement. The scope of BoNT activity has been extended to nonneuronal cells and offers the basis for novel biomolecules in the treatment of secretion disorders.

  10. Isolation and characterisation of P-EPTX-Ap1a and P-EPTX-Ar1a: pre-synaptic neurotoxins from the venom of the northern (Acanthophis praelongus) and Irian Jayan (Acanthophis rugosus) death adders.

    PubMed

    Chaisakul, Janeyuth; Konstantakopoulos, Nicki; Smith, A Ian; Hodgson, Wayne C

    2010-09-15

    The neurotoxicity observed following death adder envenoming has been thought to be solely due to the presence of potent post-synaptic neurotoxins. Clinically, these effects are often poorly reversed by death adder antivenom or anticholinesterase, particularly when patients present with established paralysis. This suggests that either the post-synaptic neurotoxins are irreversible/'pseudo' irreversible, or the venom contains pre-synaptic neurotoxins that do not respond to antivenom. To support the later hypothesis, a pre-synaptic neurotoxin (P-EPTX-Aa1a) has recently been isolated from the venom of Acanthophis antarcticus. We examined Acanthophis praelongus and Acanthophis rugosus venoms for the presence of pre-synaptic neurotoxins. P-EPTX-Ap1a (40,719Da) and P-EPTX-Ar1a (40,879Da) were isolated from A. praelongus and A. rugosus venoms, respectively. P-EPTX-Ap1a and P-EPTX-Ar1a are comprised of three different subunits, alpha, beta1 and beta2. The two toxins displayed similar levels of PLA(2) activity which was almost solely attributed to the alpha subunit in both toxins. P-EPTX-Ap1a (20-100nM) and P-EPTX-Ar1a (20-100nM) caused inhibition of indirect twitches of the skeletal muscle preparation without affecting contractile responses to nicotinic receptor agonists. Interestingly, only the alpha subunit of both toxins (300nM) displayed neurotoxic activity. Inhibition of PLA(2) activity markedly reduced the effect of the toxins on muscle twitch height. These results confirm that P-EPTX-Ap1a and P-EPTX-Ar1a are pre-synaptic neurotoxins and represent the second and third such toxins to be isolated from death adder venom. The presence of pre-synaptic neurotoxins in Acanthophis sp. venoms indicates that treatment strategies for envenoming by these snakes needs to be reassessed given the likelihood of irreversible neurotoxicity. PMID:20488165

  11. Isolation and characterisation of P-EPTX-Ap1a and P-EPTX-Ar1a: pre-synaptic neurotoxins from the venom of the northern (Acanthophis praelongus) and Irian Jayan (Acanthophis rugosus) death adders.

    PubMed

    Chaisakul, Janeyuth; Konstantakopoulos, Nicki; Smith, A Ian; Hodgson, Wayne C

    2010-09-15

    The neurotoxicity observed following death adder envenoming has been thought to be solely due to the presence of potent post-synaptic neurotoxins. Clinically, these effects are often poorly reversed by death adder antivenom or anticholinesterase, particularly when patients present with established paralysis. This suggests that either the post-synaptic neurotoxins are irreversible/'pseudo' irreversible, or the venom contains pre-synaptic neurotoxins that do not respond to antivenom. To support the later hypothesis, a pre-synaptic neurotoxin (P-EPTX-Aa1a) has recently been isolated from the venom of Acanthophis antarcticus. We examined Acanthophis praelongus and Acanthophis rugosus venoms for the presence of pre-synaptic neurotoxins. P-EPTX-Ap1a (40,719Da) and P-EPTX-Ar1a (40,879Da) were isolated from A. praelongus and A. rugosus venoms, respectively. P-EPTX-Ap1a and P-EPTX-Ar1a are comprised of three different subunits, alpha, beta1 and beta2. The two toxins displayed similar levels of PLA(2) activity which was almost solely attributed to the alpha subunit in both toxins. P-EPTX-Ap1a (20-100nM) and P-EPTX-Ar1a (20-100nM) caused inhibition of indirect twitches of the skeletal muscle preparation without affecting contractile responses to nicotinic receptor agonists. Interestingly, only the alpha subunit of both toxins (300nM) displayed neurotoxic activity. Inhibition of PLA(2) activity markedly reduced the effect of the toxins on muscle twitch height. These results confirm that P-EPTX-Ap1a and P-EPTX-Ar1a are pre-synaptic neurotoxins and represent the second and third such toxins to be isolated from death adder venom. The presence of pre-synaptic neurotoxins in Acanthophis sp. venoms indicates that treatment strategies for envenoming by these snakes needs to be reassessed given the likelihood of irreversible neurotoxicity.

  12. Positive regulation of botulinum neurotoxin gene expression by CodY in Clostridium botulinum ATCC 3502.

    PubMed

    Zhang, Zhen; Dahlsten, Elias; Korkeala, Hannu; Lindström, Miia

    2014-12-01

    Botulinum neurotoxin, produced mainly by the spore-forming bacterium Clostridium botulinum, is the most poisonous biological substance known. Here, we show that CodY, a global regulator conserved in low-G+C Gram-positive bacteria, positively regulates the botulinum neurotoxin gene expression. Inactivation of codY resulted in decreased expression of botA, encoding the neurotoxin, as well as in reduced neurotoxin synthesis. Complementation of the codY mutation in trans rescued neurotoxin synthesis, and overexpression of codY in trans caused elevated neurotoxin production. Recombinant CodY was found to bind to a 30-bp region containing the botA transcription start site, suggesting regulation of the neurotoxin gene transcription through direct interaction. GTP enhanced the binding affinity of CodY to the botA promoter, suggesting that CodY-dependent neurotoxin regulation is associated with nutritional status.

  13. Positive Regulation of Botulinum Neurotoxin Gene Expression by CodY in Clostridium botulinum ATCC 3502

    PubMed Central

    Zhang, Zhen; Dahlsten, Elias; Korkeala, Hannu

    2014-01-01

    Botulinum neurotoxin, produced mainly by the spore-forming bacterium Clostridium botulinum, is the most poisonous biological substance known. Here, we show that CodY, a global regulator conserved in low-G+C Gram-positive bacteria, positively regulates the botulinum neurotoxin gene expression. Inactivation of codY resulted in decreased expression of botA, encoding the neurotoxin, as well as in reduced neurotoxin synthesis. Complementation of the codY mutation in trans rescued neurotoxin synthesis, and overexpression of codY in trans caused elevated neurotoxin production. Recombinant CodY was found to bind to a 30-bp region containing the botA transcription start site, suggesting regulation of the neurotoxin gene transcription through direct interaction. GTP enhanced the binding affinity of CodY to the botA promoter, suggesting that CodY-dependent neurotoxin regulation is associated with nutritional status. PMID:25281376

  14. A Novel Neurotoxin from Venom of the Spider, Brachypelma albopilosum

    PubMed Central

    Yuan, Mingwei; Li, Hongli; Wang, Ping; Yuan, Minglong; Lu, Qiumin

    2014-01-01

    Spiders have evolved highly selective toxins for insects. There are many insecticidal neurotoxins in spider venoms. Although a large amount of work has been done to focus on neurotoxicity of spider components, little information, which is related with effects of spider toxins on tumor cell proliferation and cytotoxicity, is available for Brachypelma albopilosum venom. In this work, a novel spider neurotoxin (brachyin) was identified and characterized from venoms of the spider, Brachypelma albopilosum. Brachyin is composed of 41 amino acid residues with the sequence of CLGENVPCDKDRPNCCSRYECLEPTGYGWWYASYYCYKKRS. There are six cysteines in this sequence, which form three disulfided bridges. The serine residue at the C-terminus is amidated. Brachyin showed strong lethal effects on American cockroaches (Periplaneta americana) and Tenebrio molitor (common mealbeetle). This neurotoxin also showed significant analgesic effects in mice models including abdominal writhing induced by acetic acid and formalin-induced paw licking tests. It was interesting that brachyin exerted marked inhibition on tumor cell proliferation. PMID:25329070

  15. A novel neurotoxin from venom of the spider, Brachypelma albopilosum.

    PubMed

    Zhong, Yunhua; Song, Bo; Mo, Guoxiang; Yuan, Mingwei; Li, Hongli; Wang, Ping; Yuan, Minglong; Lu, Qiumin

    2014-01-01

    Spiders have evolved highly selective toxins for insects. There are many insecticidal neurotoxins in spider venoms. Although a large amount of work has been done to focus on neurotoxicity of spider components, little information, which is related with effects of spider toxins on tumor cell proliferation and cytotoxicity, is available for Brachypelma albopilosum venom. In this work, a novel spider neurotoxin (brachyin) was identified and characterized from venoms of the spider, Brachypelma albopilosum. Brachyin is composed of 41 amino acid residues with the sequence of CLGENVPCDKDRPNCCSRYECLEPTGYGWWYASYYCYKKRS. There are six cysteines in this sequence, which form three disulfided bridges. The serine residue at the C-terminus is amidated. Brachyin showed strong lethal effects on American cockroaches (Periplaneta americana) and Tenebrio molitor (common mealbeetle). This neurotoxin also showed significant analgesic effects in mice models including abdominal writhing induced by acetic acid and formalin-induced paw licking tests. It was interesting that brachyin exerted marked inhibition on tumor cell proliferation.

  16. Characteristics of the labile neurotoxin associated with nervous coccidiosis.

    PubMed Central

    Isler, C M; Bellamy, J E; Wobeser, G A

    1987-01-01

    Reported are the results of preliminary attempts to characterize the molecular weight, heat sensitivity and other features of a labile neurotoxin identified in the serum of calves exhibiting neurological signs in association with coccidial enteritis. The labile neurotoxin activity is heat labile (60 degrees C for 30 min) and is lost upon exposure to acidic pH (5.5) and cysteine (1.75 g/100 mL serum). Activity can be recovered from the precipitate of a 30% wt/vol solution of (NH4)2SO4 in serum. Ultrafiltration trials suggest that labile neurotoxin activity may be linked to a molecule of over 300,000 MW. PMID:2955866

  17. A novel neurotoxin from venom of the spider, Brachypelma albopilosum.

    PubMed

    Zhong, Yunhua; Song, Bo; Mo, Guoxiang; Yuan, Mingwei; Li, Hongli; Wang, Ping; Yuan, Minglong; Lu, Qiumin

    2014-01-01

    Spiders have evolved highly selective toxins for insects. There are many insecticidal neurotoxins in spider venoms. Although a large amount of work has been done to focus on neurotoxicity of spider components, little information, which is related with effects of spider toxins on tumor cell proliferation and cytotoxicity, is available for Brachypelma albopilosum venom. In this work, a novel spider neurotoxin (brachyin) was identified and characterized from venoms of the spider, Brachypelma albopilosum. Brachyin is composed of 41 amino acid residues with the sequence of CLGENVPCDKDRPNCCSRYECLEPTGYGWWYASYYCYKKRS. There are six cysteines in this sequence, which form three disulfided bridges. The serine residue at the C-terminus is amidated. Brachyin showed strong lethal effects on American cockroaches (Periplaneta americana) and Tenebrio molitor (common mealbeetle). This neurotoxin also showed significant analgesic effects in mice models including abdominal writhing induced by acetic acid and formalin-induced paw licking tests. It was interesting that brachyin exerted marked inhibition on tumor cell proliferation. PMID:25329070

  18. Botulinum neurotoxin treatment of palmar and plantar hyperhidrosis.

    PubMed

    Weinberg, Tessa; Solish, Nowell; Murray, Christian

    2014-10-01

    Palmar and plantar hyperhidrosis is relatively common and can have severe psychological and medical consequences for those afflicted. A multitude of treatments exist but are often inadequate especially for those with significant disease. In these cases botulinum neurotoxin provides a reliable method for reducing the symptoms and improving quality of life. Although actual administration is relatively straightforward, pain management is a crucial component that requires a mastery of several techniques. Patients have a high degree of satisfaction with botulinum neurotoxin treatment and are motivated to come back for repeat treatments, usually every 6 months.

  19. Aptamer-based electrochemical biosensor for Botulinum neurotoxin.

    PubMed

    Wei, Fang; Ho, Chih-Ming

    2009-04-01

    We have developed an aptamer-based electrochemical sensor for detection of Botulinum neurotoxin, where steric hindrance is applied to achieve specific signal amplification via conformational change of the aptamer. The incubation time and potassium concentration of the reaction buffer were found to be key parameters affecting the sensitivity of detection of the recognition of Botulinum neurotoxin by the aptamer. Under optimized experimental conditions, a high signal-to-noise ratio was obtained within 24 h with a limit of detection (LOD) of 40 pg/ml by two standard deviation cutoffs above the noise level.

  20. Structure-based drug discovery for botulinum neurotoxins.

    PubMed

    Swaminathan, Subramanyam

    2013-01-01

    Clostridium botulinum neurotoxin is the most poisonous substance known to humans. It is a potential biowarfare threat and a public health hazard. The only therapeutics available is antibody treatment which will not be effective for post-exposure therapy. There are no drugs available for post-intoxication treatment. Accordingly, it is imperative to develop effective drugs to counter botulism. Available structural information on botulinum neurotoxins both alone and in complex with their substrates offers an efficient method for designing structure-based drugs to treat botulism.

  1. Identification of Clinically Viable Quinolinol Inhibitors of Botulinum Neurotoxin A Light Chain

    PubMed Central

    2015-01-01

    Botulinum neurotoxins (BoNT) are the most potent toxins known and a significant bioterrorist threat. Few small molecule compounds have been identified that are active in cell-based or animal models, potentially due to toxin enzyme plasticity. Here we screened commercially available quinolinols, as well as synthesized hydroxyquinolines. Seventy-two compounds had IC50 values below 10 μM, with the best compound exhibiting submicromolar inhibition (IC50 = 0.8 μM). Structure–activity relationship trends showed that the enzyme tolerates various substitutions at R1 but has a clear preference for bulky aryl amide groups at R2, while methylation at R3 increased inhibitor potency. Evaluation of the most potent compounds in an ADME panel showed that these compounds possess poor solubility at pH 6.8, but display excellent solubility at low pH, suggesting that oral dosing may be possible. Our data show the potential of quinolinol compounds as BoNT therapeutics due to their good in vitro potencies and favorable ADME properties. PMID:24387280

  2. Prediction of presynaptic and postsynaptic neurotoxins by the increment of diversity.

    PubMed

    Yang, Lei; Li, Qianzhong

    2009-03-01

    Presynaptic and postsynaptic neurotoxins have very important application in basic research and drug design. The successful prediction of neurotoxin is becoming an important task in recent years. In this study, based on the concept of Chou's pseudo-amino acid compositions, an algorithm of increment of diversity (ID) is proposed for predicting presynaptic and postsynaptic neurotoxins. The results of jackknife test show that the accuracies of prediction are 90.23% for presynaptic neurotoxins and 89.40% for postsynaptic neurotoxins. In addition, toxins and non-toxins are also predicted by using this algorithm.

  3. Genomes, neurotoxins and biology of Clostridium botulinum Group I and Group II

    PubMed Central

    Carter, Andrew T.; Peck, Michael W.

    2015-01-01

    Recent developments in whole genome sequencing have made a substantial contribution to understanding the genomes, neurotoxins and biology of Clostridium botulinum Group I (proteolytic C. botulinum) and C. botulinum Group II (non-proteolytic C. botulinum). Two different approaches are used to study genomics in these bacteria; comparative whole genome microarrays and direct comparison of complete genome DNA sequences. The properties of the different types of neurotoxin formed, and different neurotoxin gene clusters found in C. botulinum Groups I and II are explored. Specific examples of botulinum neurotoxin genes are chosen for an in-depth discussion of neurotoxin gene evolution. The most recent cases of foodborne botulism are summarised. PMID:25445012

  4. Botulinum Neurotoxin Serotypes Detected by Electrochemical Impedance Spectroscopy

    PubMed Central

    Savage, Alison C.; Buckley, Nicholas; Halliwell, Jennifer; Gwenin, Christopher

    2015-01-01

    Botulinum neurotoxin is one of the deadliest biological toxins known to mankind and is able to cause the debilitating disease botulism. The rapid detection of the different serotypes of botulinum neurotoxin is essential for both diagnosis of botulism and identifying the presence of toxin in potential cases of terrorism and food contamination. The modes of action of botulinum neurotoxins are well-established in literature and differ for each serotype. The toxins are known to specifically cleave portions of the SNARE proteins SNAP-25 or VAMP; an interaction that can be monitored by electrochemical impedance spectroscopy. This study presents a SNAP-25 and a VAMP biosensors for detecting the activity of five botulinum neurotoxin serotypes (A–E) using electrochemical impedance spectroscopy. The biosensors are able to detect concentrations of toxins as low as 25 fg/mL, in a short time-frame compared with the current standard methods of detection. Both biosensors show greater specificity for their compatible serotypes compared with incompatible serotypes and denatured toxins. PMID:25954998

  5. Antibody Protection Against Botulinum Neurotoxin Intoxication In Mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Adulteration of food or feed with any of the seven serotypes of botulinum neurotoxin (BoNT) is a potential bioterrorism concern. Currently, there is strong interest in the development of detection reagents, vaccines, therapeutics and other countermeasures. A sensitive immunoassay for detecting BoNT/...

  6. Botulinum neurotoxin: Where are we with detection technologies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The poisonous nature of botulinum neurotoxin (BoNT) poses a great risk to humans and also can be exploited as a possible bioterrorism and biological warfare agent. BoNT serotypes A and B have emerged as effective treatments for a variety of neurological disorders, in addition to their applicability ...

  7. Characterization of monoclonal antibodies against Naja naja oxiana neurotoxin I.

    PubMed

    Stiles, B G; Sexton, F W; Guest, S B; Olson, M A; Hack, D C

    1994-10-01

    Seven monoclonal antibodies (mAbs) were developed against neurotoxin I (NT-1), a protein from central Asian cobra (Naja naja oxiana) venom which binds specifically to nicotinic acetylcholine receptor (AchR). All of the mAbs cross-reacted with another long-chain post-synaptic neurotoxin, Bungarus multicinctus alpha-bungarotoxin (alpha-BT), but not Naja naja kaouthia alpha-cobratoxin, in an enzyme-linked immunosorbent assay (e.l.i.s.a.). Short-chain post-synaptic neurotoxins like Naja naja atra cobrotoxin, Laticauda semifasciata erabutoxin b, or N. n. oxiana neurotoxin II did not cross-react with the NT-1 mAbs, but an antigen(s) found in Dendroaspis polylepis, Acanthophis antarcticus and Pseudechis australis venoms was immunoreactive. The e.l.i.s.a. readings for dithiothreitol-reduced NT-1 and NT-1 mAbs ranged from 13 to 27% of those for native toxin but reduced alpha-BT was not immunoreactive. Synthetic NT-1 peptides were used in epitope-mapping studies and two, non-contiguous regions (Cys15-Tyr23 and Lys25-Gly33 or Pro17-Lys25 and Asp29-Lys37) were recognized by the NT-1 mAbs. The NT-1 mAbs individually inhibited 31-71% of alpha-BT binding to AchR in vitro and afforded a slight protective effect in vivo with a toxin: antibody mole ratio of 1:1.5. This report is the first to describe mAbs which recognize and protect against a heterologous, long-chain, post-synaptic neurotoxin from snake venom.

  8. Peptide inhibitors of botulinum neurotoxin serotype A: design, inhibition, cocrystal structures, structure-activity relationship and pharmacophore modeling

    SciTech Connect

    Kumar G.; Swaminathan S.; Kumaran, D.; Ahmed, S. A.

    2012-05-01

    Clostridium botulinum neurotoxins are classified as Category A bioterrorism agents by the Centers for Disease Control and Prevention (CDC). The seven serotypes (A-G) of the botulinum neurotoxin, the causative agent of the disease botulism, block neurotransmitter release by specifically cleaving one of the three SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins and induce flaccid paralysis. Using a structure-based drug-design approach, a number of peptide inhibitors were designed and their inhibitory activity against botulinum serotype A (BoNT/A) protease was determined. The most potent peptide, RRGF, inhibited BoNT/A protease with an IC{sub 50} of 0.9 {micro}M and a K{sub i} of 358 nM. High-resolution crystal structures of various peptide inhibitors in complex with the BoNT/A protease domain were also determined. Based on the inhibitory activities and the atomic interactions deduced from the cocrystal structures, the structure-activity relationship was analyzed and a pharmacophore model was developed. Unlike the currently available models, this pharmacophore model is based on a number of enzyme-inhibitor peptide cocrystal structures and improved the existing models significantly, incorporating new features.

  9. Structures of Clostridium Botulinum Neurotoxin Serotype A Light Chain Complexed with Small-Molecule Inhibitors Highlight Active-Site Flexibility

    SciTech Connect

    Silvaggi,N.; Boldt, G.; Hixon, M.; Kennedy, J.; Tzipori, S.; Janda, K.; Allen, K.

    2007-01-01

    The potential for the use of Clostridial neurotoxins as bioweapons makes the development of small-molecule inhibitors of these deadly toxins a top priority. Recently, screening of a random hydroxamate library identified a small-molecule inhibitor of C. botulinum Neurotoxin Serotype A Light Chain (BoNT/A-LC), 4-chlorocinnamic hydroxamate, a derivative of which has been shown to have in vivo efficacy in mice and no toxicity. We describe the X-ray crystal structures of BoNT/A-LC in complexes with two potent small-molecule inhibitors. The structures of the enzyme with 4-chlorocinnamic hydroxamate or 2,4-dichlorocinnamic hydroxamate bound are compared to the structure of the enzyme complexed with L-arginine hydroxamate, an inhibitor with modest affinity. Taken together, this suite of structures provides surprising insights into the BoNT/A-LC active site, including unexpected conformational flexibility at the S1' site that changes the electrostatic environment of the binding pocket. Information gained from these structures will inform the design and optimization of more effective small-molecule inhibitors of BoNT/A-LC.

  10. Evolution of an ancient venom: recognition of a novel family of cnidarian toxins and the common evolutionary origin of sodium and potassium neurotoxins in sea anemone.

    PubMed

    Jouiaei, Mahdokht; Sunagar, Kartik; Federman Gross, Aya; Scheib, Holger; Alewood, Paul F; Moran, Yehu; Fry, Bryan G

    2015-06-01

    Despite Cnidaria (sea anemones, corals, jellyfish, and hydroids) being the oldest venomous animal lineage, structure-function relationships, phyletic distributions, and the molecular evolutionary regimes of toxins encoded by these intriguing animals are poorly understood. Hence, we have comprehensively elucidated the phylogenetic and molecular evolutionary histories of pharmacologically characterized cnidarian toxin families, including peptide neurotoxins (voltage-gated Na(+) and K(+) channel-targeting toxins: NaTxs and KTxs, respectively), pore-forming toxins (actinoporins, aerolysin-related toxins, and jellyfish toxins), and the newly discovered small cysteine-rich peptides (SCRiPs). We show that despite long evolutionary histories, most cnidarian toxins remain conserved under the strong influence of negative selection-a finding that is in striking contrast to the rapid evolution of toxin families in evolutionarily younger lineages, such as cone snails and advanced snakes. In contrast to the previous suggestions that implicated SCRiPs in the biomineralization process in corals, we demonstrate that they are potent neurotoxins that are likely involved in the envenoming function, and thus represent the first family of neurotoxins from corals. We also demonstrate the common evolutionary origin of type III KTxs and NaTxs in sea anemones. We show that type III KTxs have evolved from NaTxs under the regime of positive selection, and likely represent a unique evolutionary innovation of the Actinioidea lineage. We report a correlation between the accumulation of episodically adaptive sites and the emergence of novel pharmacological activities in this rapidly evolving neurotoxic clade.

  11. The strange case of the botulinum neurotoxin: using chemistry and biology to modulate the most deadly poison.

    PubMed

    Willis, Bert; Eubanks, Lisa M; Dickerson, Tobin J; Janda, Kim D

    2008-01-01

    In the classic novella "The Strange Case of Dr. Jekyll and Mr. Hyde", Robert Louis Stevenson paints a stark picture of the duality of good and evil within a single man. Botulinum neurotoxin (BoNT), the most potent known toxin, possesses an analogous dichotomous nature: It shows a pronounced morbidity and mortality, but it is used with great effect in much lower doses in a wide range of clinical scenarios. Recently, tremendous strides have been made in the basic understanding of the structure and function of BoNT, which have translated into widespread efforts towards the discovery of biomacromolecules and small molecules that specifically modulate BoNT activity. Particular emphasis has been placed on the identification of inhibitors that can counteract BoNT exposure in the event of a bioterrorist attack. This Review summarizes the current advances in the development of therapeutics, including vaccines, peptides, and small-molecule inhibitors, for the prevention and treatment of botulism. PMID:18844202

  12. Prediction of neurotoxins by support vector machine based on multiple feature vectors.

    PubMed

    Guang, Xuan-Min; Guo, Yan-Zhi; Wang, Xia; Li, Meng-Long

    2010-09-01

    Neurotoxin is a toxin which acts on nerve cells by interacting with membrane proteins. Different neurotoxins have different functions and sources. With much more knowledge of neurotoxins it would be greatly helpful for the development of drug design. The support vector machine (SVM) was used to predict the neurotoxin based on multiple feature vector descriptors, including the amino acid composition, length of the protein sequence, weight of the protein and the evolution information described by position specific scoring matrix (PSSM). After a five-fold cross-validation procedure, the method achieved an accuracy of 100% in discriminating neurotoxins from non-toxins. As for classifying neurotoxins based on their sources and functions, the accuracy was 99.50% and 99.38% respectively. At last, the method yielded a good performance in sub-classification of ion channels inhibitors with the total accuracy of 87.27%. These results indicate that this method outperforms previously described NTXpred method.

  13. What is the risk of aluminium as a neurotoxin?

    PubMed

    Exley, Christopher

    2014-06-01

    Aluminium is neurotoxic. Its free ion, Al(3+) (aq), is highly biologically reactive and uniquely equipped to do damage to essential cellular (neuronal) biochemistry. This unequivocal fact must be the starting point in examining the risk posed by aluminium as a neurotoxin in humans. Aluminium is present in the human brain and it accumulates with age. The most recent research demonstrates that a significant proportion of individuals older than 70 years of age have a potentially pathological accumulation of aluminium somewhere in their brain. What are the symptoms of chronic aluminium intoxication in humans? What if neurodegenerative diseases such as Alzheimer's disease are the manifestation of the risk of aluminium as a neurotoxin? How might such an (outrageous) hypothesis be tested?

  14. Isolation and Pharmacological Characterization of α-Elapitoxin-Ot1a, a Short-Chain Postsynaptic Neurotoxin from the Venom of the Western Desert Taipan, Oxyuranus temporalis

    PubMed Central

    Barber, Carmel M.; Ahmad Rusmili, Muhamad Rusdi; Hodgson, Wayne C.

    2016-01-01

    Taipans (Oxyuranus spp.) are elapids with highly potent venoms containing presynaptic (β) and postsynaptic (α) neurotoxins. O. temporalis (Western Desert taipan), a newly discovered member of this genus, has been shown to possess venom which displays marked in vitro neurotoxicity. No components have been isolated from this venom. We describe the characterization of α-elapitoxin-Ot1a (α-EPTX-Ot1a; 6712 Da), a short-chain postsynaptic neurotoxin, which accounts for approximately 30% of O. temporalis venom. α-Elapitoxin-Ot1a (0.1–1 µM) produced concentration-dependent inhibition of indirect-twitches, and abolished contractile responses to exogenous acetylcholine and carbachol, in the chick biventer cervicis nerve-muscle preparation. The inhibition of indirect twitches by α-elapitoxin-Ot1a (1 µM) was not reversed by washing the tissue. Prior addition of taipan antivenom (10 U/mL) delayed the neurotoxic effects of α-elapitoxin-Ot1a (1 µM) and markedly attenuated the neurotoxic effects of α-elapitoxin-Ot1a (0.1 µM). α-Elapitoxin-Ot1a displayed pseudo-irreversible antagonism of concentration-response curves to carbachol with a pA2 value of 8.02 ± 0.05. De novo sequencing revealed the main sequence of the short-chain postsynaptic neurotoxin (i.e., α-elapitoxin-Ot1a) as well as three other isoforms found in O. temporalis venom. α-Elapitoxin-Ot1a shows high sequence similarity (i.e., >87%) with other taipan short-chain postsynaptic neurotoxins. PMID:26938558

  15. Isolation and Pharmacological Characterization of α-Elapitoxin-Ot1a, a Short-Chain Postsynaptic Neurotoxin from the Venom of the Western Desert Taipan, Oxyuranus temporalis.

    PubMed

    Barber, Carmel M; Rusmili, Muhamad Rusdi Ahmad; Hodgson, Wayne C

    2016-03-01

    Taipans (Oxyuranus spp.) are elapids with highly potent venoms containing presynaptic (β) and postsynaptic (α) neurotoxins. O. temporalis (Western Desert taipan), a newly discovered member of this genus, has been shown to possess venom which displays marked in vitro neurotoxicity. No components have been isolated from this venom. We describe the characterization of α-elapitoxin-Ot1a (α-EPTX-Ot1a; 6712 Da), a short-chain postsynaptic neurotoxin, which accounts for approximately 30% of O. temporalis venom. α-Elapitoxin-Ot1a (0.1-1 µM) produced concentration-dependent inhibition of indirect-twitches, and abolished contractile responses to exogenous acetylcholine and carbachol, in the chick biventer cervicis nerve-muscle preparation. The inhibition of indirect twitches by α-elapitoxin-Ot1a (1 µM) was not reversed by washing the tissue. Prior addition of taipan antivenom (10 U/mL) delayed the neurotoxic effects of α-elapitoxin-Ot1a (1 µM) and markedly attenuated the neurotoxic effects of α-elapitoxin-Ot1a (0.1 µM). α-Elapitoxin-Ot1a displayed pseudo-irreversible antagonism of concentration-response curves to carbachol with a pA₂ value of 8.02 ± 0.05. De novo sequencing revealed the main sequence of the short-chain postsynaptic neurotoxin (i.e., α-elapitoxin-Ot1a) as well as three other isoforms found in O. temporalis venom. α-Elapitoxin-Ot1a shows high sequence similarity (i.e., >87%) with other taipan short-chain postsynaptic neurotoxins. PMID:26938558

  16. Aphicidal efficacy of scorpion- and spider-derived neurotoxins.

    PubMed

    Pal, Narinder; Yamamoto, Takashi; King, Glenn F; Waine, Clement; Bonning, Bryony

    2013-08-01

    Insect-specific neurotoxins that act within the insect hemocoel (body cavity) represent an untapped resource for insect pest management. On the basis of recent advances made in development of appropriate delivery systems for transport of these toxins from the insect gut, across the gut epithelium to their target site, we screened neurotoxins derived from scorpion or spider venom for efficacy against the pea aphid, Acyrthosiphon pisum, and the green peach aphid, Myzus persicae. Toxins were selected to represent different modes of electrophysiological action, including activity on voltage-gated calcium channels (ω-TRTX-Gr1a, ω-agatoxin Aa4a, ω-hexatoxin-Hv1a), calcium- and voltage-activated potassium channels (charybdotoxin, maurotoxin), chloride channels (chlorotoxin) and voltage-gated sodium channels (LqhαIT). The Bacillus thuringiensis-derived toxin Cyt1Aa was also tested as a positive control for toxicity. In per os bioassays with both aphid species, toxicity was only seen for ω-TRTX-Gr1a and Cyt1Aa. On injection into the hemocoel of A. pisum, LD₅₀ values ranged from 1 to 8 ng/mg body weight, with ω-hexatoxin-Hv1a being the most toxic (1.02 ng/mg body weight). All neurotoxins caused rapid paralysis, with charybdotoxin, maurotoxin and chlorotoxin also causing melanization of injected aphids. These data represent the first comprehensive screen of neurotoxins against aphids, and highlight the potential for practical use of the insect-specific toxin ω-hexatoxin-Hv1a in aphid management. PMID:23651761

  17. Substrate recognition mechanism of VAMP/synaptobrevin-cleaving clostridial neurotoxins.

    PubMed

    Sikorra, Stefan; Henke, Tina; Galli, Thierry; Binz, Thomas

    2008-07-25

    Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) inhibit neurotransmitter release by proteolyzing a single peptide bond in one of the three soluble N-ethylmaleimide-sensitive factor attachment protein receptors SNAP-25, syntaxin, and vesicle-associated membrane protein (VAMP)/synaptobrevin. TeNT and BoNT/B, D, F, and G of the seven known BoNTs cleave the synaptic vesicle protein VAMP/synaptobrevin. Except for BoNT/B and TeNT, they cleave unique peptide bonds, and prior work suggested that different substrate segments are required for the interaction of each toxin. Although the mode of SNAP-25 cleavage by BoNT/A and E has recently been studied in detail, the mechanism of VAMP/synaptobrevin proteolysis is fragmentary. Here, we report the determination of all substrate residues that are involved in the interaction with BoNT/B, D, and F and TeNT by means of systematic mutagenesis of VAMP/synaptobrevin. For each of the toxins, three or more residues clustered at an N-terminal site remote from the respective scissile bond are identified that affect solely substrate binding. These exosites exhibit different sizes and distances to the scissile peptide bonds for each neurotoxin. Substrate segments C-terminal of the cleavage site (P4-P4') do not play a role in the catalytic process. Mutation of residues in the proximity of the scissile bond exclusively affects the turnover number; however, the importance of individual positions at the cleavage sites varied for each toxin. The data show that, similar to the SNAP-25 proteolyzing BoNT/A and E, VAMP/synaptobrevin-specific clostridial neurotoxins also initiate substrate interaction, employing an exosite located N-terminal of the scissile peptide bond.

  18. Aphicidal efficacy of scorpion- and spider-derived neurotoxins.

    PubMed

    Pal, Narinder; Yamamoto, Takashi; King, Glenn F; Waine, Clement; Bonning, Bryony

    2013-08-01

    Insect-specific neurotoxins that act within the insect hemocoel (body cavity) represent an untapped resource for insect pest management. On the basis of recent advances made in development of appropriate delivery systems for transport of these toxins from the insect gut, across the gut epithelium to their target site, we screened neurotoxins derived from scorpion or spider venom for efficacy against the pea aphid, Acyrthosiphon pisum, and the green peach aphid, Myzus persicae. Toxins were selected to represent different modes of electrophysiological action, including activity on voltage-gated calcium channels (ω-TRTX-Gr1a, ω-agatoxin Aa4a, ω-hexatoxin-Hv1a), calcium- and voltage-activated potassium channels (charybdotoxin, maurotoxin), chloride channels (chlorotoxin) and voltage-gated sodium channels (LqhαIT). The Bacillus thuringiensis-derived toxin Cyt1Aa was also tested as a positive control for toxicity. In per os bioassays with both aphid species, toxicity was only seen for ω-TRTX-Gr1a and Cyt1Aa. On injection into the hemocoel of A. pisum, LD₅₀ values ranged from 1 to 8 ng/mg body weight, with ω-hexatoxin-Hv1a being the most toxic (1.02 ng/mg body weight). All neurotoxins caused rapid paralysis, with charybdotoxin, maurotoxin and chlorotoxin also causing melanization of injected aphids. These data represent the first comprehensive screen of neurotoxins against aphids, and highlight the potential for practical use of the insect-specific toxin ω-hexatoxin-Hv1a in aphid management.

  19. Mass spectrometry-based methods for detection and differentiation of botulinum neurotoxins

    DOEpatents

    Schmidt, Jurgen G.; Boyer, Anne E.; Kalb, Suzanne R.; Moura, Hercules; Barr, John R.; Woolfitt, Adrian R.

    2009-11-03

    The present invention is directed to a method for detecting the presence of clostridial neurotoxins in a sample by mixing a sample with a peptide that can serve as a substrate for proteolytic activity of a clostridial neurotoxin; and measuring for proteolytic activity of a clostridial neurotoxin by a mass spectroscopy technique. In one embodiment, the peptide can have an affinity tag attached at two or more sites.

  20. Injectable neurotoxins and fillers: there is no free lunch.

    PubMed

    Emer, Jason; Waldorf, Heidi

    2011-01-01

    Injection of neurotoxins and filling agents for the treatment of facial aesthetics has increased dramatically during the past few decades due to an increased interest in noninvasive aesthetic improvements. An aging but still youth-oriented population expects effective treatments with minimal recovery time and limited risk of complications. Injectable neurotoxins and soft tissue stimulators and fillers have filled this niche of "lunch-time" procedures. As demand for these procedures has increased, supply has followed with more noncore cosmetic specialty physicians, as well as unsupervised ancillary staff, becoming providers and advertising them as easy fixes. Despite an excellent record of safety and efficacy demonstrated in scores of published studies, injectable agents do carry risks of complications. These procedures require a physician with in-depth knowledge of facial anatomy and injection techniques to ensure patient safety and satisfaction. In general, adverse events are preventable and technique-dependent. Although most adverse events are minor and temporary, more serious complications can occur. The recognition, management, and treatment of poor outcomes are as important as obtaining the best aesthetic results. This review addresses important considerations regarding the complications of injectable neurotoxins and fillers used for "lunch-time" injectable procedures.

  1. Sodium channels in presynaptic nerve terminals. Regulation by neurotoxins

    PubMed Central

    1980-01-01

    Regulation of Na+ channels by neurotoxins has been studied in pinched- off nerve endings (synaptosomes) from rat brain. Activation of Na+ channels by the steroid batrachotoxin and by the alkaloid veratridine resulted in an increase in the rate of influx of 22Na into the synaptosomes. In the presence of 145 mM Na+, these agents also depolarized the synaptosomes, as indicated by increased fluorescence in the presence of a voltage-sensitive oxacarbocyanine dye [diO-C5(3)]. Polypeptide neurotoxins from the scorpion Leiurus quinquestriatus and from the sea anemone Anthopleura xanthogrammica potentiated the stimulatory effects of batrachotoxin and veratridine on the influx of 22Na into synaptosomes. Saxitoxin and tetrodotoxin blocked the stimulatory effects of batrachotoxin and veratridine, both in the presence and absence of the polypeptide toxins, but did not affect control 22Na influx or resting membrane potential. A three-state model for Na+ channel operation can account for the effects of these neurotoxins on Na+ channels as determined both by Na+ flux measurements in vitro and by electrophysiological experiments in intact nerve and muscle. PMID:6252277

  2. Injectable neurotoxins and fillers: there is no free lunch.

    PubMed

    Emer, Jason; Waldorf, Heidi

    2011-01-01

    Injection of neurotoxins and filling agents for the treatment of facial aesthetics has increased dramatically during the past few decades due to an increased interest in noninvasive aesthetic improvements. An aging but still youth-oriented population expects effective treatments with minimal recovery time and limited risk of complications. Injectable neurotoxins and soft tissue stimulators and fillers have filled this niche of "lunch-time" procedures. As demand for these procedures has increased, supply has followed with more noncore cosmetic specialty physicians, as well as unsupervised ancillary staff, becoming providers and advertising them as easy fixes. Despite an excellent record of safety and efficacy demonstrated in scores of published studies, injectable agents do carry risks of complications. These procedures require a physician with in-depth knowledge of facial anatomy and injection techniques to ensure patient safety and satisfaction. In general, adverse events are preventable and technique-dependent. Although most adverse events are minor and temporary, more serious complications can occur. The recognition, management, and treatment of poor outcomes are as important as obtaining the best aesthetic results. This review addresses important considerations regarding the complications of injectable neurotoxins and fillers used for "lunch-time" injectable procedures. PMID:22014990

  3. Molding the business end of neurotoxins by diversifying evolution.

    PubMed

    Kozminsky-Atias, Adi; Zilberberg, Noam

    2012-02-01

    A diverse range of organisms utilize neurotoxins that target specific ion channels and modulate their activity. Typically, toxins are clustered into several multigene families, providing an organism with the upper hand in the never-ending predator-prey arms race. Several gene families, including those encoding certain neurotoxins, have been subject to diversifying selection forces, resulting in rapid gene evolution. Here we sought a spatial pattern in the distribution of both diversifying and purifying selection forces common to neurotoxin gene families. Utilizing the mechanistic empirical combination model, we analyzed various toxin families from different phyla affecting various receptors and relying on diverse modes of action. Through this approach, we were able to detect clear correlations between the pharmacological surface of a toxin and rapidly evolving domains, rich in positively selected residues. On the other hand, patches of negatively selected residues were restricted to the nontoxic face of the molecule and most likely help in stabilizing the tertiary structure of the toxin. We thus propose a mutual evolutionary strategy of venomous animals in which adaptive molecular evolution is directed toward the toxin active surface. Furthermore, we propose that the binding domains of unstudied toxins could be readily predicted using evolutionary considerations.

  4. The effect of neurotoxin on rabies virus binding to mouse neuroblastoma cells.

    PubMed

    Briggs, D J; Phillips, R M

    1991-08-01

    Mouse neuroblastoma cells were exposed to alpha bungarotoxin, a neurotoxin known to inhibit rabies virus binding to the nicotinic acetylcholine receptor located at the neuromuscular junction in muscle tissue. The total amount of 3H-CVS virus that bound to neurotoxin treated cells was separated into specific and non-specific binding using a cold competition assay. Comparison of untreated and neurotoxin treated cells demonstrated that the majority of cell-associated virus in untreated cells was of a specific nature whereas the majority of the cell-associated virus in neurotoxin treated cells was due to non-specific binding.

  5. Rapid Microfluidic Assay for the Detection of Botulinum Neurotoxin in Animal Sera

    PubMed Central

    Babrak, Lmar; Lin, Alice; Stanker, Larry H.; McGarvey, Jeffery; Hnasko, Robert

    2016-01-01

    Potent Botulinum neurotoxins (BoNTs) represent a threat to public health and safety. Botulism is a disease caused by BoNT intoxication that results in muscle paralysis that can be fatal. Sensitive assays capable of detecting BoNTs from different substrates and settings are essential to limit foodborne contamination and morbidity. In this report, we describe a rapid 96-well microfluidic double sandwich immunoassay for the sensitive detection of BoNT-A from animal sera. This BoNT microfluidic assay requires only 5 μL of serum, provides results in 75 min using a standard fluorescence microplate reader and generates minimal hazardous waste. The assay has a <30 pg·mL−1 limit of detection (LOD) of BoNT-A from spiked human serum. This sensitive microfluidic BoNT-A assay offers a fast and simplified workflow suitable for the detection of BoNT-A from serum samples of limited volume in most laboratory settings. PMID:26742073

  6. Use of Monoclonal Antibodies in the Sensitive Detection and Neutralization of Botulinum Neurotoxin Serotype B.

    PubMed

    Cheng, Luisa W; Henderson, Thomas D; Lam, Tina I; Stanker, Larry H

    2015-12-01

    Botulinum neurotoxins (BoNT) are some of nature's most potent toxins. Due to potential food contamination, and bioterrorism concerns, the development of detection reagents, therapeutics and countermeasures are of urgent interest. Recently, we have developed a sensitive electrochemiluminescent (ECL) immunoassay for BoNT/B, using monoclonal antibodies (mAbs) MCS6-27 and anti-BoNT/B rabbit polyclonal antibodies as the capture and detector. The ECL assay detected as little as 1 pg/mL BoNT/B in the buffer matrix, surpassing the detection sensitivities of the gold standard mouse bioassays. The ECL assay also allowed detection of BoNT/B in sera matrices of up to 100% sera with negligible matrix effects. This highly-sensitive assay allowed the determination of the biological half-lives of BoNT/B holotoxin in vivo. We further tested the toxin neutralization potential of our monoclonal antibodies using the mouse systemic and oral intoxication models. A combination of mAbs protected mice in both pre- and post-exposure models to lethal doses of BoNT/B. MAbs were capable of increasing survival of animals when administered even 10 h post-intoxication in an oral model, suggesting a likely time for BoNT/B complexes to reach the blood stream. More sensitive detection assays and treatments against BoNT intoxication will greatly enhance efforts to combat botulism.

  7. Searching for Therapeutics Against Botulinum Neurotoxins: A True Challenge for Drug Discovery.

    PubMed

    Duplantier, Allen J; Kane, Christopher D; Bavari, Sina

    2016-01-01

    Botulinum neurotoxins (BoNTs), the most potent known toxins, cause severe muscle paralysis and death at nanogram exposures and are considered biothreat agents. BoNTs target the neuromuscular junction where they release smaller zinc metalloprotease light chains (LCs) into the neuron cytosol that selectively cleave SNARE proteins and thus block the exocytosis of acetylcholine neurotransmitters necessary for skeletal muscle contraction. The majority of efforts to develop post-symptomatic therapeutics for botulism poisoning have focused on inhibiting the LC and tremendous strides have been made in understanding how the LC binds to the SNARE proteins via X-ray crystallography. Subsequent homology modeling and structure based drug design have led to the discovery of multiple small molecule BoNT/A inhibitors in the 0.05 ~10 μΜ range, but to date none have shown significant post-symptomatic efficacy in an animal model of botulinum intoxication. With the lack of reported pharmacokinetic data, we have analyzed the BoNT/A inhibitor lead chemical matter from a physicochemical property point of view and have attempted to understand if bioavailability of drug at the neuromuscular junction is the root cause of this apparent in vitro/in vivo disconnect in the field. PMID:27072693

  8. Structure of a Bimodular Botulinum Neurotoxin Complex Provides Insights into Its Oral Toxicity

    PubMed Central

    Jin, Lei; Le, Thi Tuc Nghi; Cheng, Luisa W.; Strotmeier, Jasmin; Kruel, Anna Magdalena; Yao, Guorui; Perry, Kay; Rummel, Andreas; Jin, Rongsheng

    2013-01-01

    Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum and cause the fatal disease botulism, a flaccid paralysis of the muscle. BoNTs are released together with several auxiliary proteins as progenitor toxin complexes (PTCs) to become highly potent oral poisons. Here, we report the structure of a ∼760 kDa 14-subunit large PTC of serotype A (L-PTC/A) and reveal insight into its absorption mechanism. Using a combination of X-ray crystallography, electron microscopy, and functional studies, we found that L-PTC/A consists of two structurally and functionally independent sub-complexes. A hetero-dimeric 290 kDa complex protects BoNT, while a hetero-dodecameric 470 kDa complex facilitates its absorption in the harsh environment of the gastrointestinal tract. BoNT absorption is mediated by nine glycan-binding sites on the dodecameric sub-complex that forms multivalent interactions with carbohydrate receptors on intestinal epithelial cells. We identified monosaccharides that blocked oral BoNT intoxication in mice, which suggests a new strategy for the development of preventive countermeasures for BoNTs based on carbohydrate receptor mimicry. PMID:24130488

  9. Use of Monoclonal Antibodies in the Sensitive Detection and Neutralization of Botulinum Neurotoxin Serotype B

    PubMed Central

    Cheng, Luisa W.; Henderson, Thomas D.; Lam, Tina I.; Stanker, Larry H.

    2015-01-01

    Botulinum neurotoxins (BoNT) are some of nature’s most potent toxins. Due to potential food contamination, and bioterrorism concerns, the development of detection reagents, therapeutics and countermeasures are of urgent interest. Recently, we have developed a sensitive electrochemiluminescent (ECL) immunoassay for BoNT/B, using monoclonal antibodies (mAbs) MCS6-27 and anti-BoNT/B rabbit polyclonal antibodies as the capture and detector. The ECL assay detected as little as 1 pg/mL BoNT/B in the buffer matrix, surpassing the detection sensitivities of the gold standard mouse bioassays. The ECL assay also allowed detection of BoNT/B in sera matrices of up to 100% sera with negligible matrix effects. This highly-sensitive assay allowed the determination of the biological half-lives of BoNT/B holotoxin in vivo. We further tested the toxin neutralization potential of our monoclonal antibodies using the mouse systemic and oral intoxication models. A combination of mAbs protected mice in both pre- and post-exposure models to lethal doses of BoNT/B. MAbs were capable of increasing survival of animals when administered even 10 h post-intoxication in an oral model, suggesting a likely time for BoNT/B complexes to reach the blood stream. More sensitive detection assays and treatments against BoNT intoxication will greatly enhance efforts to combat botulism. PMID:26633496

  10. Use of Monoclonal Antibodies in the Sensitive Detection and Neutralization of Botulinum Neurotoxin Serotype B.

    PubMed

    Cheng, Luisa W; Henderson, Thomas D; Lam, Tina I; Stanker, Larry H

    2015-12-01

    Botulinum neurotoxins (BoNT) are some of nature's most potent toxins. Due to potential food contamination, and bioterrorism concerns, the development of detection reagents, therapeutics and countermeasures are of urgent interest. Recently, we have developed a sensitive electrochemiluminescent (ECL) immunoassay for BoNT/B, using monoclonal antibodies (mAbs) MCS6-27 and anti-BoNT/B rabbit polyclonal antibodies as the capture and detector. The ECL assay detected as little as 1 pg/mL BoNT/B in the buffer matrix, surpassing the detection sensitivities of the gold standard mouse bioassays. The ECL assay also allowed detection of BoNT/B in sera matrices of up to 100% sera with negligible matrix effects. This highly-sensitive assay allowed the determination of the biological half-lives of BoNT/B holotoxin in vivo. We further tested the toxin neutralization potential of our monoclonal antibodies using the mouse systemic and oral intoxication models. A combination of mAbs protected mice in both pre- and post-exposure models to lethal doses of BoNT/B. MAbs were capable of increasing survival of animals when administered even 10 h post-intoxication in an oral model, suggesting a likely time for BoNT/B complexes to reach the blood stream. More sensitive detection assays and treatments against BoNT intoxication will greatly enhance efforts to combat botulism. PMID:26633496

  11. Translocation and dissemination to target neurons of botulinum neurotoxin type B in the mouse intestinal wall.

    PubMed

    Connan, Chloé; Varela-Chavez, Carolina; Mazuet, Christelle; Molgó, Jordi; Haustant, Georges Michel; Disson, Olivier; Lecuit, Marc; Vandewalle, Alain; Popoff, Michel R

    2016-02-01

    Botulinum neurotoxins (BoNTs) are responsible for severe flaccid paralysis (botulism), which in most cases enter the organism via the digestive tract and then disseminate into the blood or lymph circulation to target autonomic and motor nerve endings. The passage way of BoNTs alone or in complex forms with associated nontoxic proteins through the epithelial barrier of the digestive tract still remains unclear. Here, we show using an in vivo model of mouse ligated intestinal loop that BoNT/B alone or the BoNT/B C-terminal domain of the heavy chain (HCcB), which interacts with cell surface receptors, translocates across the intestinal barrier. The BoNT/B or HCcB translocation through the intestinal barrier occurred via an endocytosis-dependent mechanism within 10-20 min, because Dynasore, a potent endocytosis inhibitor, significantly prevented BoNT/B as well as HCcB translocation. We also show that HCcB or BoNT/B specifically targets neuronal cells and neuronal extensions in the intestinal submucosa and musculosa expressing synaptotagmin, preferentially cholinergic neurons and to a lower extent other neuronal cell types, notably serotonergic neurons. Interestingly, rare intestinal epithelial cells accumulated HCcB suggesting that distinct cell types of the intestinal epithelium, still undefined, might mediate efficient translocation of BoNT/B. PMID:26294282

  12. Rapid Microfluidic Assay for the Detection of Botulinum Neurotoxin in Animal Sera.

    PubMed

    Babrak, Lmar; Lin, Alice; Stanker, Larry H; McGarvey, Jeffery; Hnasko, Robert

    2016-01-01

    Potent Botulinum neurotoxins (BoNTs) represent a threat to public health and safety. Botulism is a disease caused by BoNT intoxication that results in muscle paralysis that can be fatal. Sensitive assays capable of detecting BoNTs from different substrates and settings are essential to limit foodborne contamination and morbidity. In this report, we describe a rapid 96-well microfluidic double sandwich immunoassay for the sensitive detection of BoNT-A from animal sera. This BoNT microfluidic assay requires only 5 μL of serum, provides results in 75 min using a standard fluorescence microplate reader and generates minimal hazardous waste. The assay has a <30 pg·mL(-1) limit of detection (LOD) of BoNT-A from spiked human serum. This sensitive microfluidic BoNT-A assay offers a fast and simplified workflow suitable for the detection of BoNT-A from serum samples of limited volume in most laboratory settings. PMID:26742073

  13. Epitope Characterization of Sero-Specific Monoclonal Antibody to Clostridium botulinum Neurotoxin Type A

    PubMed Central

    Ballegeer, Erin; Weedmark, Kelly A.; Elias, M.D.; Al-Saleem, Fetweh H.; Ancharski, Denise M.; Simpson, Lance L.; Berry, Jody D.

    2011-01-01

    Botulinum neurotoxins (BoNTs) are extremely potent toxins that can contaminate foods and are a public health concern. Anti-BoNT antibodies have been described that are capable of detecting BoNTs; however there still exists a need for accurate and sensitive detection capabilities for BoNTs. Herein, we describe the characterization of a panel of eight monoclonal antibodies (MAbs) generated to the non-toxic receptor-binding domain of BoNT/A (HC50/A) developed using a high-throughput screening approach. In two independent hybridoma fusions, two groups of four IgG MAbs were developed against recombinant HC50/A. Of these eight, only a single MAb, F90G5-3, bound to the whole BoNT/A protein and was characterized further. The F90G5-3 MAb slightly prolonged time to death in an in vivo mouse bioassay and was mapped by pepscan to a peptide epitope in the N-terminal subdomain of HC50/A (HCN25/A) comprising amino acid residues 985WTLQDTQEIKQRVVF999, an epitope that is highly immunoreactive in humans. Furthermore, we demonstrate that F90G5-3 binds BoNT/A with nanomolar efficiency. Together, our results indicate that F90G5-3 is of potential value as a diagnostic immunoreagent for BoNT/A capture assay development and bio-forensic analysis. PMID:22149274

  14. Current methods for detecting the presence of botulinum neurotoxins in food and other biological samples

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Current methods for detecting the presence of botulinum neurotoxins in food and other biological samples Botulinum neurotoxins (BoNTs), the causative agents of botulism, are among the most lethal human bacterial toxins and the causative agent of botulism. BoNTs are also classified as Select Agents ...

  15. Clostridium botulinum neurotoxin type B is heat-stable in milk and not inactivated by pasteurization

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Foodborne botulism is caused by the ingestion of foods containing botulinum neurotoxins (BoNTs). Currently, the only accepted assay to detect active C. botulinum neurotoxin is an in vivo mouse bioassay, which raises ethical concerns with regard to the use of experimental animals. Therefore, there is...

  16. Cloning and purification of alpha-neurotoxins from king cobra (Ophiophagus hannah).

    PubMed

    He, Ying-Ying; Lee, Wei-Hui; Zhang, Yun

    2004-09-01

    Thirteen complete and three partial cDNA sequences were cloned from the constructed king cobra (Ophiophagus hannah) venom gland cDNA library. Phylogenetic analysis of nucleotide sequences of king cobra with those from other snake venoms revealed that obtained cDNAs are highly homologous to snake venom alpha-neurotoxins. Alignment of deduced mature peptide sequences of the obtained clones with those of other reported alpha-neurotoxins from the king cobra venom indicates that our obtained 16 clones belong to long-chain neurotoxins (seven), short-chain neurotoxins (seven), weak toxin (one) and variant (one), respectively. Up to now, two out of 16 newly cloned king cobra alpha-neurotoxins have identical amino acid sequences with CM-11 and Oh-6A/6B, which have been characterized from the same venom. Furthermore, five long-chain alpha-neurotoxins and two short-chain alpha-neurotoxins were purified from crude venom and their N-terminal amino acid sequences were determined. The cDNAs encoding the putative precursors of the purified native peptide were also determined based on the N-terminal amino acid sequencing. The purified alpha-neurotoxins showed different lethal activities on mice. PMID:15302536

  17. Cloning and purification of alpha-neurotoxins from king cobra (Ophiophagus hannah).

    PubMed

    He, Ying-Ying; Lee, Wei-Hui; Zhang, Yun

    2004-09-01

    Thirteen complete and three partial cDNA sequences were cloned from the constructed king cobra (Ophiophagus hannah) venom gland cDNA library. Phylogenetic analysis of nucleotide sequences of king cobra with those from other snake venoms revealed that obtained cDNAs are highly homologous to snake venom alpha-neurotoxins. Alignment of deduced mature peptide sequences of the obtained clones with those of other reported alpha-neurotoxins from the king cobra venom indicates that our obtained 16 clones belong to long-chain neurotoxins (seven), short-chain neurotoxins (seven), weak toxin (one) and variant (one), respectively. Up to now, two out of 16 newly cloned king cobra alpha-neurotoxins have identical amino acid sequences with CM-11 and Oh-6A/6B, which have been characterized from the same venom. Furthermore, five long-chain alpha-neurotoxins and two short-chain alpha-neurotoxins were purified from crude venom and their N-terminal amino acid sequences were determined. The cDNAs encoding the putative precursors of the purified native peptide were also determined based on the N-terminal amino acid sequencing. The purified alpha-neurotoxins showed different lethal activities on mice.

  18. Translocation of botulinum neurotoxin serotype a and associated proteins across the intestinal epithelia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Botulinum neurotoxins (BoNTs) are some of the most poisonous natural toxins and considered to be a major venue of bioterrorist threat. BoNTs associate with neurotoxin associated proteins (NAPs), forming large complexes. NAPs have been shown to shield the BoNT holotoxin from the harsh environment of ...

  19. Marine Toxins Potently Affecting Neurotransmitter Release

    NASA Astrophysics Data System (ADS)

    Meunier, Frédéric A.; Mattei, César; Molgó, Jordi

    Synapses are specialised structures where interneuronal communication takes place. Not only brain function is absolutely dependent on synaptic activity, but also most of our organs are intimately controlled by synaptic activity. Synapses re therefore an ideal target to act upon and poisonous species have evolved fascinating neurotoxins capable of shutting down neuronal communication by blocking or activating essential components of the synapse. By hijacking key proteins of the communication machinery, neurotoxins are therefore extremely valuable tools that have, in turn, greatly helped our understanding of synaptic biology. Moreover, analysis and understanding of the molecular strategy used by certain neurotoxins has allowed the design of entirely new classes of drugs acting on specific targets with high selectivity and efficacy. This chapter will discuss the different classes of marine neurotoxins, their effects on neurotransmitter release and how they act to incapacitate key steps in the process leading to synaptic vesicle fusion.

  20. Purification and characterization of neurotoxin complex from a dual toxin gene containing Clostridium botulinum strain PS-5

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Botulinum neurotoxins (BoNTs) are produced as a toxin complex (TC) which consists of neurotoxin (NT) and neurotoxin associated proteins (NAPs). The characterization of NT in its native state is an essential step for developing diagnostics and therapeutic countermeasures against botulism. The presenc...

  1. Molecular Structures and Functional Relationships in Clostridial Neurotoxins

    SciTech Connect

    Swaminathan S.

    2011-12-01

    The seven serotypes of Clostridium botulinum neurotoxins (A-G) are the deadliest poison known to humans. They share significant sequence homology and hence possess similar structure-function relationships. Botulinum neurotoxins (BoNT) act via a four-step mechanism, viz., binding and internalization to neuronal cells, translocation of the catalytic domain into the cytosol and finally cleavage of one of the three soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) causing blockage of neurotransmitter release leading to flaccid paralysis. Crystal structures of three holotoxins, BoNT/A, B and E, are available to date. Although the individual domains are remarkably similar, their domain organization is different. These structures have helped in correlating the structural and functional domains. This has led to the determination of structures of individual domains and combinations of them. Crystal structures of catalytic domains of all serotypes and several binding domains are now available. The catalytic domains are zinc endopeptidases and share significant sequence and structural homology. The active site architecture and the catalytic mechanism are similar although the binding mode of individual substrates may be different, dictating substrate specificity and peptide cleavage selectivity. Crystal structures of catalytic domains with substrate peptides provide clues to specificity and selectivity unique to BoNTs. Crystal structures of the receptor domain in complex with ganglioside or the protein receptor have provided information about the binding of botulinum neurotoxin to the neuronal cell. An overview of the structure-function relationship correlating the 3D structures with biochemical and biophysical data and how they can be used for structure-based drug discovery is presented here.

  2. Different mechanisms of inhibition of nerve terminals by botulinum and snake presynaptic neurotoxins.

    PubMed

    Montecucco, Cesare; Rossetto, Ornella; Caccin, Paola; Rigoni, Michela; Carli, Luca; Morbiato, Laura; Muraro, Lucia; Paoli, Marco

    2009-10-01

    The different mode of action on peripheral nerve terminals of the botulinum neurotoxins and of the snake presynaptic phospholipase A2 neurotoxins is reviewed here. These two groups of toxins are highly toxic because they are neurospecific and at the same time are enzymes that can modify many substrate molecules before being inactivated. The similarity of symptoms they cause in humans derives from the fact that both botulinum neurotoxins (seven serotypes named A-G) and snake presynaptic PLA2 neurotoxins block the nerve terminals and that peripheral cholinergic terminals are major targets. Given this general similarity of targets and clinical symptoms, the specific molecular and cellular mechanisms at the basis of their action are very different. This difference appears evident from the beginning of intoxication, i.e. neurotoxins binding to peripheral nerve terminals and proceeds with the different site of actions and molecular targets.

  3. Sensing the Deadliest Toxin: Technologies for Botulinum Neurotoxin Detection

    PubMed Central

    Čapek, Petr; Dickerson, Tobin J.

    2010-01-01

    Sensitive and rapid detection of botulinum neurotoxins (BoNTs), the most poisonous substances known to date, is essential for studies of medical applications of BoNTs and detection of poisoned food, as well as for response to potential bioterrorist threats. Currently, the most common method of BoNT detection is the mouse bioassay. While this assay is sensitive, it is slow, quite expensive, has limited throughput and requires sacrificing animals. Herein, we discuss and compare recently developed alternative in vitro detection methods and assess their ability to supplement or replace the mouse bioassay in the analysis of complex matrix samples. PMID:22069545

  4. Management of Frey syndrome using botulinum neurotoxin: a case report.

    PubMed

    Ng, Samson; Torjek, Cathy; Hovan, Allan

    2009-11-01

    Frey syndrome is manifested clinically by hemifacial flushing and sweating after a gustatory stimulus. Frey syndrome is usually secondary to traumatic injury in the parotid region and is thought to be the result of misdirected re-sprouting of damaged autonomic nerve fibres. In this case report, we highlight the clinical and psychosocial aspects of Frey syndrome from a patient"s perspective, outline the pathophysiology of the condition and current management strategies, and describe the use of botulinum neurotoxin in the treatment of Frey syndrome. PMID:19900355

  5. Serological identification of botulinum neurotoxins: A critical overview.

    PubMed

    Giménez, Domingo F

    2016-08-01

    The reasons that gave rise to the controversy over the serological method (SerM) and genetics regarding the identification of an alleged novel botulinum neurotoxin (BoNT), type H, have been concisely examined. This discussion will remain opened inasmuch as the SerM is not performed according to the recommended procedures outlined in this overview and thoroughly discussed on previous publications. If correctly performed and interpreted, the SerM will keep its preeminence in the identification, typing and taxonomy of BoNTs. PMID:27130373

  6. Labile neurotoxin in serum of calves with "nervous" coccidiosis.

    PubMed Central

    Isler, C M; Bellamy, J E; Wobeser, G A

    1987-01-01

    Mouse inoculation was used to test for the presence of a toxin in the serum, cerebrospinal fluid, and intestinal contents collected from cases of bovine enteric coccidiosis, with and without neurological signs, and from control calves. Intravenous inoculation of mice with 10 mL/kg of serum from calves showing nervous signs caused effects significantly different from those caused by the inoculation of serum from calves not showing nervous signs and from control calves. The effect was particularly evident in female mice. At this dosage severe neurological signs such as loss of righting reflex, seizures and death occurred only with serum from calves with "nervous coccidiosis". The results suggest that serum from the calves with neurological signs contains a neurotoxin. This toxin appears to be highly labile. It was not present in the cerebrospinal fluid at levels comparable to those in the serum. The significance of this labile neurotoxin with respect to the pathogenesis of the neurological signs associated with bovine enteric coccidiosis is unknown. PMID:2955865

  7. Botulinum neurotoxin A and an engineered derivate targeted secretion inhibitor (TSI) A enter cells via different vesicular compartments.

    PubMed

    Fonfria, Elena; Donald, Sarah; Cadd, Verity A

    2016-01-01

    Botulinum neurotoxins (BoNTs) are highly potent multi-domain proteins, responsible for botulism in animals and humans. The modular structural organization of BoNTs has led to the development of novel engineered bio-therapeutic proteins called targeted secretion inhibitors (TSIs). We report here that botulinum neurotoxin A (BoNT/A) and a TSI/A in which the neuronal binding domain of BoNT/A has been substituted by an epidermal growth factor (EGF) ligand, named EGFR-targeted TSI/A, exploit different routes to gain entry in the same in vitro neuroblastoma cell system, SiMa cells. We found that the EGF ligand conferred the affinity to the EGFR-targeted TSI/A at the EGF receptor when compared to an untargeted TSI/A and also the ability to internalize into the cells and cleave its cytosolic target protein SNAP-25. Using high content analysis we found that both BoNT/A and the EGFR-targeted TSI/A enter the cell in a concentration-dependent manner and in compartments which are able to translocate the proteins into the cytosol within 4 h. The EGFR-targeted TSI/A internalized into a compartment which gave a punctate staining pattern by immunofluorescence and partially overlapped with structures positive for the early endosomal marker EAA1; whereas BoNT/A did not internalize into a punctate compartment but did so in an acidifying compartment consistent with local synaptic vesicle recycling. These findings show that the BoNT/A translocation domain, common to both BoNT/A and the EGFR-targeted TSI/A, is a versatile tool for cytosolic delivery from distinct intracellular vesicular compartments. PMID:26329879

  8. Discovery of Nuclear-Encoded Genes for the Neurotoxin Saxitoxin in Dinoflagellates

    PubMed Central

    Stüken, Anke; Orr, Russell J. S.; Kellmann, Ralf; Murray, Shauna A.; Neilan, Brett A.; Jakobsen, Kjetill S.

    2011-01-01

    Saxitoxin is a potent neurotoxin that occurs in aquatic environments worldwide. Ingestion of vector species can lead to paralytic shellfish poisoning, a severe human illness that may lead to paralysis and death. In freshwaters, the toxin is produced by prokaryotic cyanobacteria; in marine waters, it is associated with eukaryotic dinoflagellates. However, several studies suggest that saxitoxin is not produced by dinoflagellates themselves, but by co-cultured bacteria. Here, we show that genes required for saxitoxin synthesis are encoded in the nuclear genomes of dinoflagellates. We sequenced >1.2×106 mRNA transcripts from the two saxitoxin-producing dinoflagellate strains Alexandrium fundyense CCMP1719 and A. minutum CCMP113 using high-throughput sequencing technology. In addition, we used in silico transcriptome analyses, RACE, qPCR and conventional PCR coupled with Sanger sequencing. These approaches successfully identified genes required for saxitoxin-synthesis in the two transcriptomes. We focused on sxtA, the unique starting gene of saxitoxin synthesis, and show that the dinoflagellate transcripts of sxtA have the same domain structure as the cyanobacterial sxtA genes. But, in contrast to the bacterial homologs, the dinoflagellate transcripts are monocistronic, have a higher GC content, occur in multiple copies, contain typical dinoflagellate spliced-leader sequences and eukaryotic polyA-tails. Further, we investigated 28 saxitoxin-producing and non-producing dinoflagellate strains from six different genera for the presence of genomic sxtA homologs. Our results show very good agreement between the presence of sxtA and saxitoxin-synthesis, except in three strains of A. tamarense, for which we amplified sxtA, but did not detect the toxin. Our work opens for possibilities to develop molecular tools to detect saxitoxin-producing dinoflagellates in the environment. PMID:21625593

  9. Monoclonal Antibodies that Inhibit the Proteolytic Activity of Botulinum Neurotoxin Serotype/B.

    PubMed

    Fan, Yongfeng; Dong, Jianbo; Lou, Jianlong; Wen, Weihua; Conrad, Fraser; Geren, Isin N; Garcia-Rodriguez, Consuelo; Smith, Theresa J; Smith, Leonard A; Ho, Mengfei; Pires-Alves, Melissa; Wilson, Brenda A; Marks, James D

    2015-09-01

    Existing antibodies (Abs) used to treat botulism cannot enter the cytosol of neurons and bind to botulinum neurotoxin (BoNT) at its site of action, and thus cannot reverse paralysis. However, Abs targeting the proteolytic domain of the toxin could inhibit the proteolytic activity of the toxin intracellularly and potentially reverse intoxication, if they could be delivered intracellularly. As such, antibodies that neutralize toxin activity could serve as potent inhibitory cargos for therapeutic antitoxins against botulism. BoNT serotype B (BoNT/B) contains a zinc endopeptidase light chain (LC) domain that cleaves synaoptobrevin-2, a SNARE protein responsible for vesicle fusion and acetylcholine vesicle release. To generate monoclonal Abs (mAbs) that could reverse paralysis, we targeted the protease domain for Ab generation. Single-chain variable fragment (scFv) libraries from immunized mice or humans were displayed on yeast, and 19 unique BoNT/B LC-specific mAbs isolated by fluorescence-activated cell sorting (FACS). The equilibrium dissociation constants (KD) of these mAbs for BoNT/B LC ranged from 0.24 nM to 14.3 nM (mean KD 3.27 nM). Eleven mAbs inhibited BoNT/B LC proteolytic activity. The fine epitopes of selected mAbs were identified by alanine-scanning mutagenesis, revealing that inhibitory mAbs bound near the active site, substrate-binding site or the extended substrate-binding site. The results provide mAbs that could prove useful for intracellular reversal of paralysis and identify epitopes that could be targeted by small molecules inhibitors.

  10. Monoclonal Antibodies that Inhibit the Proteolytic Activity of Botulinum Neurotoxin Serotype/B

    PubMed Central

    Fan, Yongfeng; Dong, Jianbo; Lou, Jianlong; Wen, Weihua; Conrad, Fraser; Geren, Isin N.; Garcia-Rodriguez, Consuelo; Smith, Theresa J.; Smith, Leonard A.; Ho, Mengfei; Pires-Alves, Melissa; Wilson, Brenda A.; Marks, James D.

    2015-01-01

    Existing antibodies (Abs) used to treat botulism cannot enter the cytosol of neurons and bind to botulinum neurotoxin (BoNT) at its site of action, and thus cannot reverse paralysis. However, Abs targeting the proteolytic domain of the toxin could inhibit the proteolytic activity of the toxin intracellularly and potentially reverse intoxication, if they could be delivered intracellularly. As such, antibodies that neutralize toxin activity could serve as potent inhibitory cargos for therapeutic antitoxins against botulism. BoNT serotype B (BoNT/B) contains a zinc endopeptidase light chain (LC) domain that cleaves synaoptobrevin-2, a SNARE protein responsible for vesicle fusion and acetylcholine vesicle release. To generate monoclonal Abs (mAbs) that could reverse paralysis, we targeted the protease domain for Ab generation. Single-chain variable fragment (scFv) libraries from immunized mice or humans were displayed on yeast, and 19 unique BoNT/B LC-specific mAbs isolated by fluorescence-activated cell sorting (FACS). The equilibrium dissociation constants (KD) of these mAbs for BoNT/B LC ranged from 0.24 nM to 14.3 nM (mean KD 3.27 nM). Eleven mAbs inhibited BoNT/B LC proteolytic activity. The fine epitopes of selected mAbs were identified by alanine-scanning mutagenesis, revealing that inhibitory mAbs bound near the active site, substrate-binding site or the extended substrate-binding site. The results provide mAbs that could prove useful for intracellular reversal of paralysis and identify epitopes that could be targeted by small molecules inhibitors. PMID:26343720

  11. Widespread Sequence Variations in VAMP1 across Vertebrates Suggest a Potential Selective Pressure from Botulinum Neurotoxins

    PubMed Central

    Peng, Lisheng; Adler, Michael; Demogines, Ann; Borrell, Andrew; Liu, Huisheng; Tao, Liang; Tepp, William H.; Zhang, Su-Chun; Johnson, Eric A.; Sawyer, Sara L.; Dong, Min

    2014-01-01

    Botulinum neurotoxins (BoNT/A-G), the most potent toxins known, act by cleaving three SNARE proteins required for synaptic vesicle exocytosis. Previous studies on BoNTs have generally utilized the major SNARE homologues expressed in brain (VAMP2, syntaxin 1, and SNAP-25). However, BoNTs target peripheral motor neurons and cause death by paralyzing respiratory muscles such as the diaphragm. Here we report that VAMP1, but not VAMP2, is the SNARE homologue predominantly expressed in adult rodent diaphragm motor nerve terminals and in differentiated human motor neurons. In contrast to the highly conserved VAMP2, BoNT-resistant variations in VAMP1 are widespread across vertebrates. In particular, we identified a polymorphism at position 48 of VAMP1 in rats, which renders VAMP1 either resistant (I48) or sensitive (M48) to BoNT/D. Taking advantage of this finding, we showed that rat diaphragms with I48 in VAMP1 are insensitive to BoNT/D compared to rat diaphragms with M48 in VAMP1. This unique intra-species comparison establishes VAMP1 as a physiological toxin target in diaphragm motor nerve terminals, and demonstrates that the resistance of VAMP1 to BoNTs can underlie the insensitivity of a species to members of BoNTs. Consistently, human VAMP1 contains I48, which may explain why humans are insensitive to BoNT/D. Finally, we report that residue 48 of VAMP1 varies frequently between M and I across seventeen closely related primate species, suggesting a potential selective pressure from members of BoNTs for resistance in vertebrates. PMID:25010769

  12. Botulinum Neurotoxin Type A Induces TLR2-Mediated Inflammatory Responses in Macrophages

    PubMed Central

    Kim, Yun Jeong; Kim, Jeong-Hee; Lee, Kwang-Jun; Choi, Myung-Min; Kim, Yeon Hee; Rhie, Gi-eun; Yoo, Cheon-Kwon; Cha, Kiweon; Shin, Na-Ri

    2015-01-01

    Botulinum neurotoxin type A (BoNT/A) is the most potent protein toxin and causes fatal flaccid muscle paralysis by blocking neurotransmission. Application of BoNT/A has been extended to the fields of therapeutics and biodefense. Nevertheless, the global response of host immune cells to authentic BoNT/A has not been reported. Employing microarray analysis, we performed global transcriptional profiling of RAW264.7 cells, a murine alveolar macrophage cell line. We identified 70 genes that were modulated following 1 nM BoNT/A treatment. The altered genes were mainly involved in signal transduction, immunity and defense, protein metabolism and modification, neuronal activities, intracellular protein trafficking, and muscle contraction. Microarray data were validated with real-time RT-PCR for seven selected genes including tlr2, tnf, inos, ccl4, slpi, stx11, and irg1. Proinflammatory mediators such as nitric oxide (NO) and tumor necrosis factor alpha (TNFα) were induced in a dose-dependent manner in BoNT/A-stimulated RAW264.7 cells. Increased expression of these factors was inhibited by monoclonal anti-Toll-like receptor 2 (TLR2) and inhibitors specific to intracellular proteins such as c-Jun N-terminal kinase (JNK), extracellular signal–regulated kinase (ERK), and p38 mitogen–activated protein kinase (MAPK). BoNT/A also suppressed lipopolysaccharide-induced NO and TNFα production from RAW264.7 macrophages at the transcription level by blocking activation of JNK, ERK, and p38 MAPK. As confirmed by TLR2-/- knock out experiments, these results suggest that BoNT/A induces global gene expression changes in host immune cells and that host responses to BoNT/A proceed through a TLR2-dependent pathway, which is modulated by JNK, ERK, and p38 MAPK. PMID:25853816

  13. Toxic and nontoxic components of botulinum neurotoxin complex are evolved from a common ancestral zinc protein

    SciTech Connect

    Inui, Ken; Sagane, Yoshimasa; Miyata, Keita; Miyashita, Shin-Ichiro; Suzuki, Tomonori; Shikamori, Yasuyuki; Ohyama, Tohru; Niwa, Koichi; Watanabe, Toshihiro

    2012-03-16

    Highlights: Black-Right-Pointing-Pointer BoNT and NTNHA proteins share a similar protein architecture. Black-Right-Pointing-Pointer NTNHA and BoNT were both identified as zinc-binding proteins. Black-Right-Pointing-Pointer NTNHA does not have a classical HEXXH zinc-coordinating motif similar to that found in all serotypes of BoNT. Black-Right-Pointing-Pointer Homology modeling implied probable key residues involved in zinc coordination. -- Abstract: Zinc atoms play an essential role in a number of enzymes. Botulinum neurotoxin (BoNT), the most potent toxin known in nature, is a zinc-dependent endopeptidase. Here we identify the nontoxic nonhemagglutinin (NTNHA), one of the BoNT-complex constituents, as a zinc-binding protein, along with BoNT. A protein structure classification database search indicated that BoNT and NTNHA share a similar domain architecture, comprising a zinc-dependent metalloproteinase-like, BoNT coiled-coil motif and concanavalin A-like domains. Inductively coupled plasma-mass spectrometry analysis demonstrated that every single NTNHA molecule contains a single zinc atom. This is the first demonstration of a zinc atom in this protein, as far as we know. However, the NTNHA molecule does not possess any known zinc-coordinating motif, whereas all BoNT serotypes possess the classical HEXXH motif. Homology modeling of the NTNHA structure implied that a consensus K-C-L-I-K-X{sub 35}-D sequence common among all NTNHA serotype molecules appears to coordinate a single zinc atom. These findings lead us to propose that NTNHA and BoNT may have evolved distinct functional specializations following their branching out from a common ancestral zinc protein.

  14. Novel therapeutics based on recombinant botulinum neurotoxins to normalize the release of transmitters and pain mediators.

    PubMed

    Dolly, J Oliver; Wang, Jiafu; Zurawski, Tomas H; Meng, Jianghui

    2011-12-01

    A major unmet clinical need exists for long-acting neurotherapeutics to alleviate chronic pain in patients unresponsive to available nonaddictive analgesics. Herein, a new strategy is described for the development of potent and specific inhibitors of the neuronal exocytosis of transmitters and pain mediators that exhibit unique antinociceptive activity. This entailed recombinant production in Escherichia coli of two serotypes of botulinum neurotoxin (BoNT) (BoNT(A) and BoNT(E) ), which are proteins that are known to block the release of transmitters by targeting and entering nerve endings, where their proteases cleave and inactivate a protein, synaptosomal protein of M(r) 25 000 (SNAP-25), that is essential for Ca(2+) -regulated exocytosis. Site-directed mutagenesis of Leu428 and Leu429 in BoNT(A) revealed that the remarkable longevity of its neuroparalytic action is attributable to a dileucine-containing motif. BoNT(E) acts transiently, because it lacks these residues, but is a superior inhibitor of transient receptor potential vanilloid type 1-mediated release of pain peptides from sensory nerves. The advantageous features of each serotype were harnessed by attaching the BoNT(E) protease moiety to an enzymically inactive mutant of BoNT(A) . The resultant purified composite protein could target motoneurons by binding to the BoNT(A) ectoacceptor and persistently produce BoNT(E) -truncated SNAP-25. As this enzyme lasted for more than 1 month (as compared with 5 days for BoNT(E) alone), such a dramatic extension in the lifetime of this BoNT(E) protease is attributable to a stabilizing influence of the BoNT(A) mutant. Most importantly, injecting this novel biotherapeutic into the foot pads of rats resulted in extended amelioration of inflammatory pain. Thus, a new generation of biotherapeutics has been created with the potential to give long-term relief of pain. PMID:21645262

  15. Botulinum neurotoxin type A induces TLR2-mediated inflammatory responses in macrophages.

    PubMed

    Kim, Yun Jeong; Kim, Jeong-Hee; Lee, Kwang-Jun; Choi, Myung-Min; Kim, Yeon Hee; Rhie, Gi-Eun; Yoo, Cheon-Kwon; Cha, Kiweon; Shin, Na-Ri

    2015-01-01

    Botulinum neurotoxin type A (BoNT/A) is the most potent protein toxin and causes fatal flaccid muscle paralysis by blocking neurotransmission. Application of BoNT/A has been extended to the fields of therapeutics and biodefense. Nevertheless, the global response of host immune cells to authentic BoNT/A has not been reported. Employing microarray analysis, we performed global transcriptional profiling of RAW264.7 cells, a murine alveolar macrophage cell line. We identified 70 genes that were modulated following 1 nM BoNT/A treatment. The altered genes were mainly involved in signal transduction, immunity and defense, protein metabolism and modification, neuronal activities, intracellular protein trafficking, and muscle contraction. Microarray data were validated with real-time RT-PCR for seven selected genes including tlr2, tnf, inos, ccl4, slpi, stx11, and irg1. Proinflammatory mediators such as nitric oxide (NO) and tumor necrosis factor alpha (TNFα) were induced in a dose-dependent manner in BoNT/A-stimulated RAW264.7 cells. Increased expression of these factors was inhibited by monoclonal anti-Toll-like receptor 2 (TLR2) and inhibitors specific to intracellular proteins such as c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 mitogen-activated protein kinase (MAPK). BoNT/A also suppressed lipopolysaccharide-induced NO and TNFα production from RAW264.7 macrophages at the transcription level by blocking activation of JNK, ERK, and p38 MAPK. As confirmed by TLR2-/- knock out experiments, these results suggest that BoNT/A induces global gene expression changes in host immune cells and that host responses to BoNT/A proceed through a TLR2-dependent pathway, which is modulated by JNK, ERK, and p38 MAPK. PMID:25853816

  16. Novel therapeutics based on recombinant botulinum neurotoxins to normalize the release of transmitters and pain mediators.

    PubMed

    Dolly, J Oliver; Wang, Jiafu; Zurawski, Tomas H; Meng, Jianghui

    2011-12-01

    A major unmet clinical need exists for long-acting neurotherapeutics to alleviate chronic pain in patients unresponsive to available nonaddictive analgesics. Herein, a new strategy is described for the development of potent and specific inhibitors of the neuronal exocytosis of transmitters and pain mediators that exhibit unique antinociceptive activity. This entailed recombinant production in Escherichia coli of two serotypes of botulinum neurotoxin (BoNT) (BoNT(A) and BoNT(E) ), which are proteins that are known to block the release of transmitters by targeting and entering nerve endings, where their proteases cleave and inactivate a protein, synaptosomal protein of M(r) 25 000 (SNAP-25), that is essential for Ca(2+) -regulated exocytosis. Site-directed mutagenesis of Leu428 and Leu429 in BoNT(A) revealed that the remarkable longevity of its neuroparalytic action is attributable to a dileucine-containing motif. BoNT(E) acts transiently, because it lacks these residues, but is a superior inhibitor of transient receptor potential vanilloid type 1-mediated release of pain peptides from sensory nerves. The advantageous features of each serotype were harnessed by attaching the BoNT(E) protease moiety to an enzymically inactive mutant of BoNT(A) . The resultant purified composite protein could target motoneurons by binding to the BoNT(A) ectoacceptor and persistently produce BoNT(E) -truncated SNAP-25. As this enzyme lasted for more than 1 month (as compared with 5 days for BoNT(E) alone), such a dramatic extension in the lifetime of this BoNT(E) protease is attributable to a stabilizing influence of the BoNT(A) mutant. Most importantly, injecting this novel biotherapeutic into the foot pads of rats resulted in extended amelioration of inflammatory pain. Thus, a new generation of biotherapeutics has been created with the potential to give long-term relief of pain.

  17. Monoclonal Antibodies that Inhibit the Proteolytic Activity of Botulinum Neurotoxin Serotype/B.

    PubMed

    Fan, Yongfeng; Dong, Jianbo; Lou, Jianlong; Wen, Weihua; Conrad, Fraser; Geren, Isin N; Garcia-Rodriguez, Consuelo; Smith, Theresa J; Smith, Leonard A; Ho, Mengfei; Pires-Alves, Melissa; Wilson, Brenda A; Marks, James D

    2015-09-01

    Existing antibodies (Abs) used to treat botulism cannot enter the cytosol of neurons and bind to botulinum neurotoxin (BoNT) at its site of action, and thus cannot reverse paralysis. However, Abs targeting the proteolytic domain of the toxin could inhibit the proteolytic activity of the toxin intracellularly and potentially reverse intoxication, if they could be delivered intracellularly. As such, antibodies that neutralize toxin activity could serve as potent inhibitory cargos for therapeutic antitoxins against botulism. BoNT serotype B (BoNT/B) contains a zinc endopeptidase light chain (LC) domain that cleaves synaoptobrevin-2, a SNARE protein responsible for vesicle fusion and acetylcholine vesicle release. To generate monoclonal Abs (mAbs) that could reverse paralysis, we targeted the protease domain for Ab generation. Single-chain variable fragment (scFv) libraries from immunized mice or humans were displayed on yeast, and 19 unique BoNT/B LC-specific mAbs isolated by fluorescence-activated cell sorting (FACS). The equilibrium dissociation constants (KD) of these mAbs for BoNT/B LC ranged from 0.24 nM to 14.3 nM (mean KD 3.27 nM). Eleven mAbs inhibited BoNT/B LC proteolytic activity. The fine epitopes of selected mAbs were identified by alanine-scanning mutagenesis, revealing that inhibitory mAbs bound near the active site, substrate-binding site or the extended substrate-binding site. The results provide mAbs that could prove useful for intracellular reversal of paralysis and identify epitopes that could be targeted by small molecules inhibitors. PMID:26343720

  18. Endogenous dynorphin protects against neurotoxin-elicited nigrostriatal dopaminergic neuron damage and motor deficits in mice

    PubMed Central

    2012-01-01

    Background The striato-nigral projecting pathway contains the highest concentrations of dynorphin in the brain. The functional role of this opioid peptide in the regulation of mesencephalic dopaminergic (DAergic) neurons is not clear. We reported previously that exogenous dynorphin exerts potent neuroprotective effects against inflammation-induced dopaminergic neurodegeneration in vitro. The present study was performed to investigate whether endogenous dynorphin has neuroprotective roles in vivo. Methods 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and methamphetamine (MA), two commonly used neurotoxins in rodent models of Parkinson’s disease, were administered to wild-type (Dyn+/+) and prodynorphin-deficient mice (Dyn−/−). We examined dopaminergic neurotoxicity by using an automated video tracking system, HPLC, immunocytochemistry, and reverse transcription and polymerase chain reaction (RT-PCR). Results Treatment with MPTP resulted in behavioral impairments in both strains. However, these impairments were more pronounced in Dyn-l- than in Dyn+/+. Dyn−/− showed more severe MPTP-induced dopaminergic neuronal loss in the substantia nigra and striatum than Dyn+/+. Similarly, the levels of dopamine and its metabolites in the striatum were depleted to a greater extent in Dyn−/− than in Dyn+/+. Additional mechanistic studies revealed that MPTP treatment caused a higher degree of microglial activation and M1 phenotype differentiation in Dyn−/− than in Dyn+/+. Consistent with these observations, prodynorphin deficiency also exacerbated neurotoxic effects induced by MA, although this effect was less pronounced than that of MPTP. Conclusions The in vivo results presented here extend our previous in vitro findings and further indicate that endogenous dynorphin plays a critical role in protecting dopaminergic neurons through its anti-inflammatory effects. PMID:22695044

  19. Complete nucleotide sequence of a plasmid containing the botulinum neurotoxin gene in Clostridium botulinum type B strain 111 isolated from an infant patient in Japan.

    PubMed

    Hosomi, Koji; Sakaguchi, Yoshihiko; Kohda, Tomoko; Gotoh, Kazuyoshi; Motooka, Daisuke; Nakamura, Shota; Umeda, Kaoru; Iida, Tetsuya; Kozaki, Shunji; Mukamoto, Masafumi

    2014-12-01

    Botulinum neurotoxins (BoNTs) are highly potent toxins that are produced by Clostridium botulinum. We determined the complete nucleotide sequence of a plasmid containing the botulinum neurotoxin gene in C. botulinum type B strain 111 in order to obtain an insight into the toxigenicity and evolution of the bont gene in C. botulinum. Group I C. botulinum type B strain 111 was isolated from the first case of infant botulism in Japan in 1995. In previous studies, botulinum neurotoxin subtype B2 (BoNT/B2) produced by strain 111 exhibited different antigenic properties from those of authentic BoNT/B1 produced by strain Okra. We have recently shown that the isolates of strain 111 that lost toxigenicity were cured of the plasmid containing the bont/B2 gene. In the present study, the plasmid (named pCB111) was circular 265,575 bp double-stranded DNA and contained 332 predicted open reading frames (ORFs). 85 gene products of these ORFs could be functionally assigned on the basis of sequence homology to known proteins. The bont/B2 complex genes were located on pCB111 and some gene products may be involved in the conjugative plasmid transfer and horizontal transfer of bont genes. pCB111 was similar to previously identified plasmids containing bont/B1, /B5, or/A3 complex genes in other group I C. botulinum strains. It was suggested that these plasmids had been derived from a common ancestor and had played important roles for the bont gene transfer between C. botulinum. PMID:25149145

  20. Variations in the pharmacological profile of post-synaptic neurotoxins isolated from the venoms of the Papuan (Oxyuranus scutellatus canni) and coastal (Oxyuranus scutellatus scutellatus) taipans.

    PubMed

    Kornhauser, Rachelle; Hart, Andrew J; Reeve, Shane; Smith, A Ian; Fry, Bryan G; Hodgson, Wayne C

    2010-03-01

    Based on murine LD(50) values, the taipans (i.e. Oxyuranus microlepidotus, Oxyuranus scutellatus and Oxyuranus scutellatus canni) are the most venomous snake genus in the world. Despite this, little is known about the toxins contained in their venoms. The aim of the present study was to isolate and characterise post-synaptic neurotoxins from the venoms of the Papuan taipan (O. s. canni) and coastal taipan (O. scutellatus), and to compare their pharmacology. A 6770Da toxin (i.e. alpha-oxytoxin 1) and a 6781Da toxin (i.e. alpha-scutoxin 1) were isolated from the venoms of O. s. canni and O. scutellatus, respectively, using reverse-phase high performance liquid chromatography. Both alpha-oxytoxin 1 (0.3-1 microM) and alpha-scutoxin 1 (0.1-1 microM) caused concentration-dependent inhibition of indirect twitches in the chick biventer cervicis nerve-muscle preparation. Contractile responses to exogenous carbachol (CCh), but not potassium chloride (KCl), were inhibited by both toxins, suggesting a post-synaptic mode of action. The inhibitory effect of alpha-oxytoxin 1 was reversed by washing. Cumulative concentration-response curves to CCh were obtained in the presence and absence of the toxins with the subsequently determined pA(2) of alpha-scutoxin 1 being 44.7-fold higher than alpha-oxytoxin 1 (i.e. 8.38+/-0.59 versus 7.62+/-0.04). The current study shows that Papuan taipan and coastal taipan venom both contain potent post-synaptic neurotoxins which exhibit different pharmacological profiles. The effect of alpha-oxytoxin 1 is atypical of most snake venom post-synaptic neurotoxins displaying a 'competitive' mode of action, whereas alpha-scutoxin 1 possesses pseudo-irreversible or non-competitive activity.

  1. Structural Analysis of Botulinum Neurotoxin Type G Receptor Binding

    SciTech Connect

    Schmitt, John; Karalewitz, Andrew; Benefield, Desire A.; Mushrush, Darren J.; Pruitt, Rory N.; Spiller, Benjamin W.; Barbieri, Joseph T.; Lacy, D. Borden

    2010-10-19

    Botulinum neurotoxin (BoNT) binds peripheral neurons at the neuromuscular junction through a dual-receptor mechanism that includes interactions with ganglioside and protein receptors. The receptor identities vary depending on BoNT serotype (A-G). BoNT/B and BoNT/G bind the luminal domains of synaptotagmin I and II, homologous synaptic vesicle proteins. We observe conditions under which BoNT/B binds both Syt isoforms, but BoNT/G binds only SytI. Both serotypes bind ganglioside G{sub T1b}. The BoNT/G receptor-binding domain crystal structure provides a context for examining these binding interactions and a platform for understanding the physiological relevance of different Syt receptor isoforms in vivo.

  2. Botulinum Neurotoxin Is Shielded by NTNHA in an Interlocked Complex

    SciTech Connect

    Gu, Shenyan; Rumpel, Sophie; Zhou, Jie; Strotmeier, Jasmin; Bigalke, Hans; Perry, Kay; Shoemaker, Charles B.; Rummel, Andreas; Jin, Rongsheng

    2012-03-28

    Botulinum neurotoxins (BoNTs) are highly poisonous substances that are also effective medicines. Accidental BoNT poisoning often occurs through ingestion of Clostridium botulinum-contaminated food. Here, we present the crystal structure of a BoNT in complex with a clostridial nontoxic nonhemagglutinin (NTNHA) protein at 2.7 angstroms. Biochemical and functional studies show that NTNHA provides large and multivalent binding interfaces to protect BoNT from gastrointestinal degradation. Moreover, the structure highlights key residues in BoNT that regulate complex assembly in a pH-dependent manner. Collectively, our findings define the molecular mechanisms by which NTNHA shields BoNT in the hostile gastrointestinal environment and releases it upon entry into the circulation. These results will assist in the design of small molecules for inhibiting oral BoNT intoxication and of delivery vehicles for oral administration of biologics.

  3. Gaining ground: assays for therapeutics against botulinum neurotoxin.

    PubMed

    Hakami, Ramin Mollaaghababa; Ruthel, Gordon; Stahl, Andrea M; Bavari, Sina

    2010-04-01

    Owing in part to recently heightened concern over bioterrorism, interest in the mechanism of action of botulinum neurotoxin (BoNT) and development of effective therapeutic strategies has dramatically increased. The emergence of BoNT as an effective treatment for a variety of neurological disorders and its growing use in the cosmetic industry have also increased interest in developing effective countermeasures. Although recent attempts to create effective vaccines appear promising, the multitude of clinical and cosmetic uses of BoNT make mass vaccination against the toxin undesirable and impractical, leading to intensified efforts to develop effective therapeutics to combat large-scale intoxications. In this review, we examine the relevant and available in vitro cell-based assays and in vivo assays for drug discovery and development, especially with regard to the potential for medium- to high-throughput automation and its use in identifying physiologically relevant inhibitors.

  4. Botulinum neurotoxin for pain management: insights from animal models.

    PubMed

    Pavone, Flaminia; Luvisetto, Siro

    2010-12-01

    The action of botulinum neurotoxins (BoNTs) at the neuromuscular junction has been extensively investigated and knowledge gained in this field laid the foundation for the use of BoNTs in human pathologies characterized by excessive muscle contractions. Although much more is known about the action of BoNTs on the peripheral system, growing evidence has demonstrated several effects also at the central level. Pain conditions, with special regard to neuropathic and intractable pain, are some of the pathological states that have been recently treated with BoNTs with beneficial effects. The knowledge of the action and potentiality of BoNTs utilization against pain, with emphasis for its possible use in modulation and alleviation of chronic pain, still represents an outstanding challenge for experimental research. This review highlights recent findings on the effects of BoNTs in animal pain models.

  5. Complexity of botulinum neurotoxins: challenges for detection technology.

    PubMed

    Dorner, Martin B; Schulz, K Melanie; Kull, Skadi; Dorner, Brigitte G

    2013-01-01

    The detection of botulinum neurotoxins (BoNT) is extremely challenging due to their high toxicity and the multiple BoNT variants. To date, seven serotypes with more than 30 subtypes have been described, and even more subtypes are expected to be discovered. The fact that the BoNT molecules are released as large complexes of different size and composition adds further complexity to the issue. Currently, in the diagnostics of botulism, the mouse bioassay (MBA) is still considered as gold standard for the detection of BoNT in complex sample materials. Over the years, different functional, immunological, and spectrometric assays or combinations thereof have been developed, supplemented by DNA-based assays for the detection of the organism. In this review, advantages and limitations of the current technologies will be discussed, highlighting some of the intricacies of real sample analysis.

  6. Peptide inhibitors of botulinum neurotoxin by mRNA display

    SciTech Connect

    Yiadom, Kwabena P.A.B.; Muhie, Seid; Yang, David C.H. . E-mail: yangdc@georgetown.edu

    2005-10-07

    Botulinum neurotoxins (BoNTs) are extremely toxic. The metalloproteases associated with the toxins cleave proteins essential for neurotransmitter secretion. Inhibitors of the metalloprotease are currently sought to control the toxicity of BoNTs. Toward that goal, we produced a synthetic cDNA for the expression and purification of the metalloprotease of BoNT/A in Escherichia coli as a biotin-ubiquitin fusion protein, and constructed a combinatorial peptide library to screen for BoNT/A light chain inhibitors using mRNA display. A protease assay was developed using immobilized intact SNAP-25 as the substrate. The new peptide inhibitors showed a 10-fold increase in affinity to BoNT/A light chain than the parent peptide. Interestingly, the sequences of the new peptide inhibitors showed abundant hydrophobic residues but few hydrophilic residues. The results suggest that mRNA display may provide a general approach in developing peptide inhibitors of BoNTs.

  7. Neurotoxins: Expanding Uses of Neuromodulators in Medicine--Headache.

    PubMed

    Nahabet, Edward; Janis, Jeffrey E; Guyuron, Bahman

    2015-11-01

    Over the course of the past 17 years, since the initial discovery of the association between botulinum toxin-A (BT-A) and the reduction of headache symptoms, the use of this neurotoxin has greatly evolved. BT-A has emerged as an alternative to prophylactic pharmacological therapies in the prevention of chronic migraine headaches, with an excellent safety profile and proven efficacy, and is Food and Drug Administration-approved for on-label use since October 2010. The mechanism of BT-A involves its effect at the neuromuscular junction, inhibition of neuropeptide and neurotransmitter release in peripheral sensory neurons, and retrograde axonal transport allowing for its direct effect on inhibiting central sensitization. Through its diagnostic and therapeutic utility, BT-A has proven to be an integral part in the treatment of chronic headache disorders.

  8. Neutralization of Botulinum Neurotoxin Type E by a Humanized Antibody.

    PubMed

    Derman, Yağmur; Selby, Katja; Miethe, Sebastian; Frenzel, André; Liu, Yvonne; Rasetti-Escargueil, Christine; Avril, Arnaud; Pelat, Thibaut; Urbain, Remi; Fontayne, Alexandre; Thullier, Philippe; Sesardic, Dorothea; Lindström, Miia; Hust, Michael; Korkeala, Hannu

    2016-01-01

    Botulinum neurotoxins (BoNTs) cause botulism and are the deadliest naturally-occurring substances known to humans. BoNTs have been classified as one of the category A agents by the Centers for Disease Control and Prevention, indicating their potential use as bioweapons. To counter bio-threat and naturally-occurring botulism cases, well-tolerated antibodies by humans that neutralize BoNTs are relevant. In our previous work, we showed the neutralizing potential of macaque (Macaca fascicularis)-derived scFv-Fc (scFv-Fc ELC18) by in vitro endopeptidase immunoassay and ex vivo mouse phrenic nerve-hemidiaphragm assay by targeting the light chain of the botulinum neurotoxin type E (BoNT/E). In the present study, we germline-humanized scFv-Fc ELC18 into a full IgG hu8ELC18 to increase its immunotolerance by humans. We demonstrated the protection and prophylaxis capacity of hu8ELC18 against BoNT/E in a mouse model. A concentration of 2.5 ng/mouse of hu8ELC18 protected against 5 mouse lethal dose (MLD) in a mouse protection assay and complete neutralization of 1 LD50 of pure BoNT/E toxin was achieved with 8 ng of hu8ELC18 in mouse paralysis assay. Furthermore, hu8ELC18 protected mice from 5 MLD if injected up to 14 days prior to intraperitoneal BoNT/E administration. This newly-developed humanized IgG is expected to have high tolerance in humans. PMID:27626446

  9. Neutralization of Botulinum Neurotoxin Type E by a Humanized Antibody

    PubMed Central

    Derman, Yağmur; Selby, Katja; Miethe, Sebastian; Frenzel, André; Liu, Yvonne; Rasetti-Escargueil, Christine; Avril, Arnaud; Pelat, Thibaut; Urbain, Remi; Fontayne, Alexandre; Thullier, Philippe; Sesardic, Dorothea; Lindström, Miia; Hust, Michael; Korkeala, Hannu

    2016-01-01

    Botulinum neurotoxins (BoNTs) cause botulism and are the deadliest naturally-occurring substances known to humans. BoNTs have been classified as one of the category A agents by the Centers for Disease Control and Prevention, indicating their potential use as bioweapons. To counter bio-threat and naturally-occurring botulism cases, well-tolerated antibodies by humans that neutralize BoNTs are relevant. In our previous work, we showed the neutralizing potential of macaque (Macaca fascicularis)-derived scFv-Fc (scFv-Fc ELC18) by in vitro endopeptidase immunoassay and ex vivo mouse phrenic nerve-hemidiaphragm assay by targeting the light chain of the botulinum neurotoxin type E (BoNT/E). In the present study, we germline-humanized scFv-Fc ELC18 into a full IgG hu8ELC18 to increase its immunotolerance by humans. We demonstrated the protection and prophylaxis capacity of hu8ELC18 against BoNT/E in a mouse model. A concentration of 2.5 ng/mouse of hu8ELC18 protected against 5 mouse lethal dose (MLD) in a mouse protection assay and complete neutralization of 1 LD50 of pure BoNT/E toxin was achieved with 8 ng of hu8ELC18 in mouse paralysis assay. Furthermore, hu8ELC18 protected mice from 5 MLD if injected up to 14 days prior to intraperitoneal BoNT/E administration. This newly-developed humanized IgG is expected to have high tolerance in humans. PMID:27626446

  10. Genetic Diversity Among Botulinum Neurotoxin Producing Clostridial Strains

    SciTech Connect

    Hill, K K; Smith, T J; Helma, C H; Ticknor, L O; Foley, B T; Svennson, R T; Brown, J L; Johnson, E A; Smith, L A; Okinaka, R T; Jackson, P J; Marks, J D

    2006-07-06

    Clostridium botulinum is a taxonomic designation for many diverse anaerobic spore forming rod-shaped bacteria which have the common property of producing botulinum neurotoxins (BoNTs). The BoNTs are exoneurotoxins that can cause severe paralysis and even death in humans and various other animal species. A collection of 174 C. botulinum strains were examined by amplified fragment length polymorphism (AFLP) analysis and by sequencing of the 16S rRNA gene and BoNT genes to examine genetic diversity within this species. This collection contained representatives of each of the seven different serotypes of botulinum neurotoxins (BoNT A-G). Analysis of the16S rRNA sequences confirmed earlier reports of at least four distinct genomic backgrounds (Groups I-IV) each of which has independently acquired one or more BoNT serotypes through horizontal gene transfer. AFLP analysis provided higher resolution, and can be used to further subdivide the four groups into sub-groups. Sequencing of the BoNT genes from serotypes A, B and E in multiple strains confirmed significant sequence variation within each serotype. Four distinct lineages within each of the BoNT A and B serotypes, and five distinct lineages of serotype E strains were identified. The nucleotide sequences of the seven serotypes of BoNT were compared and show varying degrees of interrelatedness and recombination as has been previously noted for the NTNH gene which is linked to BoNT. These analyses contribute to the understanding of the evolution and phylogeny within this species and assist in the development of improved diagnostics and therapeutics for treatment of botulism.

  11. Inhibition of presynaptic neurotoxins in taipan venom by suramin.

    PubMed

    Kuruppu, Sanjaya; Chaisakul, Janeyuth; Smith, A Ian; Hodgson, Wayne C

    2014-04-01

    Taipans are amongst the most venomous snakes in the world, and neurotoxicity is a major life-threatening symptom of envenoming by these snakes. Three species of taipans exist, and the venom from each species contains a presynaptic neurotoxin which accounts for much of the neurotoxicity observed following human envenoming. The high cost of antivenom used to treat neurotoxicity has resulted in the need to develop alternative but effective therapies. Therefore, in this study, we examined the ability of the P2Y receptor antagonist suramin to prevent the in vitro neurotoxic effects of the three presynaptic neurotoxins in taipan venoms: taipoxin, paradoxin and cannitoxin. Toxins were purchased from commercial sources or purified in house, using multiple steps of gel filtration chromatography. All three toxins (11 nM) inhibited nerve-mediated twitches in the chick biventer cervicis nerve-muscle preparation within 300 min. The presence of suramin (0.3 mM) completely blocked the taipoxin and cannitoxin-mediated inhibition of nerve-mediated twitches within the course of the experiment (P < 0.0001). However, paradoxin induced a 32 % decrease in twitch height even in the presence of suramin within 360 min. This was significantly different compared to toxin alone (P < 0.0001). We also examined the effect of suramin on the neurotoxic effects of textilotoxin and the products of phospholipase A2 action. Each toxin alone or in the presence of suramin failed to inhibit the responses to exogenous agonists ACh, CCh or KCl. Our results warrant clinical studies aimed determining the efficacy of suramin in preventing the onset of neurotoxicity following taipan envenoming.

  12. Immunological Characterization and Neutralizing Ability of Monoclonal Antibodies Directed Against Botulinum Neurotoxin Type H

    PubMed Central

    Fan, Yongfeng; Barash, Jason R.; Lou, Jianlong; Conrad, Fraser; Marks, James D.; Arnon, Stephen S.

    2016-01-01

    Background. Only Clostridium botulinum strain IBCA10-7060 produces the recently described novel botulinum neurotoxin type H (BoNT/H). BoNT/H (N-terminal two-thirds most homologous to BoNT/F and C-terminal one-third most homologous to BoNT/A) requires antitoxin to toxin ratios ≥1190:1 for neutralization by existing antitoxins. Hence, more potent and safer antitoxins against BoNT/H are needed. Methods. We therefore evaluated our existing monoclonal antibodies (mAbs) to BoNT/A and BoNT/F for BoNT/H binding, created yeast-displayed mutants to select for higher-affinity-binding mAbs by using flow cytometry, and evaluated the mAbs' ability to neutralize BoNT/H in the standard mouse bioassay. Results. Anti-BoNT/A HCC-binding mAbs RAZ1 and CR2 bound BoNT/H with high affinity. However, only 1 of 6 BoNT/F mAbs (4E17.2A) bound BoNT/H but with an affinity >800-fold lower (equilibrium dissociation binding constant [KD] = 7.56 × 10−8 M) than its BoNT/F affinity (KD = 9.1 × 10−11 M), indicating that the N-terminal two-thirds of BoNT/H is immunologically unique. The affinity of 4E17.2A for BoNT/H was increased >500-fold to KD = 1.48 × 10−10 M (mAb 4E17.2D). A combination of mAbs RAZ1, CR2, and 4E17.2D completely protected mice challenged with 280 mouse median lethal doses of BoNT/H at a mAb dose as low as 5 µg of total antibody. Conclusions. This 3-mAb combination potently neutralized BoNT/H and represents a potential human antitoxin that could be developed for the prevention and treatment of type H botulism. PMID:26936913

  13. Purification and characterization of a neurotoxin from the venom of Ophiophagus hannah (king cobra).

    PubMed

    Chang, Long-Sen; Liou, Jau-Cheng; Lin, Shinne-Ren; Huang, Hsien-Bin

    2002-06-14

    A neurotoxin, Oh9-1, from the venom of Ophiophagus hannah was isolated by a combination of ion-exchange chromatography and reverse phase HPLC. Amino acid sequence analysis revealed that Oh9-1 consists of 57 amino acids and eight cysteine residues. This protein was mainly constituted with beta-sheet as evidenced by CD spectrum. Oh9-1 inhibited carbachol-induced muscle contraction in an irreversible manner and the dose for achieving 50% inhibition was approximately fourfold that of alpha-bungarotoxin. Since the residues in alpha-neurotoxins closely involve in the binding with acetylcholine receptors are not highly conserved in this toxin molecule, Oh9-1 represents a novel type of neurotoxin structurally distinct from alpha-neurotoxins. PMID:12056805

  14. Purification and characterization of a neurotoxin from the venom of Ophiophagus hannah (king cobra).

    PubMed

    Chang, Long-Sen; Liou, Jau-Cheng; Lin, Shinne-Ren; Huang, Hsien-Bin

    2002-06-14

    A neurotoxin, Oh9-1, from the venom of Ophiophagus hannah was isolated by a combination of ion-exchange chromatography and reverse phase HPLC. Amino acid sequence analysis revealed that Oh9-1 consists of 57 amino acids and eight cysteine residues. This protein was mainly constituted with beta-sheet as evidenced by CD spectrum. Oh9-1 inhibited carbachol-induced muscle contraction in an irreversible manner and the dose for achieving 50% inhibition was approximately fourfold that of alpha-bungarotoxin. Since the residues in alpha-neurotoxins closely involve in the binding with acetylcholine receptors are not highly conserved in this toxin molecule, Oh9-1 represents a novel type of neurotoxin structurally distinct from alpha-neurotoxins.

  15. Ultrastructural confirmation of neuronal protection by melatonin against the neurotoxin 6-hydroxydopamine cell damage.

    PubMed

    Mayo, J C; Sainz, R M; Antolín, I; Rodriguez, C

    1999-02-13

    6-Hydroxydopamine (6-OHDA) is a neurotoxin used in the induction of experimental Parkinson's disease in both animals and cultured neuronal cells. Biochemical and molecular approaches showed previously that low doses of 6-OHDA induced apoptosis in PC12 cells, while high doses of this neurotoxin induced necrosis. Melatonin has been shown to protect against the neuronal programmed cell death induced by 6-OHDA, although it was not able to prevent the massive necrotic cellular death occurring after the addition of high doses of the neurotoxin. In the present work, we demonstrate by ultrastructural analysis that although low doses of 6-OHDA induced apoptosis in PC12 cells, it also damaged the non-apoptotic cells, morphologically corresponding this damage to incipient and reversible necrotic lesions. When the doses of the neurotoxin increase, there are still apoptotic cells, although most of the cells show necrotic irreversible lesions. We also found that melatonin partially prevents the incipient necrotic lesions caused by low doses of 6-OHDA. The fact that melatonin was shown in previous work to prevent apoptosis caused by low doses of 6-OHDA, but not necrosis induced by high doses of the neurotoxin, seemed to indicate that this agent is only able to protect against apoptosis. However, our present results, melatonin preventing also the incipient necrotic neuronal lesions, suggest that this hormone may provide a general protection against cell death, suggesting that higher doses should be tried in order to prevent the necrotic cell death induced by high doses of the neurotoxin.

  16. The synthesis of 2,5-Bis(4-amidinophenyl)thiophene derivatives providing submicromolar-range inhibition of the botulinum neurotoxin serotype A metalloprotease

    PubMed Central

    Opsenica, Igor; Filipovic, Vuk; Nuss, Jon E.; Gomba, Laura M.; Opsenica, Dejan; Burnett, James C.; Gussio, Rick; Solaja, Bogdan A.; Bavari, Sina

    2012-01-01

    Botulinum neurotoxins (BoNTs), composed of a family of seven serotypes (categorized A – G), are the deadliest of known biological toxins. The activity of the metalloprotease, light chain (LC) component of the toxins is responsible for causing the life-threatening paralysis associated with the disease botulism. Herein we report significantly more potent analogs of novel, lead BoNT serotype A LC inhibitor 2,5-bis(4-amidinophenyl)thiophene (Ki = 10.88 μM ± 0.90 μM). Specifically, synthetic modifications involved simultaneously replacing the lead inhibitor’s terminal bis-amidines with secondary amines and the systematic tethering of 4-amino-7-chloroquinoline substituents to provide derivatives with Ki values ranging from 0.302 μM (± 0.03 μM) – 0.889 μM (± 0.11 μM). PMID:22516424

  17. Characterization of the D/C mosaic neurotoxin produced by Clostridium botulinum associated with bovine botulism in Japan.

    PubMed

    Nakamura, Keiji; Kohda, Tomoko; Umeda, Kaoru; Yamamoto, Hideyuki; Mukamoto, Masafumi; Kozaki, Shunji

    2010-01-01

    Clostridium botulinum types C and D are related to avian and mammalian botulism. Bovine botulism occurred at various farms from 2004 to 2007 in Japan. Since culture supernatants of isolates from cases of bovine botulism were neutralized completely and partially with type D and C antitoxins, respectively, we attempted to confirm the nucleotide sequences of the neurotoxin gene in isolates. The neurotoxin gene comprised two-thirds of the type D neurotoxin gene and one-third of the type C neurotoxin gene, indicating that the neurotoxin of bovine isolates is a mosaic of type D and C neurotoxins, D/C mosaic neurotoxin. We prepared four sets of primers to differentiate the genes of the mosaic and authentic forms with PCR. The results showed that all bovine botulism-related isolates possess the gene for the D/C mosaic form. Pulsed-field gel electrophoresis analysis demonstrated that isolates from bovine botulism which had occurred between 2004 and 2007 were genetically homologous, except for the isolate from one area. We further examined the biological and antigenic properties of the D/C mosaic neurotoxin, which was found to exhibit the highest lethal activity in mice compared with other types of neurotoxins. In the D/C mosaic neurotoxin, three epitopes recognized by monoclonal antibodies that specifically react to and neutralize the toxin were located in the carboxyl-terminal domain of the heavy chain. These results indicate that D/C mosaic neurotoxin is a pathogenic agent causing bovine botulism and has unique characteristics different from other type C and D neurotoxins.

  18. Purification and Characterization of Botulinum Neurotoxin FA from a Genetically Modified Clostridium botulinum Strain

    PubMed Central

    Pellett, Sabine; Tepp, William H.; Bradshaw, Marite; Kalb, Suzanne R.; Dykes, Janet K.; Lin, Guangyun; Nawrocki, Erin M.; Pier, Christina L.; Barr, John R.; Maslanka, Susan E.

    2016-01-01

    ABSTRACT Botulinum neurotoxins (BoNTs), produced by neurotoxigenic clostridial species, are the cause of the severe disease botulism in humans and animals. Early research on BoNTs has led to their classification into seven serotypes (serotypes A to G) based upon the selective neutralization of their toxicity in mice by homologous antibodies. Recently, a report of a potential eighth serotype of BoNT, designated “type H,” has been controversial. This novel BoNT was produced together with BoNT/B2 in a dual-toxin-producing Clostridium botulinum strain. The data used to designate this novel toxin as a new serotype were derived from culture supernatant containing both BoNT/B2 and novel toxin and from sequence information, although data from two independent laboratories indicated neutralization by antibodies raised against BoNT/A1, and classification as BoNT/FA was proposed. The sequence data indicate a chimeric structure consisting of a BoNT/A1 receptor binding domain, a BoNT/F5 light-chain domain, and a novel translocation domain most closely related to BoNT/F1. Here, we describe characterization of this toxin purified from the native strain in which expression of the second BoNT (BoNT/B) has been eliminated. Mass spectrometry analysis indicated that the toxin preparation contained only BoNT/FA and confirmed catalytic activity analogous to that of BoNT/F5. The in vivo mouse bioassay indicated a specific activity of this toxin of 3.8 × 107 mouse 50% lethal dose (mLD50) units/mg, whereas activity in cultured human neurons was very high (50% effective concentration [EC50] = 0.02 mLD50/well). Neutralization assays in cells and mice both indicated full neutralization by various antibodies raised against BoNT/A1, although at 16- to 20-fold-lower efficiency than for BoNT/A1. IMPORTANCE Botulinum neurotoxins (BoNTs), produced by anaerobic bacteria, are the cause of the potentially deadly, neuroparalytic disease botulism. BoNTs have been classified into seven serotypes

  19. Purification and Characterization of Botulinum Neurotoxin FA from a Genetically Modified Clostridium botulinum Strain.

    PubMed

    Pellett, Sabine; Tepp, William H; Bradshaw, Marite; Kalb, Suzanne R; Dykes, Janet K; Lin, Guangyun; Nawrocki, Erin M; Pier, Christina L; Barr, John R; Maslanka, Susan E; Johnson, Eric A

    2016-01-01

    Botulinum neurotoxins (BoNTs), produced by neurotoxigenic clostridial species, are the cause of the severe disease botulism in humans and animals. Early research on BoNTs has led to their classification into seven serotypes (serotypes A to G) based upon the selective neutralization of their toxicity in mice by homologous antibodies. Recently, a report of a potential eighth serotype of BoNT, designated "type H," has been controversial. This novel BoNT was produced together with BoNT/B2 in a dual-toxin-producing Clostridium botulinum strain. The data used to designate this novel toxin as a new serotype were derived from culture supernatant containing both BoNT/B2 and novel toxin and from sequence information, although data from two independent laboratories indicated neutralization by antibodies raised against BoNT/A1, and classification as BoNT/FA was proposed. The sequence data indicate a chimeric structure consisting of a BoNT/A1 receptor binding domain, a BoNT/F5 light-chain domain, and a novel translocation domain most closely related to BoNT/F1. Here, we describe characterization of this toxin purified from the native strain in which expression of the second BoNT (BoNT/B) has been eliminated. Mass spectrometry analysis indicated that the toxin preparation contained only BoNT/FA and confirmed catalytic activity analogous to that of BoNT/F5. The in vivo mouse bioassay indicated a specific activity of this toxin of 3.8 × 10(7) mouse 50% lethal dose (mLD50) units/mg, whereas activity in cultured human neurons was very high (50% effective concentration [EC50] = 0.02 mLD50/well). Neutralization assays in cells and mice both indicated full neutralization by various antibodies raised against BoNT/A1, although at 16- to 20-fold-lower efficiency than for BoNT/A1. IMPORTANCE Botulinum neurotoxins (BoNTs), produced by anaerobic bacteria, are the cause of the potentially deadly, neuroparalytic disease botulism. BoNTs have been classified into seven serotypes, serotypes A

  20. Update on neurotoxins for facial rejuvenation: what they are, how they work, and how to effectively and safely use them.

    PubMed

    Brennan, Connie

    2015-01-01

    The formal aesthetic introduction of botulinum toxin (i.e., neurotoxins) more than a decade ago has revolutionized the nonsurgical aesthetic market and transformed society's view of facial rejuvenation. Understanding the similarities and differences between U.S. commercially available neurotoxins, their characteristics and composition, where they are effective, their mechanism of action, and how to safely administer them will enable the aesthetic provider to successfully utilize this powerful tool and ultimately deliver optimal facial rejuvenation outcomes. An update on neurotoxins on the horizon is also provided. Please note that this article discusses neurotoxin use in treatment areas that are considered "off label" by the Food and Drug Administration.

  1. Novel co-operation between eotaxin and substance-P in inducing eosinophil-derived neurotoxin release.

    PubMed Central

    El-Shazly, A; Ishikawa, T

    1999-01-01

    Eosinophils, chemokines, and neuropeptides are thought to play effector roles in the pathogenesis of allergic diseases such as rhinitis. Eotaxin is a novel C-C chemokine with a potent and relatively specific eosinophil chemoattractant activity that binds selectively to CCR3 receptor, however, its activity in inducing eosinophil granules proteins release is poorly characterized. This study was performed to determine whether eotaxin primes eosinophil exocytosis and whether this co-operates with the sensory neuroimmune-axis. In the present communication, we show that 10 ng/ml eotaxin primed normal human eosinophil for exaggerated eosonophil-derived neurotoxin (EDN) release stimulated by 10(-8) M Substance-P (SP). This novel priming was blocked by; 7B11 and Herbimycin A (HA), the CCR3 antagonist and the tyrosine kinase inhibitor, respectively. SDS-Page studies showed significant tyrosine phosphorylation of several protein residues induced by 10(-8) M SP only after priming with 10 ng/ml eotaxin. These results demonstrate a novel co-operation between eotaxin and SP in inducing eosinophil cytotoxicity, which at least in part involves tyrosine kinases pathway(s). PMID:10704057

  2. Effects of COX inhibitors on neurodegeneration and survival in mice exposed to the marine neurotoxin domoic acid.

    PubMed

    Ryan, James C; Cross, Cheryl A; Van Dolah, Frances M

    2011-01-01

    The marine neurotoxin domoic acid (DA) is a rigid analogue of the neurotransmitter glutamate and a potent agonist of kainate subtype glutamate receptors. Persistent activation of these receptor subtypes results in rapid excitotoxicity, calcium-dependent cell death, and neuronal degeneration in regions of the brain where glutamatergic pathways are concentrated. Previous work has shown that DA promotes the expression of inflammatory genes in the brain, such as cyclooxygenase 2 (COX2). To investigate the impact of inflammation on the development of neurodegeneration, and ultimately survival following DA administration, we used selective (L745337, Merck) and non-selective (acetylsalicylic acid (ASA)) COX inhibitors in DA exposed mice. Adult male ICR mice were given a regime of either ASA or L23547 both before and after a single LD50 dose of DA. Mice were observed immediately after toxin introduction and then sacrificed at 2 days post exposure. Our lower dose of L23547 increased survival and was most effective at decreasing neuronal degeneration in the CA1 and CA3 regions of the hippocampus, areas especially sensitive to DA excitotoxicity. This study shows that COX2 plays a role in DA induced neurodegeneration and death, and that inhibitors may be of value for treatment in human and wildlife DA exposure. PMID:20934488

  3. Identification of the amino acid residues rendering TI-VAMP insensitive toward botulinum neurotoxin B.

    PubMed

    Sikorra, Stefan; Henke, Tina; Swaminathan, Subramanyam; Galli, Thierry; Binz, Thomas

    2006-03-24

    Botulinum neurotoxins types B, D, F, and G, and tetanus neurotoxin inhibit vesicular fusion via proteolytic cleavage of VAMP/Synaptobrevin, a core component of the membrane fusion machinery. Thus, these neurotoxins became widely used tools for investigating vesicular trafficking routes. Except for VAMP-1, VAMP-2, and Cellubrevin, no other member of the VAMP family represents a substrate for these neurotoxins. The molecular basis for this discrepancy is not known. A 34 amino acid residue segment of VAMP-2 was previously suggested to mediate the interaction with botulinum neurotoxin B, but the validity of the data was later questioned. To check whether this segment alone controls the susceptibility toward botulinum neurotoxin B, it was used to replace the corresponding segment in TI-VAMP. The resulting VAMP hybrid and VAMP-2 were hydrolysed at virtually identical rates. Resetting the VAMP-2 portion in the hybrid from either end to TI-VAMP residues gradually reduced the cleavability. A hybrid encompassing merely the VAMP-2 segment 71-80 around the Gln76/Phe77 scissile bond was still hydrolysed, albeit at a approximately tenfold lower cleavage rate. The contribution of each non-conserved amino acid of the whole 34-mer segment to the interaction was investigated employing VAMP-2. We find that the eight non-conserved residues of the 71-80 segment are all necessary for efficient cleavage. Mutation of an additional six residues located upstream and downstream of this segment affects substrate hydrolysis as well. Vice versa, a readily cleavable TI-VAMP molecule requires at the least the replacement of Ile158, Thr161, and the section 165-174 by Asp64, Ala67, and the 71-80 segment of VAMP-2, respectively. However, the insensitivity of TI-VAMP to botulinum neurotoxin B relies on at least 12 amino acid changes versus VAMP-2. These are scattered along an interface of 22 amino acid residues in length.

  4. Use of pharmacologic antagonists to deduce commonalities of biologic activity among clostridial neurotoxins.

    PubMed

    Simpson, L L

    1988-06-01

    The pharmacologic activity of several clostridial neurotoxins was assayed on the mouse phrenic nerve-hemidiaphragm preparation. The substances that were assayed included botulinum neurotoxin types A, B, C and E and tetanus toxin. Experiments were done in the presence or absence of antagonists that inhibit either the internalization of toxins or intracellular expression of toxicity. Ammonium chloride and methylamine hydrochloride, agents that inhibit toxins that enter cells by receptor-mediated endocytosis, antagonized botulinum and tetanus neurotoxins. The magnitude of antagonism was substantial for all toxins. Calcium, 3,4-diaminopyridine and guanidine, agents that alter the intracellular expression of toxicity, produced a variable result. They were effective antagonists of botulinum neurotoxin type A, but they were less effective or inactive against the other neurotoxins. The ability of 3,4-diaminopyridine and guanidine to antagonize botulinum neurotoxin type A was highly calcium dependent. When ambient levels of the cation were reduced from 1.8 to 1.0 mM, the activity of the drugs was substantially reduced. The ability of these drugs to produce antagonism was also time dependent. When added simultaneously with toxin, they were maximally active; when added at later times, activity was diminished. A host of agents that alter intracellular levels of cyclic AMP, including theophylline, forskolin, isobutylmethylxanthine and cholera toxin, were evaluated as potential neurotoxin antagonists. Theophylline and isobutylmethylxanthine produced a transient increase in nerve-evoked muscle twitch. None of the drugs that alter tissue levels of cyclic AMP had a universal effect in antagonizing clostridial toxins. The data here have been compared with published data on drugs that antagonize binding of botulinum toxin and tetanus toxin.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2455038

  5. Time-dependent botulinum neurotoxin serotype A metalloprotease inhibitors

    PubMed Central

    Cardinale, Steven C.; Butler, Michelle M.; Pai, Ramdas; Nuss, Jonathan E.; Peet, Norton P.; Bavari, Sina; Bowlin, Terry L.

    2011-01-01

    Botulinum neurotoxins (BoNTs) are the most lethal of biological substances, and are categorized as class A biothreat agents by the Centers for Disease Control and Prevention. There are currently no drugs to treat the deadly flaccid paralysis resulting from BoNT intoxication. Among the seven BoNT serotypes, the development of therapeutics to counter BoNT/A is a priority (due to its long half-life in the neuronal cytosol and its ease of production). In this regard, the BoNT/A enzyme light chain (LC) component, a zinc metalloprotease responsible for the intracellular cleavage of synaptosomal-associated protein of 25 kDa, is a desirable target for developing post-BoNT/A intoxication rescue therapeutics. In an earlier study, we reported the high throughput screening of a library containing 70,000 compounds, and uncovered a novel class of benzimidazole acrylonitrile-based BoNT/A LC inhibitors. Herein, we present both structure-activity relationships and a proposed mechanism of action for this novel inhibitor chemotype. PMID:22082667

  6. Botulinum neurotoxins in the treatment of refractory pain.

    PubMed

    Jabbari, Bahman

    2008-12-01

    The proper management of pain is a critical issue in the practice of medicine. Despite the availability of a large number of analgesic medications, management of pain that is refractory to conventional treatments remains a challenge for both clinicians and surgeons. Botulinum neurotoxin (BoNT) has recently emerged as a potential novel approach to control pain. Animal studies have revealed a number of mechanisms by which BoNTs can influence and alleviate chronic pain, including inhibition of pain peptide release from nerve terminals and sensory ganglia, anti-inflammatory and antiglutaminergic effects, reduction of sympathetic neural discharge, and inhibition of muscle spindle discharge. In humans, prospective, placebo-controlled, double-blind studies have also provided evidence for effectiveness of BoNT therapy in a number of painful disorders. These include cervical dystonia, pelvic pain, low back pain, plantar fasciitis, postsurgical painful spasms, myofascial pain syndromes, migraine, and chronic daily headaches. Long-term studies on cervical dystonia and low back pain have demonstrated safety and sustained efficacy after repeated injections. This Review focuses on the analgesic effects of BoNT and the mechanisms of its pain control as revealed by animal models, and provides evidence-based data on the efficacy of BoNT therapy in various pain syndromes in humans. PMID:19043424

  7. Using llama derived single domain antibodies to target botulinum neurotoxins

    NASA Astrophysics Data System (ADS)

    Swain, Marla D.; Anderson, George P.; Bernstein, Rachael D.; Liu, Jinny L.; Goldman, Ellen R.

    2010-04-01

    Llama serum contains both conventional IgG as well as unique forms of antibody that contain only heavy chains where antigen binding is mediated through a single variable domain. These variable domains can be expressed recombinantly and are referred to as single domain antibodies (sdAb). SdAb are among the smallest known naturally derived antigen binding fragments, possess good solubility, thermal stability, and can refold after heat and chemical denaturation. Llamas were immunized with either BoNT A or B toxoid and phage display libraries prepared. Single domain antibodies (sdAb) that were able to detect botulinum neurotoxin (BoNT) serotypes A and B were selected from their respective libraries. Here, the binders obtained by panning the BoNT B library on either BoNT B toxoid or BoNT B complex toxoid coated plates or BoNT B toxin coupled microspheres are described. Using these panning methods, we selected for binders that showed specificity for BoNT B. Phage displayed binders were screened, moved to a protein expression vector and soluble sdAb was produced. Using a Luminex flow cytometer binders were evaluated in direct binding assays. We have exploited the unique properties of sdAb and used them as biological recognition elements in immuno-based sensors that can detect BoNT B.

  8. The blockade of the neurotransmitter release apparatus by botulinum neurotoxins.

    PubMed

    Pantano, Sergio; Montecucco, Cesare

    2014-03-01

    The high toxicity of the seven serotypes of botulinum neurotoxins (BoNT/A to G), together with their specificity and reversibility, includes them in the list A of potential bioterrorism weapons and, at the same time, among the therapeutics of choice for a variety of human syndromes. They invade nerve terminals and cleave specifically the three proteins which form the heterotrimeric SNAP REceptors (SNARE) complex that mediates neurotransmitter release. The BoNT-induced cleavage of the SNARE proteins explains by itself the paralysing activity of the BoNTs because the truncated proteins cannot form the SNARE complex. However, in the case of BoNT/A, the most widely used toxin in therapy, additional factors come into play as it only removes a few residues from the synaptosomal associate protein of 25 kDa C-terminus and this results in a long duration of action. To explain these facts and other experimental data, we present here a model for the assembly of the neuroexocytosis apparatus in which Synaptotagmin and Complexin first assist the zippering of the SNARE complex, and then stabilize and clamp an octameric radial assembly of the SNARE complexes.

  9. Synaptic vesicle proteins: targets and routes for botulinum neurotoxins.

    PubMed

    Ahnert-Hilger, Gudrun; Münster-Wandowski, Agnieszka; Höltje, Markus

    2013-01-01

    Synaptic vesicles (SV) are key organelles of neuronal communication. SV are responsible for the storage of neurotransmitters, which are released by Ca(2+)-dependent exocytosis. After release and interaction with postsynaptic receptors, transmitters rapidly diffuse out of the synaptic cleft and are sequestered by plasma membrane transporters (in some cases following enzymatic conversion). SVs undergo endocytosis and are refilled by specific vesicular transmitter transporters different in the various neuronal subtypes. Besides these differences, SVs in general are equipped with a remarkable common set of proteins. Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release from almost all types of neurons by cleaving proteins required for membrane fusion localized either to SVs (synaptobrevin) or to the plasma membrane (SNAP-25 and syntaxin) depending on the BoNT serotype. To enter the neuronal cytoplasm, BoNTs specifically interact with the luminal domain of SV proteins (synaptotagmin or SV2, depending on serotype) transiently exposed during exocytotic membrane fusion and occurring in almost every neuron. Thus, the highly specific interaction with luminal domains of SV proteins commonly expressed on all SV types is one reason why BoNTs exhibit such a high neuronal specificity but attack almost every neuron type.

  10. Snake and Spider Toxins Induce a Rapid Recovery of Function of Botulinum Neurotoxin Paralysed Neuromuscular Junction

    PubMed Central

    Duregotti, Elisa; Zanetti, Giulia; Scorzeto, Michele; Megighian, Aram; Montecucco, Cesare; Pirazzini, Marco; Rigoni, Michela

    2015-01-01

    Botulinum neurotoxins (BoNTs) and some animal neurotoxins (β-Bungarotoxin, β-Btx, from elapid snakes and α-Latrotoxin, α-Ltx, from black widow spiders) are pre-synaptic neurotoxins that paralyse motor axon terminals with similar clinical outcomes in patients. However, their mechanism of action is different, leading to a largely-different duration of neuromuscular junction (NMJ) blockade. BoNTs induce a long-lasting paralysis without nerve terminal degeneration acting via proteolytic cleavage of SNARE proteins, whereas animal neurotoxins cause an acute and complete degeneration of motor axon terminals, followed by a rapid recovery. In this study, the injection of animal neurotoxins in mice muscles previously paralyzed by BoNT/A or /B accelerates the recovery of neurotransmission, as assessed by electrophysiology and morphological analysis. This result provides a proof of principle that, by causing the complete degeneration, reabsorption, and regeneration of a paralysed nerve terminal, one could favour the recovery of function of a biochemically- or genetically-altered motor axon terminal. These observations might be relevant to dying-back neuropathies, where pathological changes first occur at the neuromuscular junction and then progress proximally toward the cell body. PMID:26670253

  11. Sequence characterization of a novel alpha-neurotoxin from the king cobra (Ophiophagus hannah) venom.

    PubMed

    Chang, C C; Huang, T Y; Kuo, K W; Chen, S W; Huang, K F; Chiou, S H

    1993-02-26

    Several postsynaptic neurotoxins (alpha-neurotoxins) with distinct pharmacological and biochemical properties were isolated and purified from the King cobra venom (Ophiophagus hannah) by employing sequentially preparative-scale cation-exchange chromatography on SP-Sephadex C-25 coupled with gel filtration and reversed-phase HPLC. The complete sequence of one neurotoxin was determined by N-terminal Edman degradation with the automatic pulsed-liquid phase sequencer on some peptide fragments generated from the endopeptidases, i.e. trypsin, S. aureus V8 protease and lysyl endopeptidase. This novel neurotoxin is a basic polypeptide of pI 9.05, consisting of 72 amino-acid residues with 10 cysteine residues. It is found to share about 60% sequence homology with Toxins a and b isolated from the same venom and the well established alpha-bungarotoxin, a major postsynaptic toxic ligand for acetylcholine receptor isolated from Bungarus multicinctus. The characterized alpha-neurotoxin molecules were also shown to bind specifically with nicotinic acetylcholine receptors of the electric eel, Torpedo californica.

  12. Structural and functional characterization of a novel homodimeric three-finger neurotoxin from the venom of Ophiophagus hannah (king cobra).

    PubMed

    Roy, Amrita; Zhou, Xingding; Chong, Ming Zhi; D'hoedt, Dieter; Foo, Chun Shin; Rajagopalan, Nandhakishore; Nirthanan, Selvanayagam; Bertrand, Daniel; Sivaraman, J; Kini, R Manjunatha

    2010-03-12

    Snake venoms are a mixture of pharmacologically active proteins and polypeptides that have led to the development of molecular probes and therapeutic agents. Here, we describe the structural and functional characterization of a novel neurotoxin, haditoxin, from the venom of Ophiophagus hannah (King cobra). Haditoxin exhibited novel pharmacology with antagonism toward muscle (alphabetagammadelta) and neuronal (alpha(7), alpha(3)beta(2), and alpha(4)beta(2)) nicotinic acetylcholine receptors (nAChRs) with highest affinity for alpha(7)-nAChRs. The high resolution (1.5 A) crystal structure revealed haditoxin to be a homodimer, like kappa-neurotoxins, which target neuronal alpha(3)beta(2)- and alpha(4)beta(2)-nAChRs. Interestingly however, the monomeric subunits of haditoxin were composed of a three-finger protein fold typical of curaremimetic short-chain alpha-neurotoxins. Biochemical studies confirmed that it existed as a non-covalent dimer species in solution. Its structural similarity to short-chain alpha-neurotoxins and kappa-neurotoxins notwithstanding, haditoxin exhibited unique blockade of alpha(7)-nAChRs (IC(50) 180 nm), which is recognized by neither short-chain alpha-neurotoxins nor kappa-neurotoxins. This is the first report of a dimeric short-chain alpha-neurotoxin interacting with neuronal alpha(7)-nAChRs as well as the first homodimeric three-finger toxin to interact with muscle nAChRs. PMID:20071329

  13. Structural and functional characterization of a novel homodimeric three-finger neurotoxin from the venom of Ophiophagus hannah (king cobra).

    PubMed

    Roy, Amrita; Zhou, Xingding; Chong, Ming Zhi; D'hoedt, Dieter; Foo, Chun Shin; Rajagopalan, Nandhakishore; Nirthanan, Selvanayagam; Bertrand, Daniel; Sivaraman, J; Kini, R Manjunatha

    2010-03-12

    Snake venoms are a mixture of pharmacologically active proteins and polypeptides that have led to the development of molecular probes and therapeutic agents. Here, we describe the structural and functional characterization of a novel neurotoxin, haditoxin, from the venom of Ophiophagus hannah (King cobra). Haditoxin exhibited novel pharmacology with antagonism toward muscle (alphabetagammadelta) and neuronal (alpha(7), alpha(3)beta(2), and alpha(4)beta(2)) nicotinic acetylcholine receptors (nAChRs) with highest affinity for alpha(7)-nAChRs. The high resolution (1.5 A) crystal structure revealed haditoxin to be a homodimer, like kappa-neurotoxins, which target neuronal alpha(3)beta(2)- and alpha(4)beta(2)-nAChRs. Interestingly however, the monomeric subunits of haditoxin were composed of a three-finger protein fold typical of curaremimetic short-chain alpha-neurotoxins. Biochemical studies confirmed that it existed as a non-covalent dimer species in solution. Its structural similarity to short-chain alpha-neurotoxins and kappa-neurotoxins notwithstanding, haditoxin exhibited unique blockade of alpha(7)-nAChRs (IC(50) 180 nm), which is recognized by neither short-chain alpha-neurotoxins nor kappa-neurotoxins. This is the first report of a dimeric short-chain alpha-neurotoxin interacting with neuronal alpha(7)-nAChRs as well as the first homodimeric three-finger toxin to interact with muscle nAChRs.

  14. Antibody protection against botulinum neurotoxin intoxication in mice.

    PubMed

    Cheng, Luisa W; Stanker, Larry H; Henderson, Thomas D; Lou, Jianlong; Marks, James D

    2009-10-01

    Adulteration of food or feed with any of the seven serotypes of botulinum neurotoxin (BoNT) is a potential bioterrorism concern. Currently, there is strong interest in the development of detection reagents, vaccines, therapeutics, and other countermeasures. A sensitive immunoassay for detecting BoNT serotype A (BoNT/A), based on monoclonal antibodies (MAbs) F1-2 and F1-40, has been developed and used in complex matrices. The epitope for F1-2 has been mapped to the heavy chain of BoNT/A, and the epitope of F1-40 has been mapped to the light chain. The ability of these MAbs to provide therapeutic protection against BoNT/A intoxication in mouse intravenous and oral intoxication models was tested. High dosages of individual MAbs protected mice well both pre- and postexposure to BoNT/A holotoxin. A combination therapy consisting of antibodies against both the light and heavy chains of the toxin, however, significantly increased protection, even at a lower MAb dosage. An in vitro peptide assay for measuring toxin activity showed that pretreatment of toxin with these MAbs did not block catalytic activity but instead blocked toxin entry into primary and cultured neuronal cells. The timing of antibody rescue in the mouse intoxication models revealed windows of opportunity for antibody therapeutic treatment that correlated well with the biologic half-life of the toxin in the serum. Knowledge of BoNT intoxication and antibody clearance in these mouse models and understanding of the pharmacokinetics of BoNT are invaluable for future development of antibodies and therapeutics against intoxication by BoNT.

  15. Proconvulsant actions of intrahippocampal botulinum neurotoxin B in the rat.

    PubMed

    Bröer, S; Zolkowska, D; Gernert, M; Rogawski, M A

    2013-11-12

    Botulinum neurotoxins (BoNTs) may affect the excitability of brain circuits by inhibiting neurotransmitter release at central synapses. There is evidence that local delivery of BoNT serotypes A and E, which target SNAP-25, a component of the release machinery specific to excitatory synapses, can inhibit seizure generation. BoNT serotype B (BoNT/B) targets VAMP2, which is expressed in both excitatory and inhibitory terminals. Here we assessed the effects of unilateral intrahippocampal infusion of BoNT/B in the rat on intravenous pentylenetetrazol (PTZ) seizure thresholds, and on the expression of spontaneous behavioral and electrographic seizures. Infusion of BoNT/B (500 and 1,000 unit) by convection-enhanced delivery caused a reduction in myoclonic twitch and clonic seizure thresholds in response to intravenous PTZ beginning about 6 days after the infusion. Handling-evoked and spontaneous convulsive seizures were observed in many BoNT/B-treated animals but not in vehicle-treated controls. Spontaneous electrographic seizure discharges were recorded in the dentate gyrus of animals that received local BoNT/B infusion. In addition, there was an increased frequency of interictal epileptiform spikes and sharp waves at the same recording site. BoNT/B-treated animals also exhibited tactile hyperresponsivity in comparison with vehicle-treated controls. This is the first demonstration that BoNT/B causes a delayed proconvulsant action when infused into the hippocampus. Local infusion of BoNT/B could be useful as a focal epilepsy model.

  16. Botulinum neurotoxins and botulism: a novel therapeutic approach.

    PubMed

    Thanongsaksrikul, Jeeraphong; Chaicumpa, Wanpen

    2011-05-01

    Specific treatment is not available for human botulism. Current remedial mainstay is the passive administration of polyclonal antibody to botulinum neurotoxin (BoNT) derived from heterologous species (immunized animal or mouse hybridoma) together with supportive and symptomatic management. The antibody works extracellularly, probably by blocking the binding of receptor binding (R) domain to the neuronal receptors; thus inhibiting cellular entry of the holo-BoNT. The antibody cannot neutralize the intracellular toxin. Moreover, a conventional antibody with relatively large molecular size (150 kDa) is not accessible to the enzymatic groove and, thus, cannot directly inhibit the BoNT zinc metalloprotease activity. Recently, a 15-20 kDa single domain antibody (V(H)H) that binds specifically to light chain of BoNT serotype A was produced from a humanized-camel VH/V(H)H phage display library. The V(H)H has high sequence homology (>80%) to the human VH and could block the enzymatic activity of the BoNT. Molecular docking revealed not only the interface binding between the V(H)H and the toxin but also an insertion of the V(H)H CDR3 into the toxin enzymatic pocket. It is envisaged that, by molecular linking the V(H)H to a cell penetrating peptide (CPP), the CPP-V(H)H fusion protein would be able to traverse the hydrophobic cell membrane into the cytoplasm and inhibit the intracellular BoNT. This presents a novel and safe immunotherapeutic strategy for botulism by using a cell penetrating, humanized-single domain antibody that inhibits the BoNT by means of a direct blockade of the groove of the menace enzyme.

  17. Molecular structure and conformations of caramboxin, a natural neurotoxin from the star fruit: A computational study

    NASA Astrophysics Data System (ADS)

    Pichierri, Fabio

    2015-01-01

    Using density functional theory calculations we investigate the molecular structure and conformations of caramboxin, a neurotoxin recently isolated from the star fruit Averroha carambola. Among the seven conformers that exist within an energy window of ∼16.0 kcal/mol, two of them are the most favored ones with an energy difference of less than 2.0 kcal/mol. The computed chemical shifts of these two low-energy conformers are in good agreement with the experimental values determined in deuterated dimethylsulfoxide thus confirming the 2D chemical structure assigned to the neurotoxin. A topological analysis of the theoretical electronic charge density of four caramboxin conformers reveals the existence of intramolecular CH⋯O/N interactions which, in addition to the classical OH⋯O/N H-bonding interactions, contribute to decrease the conformational freedom of the neurotoxin.

  18. Calpains participate in nerve terminal degeneration induced by spider and snake presynaptic neurotoxins.

    PubMed

    Duregotti, Elisa; Tedesco, Erik; Montecucco, Cesare; Rigoni, Michela

    2013-03-15

    α-latrotoxin and snake presynaptic phospholipases A2 neurotoxins target the presynaptic membrane of axon terminals of the neuromuscular junction causing paralysis. These neurotoxins display different biochemical activities, but similarly alter the presynaptic membrane permeability causing Ca(2+) overload within the nerve terminals, which in turn induces nerve degeneration. Using different methods, here we show that the calcium-activated proteases calpains are involved in the cytoskeletal rearrangements that we have previously documented in neurons exposed to α-latrotoxin or to snake presynaptic phospholipases A2 neurotoxins. These results indicate that calpains, activated by the massive calcium influx from the extracellular medium, target fundamental components of neuronal cytoskeleton such as spectrin and neurofilaments, whose cleavage is functional to the ensuing nerve terminal fragmentation.

  19. Three-finger snake neurotoxins and Ly6 proteins targeting nicotinic acetylcholine receptors: pharmacological tools and endogenous modulators.

    PubMed

    Tsetlin, Victor I

    2015-02-01

    Snake venom neurotoxins and lymphocyte antigen 6 (Ly6) proteins, most of the latter being membrane tethered by a glycosylphosphatidylinositol (GPI) anchor, have a variety of biological activities, but their three-finger (3F) folding combines them in one Ly6/neurotoxin family. Subsets of two groups, represented by α-neurotoxins and Lynx1, respectively, interact with nicotinic acetylcholine receptors (nAChR) and, hence, are of therapeutic interest for the treatment of neurodegenerative diseases, pain, and cancer. Information on the mechanisms of action and 3D structure of the binding sites, which is required for drug design, is available from the 3D structure of α-neurotoxin complexes with nAChR models. Here, I compare the structural and functional features of α-neurotoxins versus Lynx1 and its homologs to get a clearer picture of Lynx1-nAChR interactions that is necessary for fundamental science and practical applications.

  20. Prevention of paralytic neurotoxin action on voltage-sensitive sodium channels. Final report

    SciTech Connect

    Catterall, W.A.

    1993-10-11

    This final report summarizes the conclusions from research in the first half of this contract that was presented in detail in the midterm report, presents a detailed description of the research carried out in the second half of this contract, and, where appropriate proposes potentially fruitful directions for future research on the mechanisms of action of paralytic neurotoxins and on approaches to prevention of their action. The presentation is organized in sequence according to the ten Tasks undertaken as proposed in the original contract. Sections describing Experimental Procedures, Results, Discussion, and Figures are presented for each Task undertaken. RA I, Lab animals, Rats, Rabbits, Synthetic peptides, Neurotoxins, Sodium channels, Receptor sites.

  1. Clinical differences between botulinum neurotoxin type A and B.

    PubMed

    Bentivoglio, Anna Rita; Del Grande, Alessandra; Petracca, Martina; Ialongo, Tamara; Ricciardi, Lucia

    2015-12-01

    In humans, the therapeutic use of botulinum neurotoxin A (BoNT/A) is well recognized and continuously expanding. Four BoNTs are widely available for clinical practice: three are serotype A and one is serotype B: onabotulinumtoxinA (A/Ona), abobotulinumtoxinA (A/Abo) and incobotulinumtoxinA (A/Inco), rimabotulinumtoxinB (B/Rima). A/Abo, A/Inco, A/Ona and B/Rima are all licensed worldwide for cervical dystonia. In addition, the three BoNT/A products are approved for blepharospasm and focal dystonias, spasticity, hemifacial spasm, hyperhidrosis and facial lines, with remarkable regional differences. These toxin brands differ for specific activity, packaging, constituents, excipient, and storage. Comparative literature assessing the relative safety and efficacy of different BoNT products is limited, most data come from reports on small samples, and only a few studies meet criteria of evidence-based medicine. One study compared the effects of BoNT/A and BoNT/B on muscle activity of healthy volunteers, showing similar neurophysiological effects with a dose ratio of 1:100. In cervical dystonia, when comparing the effects of BoNT/A and BoNT/B, results are more variable, some studies reporting roughly similar peak effect and overall duration (at a ratio of 1:66, others reporting substantially shorter duration of BoNT/B than BoNT/A (at a ratio 1/24). Although the results of clinical studies are difficult to compare for methodological differences (dose ratio, study design, outcome measures), it is widely accepted that: BoNT/B is clinically effective using appropriate doses as BoNT/A (1:40-50), injections are generally more painful, in most of the studies on muscular conditions, efficacy is shorter, and immunogenicity higher. Since the earliest clinical trials, it has been reported that autonomic side effects are more frequent after BoNT/B injections, and this observation encouraged the use of BoNT/B for sialorrhea, hyperhidrosis and other non-motor symptoms. In these

  2. Neurotoxins from Snake Venoms and α-Conotoxin ImI Inhibit Functionally Active Ionotropic γ-Aminobutyric Acid (GABA) Receptors*

    PubMed Central

    Kudryavtsev, Denis S.; Shelukhina, Irina V.; Son, Lina V.; Ojomoko, Lucy O.; Kryukova, Elena V.; Lyukmanova, Ekaterina N.; Zhmak, Maxim N.; Dolgikh, Dmitry A.; Ivanov, Igor A.; Kasheverov, Igor E.; Starkov, Vladislav G.; Ramerstorfer, Joachim; Sieghart, Werner; Tsetlin, Victor I.; Utkin, Yuri N.

    2015-01-01

    Ionotropic receptors of γ-aminobutyric acid (GABAAR) regulate neuronal inhibition and are targeted by benzodiazepines and general anesthetics. We show that a fluorescent derivative of α-cobratoxin (α-Ctx), belonging to the family of three-finger toxins from snake venoms, specifically stained the α1β3γ2 receptor; and at 10 μm α-Ctx completely blocked GABA-induced currents in this receptor expressed in Xenopus oocytes (IC50 = 236 nm) and less potently inhibited α1β2γ2 ≈ α2β2γ2 > α5β2γ2 > α2β3γ2 and α1β3δ GABAARs. The α1β3γ2 receptor was also inhibited by some other three-finger toxins, long α-neurotoxin Ls III and nonconventional toxin WTX. α-Conotoxin ImI displayed inhibitory activity as well. Electrophysiology experiments showed mixed competitive and noncompetitive α-Ctx action. Fluorescent α-Ctx, however, could be displaced by muscimol indicating that most of the α-Ctx-binding sites overlap with the orthosteric sites at the β/α subunit interface. Modeling and molecular dynamic studies indicated that α-Ctx or α-bungarotoxin seem to interact with GABAAR in a way similar to their interaction with the acetylcholine-binding protein or the ligand-binding domain of nicotinic receptors. This was supported by mutagenesis studies and experiments with α-conotoxin ImI and a chimeric Naja oxiana α-neurotoxin indicating that the major role in α-Ctx binding to GABAAR is played by the tip of its central loop II accommodating under loop C of the receptors. PMID:26221036

  3. Neuronal targeting, internalization, and biological activity of a recombinant atoxic derivative of botulinum neurotoxin A

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Non-toxic derivatives of Botulinum neurotoxin A (BoNT/A) have potential use as neuron-targeting delivery vehicles, and as reagents to study intracellular trafficking. We have designed and expressed an atoxic derivative of BoNT/A (BoNT/A ad) as a full-length 150kDa molecule consisting of a 50 kDa lig...

  4. Correcting age-related changes in the face by use of injectable fillers and neurotoxins.

    PubMed

    Rubin, Mark G; Cox, Sue Ellen; Kaminer, Michael S; Solish, Nowell

    2014-06-01

    Many patients seeking rejuvenation treatment have readily apparent age-related changes in facial features. Others exhibit more subtle changes that nonetheless can be corrected to achieve a more youthful appearance. In the following article, four specialists in aesthetic dermatology discuss how injectable hyaluronic acid-based fillers and neurotoxins can achieve rejuvenation without surgery.

  5. A monoclonal antibody based capture ELISA for botulinum neurotoxin serotype B: toxin detection in food

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Botulism is a serious foodborne neuroparalyic disease caused by botulinum neurotoxin (BoNT) produced by the anaerobic bacterium Clostridium botulinum. Seven toxin serotypes (A-H) have been described. The majority of human cases of botulism are caused by serotypes A and B followed by E and F. We repo...

  6. Inhibiting oral intoxication of botulinum neurotoxin A by carbohydrate receptor mimics

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Botulinum neurotoxins (BoNTs) cause the disease botulism manifested by flaccid paralysis that could be fatal to humans and animals. Oral ingestion of the toxin with contaminated food is one of the most common routes of BoNT intoxication, where BoNT assembles with several auxiliary proteins to surviv...

  7. Translocation of botulinum neurotoxins and associated proteins across intestinal epithelial cells(Abstract)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Botulinum neurotoxins(BoNTs)secreted by Clostridium botulinum are some of the most poisonous toxins in nature and considered to be major bioterrorism threats. To date, seven BoNT subtypes (A to G) have been identified. When secreted from bacteria, some BoNTs associate with a non-toxic, non hemagglu...

  8. Complete Assignment of (1)H-NMR Resonances of the King Cobra Neurotoxin CM-11.

    PubMed

    Pang, Yu-Xi; Liu, Wei-Dong; Liu, Ai-Zhuo; Pei, Feng-Kui

    1997-01-01

    The king cobra (Ophiophagus Hannah) neurotoxin CM-Il is long-chain peptide with 72 amino acid residues. Its complete assignment of (1)H-NMR resonances was obtained using various 2D-NMR technologies, including DQF-COSY, clean-TOCSY and NOESY.

  9. Zebrafish (Danio rerio) bioassay for visceral toxicosis of catfish and botulinum neurotoxin serotype E

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Visceral toxicosis of catfish (VTC), a sporadic disease of cultured channel catfish (Ictalurus punctatus) often with high mortality, is caused by botulinum neurotoxin serotype E (BoNT/E). Presumptive diagnosis of VTC is based on characteristic clinical signs and lesions, and the production of these ...

  10. Clostridium botulinum neurotoxin type B is heat-stable in milk and not inactivated by pasteurization.

    PubMed

    Rasooly, Reuven; Do, Paula M

    2010-12-01

    Foodborne botulism is caused by the ingestion of foods containing botulinum neurotoxins (BoNTs). To study the heat stability of Clostridium botulinum neurotoxins, we needed to measure and compare the activity of botulinum neurotoxins, serotypes A and B, under various pasteurization conditions. Currently, the only accepted assay to detect active C. botulinum neurotoxin is an in vivo mouse bioassay, which raises ethical concerns with regard to the use of experimental animals. In this study, noninvasive methods were used to simultaneously detect and distinguish between active BoNT serotypes A and B in one reaction and sample. We developed an enzymatic activity assay employing internally quenched fluorogenic peptides corresponding to SNAP-25, for BoNT-A, and VAMP2, for BoNT-B, as an alternative method to the mouse bioassay. Because each peptide is labeled with different fluorophores, we were able to distinguish between these two toxins. We used this method to analyze the heat stability of BoNT-A and BoNT-B. This study reports that conventional milk pasteurization (63 °C, 30 min) inactivated BoNT serotype A; however, serotype B is heat-stable in milk and not inactivated by pasteurization. Using this activity assay, we also showed that the commonly used food processes such as acidity and pasteurization, which are known to inhibit C. botulinum growth and toxin production, are more effective in inactivating BoNT serotype A than serotype B when conventional pasteurization (63 °C, 30 min) is used.

  11. Development of a quail embryo model for the detection of botulinum neurotoxin activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium botulinum is a ubiquitous microorganism that under anaerobic conditions produces botulinum neurotoxins. In regards to both food-borne illness and the potential use of botulinum toxin as a biological weapon, the capability to assess the amount of toxin in a food or environmental sample e...

  12. Atoxic derivative of botulinum neurotoxin A as a prototype vehicle for targeted delivery to neuronal cytoplasm

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have previously described genetic constructs and expression systems that enable facile production of recombinant derivatives of botulinum neurotoxins (BoNTs) that retain the structural and trafficking properties of wt BoNTs. In this report we describe the properties of one such derivative, BoNT/A...

  13. Comparison of Toxicological Properties of Botulinum Neurotoxin Serotypes A and B in Mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Botulinum neurotoxins (BoNTs) are among the most toxic biological toxins for humans. Of the seven known serotypes (A-G) of BoNT, serotypes A, B and E cause most of the human foodborne intoxications. In this study, we compared the toxicological properties of BoNT serotype A and B holotoxins and compl...

  14. Substrates and controls for the quantitative detection of active botulinum neurotoxin in protease-containing samples

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Botulinum neurotoxins (BoNTs) enjoy a wide variety of medical applications. However, limited pharmacokinetic data on active BoNT is available. Monitoring BoNT activity in the circulation is a challenging task, due to BoNT’s enormous toxicity, rapid neuronal uptake, and removal from the bloodstream. ...

  15. Neuronal targeting, internalization, and biological activity of a recombinant atoxic derivative of botulinum neurotoxin A

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Botulinum neurotoxins (BoNT) have the unique capacity to cross epithelial barriers, target neuromuscular junctions, and translocate active metalloprotease component to the cytosol of motor neurons. We have taken advantage of the molecular carriers responsible for this trafficking to create a family ...

  16. Antibody-mediated neutralization and binding-reversal studies on alpha-neurotoxins from Micrurus nigrocinctus nigrocinctus (coral snake) venom.

    PubMed

    Alape-Giron, A; Stiles, B G; Gutierrez, J M

    1996-03-01

    An ELISA based, non-radioactive acetylcholine receptor (AchR) binding assay was used to detect the alpha-neurotoxins present in Micrurus nigrocinctus nigrocinctus venom. Sera from horses hyperimmunized against M. nigrocinctus venom contain antibodies which inhibit the binding of M. n. nigrocinctus alpha-neurotoxins to AchR and reverse the binding of toxins already complexed with the receptor. This result supports the importance of using antivenom therapeutically in M. n. nigrocinctus envenomations even after the onset of neurological symptoms. M. nigrocinctus antivenoms cross-reacted in an ELISA with several elapid alpha-neurotoxins and inhibited the binding of Bungarus multicinctus alpha-bungarotoxin and Naja naja oxiana neurotoxin II to AchR in vitro, suggesting the presence of short-chain and long-chain alpha-neurotoxins in M. nigrocinctus venom. In vivo neutralization experiments with M. nigrocinctus antivenom demonstrate that M. nigrocinctus venom contains short-chain alpha-neurotoxin(s) which share common neutralizing epitope(s) with Naja naja oxiana neurotoxin II.

  17. Botulinum Neurotoxin Serotype A Recognizes Its Protein Receptor SV2 by a Different Mechanism than Botulinum Neurotoxin B Synaptotagmin

    PubMed Central

    Weisemann, Jasmin; Stern, Daniel; Mahrhold, Stefan; Dorner, Brigitte G.; Rummel, Andreas

    2016-01-01

    Botulinum neurotoxins (BoNTs) exhibit extraordinary potency due to their exquisite neurospecificity, which is achieved by dual binding to complex polysialo-gangliosides and synaptic vesicle proteins. The luminal domain 4 (LD4) of the three synaptic vesicle glycoprotein 2 isoforms, SV2A‐C, identified as protein receptors for the most relevant serotype BoNT/A, binds within the 50 kDa cell binding domain HC of BoNT/A. Here, we deciphered the BoNT/A‐SV2 interactions in more detail. In pull down assays, the binding of HCA to SV2-LD4 isoforms decreases from SV2C >> SV2A > SV2B. A binding constant of 200 nM was determined for BoNT/A to rat SV2C-LD4 in GST pull down assay. A similar binding constant was determined by surface plasmon resonance for HCA to rat SV2C and to human SV2C, the latter being slightly lower due to the substitution L563F in LD4. At pH 5, as measured in acidic synaptic vesicles, the binding constant of HCA to hSV2C is increased more than 10-fold. Circular dichroism spectroscopy reveals that the quadrilateral helix of SV2C-LD4 already exists in solution prior to BoNT/A binding. Hence, the BoNT/A‐SV2C interaction is of different nature compared to BoNT/B‐Syt-II. In particular, the preexistence of the quadrilateral β-sheet helix of SV2 and its pH-dependent binding to BoNT/A via backbone–backbone interactions constitute major differences. Knowledge of the molecular details of BoNT/A‐SV2 interactions drives the development of high affinity peptides to counteract BoNT/A intoxications or to capture functional BoNT/A variants in innovative detection systems for botulism diagnostic. PMID:27196927

  18. Botulinum Neurotoxin Serotype A Recognizes Its Protein Receptor SV2 by a Different Mechanism than Botulinum Neurotoxin B Synaptotagmin.

    PubMed

    Weisemann, Jasmin; Stern, Daniel; Mahrhold, Stefan; Dorner, Brigitte G; Rummel, Andreas

    2016-01-01

    Botulinum neurotoxins (BoNTs) exhibit extraordinary potency due to their exquisite neurospecificity, which is achieved by dual binding to complex polysialo-gangliosides and synaptic vesicle proteins. The luminal domain 4 (LD4) of the three synaptic vesicle glycoprotein 2 isoforms, SV2A-C, identified as protein receptors for the most relevant serotype BoNT/A, binds within the 50 kDa cell binding domain HC of BoNT/A. Here, we deciphered the BoNT/A-SV2 interactions in more detail. In pull down assays, the binding of HCA to SV2-LD4 isoforms decreases from SV2C > SV2A > SV2B. A binding constant of 200 nM was determined for BoNT/A to rat SV2C-LD4 in GST pull down assay. A similar binding constant was determined by surface plasmon resonance for HCA to rat SV2C and to human SV2C, the latter being slightly lower due to the substitution L563F in LD4. At pH 5, as measured in acidic synaptic vesicles, the binding constant of HCA to hSV2C is increased more than 10-fold. Circular dichroism spectroscopy reveals that the quadrilateral helix of SV2C-LD4 already exists in solution prior to BoNT/A binding. Hence, the BoNT/A-SV2C interaction is of different nature compared to BoNT/B-Syt-II. In particular, the preexistence of the quadrilateral β-sheet helix of SV2 and its pH-dependent binding to BoNT/A via backbone-backbone interactions constitute major differences. Knowledge of the molecular details of BoNT/A-SV2 interactions drives the development of high affinity peptides to counteract BoNT/A intoxications or to capture functional BoNT/A variants in innovative detection systems for botulism diagnostic. PMID:27196927

  19. Presynaptic snake beta-neurotoxins produce tetanic fade and endplate potential run-down during neuromuscular blockade in mouse diaphragm.

    PubMed

    Wilson, H I; Nicholson, G M

    1997-11-01

    The present study investigated the ability of a number of presynaptic snake neurotoxins (snake beta-neurotoxins) to produce nerve-evoked train-of-four fade, tetanic fade and endplate potential run-down during the development of neuromuscular blockade in the isolated mouse phrenic-hemidiaphragm nerve-muscle preparation. All the snake beta-neurotoxins tested, with the exception of notexin, produced train-of-four and tetanic fade of nerve-evoked isometric muscle contractions. Train-of-four fade was not present during the initial depressant or facilitatory phases of muscle tension produced by the snake beta-neurotoxins but developed progressively during the final depressant phase that precedes complete neuromuscular blockade. The 'non-neurotoxic' bovine pancreatic phospholipase A2 and the 'low-toxicity' phospholipase A2 from Naja naja atra venom failed to elicit train-of-four fade, indicating that the phospholipase activity of the snake beta-neurotoxins is not responsible for the development of fade. Intracellular recording of endplate potentials (EPPs) elicited by nerve-evoked trains of stimuli showed a progressive run-down in EPP amplitude during the train following incubation with all snake beta-neurotoxins except notexin. Again this run-down in EPP amplitude was confined to the final depressant phase of snake beta-neurotoxin action. However when EPP amplitude fell to near uniquantal levels (< 3 mV) the extent of toxin induced-fade was reduced. Unlike postjunctional snake alpha-neurotoxins, prejunctional snake beta-neurotoxins interfere with acetylcholine release at the neuromuscular junction during the development of neuromuscular blockade. This study provides further support for the hypothesis that fade in twitch and tetanic muscle tension is due to an underlying rundown in EPP amplitude resulting from a prejunctional alteration of transmitter release rather than a use-dependent block of postjunctional nicotinic receptors.

  20. Peptide neurotoxins that affect voltage-gated calcium channels: a close-up on ω-agatoxins.

    PubMed

    Pringos, Emilie; Vignes, Michel; Martinez, Jean; Rolland, Valerie

    2011-01-01

    Peptide neurotoxins found in animal venoms have gained great interest in the field of neurotransmission. As they are high affinity ligands for calcium, potassium and sodium channels, they have become useful tools for studying channel structure and activity. Peptide neurotoxins represent the clinical potential of ion-channel modulators across several therapeutic fields, especially in developing new strategies for treatment of ion channel-related diseases. The aim of this review is to overview the latest updates in the domain of peptide neurotoxins that affect voltage-gated calcium channels, with a special focus on ω-agatoxins.

  1. Identification and Characterization of Botulinum Neurotoxin A Substrate Binding Pockets and Their Re-Engineering for Human SNAP-23.

    PubMed

    Sikorra, Stefan; Litschko, Christa; Müller, Carina; Thiel, Nadine; Galli, Thierry; Eichner, Timo; Binz, Thomas

    2016-01-29

    Botulinum neurotoxins (BoNTs) are highly potent bacterial proteins that block neurotransmitter release at the neuromuscular junction by cleaving SNAREs (soluble N-ethyl maleimide sensitive factor attachment protein receptors). However, their serotype A (BoNT/A) that cleaves SNAP-25 (synaptosomal-associated protein of 25 kDa) has also been an established pharmaceutical for treatment of medical conditions that rely on hyperactivity of cholinergic nerve terminals for 25 years. The expansion of its use to a variety of further medical conditions associated with hypersecretion components is prevented partly because the involved SNARE isoforms are not cleaved. Therefore, we examined by mutational analyses the reason for the resistance of human SNAP-23, an isoform of SNAP-25. We show that replacement of 10 SNAP-23 residues with their SNAP-25 counterparts effects SNAP-25-like cleavability. Conversely, transfer of each of the replaced SNAP-23 residues to SNAP-25 drastically decreased the cleavability of SNAP-25. By means of the existing SNAP-25-toxin co-crystal structure, molecular dynamics simulations, and corroborative mutagenesis studies, the appropriate binding pockets for these residues in BoNT/A were characterized. Systematic mutagenesis of two major BoNT/A binding pockets was conducted in order to adapt these pockets to corresponding amino acids of human SNAP-23. Human SNAP-23 cleaving mutants were isolated using a newly established yeast-based screening system. This method may be useful for engineering novel BoNT/A pharmaceuticals for the treatment of diseases that rely on SNAP-23-mediated hypersecretion.

  2. A microscale neuron and Schwann cell coculture model for increasing detection sensitivity of botulinum neurotoxin type A.

    PubMed

    Hong, Won S; Young, Edmond W K; Tepp, William H; Johnson, Eric A; Beebe, David J

    2013-07-01

    Botulinum neurotoxin (BoNT) is a potent and specific biomolecule that is both implicated as a potential threat in bioterrorism and used in therapeutics. Highly sensitive and robust assays that measure BoNT activity are needed to manage outbreak or controlled distribution of BoNT. Current in vivo and in vitro assays have limitations, including high costs and variability for mouse bioassays, extensive preparations for primary and stem cell-derived neurons, and inherent low sensitivity for cell lines. Sensitivity of cell lines can be increased by direct differentiation and with their physiological relevance (compared with cell-free strategies) and robustness (compared with primary cell strategies); adopting cell lines is an attractive alternative to in vivo assays. Here, we present two distinct strategies that improved sensitivity of a cell line to BoNT serotype A (BoNT/A) without direct differentiation. We developed a cell-based BoNT assay using microscale culture and coculture of neuronal and Schwann cell lines, NG108-15 and S16, respectively, to improve both sensitivity and physiological relevance. Results showed that NG108-15 and S16 coculture decreased EC50 from 12.5 to 0.8ng/µl (p < 0.001) in macroscale and from 2.6 to 1.1ng/µl (p = 0.006) in microscale. In addition, NG108-15 monoculture at microscale decreased EC50 from 12.5 to 2.6ng/µl (p < 0.001) compared with macroscale. Finally, controlling the spatial arrangement of microscale coculture revealed that S16-derived soluble factors can increase sensitivity. Thus, our study demonstrates two distinct strategies for increasing the sensitivity of a cell line to BoNT using coculture and microscale culture, thereby advancing assay technology for BoNT detection. PMID:23564642

  3. Accelerated intoxication of GABAergic synapses by botulinum neurotoxin A disinhibits stem cell-derived neuron networks prior to network silencing

    PubMed Central

    Beske, Phillip H.; Scheeler, Stephen M.; Adler, Michael; McNutt, Patrick M.

    2015-01-01

    Botulinum neurotoxins (BoNTs) are extremely potent toxins that specifically cleave SNARE proteins in peripheral synapses, preventing neurotransmitter release. Neuronal responses to BoNT intoxication are traditionally studied by quantifying SNARE protein cleavage in vitro or monitoring physiological paralysis in vivo. Consequently, the dynamic effects of intoxication on synaptic behaviors are not well-understood. We have reported that mouse embryonic stem cell-derived neurons (ESNs) are highly sensitive to BoNT based on molecular readouts of intoxication. Here we study the time-dependent changes in synapse- and network-level behaviors following addition of BoNT/A to spontaneously active networks of glutamatergic and GABAergic ESNs. Whole-cell patch-clamp recordings indicated that BoNT/A rapidly blocked synaptic neurotransmission, confirming that ESNs replicate the functional pathophysiology responsible for clinical botulism. Quantitation of spontaneous neurotransmission in pharmacologically isolated synapses revealed accelerated silencing of GABAergic synapses compared to glutamatergic synapses, which was consistent with the selective accumulation of cleaved SNAP-25 at GAD1+ pre-synaptic terminals at early timepoints. Different latencies of intoxication resulted in complex network responses to BoNT/A addition, involving rapid disinhibition of stochastic firing followed by network silencing. Synaptic activity was found to be highly sensitive to SNAP-25 cleavage, reflecting the functional consequences of the localized cleavage of the small subpopulation of SNAP-25 that is engaged in neurotransmitter release in the nerve terminal. Collectively these findings illustrate that use of synaptic function assays in networked neurons cultures offers a novel and highly sensitive approach for mechanistic studies of toxin:neuron interactions and synaptic responses to BoNT. PMID:25954159

  4. Celebrity Patients, VIPs, and Potentates

    PubMed Central

    Groves, James E.; Dunderdale, Barbara A.; Stern, Theodore A.

    2002-01-01

    Background: During the second half of the 20th century, the literature on the doctor-patient relationship mainly dealt with the management of “difficult” (personality-disordered) patients. Similar problems, however, surround other types of “special” patients. Method: An overview and analysis of the literature were conducted. As a result, such patients can be subcategorized by their main presentations; each requires a specific management strategy. Results: Three types of “special” patients stir up irrational feelings in their caregivers. Sick celebrities threaten to focus public scrutiny on the private world of medical caregivers. VIPs generate awe in caregivers, with loss of the objectivity essential to the practice of scientific medicine. Potentates unearth narcissism in the caregiver-patient relationship, which triggers a struggle between power and shame. Pride, privacy, and the staff's need to be in control are all threatened by introduction of the special patient into medicine's closed culture. Conclusion: The privacy that is owed to sick celebrities should be extended to protect overexposed staff. The awe and loss of medical objectivity that VIPs generate are counteracted by team leadership dedicated to avoiding any deviation from standard clinical procedure. Moreover, the collective ill will surrounding potentates can be neutralized by reassuring them that they are “special”—and by caregivers mending their own vulnerable self-esteem. PMID:15014712

  5. Neuroprotective Effects of the Triterpenoid, CDDO Methyl Amide, a Potent Inducer of Nrf2-Mediated Transcription

    PubMed Central

    Yang, Lichuan; Calingasan, Noel Y.; Thomas, Bobby; Chaturvedi, Rajnish K.; Kiaei, Mahmoud; Wille, Elizabeth J.; Liby, Karen T.; Williams, Charlotte; Royce, Darlene; Risingsong, Renee; Musiek, Eric S.; Morrow, Jason D.; Sporn, Michael; Beal, M. Flint

    2009-01-01

    The NF-E2-related factor-2 (Nrf2)/antioxidant response element (ARE) signaling pathway regulates phase 2 detoxification genes, including a variety of antioxidative enzymes. We tested neuroprotective effects of the synthetic triterpenoid CDDO-MA, a potent activator of the Nrf2/ARE signaling. CDDO-MA treatment of neuroblastoma SH-SY5Y cells resulted in Nrf2 upregulation and translocation from cytosol to nucleus and subsequent activation of ARE pathway genes. CDDO-MA blocked t-butylhydroperoxide-induced production of reactive oxygen species (ROS) by activation of ARE genes only in wild type, but not Nrf2 knockout mouse embryonic fibroblasts. Oral administration of CDDO-MA resulted in significant protection against MPTP-induced nigrostriatal dopaminergic neurodegeneration, pathological alpha-synuclein accumulation and oxidative damage in mice. Additionally, CDDO-MA treatment in rats produced significant rescue against striatal lesions caused by the neurotoxin 3-NP, and associated increases in the oxidative damage markers malondialdehyde, F2-Isoprostanes, 8-hydroxy-2-deoxyguanosine, 3-nitrotyrosine, and impaired glutathione homeostasis. Our results indicate that the CDDO-MA renders its neuroprotective effects through its potent activation of the Nrf2/ARE pathway, and suggest that triterpenoids may be beneficial for the treatment of neurodegenerative diseases like Parkinson's disease and Huntington's disease. PMID:19484125

  6. Multivalent Chromosomal Expression of the Clostridium botulinum Serotype A Neurotoxin Heavy-Chain Antigen and the Bacillus anthracis Protective Antigen in Lactobacillus acidophilus

    PubMed Central

    Klaenhammer, Todd R.

    2016-01-01

    ABSTRACT Clostridium botulinum and Bacillus anthracis produce potent toxins that cause severe disease in humans. New and improved vaccines are needed for both of these pathogens. For mucosal vaccine delivery using lactic acid bacteria, chromosomal expression of antigens is preferred over plasmid-based expression systems, as chromosomal expression circumvents plasmid instability and the need for antibiotic pressure. In this study, we constructed three strains of Lactobacillus acidophilus NCFM expressing from the chromosome (i) the nontoxic host receptor-binding domain of the heavy chain of Clostridium botulinum serotype A neurotoxin (BoNT/A-Hc), (ii) the anthrax protective antigen (PA), and (iii) both the BoNT/A-Hc and the PA. The BoNT/A-Hc vaccine cassette was engineered to contain the signal peptide from the S-layer protein A from L. acidophilus and a dendritic-cell-targeting peptide. A chromosomal region downstream of lba0889 carrying a highly expressed enolase gene was selected for insertion of the vaccine cassettes. Western blot analysis confirmed the heterologous expression of the two antigens from plasmid and chromosome locations. Stability assays demonstrated loss of the vaccine cassettes from expression plasmids without antibiotic maintenance. RNA sequencing showed high expression of each antigen and that insertion of the vaccine cassettes had little to no effect on the transcription of other genes in the chromosome. This study demonstrated that chromosomal integrative recombinant strains are promising vaccine delivery vehicles when targeted into high-expression chromosomal regions. Levels of expression match high-copy-number plasmids and eliminate the requirement for antibiotic selective maintenance of recombinant plasmids. IMPORTANCE Clostridium botulinum and Bacillus anthracis produce potent neurotoxins that pose a biochemical warfare concern; therefore, effective vaccines against these bacteria are required. Chromosomal expression of antigens is

  7. Architecture of the botulinum neurotoxin complex: a molecular machine for protection and delivery

    PubMed Central

    Lam, Kwok-Ho; Jin, Rongsheng

    2015-01-01

    Botulinum neurotoxins (BoNTs) are extremely poisonous protein toxins that cause the fatal paralytic disease botulism. They are naturally produced in bacteria with several nontoxic neurotoxin-associated proteins (NAPs) and together they form a progenitor toxin complex (PTC), the largest bacterial toxin complex known. In foodborne botulism, the PTC functions as a molecular machine that helps BoNT breach the host defense in the gut. Here, we discuss the substantial recent advance in elucidating the atomic structures and assembly of the 14-subunit PTC, including structures of BoNT and four NAPs. These structural studies shed light on the molecular mechanisms by which BoNT is protected against the acidic environment and proteolytic destruction in the gastrointestinal tract, and how it is delivered across the intestinal epithelial barrier. PMID:25889616

  8. VAMP/synaptobrevin cleavage by tetanus and botulinum neurotoxins is strongly enhanced by acidic liposomes.

    PubMed

    Caccin, Paola; Rossetto, Ornella; Rigoni, Michela; Johnson, Eric; Schiavo, Giampietro; Montecucco, Cesare

    2003-05-01

    Tetanus and botulinum neurotoxins (TeNT and BoNTs) block neuroexocytosis via specific cleavage and inactivation of SNARE proteins. Such activity is exerted by the N-terminal 50 kDa light chain (L) domain, which is a zinc-dependent endopeptidase. TeNT, BoNT/B, /D, /F and /G cleave vesicle associated membrane protein (VAMP), a protein of the neurotransmitter-containing small synaptic vesicles, at different single peptide bonds. Since the proteolytic activity of these metalloproteases is higher on native VAMP inserted in synaptic vesicles than on recombinant VAMP, we have investigated the influence of liposomes of different lipid composition on this activity. We found that the rate of VAMP cleavage with all neurotoxins tested here is strongly enhanced by negatively charged lipid mixtures. This effect is at least partially due to the binding of the metalloprotease to the lipid membranes, with electrostatic interactions playing an important role.

  9. [Analysis of Phytoplankton in Tsimlyansk Reservoir (RUSSIA) for the Presence of Cyanobacterial Hepato- and Neurotoxins].

    PubMed

    Sidelev, S I; Golokolenova, T B; Chernova, E N; Russkikh, I V

    2015-01-01

    Although the water bodies of southern Russia experience the most extreme effects of cyanobacterial blooms, molecular genetic data on the composition of toxigenic cyanobacteria in this region have been absent. Screening for the genes responsible for the synthesis of hepatotoxins (microcystins and cylindrospermopsin) and neurotoxins (anatoxin-a and saxitoxins) in cyanobacteria from the Tsimlyansk reservoir on the Don River was carried out. The presence of microcystin-producing Microcystis and Planktothrix populations, as well as of cyanobacteria capable of synthesis of a neurotoxin anatoxin-a was revealed by polymerase chain reaction (PCR). A hypothesis of the presence of anatoxin-a-producing Planktothrix rubescens population in the phytoplankton of the Tsimlyansk reservoir is proposed. The obtained PCR data were confirmed by the results of enzyme-linked immunosorbent assay (ELISA) and liquid chromatography/mass-spectrometry (LC/MS). Anatoxin-a and five microcystin variants were identified in the phytoplankton biomass.

  10. Scorpion Potassium Channel-blocking Defensin Highlights a Functional Link with Neurotoxin.

    PubMed

    Meng, Lanxia; Xie, Zili; Zhang, Qian; Li, Yang; Yang, Fan; Chen, Zongyun; Li, Wenxin; Cao, Zhijian; Wu, Yingliang

    2016-03-25

    The structural similarity between defensins and scorpion neurotoxins suggests that they might have evolved from a common ancestor. However, there is no direct experimental evidence demonstrating a functional link between scorpion neurotoxins and defensins. The scorpion defensin BmKDfsin4 from Mesobuthus martensiiKarsch contains 37 amino acid residues and a conserved cystine-stabilized α/β structural fold. The recombinant BmKDfsin4, a classical defensin, has been found to have inhibitory activity against Gram-positive bacteria such as Staphylococcus aureus, Bacillus subtilis, and Micrococcus luteusas well as methicillin-resistant Staphylococcus aureus Interestingly, electrophysiological experiments showed that BmKDfsin4,like scorpion potassium channel neurotoxins, could effectively inhibit Kv1.1, Kv1.2, and Kv1.3 channel currents, and its IC50value for the Kv1.3 channel was 510.2 nm Similar to the structure-function relationships of classical scorpion potassium channel-blocking toxins, basic residues (Lys-13 and Arg-19) of BmKDfsin4 play critical roles in peptide-Kv1.3 channel interactions. Furthermore, mutagenesis and electrophysiological experiments demonstrated that the channel extracellular pore region is the binding site of BmKDfsin4, indicating that BmKDfsin4 adopts the same mechanism for blocking potassium channel currents as classical scorpion toxins. Taken together, our work identifies scorpion BmKDfsin4 as the first invertebrate defensin to block potassium channels. These findings not only demonstrate that defensins from invertebrate animals are a novel type of potassium channel blockers but also provide evidence of a functional link between defensins and neurotoxins.

  11. A Protein Microarray ELISA for the Detection of Botulinum neurotoxin A

    SciTech Connect

    Varnum, Susan M.

    2007-06-01

    An enzyme-linked immunosorbent assay (ELISA) microarray was developed for the specific and sensitive detection of botulinum neurotoxin A (BoNT/A), using high-affinity recombinant monoclonal antibodies against the receptor binding domain of the heavy chain of BoNT/A. The ELISA microarray assay, because of its sensitivity, offers a screening test with detection limits comparable to the mouse bioassay, with results available in hours instead of days.

  12. Lectins from Triticum vulgaris and Limax flavus are universal antagonists of botulinum neurotoxin and tetanus toxin.

    PubMed

    Bakry, N; Kamata, Y; Simpson, L L

    1991-09-01

    Lectins from Anguilla anguilla, Artocarpus integrifolia, Canavalia ensiformis, Datora stramonium, Glycine max, Limax flavus, Ricinus communis and Triticum vulgaris were tested for their abilities to antagonize the binding of botulinum neurotoxin and tetanus toxin to rat brain membranes and to antagonize the ability of these toxins to block neuromuscular transmission in mouse phrenic nerve-hemidiaphragm preparations. Lectins from Limax flavus and Triticum vulgaris, both of which have affinity for sialic acid, were antagonists of the various serotypes of botulinum neurotoxin and tetanus toxin. When tested against the high affinity binding site for botulinum neurotoxin type B, the lectin from Limax flavus had a Ki of 3.1 x 10(-7) M and the lectin from Triticum vulgaris had a Ki of 3.75 x 10(-7) M. When tested against the high affinity binding site for tetanus toxin, the lectins from Limax flavus and Triticum vulgaris had Ki values of 1.5 x 10(-7) and 1 x 10(-6) M, respectively. In all cases the lectins behaved as competitive antagonists. In reverse experiments, neither botulinum toxin nor tetanus toxin was a very effective antagonist of lectin binding to brain membranes. Studies on isolated neuromuscular preparations showed that the lectin from Triticum vulgaris did not affect transmission at concentrations of 10(-6) to 10(-3) M, but at a concentration of 3 x 10(-5) M the lectin produced highly statistically significant antagonism of the neuromuscular blocking properties of botulinum neurotoxin types A, B, C, D, E and F as well as tetanus toxin. The lectin did not antagonize beta-bungarotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)

  13. Substrate Recognition Mechanism of VAMP/Synaptobrevin-cleaving Clostridial Neurotoxins*S⃞

    PubMed Central

    Sikorra, Stefan; Henke, Tina; Galli, Thierry; Binz, Thomas

    2008-01-01

    Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) inhibit neurotransmitter release by proteolyzing a single peptide bond in one of the three soluble N-ethylmaleimide-sensitive factor attachment protein receptors SNAP-25, syntaxin, and vesicle-associated membrane protein (VAMP)/synaptobrevin. TeNT and BoNT/B, D, F, and G of the seven known BoNTs cleave the synaptic vesicle protein VAMP/synaptobrevin. Except for BoNT/B and TeNT, they cleave unique peptide bonds, and prior work suggested that different substrate segments are required for the interaction of each toxin. Although the mode of SNAP-25 cleavage by BoNT/A and E has recently been studied in detail, the mechanism of VAMP/synaptobrevin proteolysis is fragmentary. Here, we report the determination of all substrate residues that are involved in the interaction with BoNT/B, D, and F and TeNT by means of systematic mutagenesis of VAMP/synaptobrevin. For each of the toxins, three or more residues clustered at an N-terminal site remote from the respective scissile bond are identified that affect solely substrate binding. These exosites exhibit different sizes and distances to the scissile peptide bonds for each neurotoxin. Substrate segments C-terminal of the cleavage site (P4-P4′) do not play a role in the catalytic process. Mutation of residues in the proximity of the scissile bond exclusively affects the turnover number; however, the importance of individual positions at the cleavage sites varied for each toxin. The data show that, similar to the SNAP-25 proteolyzing BoNT/A and E, VAMP/synaptobrevin-specific clostridial neurotoxins also initiate substrate interaction, employing an exosite located N-terminal of the scissile peptide bond. PMID:18511418

  14. Lasers, fillers, and neurotoxins: avoiding complications in the cosmetic facial practice.

    PubMed

    Hassouneh, Basil; Newman, James P

    2013-11-01

    Lasers, injectable fillers, and neurotoxins are widely used in facial restoration and rejuvenation by a variety of practitioners. Although they are less invasive than traditional surgical modalities, they still carry risks for both transient as well as permanent complications. It is paramount for the practitioner to understand these complications, optimize their prevention, and initiate appropriate treatment when they are encountered. This article reviews early, often transient, complications as well as delayed, often prolonged or permanent, complications, with particular focus on prevention and management.

  15. Novel arylalkenylpropargylamines as neuroprotective, potent, and selective monoamine oxidase B inhibitors for the treatment of Parkinson's disease.

    PubMed

    Huleatt, Paul B; Khoo, Mui Ling; Chua, Yi Yuan; Tan, Tiong Wei; Liew, Rou Shen; Balogh, Balázs; Deme, Ruth; Gölöncsér, Flóra; Magyar, Kalman; Sheela, David P; Ho, Han Kiat; Sperlágh, Beáta; Mátyus, Péter; Chai, Christina L L

    2015-02-12

    To develop novel neuroprotective agents, a library of novel arylalkenylpropargylamines was synthesized and tested for inhibitory activities against monoamine oxidases. From this, a number of highly potent and selective monoamine oxidase B inhibitors were identified. Selected compounds were also tested for neuroprotection in in vitro studies with PC-12 cells treated with 6-OHDA and rotenone, respectively. It was observed that some of the compounds tested yielded a marked increase in survival in PC-12 cells treated with the neurotoxins. This indicates that these propargylamines are able to confer protection against the effects of the toxins and may also be considered as novel disease-modifying anti-Parkinsonian agents, which are much needed for the therapy of Parkinson's disease.

  16. Geochemical legacies and the future health of cities: A tale of two neurotoxins in urban soils

    USGS Publications Warehouse

    Fillipelli, Gabriel M.; Risch, Martin R.; Laidlaw, Mark A. S.; Nichols, Deborah E.; Crewe, Julie

    2015-01-01

    Acute exposure to lead (Pb), a powerful neurotoxin to which children are particularly susceptible, has largely been eliminated in the U.S. and other countries through policy-based restrictions on leaded gasoline and lead-based paints. But the legacy of these sources remains in the form of surface soil Pb contamination, a common problem in cities and one that has only recently emerged as a widespread chronic exposure mechanism in cities. Some urban soils are also contaminated with another neurotoxin, mercury (Hg). The greatest human exposure to Hg is through fish consumption, so eating fish caught in urban areas presents risks for toxic Hg exposure. The potential double impact of chronic exposure to these two neurotoxins is pronounced in cities. Overall, there is a paradigmatic shift from reaction to and remediation of acute exposures towards a more nuanced understanding of the dynamic cycling of persistent environmental contaminants with resultant widespread and chronic exposure of inner-city dwellers, leading to chronic toxic illness and disability at substantial human and social cost.

  17. Neurotoxin-conjugated upconversion nanoprobes for direct visualization of tumors under near-infrared irradiation.

    PubMed

    Yu, Xue-Feng; Sun, Zhengbo; Li, Min; Xiang, Yang; Wang, Qu-Quan; Tang, Fenfen; Wu, Yingliang; Cao, Zhijian; Li, Wenxin

    2010-11-01

    We report the development of neurotoxin-mediated upconversion nanoprobes for tumor targeting and visualization in living animals. The nanoprobes were synthesized by preparing polyethylenimine-coated hexagonal-phase NaYF(4):Yb,Er/Ce nanoparticles and conjugating them with recombinant chlorotoxin, a typical peptide neurotoxin that could bind with high specificity to many types of cancer cells. Nanoprobes that specifically targeted glioma cells were visualized by laser scanning upconversion fluorescence microscopy. Good probe biocompatibility was displayed with cellular and animal toxicity determinations. Animal studies were performed using Balb-c nude mice injected intravenously with the nanoprobes. The obtained high-contrast images demonstrated highly specific tumor binding and direct tumor visualization with bright red fluorescence under 980-nm near-infrared irradiation. The high sensitivity and high specificity of the neurotoxin-mediated upconversion nanoprobes and the simplification of the required optical device for tumor visualization suggest an approach that may help improve the effectiveness of the diagnostic and therapeutic modalities available for tumor patients.

  18. An Overview on the Marine Neurotoxin, Saxitoxin: Genetics, Molecular Targets, Methods of Detection and Ecological Functions

    PubMed Central

    Cusick, Kathleen D.; Sayler, Gary S.

    2013-01-01

    Marine neurotoxins are natural products produced by phytoplankton and select species of invertebrates and fish. These compounds interact with voltage-gated sodium, potassium and calcium channels and modulate the flux of these ions into various cell types. This review provides a summary of marine neurotoxins, including their structures, molecular targets and pharmacologies. Saxitoxin and its derivatives, collectively referred to as paralytic shellfish toxins (PSTs), are unique among neurotoxins in that they are found in both marine and freshwater environments by organisms inhabiting two kingdoms of life. Prokaryotic cyanobacteria are responsible for PST production in freshwater systems, while eukaryotic dinoflagellates are the main producers in marine waters. Bioaccumulation by filter-feeding bivalves and fish and subsequent transfer through the food web results in the potentially fatal human illnesses, paralytic shellfish poisoning and saxitoxin pufferfish poisoning. These illnesses are a result of saxitoxin’s ability to bind to the voltage-gated sodium channel, blocking the passage of nerve impulses and leading to death via respiratory paralysis. Recent advances in saxitoxin research are discussed, including the molecular biology of toxin synthesis, new protein targets, association with metal-binding motifs and methods of detection. The eco-evolutionary role(s) PSTs may serve for phytoplankton species that produce them are also discussed. PMID:23535394

  19. Pharmacological characterization of α-elapitoxin-Al2a from the venom of the Australian pygmy copperhead (Austrelaps labialis): an atypical long-chain α-neurotoxin with only weak affinity for α7 nicotinic receptors.

    PubMed

    Marcon, Francesca; Leblanc, Mathieu; Vetter, Irina; Lewis, Richard J; Escoubas, Pierre; Nicholson, Graham M

    2012-09-15

    Despite the in vivo lethality of venom, neurotoxicity has not previously been considered a significant complication of envenoming by the Australian pygmy copperhead (Austrelaps labialis). However, recent evidence has emerged demonstrating that this venom contains potent presynaptic and postsynaptic neurotoxicity. The present study describes the isolation and pharmacological characterization of the first postsynaptic neurotoxin, α-EPTX-Al2a, from the venom of A. labialis. α-EPTX-Al2a (8072.77 Da) caused a concentration-dependent block of twitch contractions and a complete block of responses to cholinergic agonists in the chick biventer cervicis nerve-muscle preparation. This action is consistent with postjunctional neurotoxicity. Monovalent tiger snake antivenom prevented the onset of neurotoxicity if applied prior to toxin administration, but was only able to partially reverse neurotoxicity once muscle paralysis had developed. α-EPTX-Al2a produced a potent pseudo-irreversible antagonism of chick muscle nicotinic acetylcholine receptors (nAChRs), with an estimated pA(2) value of 7.902 (K(B) = 12.5 nM). Interestingly, the toxin only produced a modest block of neuronal α7 nAChRs, with an IC(50) of 1.2 μM, and failed to inhibit ganglionic α3β2/α3β4 nAChRs in a fluorescence-based FLIPR assay using SH-SY5Y cells. α-EPTX-Al2a contained 75 amino acid residues with five disulfide bonds that had significant homology to classical long-chain α-neurotoxins. While α-EPTX-Al2a retains most pharmacophore residues critical for binding to muscle-type (α1)(2)βγδ nAChRs it lacks the key Ala(28) and Arg(36) residues important for α7 nAChR affinity. Given that A. labialis venom contains both irreversible presynaptic and postsynaptic neurotoxins, clinicians need to be aware of potential neurotoxic complications associated with pygmy copperhead envenomation. PMID:22771828

  20. Isolation and functional characterization of the novel Clostridium botulinum neurotoxin A8 subtype.

    PubMed

    Kull, Skadi; Schulz, K Melanie; Weisemann, Jasmin; Kirchner, Sebastian; Schreiber, Tanja; Bollenbach, Alexander; Dabrowski, P Wojtek; Nitsche, Andreas; Kalb, Suzanne R; Dorner, Martin B; Barr, John R; Rummel, Andreas; Dorner, Brigitte G

    2015-01-01

    Botulism is a severe neurological disease caused by the complex family of botulinum neurotoxins (BoNT). Based on the different serotypes known today, a classification of serotype variants termed subtypes has been proposed according to sequence diversity and immunological properties. However, the relevance of BoNT subtypes is currently not well understood. Here we describe the isolation of a novel Clostridium botulinum strain from a food-borne botulism outbreak near Chemnitz, Germany. Comparison of its botulinum neurotoxin gene sequence with published sequences identified it to be a novel subtype within the BoNT/A serotype designated BoNT/A8. The neurotoxin gene is located within an ha-orfX+ cluster and showed highest homology to BoNT/A1, A2, A5, and A6. Unexpectedly, we found an arginine insertion located in the HC domain of the heavy chain, which is unique compared to all other BoNT/A subtypes known so far. Functional characterization revealed that the binding characteristics to its main neuronal protein receptor SV2C seemed unaffected, whereas binding to membrane-incorporated gangliosides was reduced in comparison to BoNT/A1. Moreover, we found significantly lower enzymatic activity of the natural, full-length neurotoxin and the recombinant light chain of BoNT/A8 compared to BoNT/A1 in different endopeptidase assays. Both reduced ganglioside binding and enzymatic activity may contribute to the considerably lower biological activity of BoNT/A8 as measured in a mouse phrenic nerve hemidiaphragm assay. Despite its reduced activity the novel BoNT/A8 subtype caused severe botulism in a 63-year-old male. To our knowledge, this is the first description and a comprehensive characterization of a novel BoNT/A subtype which combines genetic information on the neurotoxin gene cluster with an in-depth functional analysis using different technical approaches. Our results show that subtyping of BoNT is highly relevant and that understanding of the detailed toxin function might

  1. Three Classes of Plasmid (47–63 kb) Carry the Type B Neurotoxin Gene Cluster of Group II Clostridium botulinum

    PubMed Central

    Carter, Andrew T.; Austin, John W.; Weedmark, Kelly A.; Corbett, Cindi; Peck, Michael W.

    2014-01-01

    Pulsed-field gel electrophoresis and DNA sequence analysis of 26 strains of Group II (nonproteolytic) Clostridium botulinum type B4 showed that 23 strains carried their neurotoxin gene cluster on a 47–63 kb plasmid (three strains lacked any hybridization signal for the neurotoxin gene, presumably having lost their plasmid). Unexpectedly, no neurotoxin genes were found on the chromosome. This apparent constraint on neurotoxin gene transfer to the chromosome stands in marked contrast to Group I C. botulinum, in which neurotoxin gene clusters are routinely found in both locations. The three main classes of type B4 plasmid identified in this study shared different regions of homology, but were unrelated to any Group I or Group III plasmid. An important evolutionary aspect firmly links plasmid class to geographical origin, with one class apparently dominant in marine environments, whereas a second class is dominant in European terrestrial environments. A third class of plasmid is a hybrid between the other two other classes, providing evidence for contact between these seemingly geographically separated populations. Mobility via conjugation has been previously demonstrated for the type B4 plasmid of strain Eklund 17B, and similar genes associated with conjugation are present in all type B4 plasmids now described. A plasmid toxin–antitoxin system pemI gene located close to the neurotoxin gene cluster and conserved in each type B4 plasmid class may be important in understanding the mechanism which regulates this unique and unexpected bias toward plasmid-borne neurotoxin genes in Group II C. botulinum type B4. PMID:25079343

  2. Novel chimeras of botulinum neurotoxins A and E unveil contributions from the binding, translocation, and protease domains to their functional characteristics.

    PubMed

    Wang, Jiafu; Meng, Jianghui; Lawrence, Gary W; Zurawski, Tomas H; Sasse, Astrid; Bodeker, MacDara O; Gilmore, Marcella A; Fernández-Salas, Ester; Francis, Joseph; Steward, Lance E; Aoki, K Roger; Dolly, J Oliver

    2008-06-20

    Hyperexcitability disorders of cholinergically innervated muscles are treatable with botulinum neurotoxin (BoNT) A. The seven serotypes (A-G) potently block neurotransmission by binding to presynaptic receptors, undergoing endocytosis, transferring to the cytosol, and inactivating proteins essential for vesicle fusion. Although BoNT/A and BoNT/E cleave SNAP-25, albeit at distinct sites, BoNT/E blocks neurotransmission faster and more potently. To identify the domains responsible for these characteristics, the C-terminal heavy chain portions of BoNT/A and BoNT/E were exchanged to create chimeras AE and EA. After high yield expression in Escherichia coli, these single chain chimeras were purified by two-step chromatography and activated by conversion to disulfide-linked dichains. In vitro, each entered neurons, cleaved SNAP-25, and blocked neuromuscular transmission while causing flaccid paralysis in vivo. Acidification-dependent translocation of the light chain to the cytosol occurred more rapidly for BoNT/E and EA than for BoNT/A and AE because the latter pair remained susceptible for longer to inhibitors of the vesicular proton pump, and BoNT/A proved less sensitive. The receptor-binding and protease domains do not seem to be responsible for the speeds of intoxication; rather the N-terminal halves of their heavy chains are implicated, with dissimilar rates of cytosolic transfer of the light chains being due to differences in pH sensitivity. AE produced the most persistent muscle weakening and therefore has therapeutic potential. Thus, proof of principle is provided for tailoring the pharmacological properties of these toxins by protein engineering.

  3. Brevetoxicosis: red tides and marine mammal mortalities.

    PubMed

    Flewelling, Leanne J; Naar, Jerome P; Abbott, Jay P; Baden, Daniel G; Barros, Nélio B; Bossart, Gregory D; Bottein, Marie-Yasmine D; Hammond, Daniel G; Haubold, Elsa M; Heil, Cynthia A; Henry, Michael S; Jacocks, Henry M; Leighfield, Tod A; Pierce, Richard H; Pitchford, Thomas D; Rommel, Sentiel A; Scott, Paula S; Steidinger, Karen A; Truby, Earnest W; Van Dolah, Frances M; Landsberg, Jan H

    2005-06-01

    Potent marine neurotoxins known as brevetoxins are produced by the 'red tide' dinoflagellate Karenia brevis. They kill large numbers of fish and cause illness in humans who ingest toxic filter-feeding shellfish or inhale toxic aerosols. The toxins are also suspected of having been involved in events in which many manatees and dolphins died, but this has usually not been verified owing to limited confirmation of toxin exposure, unexplained intoxication mechanisms and complicating pathologies. Here we show that fish and seagrass can accumulate high concentrations of brevetoxins and that these have acted as toxin vectors during recent deaths of dolphins and manatees, respectively. Our results challenge claims that the deleterious effects of a brevetoxin on fish (ichthyotoxicity) preclude its accumulation in live fish, and they reveal a new vector mechanism for brevetoxin spread through food webs that poses a threat to upper trophic levels. PMID:15944690

  4. A Distinct Functional Site in Ω-Neurotoxins: Novel Antagonists of Nicotinic Acetylcholine Receptors from Snake Venom.

    PubMed

    Hassan-Puttaswamy, Varuna; Adams, David J; Kini, R Manjunatha

    2015-12-18

    Snake venom α-neurotoxins from the three-finger toxin (3FTx) family are competitive antagonists with nanomolar affinity and high selectivity for nicotinic acetylcholine receptors (nAChR). Here, we report the characterization of a new group of competitive nAChR antagonists: Ω-neurotoxins. Although they belong to the 3FTx family, the characteristic functional residues of α-neurotoxins are not conserved. We evaluated the subtype specificity and structure-function relationships of Oh9-1, an Ω-neurotoxin from Ophiophagus hannah venom. Recombinant Oh9-1 showed reversible postsynaptic neurotoxicity in the micromolar range. Experiments with different nAChR subtypes expressed in Xenopus oocytes indicated Oh9-1 is selective for rat muscle type α1β1εδ (adult) and α1β1γδ (fetal) and rat neuronal α3β2 subtypes. However, Oh9-1 showed low or no affinity for other human and rat neuronal subtypes. Twelve individual alanine-scan mutants encompassing all three loops of Oh9-1 were evaluated for binding to α1β1εδ and α3β2 subtypes. Oh9-1's loop-II residues (M25, F27) were the most critical for interactions and formed the common binding core. Mutations at T23 and F26 caused a significant loss in activity at α1β1εδ receptors but had no effect on the interaction with the α3β2 subtype. Similarly, mutations at loop-II (H7, K22, H30) and -III (K45) of Oh9-1 had a distinctly different impact on its activity with these subtypes. Thus, Oh9-1 interacts with these nAChRs via distinct residues. Unlike α-neurotoxins, the tip of loop-II is not involved. We reveal a novel mode of interaction, where both sides of the β-strand of Oh9-1's loop-II interact with α1β1εδ, but only one side interacts with α3β2. Phylogenetic analysis revealed functional organization of the Ω-neurotoxins independent of α-neurotoxins. Thus, Ω-neurotoxin: Oh9-1 may be a new, structurally distinct class of 3FTxs that, like α-neurotoxins, antagonize nAChRs. However, Oh9-1 binds to the ACh

  5. Geochemical Legacies and the Future Health of Cities: An Analysis of two Neurotoxins in Urban Soils

    NASA Astrophysics Data System (ADS)

    Filippelli, G. M.; Risch, M.

    2015-12-01

    The past and future of cities are inextricably linked, a linkage that can be seen clearly in the long-term impacts of urban geochemical legacies. As loci of population as well as the means of employment and industry to support these populations, cities have a long history of co-locating contaminating practices and people, sometimes with negative implications for human health. Working at the intersection between geochemical processes, communities, and human health is critical to grapple with environmental legacies and to support healthy, sustainable, and growing urban populations. An emerging area of environmental health research is to understand the impacts of chronic exposures and exposure mixtures—these impacts are very poorly studied, yet have materialized as perhaps the greatest threat to large-scale population health. Acute exposure to lead (Pb), a powerful neurotoxin to which children are particularly susceptible, has largely been eliminated in the U.S. and other countries through policy-based restrictions on leaded gasoline and lead-based paints. But these legacy Pb sources are still around in the form of surface soil Pb contamination, a common problem in cities and one that has only recently emerged as a pernicious and widespread chronic exposure mechanism in cities. Some urban soils are also contaminated with another neurotoxin, mercury (Hg), although very little work has been done to understand human exposures to low levels of this element in soils. The most documented human exposure to Hg is through fish consumption, so eating fish caught in urban areas presents risks for above average dietary Hg exposure. The potential double impact of chronic exposure to these two neurotoxins is pronounced in cities. Many aspects of the dose-response curves for individual elements and mixtures are poorly understood, especially at lower levels, leaving unanswered several interesting and provocative questions about environmental impacts on neurological and

  6. Structural and Functional Interactions between Transient Receptor Potential Vanilloid Subfamily 1 and Botulinum Neurotoxin Serotype A

    PubMed Central

    2016-01-01

    Background Botulinum neurotoxins are produced by Clostridium botulinum bacteria. There are eight serologically distinct botulinum neurotoxin isoforms (serotypes A–H). Currently, botulinum neurotoxin serotype A (BoNT⁄A) is commonly used for the treatment of many disorders, such as hyperactive musculoskeletal disorders, dystonia, and pain. However, the effectiveness of BoNT⁄A for pain alleviation and the mechanisms that mediate the analgesic effects of BoNT⁄A remain unclear. To define the antinociceptive mechanisms by which BoNT/A functions, the interactions between BoNT⁄A and the transient receptor potential vanilloid subfamily 1 (TRPV1) were investigated using immunofluorescence, co-immunoprecipitation, and western blot analysis in primary mouse embryonic dorsal root ganglion neuronal cultures. Results 1) Three-week-old cultured dorsal root ganglion neurons highly expressed transient TRPV1, synaptic vesicle 2A (SV2A) and synaptosomal-associated protein 25 (SNAP-25). SV2A and SNAP-25 are the binding receptor and target protein, respectively, of BoNT⁄A. 2) TRPV1 colocalized with both BoNT⁄A and cleaved SNAP-25 when BoNT⁄A was added to dorsal root ganglia neuronal cultures. 3) After 24 hours of BoNT⁄A treatment (1 nmol⁄l), both TRPV1 and BoNT⁄A positive bands were detected in western blots of immunoprecipitated pellets. 4) Blocking TRPV1 with a specific antibody decreased the cleavage of SNAP-25 by BoNT⁄A. Conclusion BoNT/A interacts with TRPV1 both structurally and functionally in cultured mouse embryonic dorsal root ganglion neurons. These results suggest that an alternative mechanism is used by BoNT⁄A to mediate pain relief. PMID:26745805

  7. Botulinum neurotoxin C1 blocks neurotransmitter release by means of cleaving HPC-1/syntaxin.

    PubMed Central

    Blasi, J; Chapman, E R; Yamasaki, S; Binz, T; Niemann, H; Jahn, R

    1993-01-01

    The anaerobic bacterium Clostridium botulinum produces several related neurotoxins that block exocytosis of synaptic vesicles in nerve terminals and that are responsible for the clinical manifestations of botulism. Recently, it was reported that botulinum neurotoxin type B as well as tetanus toxin act as zinc-dependent proteases that specifically cleave synaptobrevin, a membrane protein of synaptic vesicles (Link et al., Biochem. Biophys. Res. Commun., 189, 1017-1023; Schiavo et al., Nature, 359, 832-835). Here we report that inhibition of neurotransmitter release by botulinum neurotoxin type C1 was associated with the proteolysis of HPC-1 (= syntaxin), a membrane protein present in axonal and synaptic membranes. Breakdown of HPC-1/syntaxin was selective since no other protein degradation was detectable. In vitro studies showed that the breakdown was due to a direct interaction between HPC-1/syntaxin and the toxin light chain which acts as a metallo-endoprotease. Toxin-induced cleavage resulted in the generation of a soluble fragment of HPC-1/syntaxin that is 2-4 kDa smaller than the native protein. When HPC-1/syntaxin was translated in vitro, cleavage occurred only when translation was performed in the presence of microsomes, although a full-length product was obtained in the absence of membranes. However, susceptibility to toxin cleavage was restored when the product of membrane-free translation was subsequently incorporated into artificial proteoliposomes. In addition, a translated form of HPC-1/syntaxin, which lacked the putative transmembrane domain at the C-terminus, was soluble and resistant to toxin action. We conclude that HPC-1/syntaxin is involved in exocytotic membrane fusion.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:7901002

  8. Interactions of the Neurotoxin Vipoxin in Solution Studied by Dynamic Light Scattering

    PubMed Central

    Georgieva, Dessislava Nikolova; Genov, Nicolay; Hristov, Krassimir; Dierks, Karsten; Betzel, Christian

    2004-01-01

    The neurotoxin vipoxin is the lethal component of the venom of Vipera ammodytes meridionalis. It is a heterodimer of a basic toxic His-48 phospholipase A2 (PLA2) and an acidic nontoxic Gln-48 PLA2. The shape of the neurotoxin and its separated components in solution as well as their interactions with calcium, the brain phospholipid phosphatidylcholine, and two inhibitors, elaidoylamide and vitamin E, were investigated by dynamic light scattering. Calcium binding is connected with a conformational change in vipoxin observed as a change of the hydrodynamic shape from oblate ellipsoid to a shape closer to a sphere. The Ca2+-bound form of vipoxin, which is catalytically active, is more compact and symmetric than the calcium-free heterodimer. Similar changes were observed as a result of the Ca2+-binding to the two separated subunits. In the presence of aggregated phosphatidylcholine, the neurotoxic complex dissociates to subunits. It is supposed that only the toxic component binds to the substrate, and the other subunit, which plays a chaperone function, remains in solution. The inhibition of vipoxin with the synthetic inhibitor elaidoylamide and the natural compound vitamin E changes the shape of the toxin from oblate to prolate ellipsoid. The inhibited toxin is more asymmetric in comparison to the native one. Similar, but not so pronounced, effects were observed after the inhibition of the monomeric and homodimeric forms of the toxic His-48 PLA2. Circular dichroism measurements in the presence of urea, methylurea, and ethylurea indicate a strong hydrophobic stabilization of the neurotoxin. Hydrophobic interactions stabilize not only the folded regions but also the regions of intersubunit contacts. PMID:14695289

  9. Properties and use of botulinum toxin and other microbial neurotoxins in medicine.

    PubMed Central

    Schantz, E J; Johnson, E A

    1992-01-01

    Crystalline botulinum toxin type A was licensed in December 1989 by the Food and Drug Administration for treatment of certain spasmodic muscle disorders following 10 or more years of experimental treatment on human volunteers. Botulinum toxin exerts its action on a muscle indirectly by blocking the release of the neurotransmitter acetylcholine at the nerve ending, resulting in reduced muscle activity or paralysis. The injection of only nanogram quantities (1 ng = 30 mouse 50% lethal doses [U]) of the toxin into a spastic muscle is required to bring about the desired muscle control. The type A toxin produced in anaerobic culture and purified in crystalline form has a specific toxicity in mice of 3 x 10(7) U/mg. The crystalline toxin is a high-molecular-weight protein of 900,000 Mr and is composed of two molecules of neurotoxin (ca. 150,000 Mr) noncovalently bound to nontoxic proteins that play an important role in the stability of the toxic unit and its effective toxicity. Because the toxin is administered by injection directly into neuromuscular tissue, the methods of culturing and purification are vital. Its chemical, physical, and biological properties as applied to its use in medicine are described. Dilution and drying of the toxin for dispensing causes some detoxification, and the mouse assay is the only means of evaluation for human treatment. Other microbial neurotoxins may have uses in medicine; these include serotypes of botulinum toxins and tetanus toxin. Certain neurotoxins produced by dinoflagellates, including saxitoxin and tetrodotoxin, cause muscle paralysis through their effect on the action potential at the voltage-gated sodium channel. Saxitoxin used with anaesthetics lengthens the effect of the anaesthetic and may enhance the effectiveness of other medical drugs. Combining toxins with drugs could increase their effectiveness in treatment of human disease. PMID:1579114

  10. Differential role of molten globule and protein folding in distinguishing unique features of botulinum neurotoxin.

    PubMed

    Kumar, Raj; Kukreja, Roshan V; Cai, Shuowei; Singh, Bal R

    2014-06-01

    Botulinum neurotoxins (BoNTs) are proteins of great interest not only because of their extreme toxicity but also paradoxically for their therapeutic applications. All the known serotypes (A-G) have varying degrees of longevity and potency inside the neuronal cell. Differential chemical modifications such as phosphorylation and ubiquitination have been suggested as possible mechanisms for their longevity, but the molecular basis of the longevity remains unclear. Since the endopeptidase domain (light chain; LC) of toxin apparently survives inside the neuronal cells for months, it is important to examine the structural features of this domain to understand its resistance to intracellular degradation. Published crystal structures (both botulinum neurotoxins and endopeptidase domain) have not provided adequate explanation for the intracellular longevity of the domain. Structural features obtained from spectroscopic analysis of LCA and LCB were similar, and a PRIME (PReImminent Molten Globule Enzyme) conformation appears to be responsible for their optimal enzymatic activity at 37°C. LCE, on the other hand, was although optimally active at 37°C, but its active conformation differed from the PRIME conformation of LCA and LCB. This study establishes and confirms our earlier finding that an optimally active conformation of these proteins in the form of PRIME exists for the most poisonous poison, botulinum neurotoxin. There are substantial variations in the structural and functional characteristics of these active molten globule related structures among the three BoNT endopeptidases examined. These differential conformations of LCs are important in understanding the fundamental structural features of proteins, and their possible connection to intracellular longevity could provide significant clues for devising new countermeasures and effective therapeutics. PMID:24568862

  11. Early onset of neurological symptoms in fragile X premutation carriers exposed to neurotoxins

    PubMed Central

    Paul, Ripon; Pessah, Isaac N.; Gane, Louise; Ono, Michele; Hagerman, Paul J.; Brunberg, James A.; Tassone, Flora; Bourgeois, James A.; Adams, Patrick E.; Nguyen, Danh V.; Hagerman, Randi

    2014-01-01

    We present four cases of fragile X premutation carriers with early neurological symptoms, including symptoms consistent with multiple sclerosis (MS) and fragile X-associated tremor/ataxia syndrome (FXTAS). Each patient had significant exposure to one or more environmental neurotoxicants that have documented neurotoxicity (i.e. hexachlorocyclopentadiene or C56, Agent Orange, and 2,4- or 2,6-toluene diisocyanate and dichlormate). We hypothesize that premutation carriers are a vulnerable group to neurotoxins because elevated mRNA in the premutation can lead to early cell death and brain disease, leading to neuropsychiatric and neurological symptoms consistent with FXTAS. PMID:20466021

  12. Separation of Betti Reaction Product Enantiomers: Absolute Configuration and Inhibition of Botulinum Neurotoxin A

    PubMed Central

    2011-01-01

    The racemic product of the Betti reaction of 5-chloro-8-hydroxyquinoline, benzaldehyde, and 2-aminopyridine was separated by chiral HPLC to determine which enantiomer inhibited botulinum neurotoxin serotype A. When the enantiomers unexpectedly proved to have comparable activity, the absolute structures of (+)-(R)-1 and (−)-(S)-1 were determined by comparison of calculated and observed circular dichroism spectra. Molecular modeling studies were undertaken in an effort to understand the observed bioactivity and revealed different ensembles of binding modes, with roughly equal binding energies, for the two enantiomers. PMID:22102940

  13. Presence of antibotulinum neurotoxin antibodies in selected wild canids in Israel.

    PubMed

    Steinman, Amir; Millet, Neta; Frenkel, Chana; King, Roni; Shpigel, Nahum Y

    2007-07-01

    Serum samples from 35 golden jackals (Canis aureus syriacus), eight wolves (Canis lupus), and four red foxes (Vulpes vulpes) from various regions of Israel were collected during the years 2001-04 and tested for antibodies to Clostridium botulinum neurotoxin (BoNT) types C and D. Antibodies against BoNT types C and D were detected in 10 (29%) and in 3 (9%) of 35 golden jackals, respectively, using enzyme-linked immunosorbent assay. This report describes detection of anti BoNT antibodies in wild canids other than coyotes (Canis latrans) for the first time and demonstrates that C. botulinum type C is prevalent in Israel. PMID:17699099

  14. Early onset of neurological symptoms in fragile X premutation carriers exposed to neurotoxins.

    PubMed

    Paul, Ripon; Pessah, Isaac N; Gane, Louise; Ono, Michele; Hagerman, Paul J; Brunberg, James A; Tassone, Flora; Bourgeois, James A; Adams, Patrick E; Nguyen, Danh V; Hagerman, Randi

    2010-08-01

    We present four cases of fragile X premutation carriers with early neurological symptoms, including symptoms consistent with multiple sclerosis (MS) and fragile X-associated tremor/ataxia syndrome (FXTAS). Each patient had significant exposure to one or more environmental neurotoxicants that have documented neurotoxicity (i.e. hexachlorocyclopentadiene or C56, Agent Orange, and 2,4- or 2,6-toluene diisocyanate and dichlormate). We hypothesize that premutation carriers are a vulnerable group to neurotoxins because elevated mRNA in the premutation can lead to early cell death and brain disease, leading to neuropsychiatric and neurological symptoms consistent with FXTAS. PMID:20466021

  15. Computer modeling of the neurotoxin binding site of acetylcholine receptor spanning residues 185 through 196

    NASA Technical Reports Server (NTRS)

    Garduno-Juarez, R.; Shibata, M.; Zielinski, T. J.; Rein, R.

    1987-01-01

    A model of the complex between the acetylcholine receptor and the snake neurotoxin, cobratoxin, was built by molecular model building and energy optimization techniques. The experimentally identified functionally important residues of cobratoxin and the dodecapeptide corresponding to the residues 185-196 of acetylcholine receptor alpha subunit were used to build the model. Both cis and trans conformers of cyclic L-cystine portion of the dodecapeptide were examined. Binding residues independently identified on cobratoxin are shown to interact with the dodecapeptide AChR model.

  16. Presence of antibotulinum neurotoxin antibodies in selected wild canids in Israel.

    PubMed

    Steinman, Amir; Millet, Neta; Frenkel, Chana; King, Roni; Shpigel, Nahum Y

    2007-07-01

    Serum samples from 35 golden jackals (Canis aureus syriacus), eight wolves (Canis lupus), and four red foxes (Vulpes vulpes) from various regions of Israel were collected during the years 2001-04 and tested for antibodies to Clostridium botulinum neurotoxin (BoNT) types C and D. Antibodies against BoNT types C and D were detected in 10 (29%) and in 3 (9%) of 35 golden jackals, respectively, using enzyme-linked immunosorbent assay. This report describes detection of anti BoNT antibodies in wild canids other than coyotes (Canis latrans) for the first time and demonstrates that C. botulinum type C is prevalent in Israel.

  17. Infant botulism with prolonged faecal excretion of botulinum neurotoxin and Clostridium botulinum for 7 months.

    PubMed

    Derman, Y; Korkeala, H; Salo, E; Lönnqvist, T; Saxen, H; Lindström, M

    2014-02-01

    In Finland in April 2010, a 3-month old baby was diagnosed with type A infant botulism. He excreted botulinum neurotoxin and/or Clostridium botulinum in his faeces until November 2010. Five months of excretion was after clinical recovery and discharge from hospital. C. botulinum isolates recovered from the household dust in the patient's home were genetically identical to those found in the infant's stool samples. Long-term faecal excretion of C. botulinum may pose a possible health risk for the parents and others in close contact with the infant.

  18. Toxicity of the cyanobacterial neurotoxin beta-N-methylamino-L-alanine to three aquatic animal species.

    PubMed

    Purdie, Esme L; Metcalf, James S; Kashmiri, Shereen; Codd, Geoffrey A

    2009-01-01

    Beta-N-methylamino-L-alanine (BMAA), a neurotoxin and candidate contributory cause of neurodegenerative diseases including amyotrophic lateral sclerosis, is produced by aquatic and terrestrial cyanobacteria. We have determined BMAA toxicity to three aquatic animal species: zebra fish (Danio rerio), brine shrimp (Artemia salina) and the protozoan Nassula sorex. Responses included: clonus convulsions and abnormal spinal axis formation (D. rerio), loss of phototaxis (A. salina) and mortalities (all species). These systems offer potential to further understand BMAA toxicity and the bioaccumulation and fates of BMAA in aquatic food chains leading to potential human exposure.

  19. Acute sialadenitis secondary to submandibular calculi after botulinum neurotoxin injection for sialorrhea in a child with cerebral palsy.

    PubMed

    Yuan, Mike; Shelton, Jean

    2011-12-01

    Children with cerebral palsy and other neurologic diseases often present with sialorrhea. Intraglandular botulinum neurotoxin is being increasingly reported to be clinically effective for the treatment of sialorrhea. This treatment is becoming more popular in recent years because of being less invasive than surgical procedures. In addition, fewer adverse effects have been documented compared with oral or topical anticholinergic medication. We report the first case in a child with cerebral palsy who developed serious acute sialadenitis with submandibular sialolithiasis after intraglandular botulinum neurotoxin injection for sialorrhea.

  20. Recent developments with metalloprotease inhibitor class of drug candidates for Botulinum neurotoxins

    SciTech Connect

    Kumar, Gyanendra; Swaminathan, Subramanyam

    2015-03-01

    Botulinum Neurotoxins are the most poisonous of all toxins with lethal dose in nanogram quantities. They are also potential biological warfare and bioterrorism agents due to their high toxicity and ease of preparation. On the other hand BoNTs are also being increasingly used for therapeutic and cosmetic purposes, and with that the chances of accidental overdose are increasing. And despite the potential damage they could cause to human health, there are no post-intoxication drugs available so far. But progress is being made in this direction. The crystal structures in native form and bound with substrate peptides have been determined, and these are enabling structure-based drug discovery possible. High throughput assays have also been designed to speed up the screening progress. Substrate-based and small molecule inhibitors have been identified. But turning high affinity inhibitors into clinically viable drug candidates has remained a challenge. We discuss here the latest developments and the future challenges in drug discovery for Botulinum neurotoxins.

  1. Domain Organization in Clostridium botulinum Neurotoxin Type E is Unique: Its Implication in Faster Translocation

    SciTech Connect

    Kumaran, D.; Eswaramoorthy, S; Furey, W; Navaza, J; Sax, M; Swaminathan, S

    2009-01-01

    Clostridium botulinum produces seven antigenically distinct neurotoxins [C. botulinum neurotoxins (BoNTs) A-G] sharing a significant sequence homology. Based on sequence and functional similarity, it was believed that their three-dimensional structures will also be similar. Indeed, the crystal structures of BoNTs A and B exhibit similar fold and domain association where the translocation domain is flanked on either side by binding and catalytic domains. Here, we report the crystal structure of BoNT E holotoxin and show that the domain association is different and unique, although the individual domains are similar to those of BoNTs A and B. In BoNT E, both the binding domain and the catalytic domain are on the same side of the translocation domain, and all three have mutual interfaces. This unique association may have an effect on the rate of translocation, with the molecule strategically positioned in the vesicle for quick entry into cytosol. Botulism, the disease caused by BoNT E, sets in faster than any other serotype because of its speedy internalization and translocation, and the present structure offers a credible explanation. We propose that the translocation domain in other BoNTs follows a two-step process to attain translocation-competent conformation as in BoNT E. We also suggest that this translocation-competent conformation in BoNT E is a probable reason for its faster toxic rate compared to BoNT A. However, this needs further experimental elucidation.

  2. Inhibition of botulinum neurotoxins interchain disulfide bond reduction prevents the peripheral neuroparalysis of botulism.

    PubMed

    Zanetti, Giulia; Azarnia Tehran, Domenico; Pirazzini, Marcon; Binz, Thomas; Shone, Clifford C; Fillo, Silvia; Lista, Florigio; Rossetto, Ornella; Montecucco, Cesare

    2015-12-01

    Botulinum neurotoxins (BoNTs) form a growing family of metalloproteases with a unique specificity either for VAMP, SNAP25 or syntaxin. The BoNTs are grouped in seven different serotypes indicated by letters from A to G. These neurotoxins enter the cytosol of nerve terminals via a 100 kDa chain which binds to the presynaptic membrane and assists the translocation of a 50 kDa metalloprotease chain. These two chains are linked by a single disulfide bridge which plays an essential role during the entry of the metalloprotease chain in the cytosol, but thereafter it has to be reduced to free the proteolytic activity. Its reduction is mediated by thioredoxin which is continuously regenerated by its reductase. Here we show that inhibitors of thioredoxin reductase or of thioredoxin prevent the specific proteolysis of VAMP by the four VAMP-specific BoNTs: type B, D, F and G. These compounds are effective not only in primary cultures of neurons, but also in preventing the in vivo mouse limb neuroparalysis. In addition, one of these inhibitors, Ebselen, largely protects mice from the death caused by a systemic injection. Together with recent results obtained with BoNTs specific for SNAP25 and syntaxin, the present data demonstrate the essential role of the thioredoxin-thioredoxin reductase system in reducing the interchain disulfide during the nerve intoxication mechanism of all serotypes. Therefore its inhibitors should be considered for a possible use to prevent botulism and for treating infant botulism.

  3. Unconventional amino acid sequence of the sun anemone (Stoichactis helianthus) polypeptide neurotoxin

    SciTech Connect

    Kem, W.; Dunn, B.; Parten, B.; Pennington, M.; Price, D.

    1986-05-01

    A 5000 dalton polypeptide neurotoxin (Sh-NI) purified by G50 Sephadex, P-cellulose, and SP-Sephadex chromatography was homogeneous by isoelectric focusing. Sh-NI was highly toxic to crayfish (LD/sub 50/ 0.6 ..mu..g/kg) but without effect upon mice at 15,000 ..mu..g/kg (i.p. injection). The reduced, /sup 3/H-carboxymethylated toxin and its fragments were subjected to automatic Edman degradation and the resulting PTH-amino acids were identified by HPLC, back hydrolysis, and scintillation counting. Peptides resulting from proteolytic (clostripain, staphylococcal protease) and chemical (tryptophan) cleavage were sequenced. The sequence is: AACKCDDEGPDIRTAPLTGTVDLGSCNAGWEKCASYYTIIADCCRKKK. This sequence differs considerably from the homologous Anemonia and Anthopleura toxins; many of the identical residues (6 half-cystines, G9, P10, R13, G19, G29, W30) are probably critical for folding rather than receptor recognition. However, the Sh-NI sequence closely resembles Radioanthus macrodactylus neurotoxin III and r. paumotensis II. The authors propose that Sh-NI and related Radioanthus toxins act upon a different site on the sodium channel.

  4. Botulinum neurotoxin type C protease induces apoptosis in differentiated human neuroblastoma cells.

    PubMed

    Rust, Aleksander; Leese, Charlotte; Binz, Thomas; Davletov, Bazbek

    2016-05-31

    Neuroblastomas constitute a major cause of cancer-related deaths in young children. In recent years, a number of translation-inhibiting enzymes have been evaluated for killing neuroblastoma cells. Here we investigated the potential vulnerability of human neuroblastoma cells to protease activity derived from botulinum neurotoxin type C. We show that following retinoic acid treatment, human neuroblastoma cells, SiMa and SH-SY5Y, acquire a neuronal phenotype evidenced by axonal growth and expression of neuronal markers. Botulinum neurotoxin type C which cleaves neuron-specific SNAP25 and syntaxin1 caused apoptotic death only in differentiated neuroblastoma cells. Direct comparison of translation-inhibiting enzymes and the type C botulinum protease revealed one order higher cytotoxic potency of the latter suggesting a novel neuroblastoma-targeting pathway. Our mechanistic insights revealed that loss of ubiquitous SNAP23 due to differentiation coupled to SNAP25 cleavage due to botulinum activity may underlie the apoptotic death of human neuroblastoma cells. PMID:27121208

  5. Chemical modification of tryptophan residues in alpha-neurotoxins from Ophiophagus hannah (king cobra) venom.

    PubMed

    Chang, C C; Lin, P M; Chang, L S; Kuo, K W

    1995-02-01

    Two alpha-neurotoxins, Oh-4 and Oh-7, from the king cobra (Ophiophagus hannah) venom were subjected to Trp modification with 2-nitrophenylsulfenyl chloride (NPS-Cl). One major NPS derivative was isolated from the modified mixtures of Oh-4 and two from Oh-7 by HPLC. Amino acid analysis and sequence determination revealed that Trp-27 in Oh-4, and Trp-30 and Trp-26 and 30 in the two Oh-7 derivatives, were modified, respectively. Sulfenylation of Trp-27 in Oh-4 caused about 70% drop in lethal toxicity and nicotinic acetylcholine receptor-binding activity. Modification of Trp-30 in Oh-7 resulted in the decrease of lethal toxicity by 36% and binding activity by 61%. The activities were further lost when the conserved Trp-26 in Oh-7 was modified. Sulfenylation of the Trp residues did not significantly affect the secondary structure of the toxins as revealed by the CD spectra. These results indicate that the Trp residues in these two long alpha-neurotoxins may be involved in the receptor binding.

  6. Acute Radiation Disease : Cutaneous Syndrome and Toxic properties of Radiomimetics -Radiation Neurotoxins and Hematotoxins.

    NASA Astrophysics Data System (ADS)

    Popov, Dmitri; Maliev, Slava

    Cutaneous injury is an important complication of a general or local acute irradiation. A type of a skin and tissues lesions depends on a type, intensity, and period of irradiation. Also, the clinical picture, signs, and manifestations of the cutaneous syndrome depend on a type of the radiation toxins circulated in lymph and blood of irradiated mammals. Radiation Toxins were isolated from lymph of the mammals that were irradiated and developed different forms of the Acute Radiation Syndromes (ARS) -Cerebrovascular, Cardiovascular, Gastrointestinal, and Hematopoietic. Radiation Toxins can be divided into the two important types of toxins (Neu-rotoxins and Hematotoxins) or four groups. The effects of Radiation Neurotoxins include severe damages and cell death of brain, heart, gastrointestinal tissues and endothelial cells of blood and lymphatic vessels. The hematotoxicity of Hematotoxic Radiation Toxins includes lym-phopenia, leukopenia, thrombocytopenia, and anemia in the blood circulation and transitory lymphocytosis and leukocytosis in the Central Lymphatic System. In all cases, administration of the Radiomimetics (Radiation Toxins) intramuscularly or intravenously to healthy, radiation naive mammals had induced and developed the typical clinical manifestations of the ARS. In all cases, administration of Radiomimetics by subtoxic doses had demonstrated development of typical clinical signs of the cutaneous syndrome such as hair loss, erythema, swelling, desqua-mation, blistering and skin necrosis. In animal-toxic models, we have activated development of the local skin and tissue injury after injection of Radiation Toxins with cytoxic properties.

  7. Sialorrhea in patients with Parkinson's disease: safety and administration of botulinum neurotoxin.

    PubMed

    Egevad, Gustav; Petkova, Valentina Yankova; Vilholm, Ole Jakob

    2014-01-01

    Sialorrhea may present as a troublesome symptom in patients suffering from Parkinson's disease. Current options for treatment include anticholinergic drugs, irradiation, surgery, oral-motor and behavioural therapies, and injection of botulinum neurotoxin (BoNT) in the salivary glands. The aim of this study is to evaluate the safety and administration of BoNT as a treatment for sialorrhea in patients with Parkinson's disease (PD) based on a review of the studies conducted so far in this field. A PubMed search was conducted using the major keywords sialorrhea, botulinum neurotoxin, botulinum toxin and Parkinson's disease. The literature search identified 12 articles, which were selected for further analysis. Few adverse effects were described in the studies included in this present review. Various treatment strategies, including different medication dosages, were applied in the studies. BoNT treatment is safe for sialorrhea in patients with PD. Positive effect is well documented, and there have been relatively few reported adverse effects, which have been mild and transient. Based on this review, a treatment algorithm is proposed. Ultrasound guidance may not be necessary when injecting the parotid gland but may improve the effect and safety of administration, especially when injecting the submandibular glands.

  8. Recent developments with metalloprotease inhibitor class of drug candidates for Botulinum neurotoxins

    DOE PAGESBeta

    Kumar, Gyanendra; Swaminathan, Subramanyam

    2015-03-01

    Botulinum Neurotoxins are the most poisonous of all toxins with lethal dose in nanogram quantities. They are also potential biological warfare and bioterrorism agents due to their high toxicity and ease of preparation. On the other hand BoNTs are also being increasingly used for therapeutic and cosmetic purposes, and with that the chances of accidental overdose are increasing. And despite the potential damage they could cause to human health, there are no post-intoxication drugs available so far. But progress is being made in this direction. The crystal structures in native form and bound with substrate peptides have been determined, andmore » these are enabling structure-based drug discovery possible. High throughput assays have also been designed to speed up the screening progress. Substrate-based and small molecule inhibitors have been identified. But turning high affinity inhibitors into clinically viable drug candidates has remained a challenge. We discuss here the latest developments and the future challenges in drug discovery for Botulinum neurotoxins.« less

  9. Botulinum neurotoxin type C protease induces apoptosis in differentiated human neuroblastoma cells

    PubMed Central

    Rust, Aleksander; Leese, Charlotte; Binz, Thomas; Davletov, Bazbek

    2016-01-01

    Neuroblastomas constitute a major cause of cancer-related deaths in young children. In recent years, a number of translation-inhibiting enzymes have been evaluated for killing neuroblastoma cells. Here we investigated the potential vulnerability of human neuroblastoma cells to protease activity derived from botulinum neurotoxin type C. We show that following retinoic acid treatment, human neuroblastoma cells, SiMa and SH-SY5Y, acquire a neuronal phenotype evidenced by axonal growth and expression of neuronal markers. Botulinum neurotoxin type C which cleaves neuron-specific SNAP25 and syntaxin1 caused apoptotic death only in differentiated neuroblastoma cells. Direct comparison of translation-inhibiting enzymes and the type C botulinum protease revealed one order higher cytotoxic potency of the latter suggesting a novel neuroblastoma-targeting pathway. Our mechanistic insights revealed that loss of ubiquitous SNAP23 due to differentiation coupled to SNAP25 cleavage due to botulinum activity may underlie the apoptotic death of human neuroblastoma cells. PMID:27121208

  10. Two protein trafficking processes at motor nerve endings unveiled by botulinum neurotoxin E.

    PubMed

    Lawrence, Gary; Wang, Jiafu; Chion, C K N Kwo; Aoki, K Roger; Dolly, J Oliver

    2007-01-01

    The unique ability of a family of botulinum neurotoxins to block neuroexocytosis specifically-by selective interaction with peripheral cholinergic nerve endings, endocytotic uptake, translocation to the cytosol, and enzymic cleavage of essential proteins-underlies their increasing therapeutic applications. Although clinical use of type A is most widespread due to its prolonged inactivation of the synaptosomal-associated protein of 25 kDa, botulinum neurotoxin E cleaves this same target but at a different bond and exhibits faster onset of neuromuscular paralysis. Herein, insights were gained into the different dynamics of action of types A and E toxins, which could help in designing variants with new pharmacological profiles. Natural and recombinant type E dichain forms showed similar proteolytic and neuromuscular paralytic activities. The neuroparalysis induced by type E toxin was accelerated between 21 and 35 degrees C and attenuated by bafilomycin A1. Temperature elevation also revealed an unanticipated bipartite dose response indicative of two distinct internalization processes, one being independent of temperature and the other dependent. Although elevating the temperature also hastened intoxication by type A, a second uptake mechanism was not evident. Increasing the frequency of nerve stimulation raised the uptake of type E via both processes, but the enhanced trafficking through the temperature-dependent pathway was only seen at 35 degrees C. These novel observations reveal that two membrane retrieval mechanisms are operative at motor nerve terminals which type E toxin exploits to gain entry via an acidification-dependent step, whereas A uses only one.

  11. Extravesicular intraneuronal migration of internalized botulinum neurotoxins without detectable inhibition of distal neurotransmission.

    PubMed

    Lawrence, Gary W; Ovsepian, Saak V; Wang, Jiafu; Aoki, K Roger; Dolly, J Oliver

    2012-01-01

    Intracellular protein transport routes can be studied using toxins that exploit these to enter cells. BoNTA (botulinum neurotoxin type A) is a protease that binds to peripheral nerve terminals, becomes endocytosed and causes prolonged blockade of transmitter release by cleaving SNAP-25 (synaptosome-associated protein of 25 kDa). Retrograde transport of the toxin has been suggested, but not of the transient muscle relaxant, BoNTE (botulinum neurotoxin type E). In the present study, dispersal of these proteases in compartmented cultures of rat sympathetic neurons was examined after focal application of BoNTA or BoNTE to neurites. A majority of cleaved SNAP-25 was seen locally, but some appeared along neurites and accumulated in the soma over several weeks. BoNTE yielded less cleaved SNAP-25 at distal sites due to shorter-lived enzymic activity. Neurite transection prevented movement of BoNTA. The BoNTA protease could be detected only in the supernatants of neurites or cell body lysates, hence these proteases must move along neuronal processes in the axoplasm or are reversibly associated with membranes. Substitution into BoNTE of the BoNTA acceptor-binding domain did not alter its potency or mobility. Spontaneous or evoked transmission to cell bodies were not inhibited by retrogradely migrated BoNTA except with high doses, concurring with the lack of evidence for a direct central action when used clinically.

  12. Current status and future directions of botulinum neurotoxins for targeting pain processing.

    PubMed

    Pellett, Sabine; Yaksh, Tony L; Ramachandran, Roshni

    2015-11-04

    Current evidence suggests that botulinum neurotoxins (BoNTs) A1 and B1, given locally into peripheral tissues such as skin, muscles, and joints, alter nociceptive processing otherwise initiated by inflammation or nerve injury in animal models and humans. Recent data indicate that such locally delivered BoNTs exert not only local action on sensory afferent terminals but undergo transport to central afferent cell bodies (dorsal root ganglia) and spinal dorsal horn terminals, where they cleave SNAREs and block transmitter release. Increasing evidence supports the possibility of a trans-synaptic movement to alter postsynaptic function in neuronal and possibly non-neuronal (glial) cells. The vast majority of these studies have been conducted on BoNT/A1 and BoNT/B1, the only two pharmaceutically developed variants. However, now over 40 different subtypes of botulinum neurotoxins (BoNTs) have been identified. By combining our existing and rapidly growing understanding of BoNT/A1 and /B1 in altering nociceptive processing with explorations of the specific characteristics of the various toxins from this family, we may be able to discover or design novel, effective, and long-lasting pain therapeutics. This review will focus on our current understanding of the molecular mechanisms whereby BoNTs alter pain processing, and future directions in the development of these agents as pain therapeutics.

  13. Subtyping botulinum neurotoxins by sequential multiple endoproteases in-gel digestion coupled with mass spectrometry.

    PubMed

    Wang, Dongxia; Baudys, Jakub; Rees, Jon; Marshall, Kristin M; Kalb, Suzanne R; Parks, Bryan A; Nowaczyk, Louis; Pirkle, James L; Barr, John R

    2012-06-01

    Botulinum neurotoxin (BoNT) is one of the most toxic substances known. BoNT is classified into seven distinct serotypes labeled A-G. Among individual serotypes, researchers have identified subtypes based on amino acid variability within a serotype and toxin variants with minor amino acid sequence differences within a subtype. BoNT subtype identification is valuable for tracing and tracking bacterial pathogens. A proteomics approach is useful for BoNT subtyping since botulism is caused by botulinum neurotoxin and does not require the presence of the bacteria or its DNA. Enzymatic digestion and peptide identification using tandem mass spectrometry determines toxin protein sequences. However, with the conventional one-step digestion method, producing sufficient numbers of detectable peptides to cover the entire protein sequence is difficult, and incomplete sequence coverage results in uncertainty in distinguishing BoNT subtypes and toxin variants because of high sequence similarity. We report here a method of multiple enzymes and sequential in-gel digestion (MESID) to characterize the BoNT protein sequence. Complementary peptide detection from toxin digestions has yielded near-complete sequence coverage for all seven BoNT serotypes. Application of the method to a BoNT-contaminated carrot juice sample resulted in the identification of 98.4% protein sequence which led to a confident determination of the toxin subtype.

  14. Recent developments with metalloprotease inhibitor class of drug candidates for botulinum neurotoxins.

    PubMed

    Kumar, Gyanendra; Swaminathan, Subramanyam

    2015-01-01

    Botulinum Neurotoxins are the most poisonous of all toxins with lethal dose in nanogram quantities. They are potential biological warfare and bioterrorism agents due to their high toxicity and ease of preparation. On the other hand BoNTs are also being increasingly used for therapeutic and cosmetic purposes, and with that the chances of accidental overdose are increasing. And despite the potential damage they could cause to human health, there are no post-intoxication drugs available so far. But progress is being made in this direction. The crystal structures in native form and bound with substrate peptides have been determined, and these are enabling structure-based drug discovery possible. High throughput assays have also been designed to speed up the screening progress. Substrate-based and small molecule inhibitors have been identified. But turning high affinity inhibitors into clinically viable drug candidates has remained a challenge. We discuss here the latest developments and the future challenges in drug discovery for Botulinum neurotoxins.

  15. Inhibition of botulinum neurotoxins interchain disulfide bond reduction prevents the peripheral neuroparalysis of botulism.

    PubMed

    Zanetti, Giulia; Azarnia Tehran, Domenico; Pirazzini, Marcon; Binz, Thomas; Shone, Clifford C; Fillo, Silvia; Lista, Florigio; Rossetto, Ornella; Montecucco, Cesare

    2015-12-01

    Botulinum neurotoxins (BoNTs) form a growing family of metalloproteases with a unique specificity either for VAMP, SNAP25 or syntaxin. The BoNTs are grouped in seven different serotypes indicated by letters from A to G. These neurotoxins enter the cytosol of nerve terminals via a 100 kDa chain which binds to the presynaptic membrane and assists the translocation of a 50 kDa metalloprotease chain. These two chains are linked by a single disulfide bridge which plays an essential role during the entry of the metalloprotease chain in the cytosol, but thereafter it has to be reduced to free the proteolytic activity. Its reduction is mediated by thioredoxin which is continuously regenerated by its reductase. Here we show that inhibitors of thioredoxin reductase or of thioredoxin prevent the specific proteolysis of VAMP by the four VAMP-specific BoNTs: type B, D, F and G. These compounds are effective not only in primary cultures of neurons, but also in preventing the in vivo mouse limb neuroparalysis. In addition, one of these inhibitors, Ebselen, largely protects mice from the death caused by a systemic injection. Together with recent results obtained with BoNTs specific for SNAP25 and syntaxin, the present data demonstrate the essential role of the thioredoxin-thioredoxin reductase system in reducing the interchain disulfide during the nerve intoxication mechanism of all serotypes. Therefore its inhibitors should be considered for a possible use to prevent botulism and for treating infant botulism. PMID:26449594

  16. Current Status and Future Directions of Botulinum Neurotoxins for Targeting Pain Processing

    PubMed Central

    Pellett, Sabine; Yaksh, Tony L.; Ramachandran, Roshni

    2015-01-01

    Current evidence suggests that botulinum neurotoxins (BoNTs) A1 and B1, given locally into peripheral tissues such as skin, muscles, and joints, alter nociceptive processing otherwise initiated by inflammation or nerve injury in animal models and humans. Recent data indicate that such locally delivered BoNTs exert not only local action on sensory afferent terminals but undergo transport to central afferent cell bodies (dorsal root ganglia) and spinal dorsal horn terminals, where they cleave SNAREs and block transmitter release. Increasing evidence supports the possibility of a trans-synaptic movement to alter postsynaptic function in neuronal and possibly non-neuronal (glial) cells. The vast majority of these studies have been conducted on BoNT/A1 and BoNT/B1, the only two pharmaceutically developed variants. However, now over 40 different subtypes of botulinum neurotoxins (BoNTs) have been identified. By combining our existing and rapidly growing understanding of BoNT/A1 and /B1 in altering nociceptive processing with explorations of the specific characteristics of the various toxins from this family, we may be able to discover or design novel, effective, and long-lasting pain therapeutics. This review will focus on our current understanding of the molecular mechanisms whereby BoNTs alter pain processing, and future directions in the development of these agents as pain therapeutics. PMID:26556371

  17. Characterization of alpha-neurotoxin and phospholipase A2 activities from Micrurus venoms. Determination of the amino acid sequence and receptor-binding ability of the major alpha-neurotoxin from Micrurus nigrocinctus nigrocinctus.

    PubMed

    Rosso, J P; Vargas-Rosso, O; Gutiérrez, J M; Rochat, H; Bougis, P E

    1996-05-15

    New World elapids are coral snakes that belong to the genus Micrurus, and for which the venom biochemistry is mostly unknown. Analysis has been difficult because the coral snakes produce small quantities of venom. Clinical observations following bites show mainly neurotoxic effects. Experimentally, cardiotoxic, haemolytic and myotoxic activities are also reported. An experimental approach, using reverse-phase high-performance liquid chromatography and specific assays for alpha-neurotoxin and phospholipase A2 activities, was conducted on milligram quantities of venoms from three Micrurus species from Costa Rica; M. nigrocinctus nigrocinctus, M. alleni yatesi and M. multifasciatus. Neurotoxicity was determined by competition binding experiments with the Torpedo marmorata acetylcholine receptor. Phospholipase A2 activity was measured by fluorimetry using a pyrene lipid substrate. In this way, we purified and characterized seven alpha-neurotoxins, five phospholipases A2 and four toxin homologs. The amino acid sequence of the major alpha-neurotoxin from M. nigrocinctus nigrocinctus venom was fully determined and compared to Old Word representatives. Distance matrix data were generated to set up phylogeny relationships among elapid short-chain alpha-neurotoxins, which proved to be in accordance with the taxonomic classification and geographical distribution of snake species.

  18. Cerebrovascular Acute Radiation Syndrome : Radiation Neurotoxins, Mechanisms of Toxicity, Neuroimmune Interactions.

    NASA Astrophysics Data System (ADS)

    Popov, Dmitri; Maliev, Slava

    Introduction: Cerebrovascular Acute Radiation Syndrome (CvARS) is an extremely severe in-jury of Central Nervous System (CNS) and Peripheral Nervous System (PNS). CvARS can be induced by the high doses of neutron, heavy ions, or gamma radiation. The Syndrome clinical picture depends on a type, timing, and the doses of radiation. Four grades of the CvARS were defined: mild, moderate, severe, and extremely severe. Also, four stages of CvARS were developed: prodromal, latent, manifest, outcome -death. Duration of stages depends on the types, doses, and time of radiation. The CvARS clinical symptoms are: respiratory distress, hypotension, cerebral edema, severe disorder of cerebral blood microcirculation, and acute motor weakness. The radiation toxins, Cerebro-Vascular Radiation Neurotoxins (SvARSn), determine development of the acute radiation syndrome. Mechanism of action of the toxins: Though pathogenesis of radiation injury of CNS remains unknown, our concept describes the Cv ARS as a result of Neurotoxicity and Excitotoxicity, cell death through apoptotic necrosis. Neurotoxicity occurs after the high doses radiation exposure, formation of radiation neuro-toxins, possible bioradicals, or group of specific enzymes. Intracerebral hemorrhage can be a consequence of the damage of endothelial cells caused by radiation and the radiation tox-ins. Disruption of blood-brain barrier (BBB)and blood-cerebrospinal fluid barrier (BCFB)is possibly the most significant effect of microcirculation disorder and metabolic insufficiency. NMDA-receptors excitotoxic injury mediated by cerebral ischemia and cerebral hypoxia. Dam-age of the pyramidal cells in layers 3 and 5 and Purkinje cell layer the cerebral cortex , damage of pyramidal cells in the hippocampus occur as a result of cerebral ischemia and intracerebral bleeding. Methods: Radiation Toxins of CV ARS are defined as glycoproteins with the molec-ular weight of RT toxins ranges from 200-250 kDa and with high enzymatic activity

  19. Solution structure of toxin b, a long neurotoxin from the venom of the king cobra (Ophiophagus hannah).

    PubMed

    Peng, S S; Kumar, T K; Jayaraman, G; Chang, C C; Yu, C

    1997-03-21

    The solution structure of toxin b, a long neurotoxin (73 amino acids and 5 disulfides) from the venom of Ophiophagus hannah (king cobra), has been determined using 1H NMR and dynamical simulated annealing techniques. The structures were calculated using 485 distance constraints and 52 dihedral angle restraints. The 21 structures that were obtained satisfy the experimental restraints and possess good nonbonded contacts. Analysis of the converged structures revealed that the protein consists of a core region from which three finger-like loops extend outwards. The regular secondary structure in toxin b includes a double and a triple stranded antiparallel beta sheet. Comparison with the solution structures of other long neurotoxins reveals that although the structure of toxin b is similar to those of previously reported long neurotoxins, clear local structural differences are observed in regions proposed to be involved in binding to the acetylcholine receptor. A positively charged cluster is found in the C-terminal tail, in Loop III, and in the tip of Loop II. This cationic cluster could be crucial for the binding of the long neurotoxins to the acetylcholine receptor.

  20. Oligosaccharide composition of the neurotoxin responsive Na/sup +/ channel and the requirement of sialic acid for activity

    SciTech Connect

    Negishi, M.; Shaw, G.W.; Glick, M.C.

    1986-05-01

    The neurotoxin responsive Na/sup +/ channel was purified to homogeneity in an 18% yield from a clonal cell line of mouse neuroblastoma, N-18, metabolically labeled with L-(/sup 3/H)fucose. The Na/sup +/ channel, a glycoprotein, M/sub r/=200,000 (gradient 7-14% PAGE) was digested with Pronase and the glycopeptides were characterized by serial lectin affinity chromatography. greater than 90% of the oligosaccharides contained sialic acid and 18% were biantennary, 39% were triantennary and 30% tetraantennary. The glycoprotein was reconstituted into artificial phospholipid vesicles and /sup 86/Rb flux was stimulated (65%) by 200 ..mu..M veratridine and 1.2 ..mu..g of scorpion venom and was inhibited (95%) by 5 ..mu..M tetrodotoxin. The requirement of sialic acid for Na/sup +/ channel activity was demonstrated since neuraminidase (0.01 U) treatment of the reconstituted glycoprotein eliminated the response of /sup 86/Rb flux to the stimulating neurotoxins. In other experiments, treatment of N-18 cells with 10 ..mu..M swainsonine, an inhibitor of glycoprotein processing, altered the oligosaccharide composition of the Na/sup +/ channel. When the abnormally glycosylated Na/sup +/ channel was reconstituted into artificial phospholipid vesicles, /sup 86/Rb flux in response to neurotoxins was impaired. Thus, glycosylation of the polypeptide with oligosaccharides of specific composition and structure is essential for expression of the biological activity of the neurotoxin responsive Na/sup +/ channel.

  1. Alpha-synuclein overexpression induced mitochondrial damage by the generation of endogenous neurotoxins in PC12 cells.

    PubMed

    Zhang, Yanyan; Ma, Hong; Xie, Bingjie; Han, Chao; Wang, Chen; Qing, Hong; Deng, Yulin

    2013-06-28

    Alpha-synuclein is one of the important components of Lewy body which involved in neuropathology of Parkinson's disease (PD). The relationship between α-synuclein and cell death is still unclear. In the study, PC12 cell, stably over expressing α-synuclein model was used, and we investigated the level of intracellular oxidative stress, dopamine and endogenous neurotoxin. The results showed that the level of oxidative stress and intracytoplasmic dopamine (DA) was increased in cells over expressing α-synuclein compared with normal PC12 cells. Simultaneously, additional generation of endogenous neurotoxins 1-methyl-4-phenyl-1,2,3,4-tetrahydroisoquinoline (salsolinol) and 1(R),2(N)-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinolin (NM-salsolinol) was detected and this phenomenon was exacerbated after exposed to H₂O₂ for 24 h, but mitigated when treated with dopamine synthesis inhibitors. The presence of endogenous neurotoxins exacerbated α-synuclein induced mitochondrial damage. These results suggest that the endogenous neurotoxins may become a bridge between α-synuclein and cell death.

  2. Structural dynamics of the alpha-neurotoxin-acetylcholine-binding protein complex: hydrodynamic and fluorescence anisotropy decay analyses.

    PubMed

    Hibbs, Ryan E; Johnson, David A; Shi, Jianxin; Hansen, Scott B; Taylor, Palmer

    2005-12-20

    The three-fingered alpha-neurotoxins have played a pivotal role in elucidating the structure and function of the muscle-type and neuronal alpha7 nicotinic acetylcholine receptors (nAChRs). To advance our understanding of the alpha-neurotoxin-nAChR interaction, we examined the flexibility of alpha-neurotoxin bound to the acetylcholine-binding protein (AChBP), which shares structural similarity and sequence identities with the extracellular domain of nAChRs. Because the crystal structure of five alpha-cobratoxin molecules bound to AChBP shows the toxins projecting radially like propeller "blades" from the perimeter of the donut-shaped AChBP, the toxin molecules should increase the frictional resistance and thereby alter the hydrodynamic properties of the complex. alpha-Bungarotoxin binding had little effect on the frictional coefficients of AChBP measured by analytical ultracentrifugation, suggesting that the bound toxins are flexible. To support this conclusion, we measured the anisotropy decay of four site-specifically labeled alpha-cobratoxins (conjugated at positions Lys(23), Lys(35), Lys(49), and Lys(69)) bound to AChBP and free in solution and compared their anisotropy decay properties with fluorescently labeled cysteine mutants of AChBP. The results indicated that the core of the toxin molecule is relatively flexible when bound to AChBP. When hydrodynamic and anisotropy decay analyses are taken together, they establish that only one face of the second loop of the alpha-neurotoxin is immobilized significantly by its binding. The results indicate that bound alpha-neurotoxin is not rigidly oriented on the surface of AChBP but rather exhibits segmental motion by virtue of flexibility in its fingerlike structure.

  3. Evolution of Chromosomal Clostridium botulinum Type E Neurotoxin Gene Clusters: Evidence Provided by Their Rare Plasmid-Borne Counterparts

    PubMed Central

    Carter, Andrew T.; Austin, John W.; Weedmark, Kelly A.; Peck, Michael W.

    2016-01-01

    Analysis of more than 150 Clostridium botulinum Group II type E genomes identified a small fraction (6%) where neurotoxin-encoding genes were located on plasmids. Seven closely related (134–144 kb) neurotoxigenic plasmids of subtypes E1, E3, and E10 were characterized; all carried genes associated with plasmid mobility via conjugation. Each plasmid contained the same 24-kb neurotoxin cluster cassette (six neurotoxin cluster and six flanking genes) that had split a helicase gene, rather than the more common chromosomal rarA. The neurotoxin cluster cassettes had evolved as separate genetic units which had either exited their chromosomal rarA locus in a series of parallel events, inserting into the plasmid-borne helicase gene, or vice versa. A single intact version of the helicase gene was discovered on a nonneurotoxigenic form of this plasmid. The observed low frequency for the plasmid location may reflect one or more of the following: 1) Less efficient recombination mechanism for the helicase gene target, 2) lack of suitable target plasmids, and 3) loss of neurotoxigenic plasmids. Type E1 and E10 plasmids possessed a Clustered Regularly Interspaced Short Palindromic Repeats locus with spacers that recognized C. botulinum Group II plasmids, but not C. botulinum Group I plasmids, demonstrating their long-term separation. Clostridium botulinum Group II type E strains also carry nonneurotoxigenic plasmids closely related to C. botulinum Group II types B and F plasmids. Here, the absence of neurotoxin cassettes may be because recombination requires both a specific mechanism and specific target sequence, which are rarely found together. PMID:26936890

  4. Inhibition of the Nicotinic Acetylcholine Receptors by Cobra Venom α-Neurotoxins: Is There a Perspective in Lung Cancer Treatment?

    PubMed Central

    Alama, Angela; Bruzzo, Cristina; Cavalieri, Zita; Forlani, Alessandra; Utkin, Yuri; Casciano, Ida; Romani, Massimo

    2011-01-01

    Nicotine exerts its oncogenic effects through the binding to nicotinic acetylcholine receptors (nAChRs) and the activation of downstream pathways that block apoptosis and promote neo-angiogenesis. The nAChRs of the α7 subtype are present on a wide variety of cancer cells and their inhibition by cobra venom neurotoxins has been proposed in several articles and reviews as a potential innovative lung cancer therapy. However, since part of the published results was recently retracted, we believe that the antitumoral activity of cobra venom neurotoxins needs to be independently re-evaluated. We determined the activity of α-neurotoxins from Naja atra (short-chain neurotoxin, α-cobrotoxin) and Naja kaouthia (long-chain neurotoxin, α-cobratoxin) in vitro by cytotoxicity measurements in 5 lung cancer cell lines, by colony formation assay with α7nAChRs expressing and non-expressing cell lines and in vivo by assessing tumor growth in an orthotopic Non-Obese Diabetic/Severe Combined Immunodeficient (NOD/SCID) mouse model system utilizing different treatment schedules and dosages. No statistically significant reduction in tumor growth was observed in the treatment arms in comparison to the control for both toxins. Paradoxically α-cobrotoxin from Naja atra showed the tendency to enhance tumor growth although, even in this case, the statistical significance was not reached. In conclusion our results show that, in contrast with other reports, the nAChR inhibitors α-cobratoxin from N. kaouthia and α-cobrotoxin from N. atra neither suppressed tumor growth nor prolonged the survival of the treated animals. PMID:21695184

  5. Exploring the obscure profiles of pharmacological binding sites on voltage-gated sodium channels by BmK neurotoxins.

    PubMed

    Liu, Zhi-Rui; Ye, Pin; Ji, Yong-Hua

    2011-06-01

    Diverse subtypes of voltage-gated sodium channels (VGSCs) have been found throughout tissues of the brain, muscles and the heart. Neurotoxins extracted from the venom of the Asian scorpion Buthus martensi Karsch (BmK) act as sodium channel-specific modulators and have therefore been widely used to study VGSCs. α-type neurotoxins, named BmK I, BmK αIV and BmK abT, bind to receptor site-3 on VGSCs and can strongly prolong the inactivation phase of VGSCs. In contrast, β-type neurotoxins, named BmK AS, BmK AS-1, BmK IT and BmK IT2, occupy receptor site-4 on VGSCs and can suppress peak currents and hyperpolarize the activation kinetics of sodium channels. Accumulating evidence from binding assays of scorpion neurotoxins on VGSCs, however, indicate that pharmacological sensitivity of VGSC subtypes to different modulators is much more complex than that suggested by the simple α-type and β-type neurotoxin distinction. Exploring the mechanisms of possible dynamic interactions between site 3-/4-specific modulators and region- and/or species-specific subtypes of VGSCs would therefore greatly expand our understanding of the physiological and pharmacological properties of diverse VGSCs. In this review, we discuss the pharmacological and structural diversity of VGSCs as revealed by studies exploring the binding properties and cross-competitive binding of site 3- or site 4-specific modulators in VGSC subtypes in synaptosomes from distinct tissues of diverse species.

  6. Evolution of Chromosomal Clostridium botulinum Type E Neurotoxin Gene Clusters: Evidence Provided by Their Rare Plasmid-Borne Counterparts.

    PubMed

    Carter, Andrew T; Austin, John W; Weedmark, Kelly A; Peck, Michael W

    2016-03-01

    Analysis of more than 150 Clostridium botulinum Group II type E genomes identified a small fraction (6%) where neurotoxin-encoding genes were located on plasmids. Seven closely related (134-144 kb) neurotoxigenic plasmids of subtypes E1, E3, and E10 were characterized; all carried genes associated with plasmid mobility via conjugation. Each plasmid contained the same 24-kb neurotoxin cluster cassette (six neurotoxin cluster and six flanking genes) that had split a helicase gene, rather than the more common chromosomal rarA. The neurotoxin cluster cassettes had evolved as separate genetic units which had either exited their chromosomal rarA locus in a series of parallel events, inserting into the plasmid-borne helicase gene, or vice versa. A single intact version of the helicase gene was discovered on a nonneurotoxigenic form of this plasmid. The observed low frequency for the plasmid location may reflect one or more of the following: 1) Less efficient recombination mechanism for the helicase gene target, 2) lack of suitable target plasmids, and 3) loss of neurotoxigenic plasmids. Type E1 and E10 plasmids possessed a Clustered Regularly Interspaced Short Palindromic Repeats locus with spacers that recognized C. botulinum Group II plasmids, but not C. botulinum Group I plasmids, demonstrating their long-term separation. Clostridium botulinum Group II type E strains also carry nonneurotoxigenic plasmids closely related to C. botulinum Group II types B and F plasmids. Here, the absence of neurotoxin cassettes may be because recombination requires both a specific mechanism and specific target sequence, which are rarely found together. PMID:26936890

  7. Recognition of Vipera ammodytes meridionalis neurotoxin vipoxin and its components using phage-displayed scFv and polyclonal antivenom sera.

    PubMed

    Stoyanova, Vishnya; Aleksandrov, Radoslav; Lukarska, Maria; Duhalov, Deyan; Atanasov, Vasil; Petrova, Svetla

    2012-10-01

    Vipoxin is a potent postsynaptic heterodimeric neurotoxin isolated from the venom of the Bulgarian snake Vipera ammodytes meridionalis, whose snakebites cause different and strongly manifested pathophysiological effects (neurotoxic, hemolytic, anticoagulant, convulsant, hypotensive, hyperglycemic etc.). The neutralization of snake toxins calls for extensive research through the application of different approaches: antibodies, non-immunologic inhibitors, natural products derived from plants and animals, as well as synthetic drugs. In this study, we applied naive Tomlinson I + J (Cambridge, UK) libraries to obtain recombinant human scFv antibodies against the vipoxin's two subunits--basic and toxic phospholipase A₂ (PLA₂) and acidic, non-toxic component. We found that 33 of more than hundred tested clones were positive and recognized vipoxin and its subunits. Enriched scFv-phage samples (1.2 × 10⁹ pfu/ml) were analyzed for their binding (ELISA) and enzyme-inhibiting abilities. Single chain Fv-phage clones--D₁₂, E₃, F₆, D₁₀ and G₅ exhihest binding affinity for the toxic component. Clones A₁, D₁₂ and C₁₂ recognized preferentially vipoxin's acidic component. Clones E₃, G₅ and H₄ inhibited the enzymatic activity of both vipoxin and its purified and separated toxic subunit to the highest extent. Six of the selected clones (E₃, G₅, H₄, C₁₂, D₁₀ and A₁₁) inhibited direct hemolytic activity of vipoxin and its pure PLA₂ subunit. The obtained specific scFv antibodies will be used for epitope mapping studies required to shed light on the role of the phospholipase A₂ activity for the vipoxin toxicity and its effective neutralization.

  8. Excitatory cholinergic and purinergic signaling in bladder are equally susceptible to botulinum neurotoxin a consistent with co-release of transmitters from efferent fibers.

    PubMed

    Lawrence, Gary W; Aoki, K Roger; Dolly, J Oliver

    2010-09-01

    Mediators of neuromuscular transmission in rat bladder strips were dissected pharmacologically to examine their susceptibilities to inhibition by botulinum neurotoxins (BoNTs) and elucidate a basis for the clinical effectiveness of BoNT/A in alleviating smooth muscle spasms associated with overactive bladder. BoNT/A, BoNT/C1, or BoNT/E reduced peak and average force of muscle contractions induced by electric field stimulation (EFS) in dose-dependent manners by acting only on neurogenic, tetrodotoxin-sensitive responses. BoNTs that cleaved vesicle-associated membrane protein proved to be much less effective. Acetylcholine (ACh) and ATP were found to provide virtually all excitatory input, because EFS-evoked contractions were abolished by the muscarinic receptor antagonist, atropine, combined with either a desensitizing agonist of P2X(1) and P2X(3) or a nonselective ATP receptor antagonist. Both transmitters were released in the innervated muscle layer and, thus, persisted after removal of urothelium. Atropine or a desensitizer of the P2X(1) or P2X(3) receptors did not alter the rate at which muscle contractions were weakened by BoNT/A. Moreover, although cholinergic and purinergic signaling could be partially delineated by using high-frequency EFS (which intensified a transient, largely atropine-resistant spike in muscle contractions that was reduced after P2X receptor desensitization), they proved equally susceptible to BoNT/A. Thus, equi-potent blockade of ATP co-released with ACh from muscle efferents probably contributes to the effectiveness of BoNT/A in treating bladder overactivity, including nonresponders to anticholinergic drugs. Because purinergic receptors are known mediators of sensory afferent excitation, inhibition of efferent ATP release by BoNT/A could also help to ameliorate acute pain and urgency sensation reported by some recipients.

  9. Long-lasting attenuation of amygdala-kindled seizures after convection-enhanced delivery of botulinum neurotoxins a and B into the amygdala in rats.

    PubMed

    Gasior, Maciej; Tang, Rebecca; Rogawski, Michael A

    2013-09-01

    Botulinum neurotoxins (BoNTs) are well recognized to cause potent, selective, and long-lasting neuroparalytic actions by blocking cholinergic neurotransmission to muscles and glands. There is evidence that BoNT isoforms can also inhibit neurotransmission in the brain. In this study, we examined whether locally delivered BoNT/A and BoNT/B can attenuate kindling measures in amygdala-kindled rats. Male rats were implanted with a combination infusion cannula-stimulating electrode assembly into the right basolateral amygdala. Fully kindled animals received a single infusion of vehicle or BoNT/A or BoNT/B at doses of 1, 3.2, or 10 ng over a 20-minute period by convection-enhanced delivery. Electrographic (EEG) and behavioral kindling measures were determined at selected times during the 3- to 64-day period after the infusion. BoNT/B produced a dose-dependent elevation in after-discharge threshold and duration and a reduction in the seizure stage and duration of behavioral seizures that lasted for up to 50 days after infusion. BoNT/A had similar effects on EEG measures; behavioral seizure measures were also reduced, but the effect did not reach statistical significance. The effects of both toxins on EEG and behavioral measures progressively resolved during the latter half of the observation period. Animals gained weight normally, maintained normal body temperature, and did not show altered behavior. This study demonstrates for the first time that locally delivered BoNTs can produce prolonged inhibition of brain excitability, indicating that they could be useful for the treatment of brain disorders, including epilepsy, that would benefit from long-lasting suppression of neurotransmission within a circumscribed brain region.

  10. Long-Lasting Attenuation of Amygdala-Kindled Seizures after Convection-Enhanced Delivery of Botulinum Neurotoxins A and B into the Amygdala in Rats

    PubMed Central

    Gasior, Maciej; Tang, Rebecca

    2013-01-01

    Botulinum neurotoxins (BoNTs) are well recognized to cause potent, selective, and long-lasting neuroparalytic actions by blocking cholinergic neurotransmission to muscles and glands. There is evidence that BoNT isoforms can also inhibit neurotransmission in the brain. In this study, we examined whether locally delivered BoNT/A and BoNT/B can attenuate kindling measures in amygdala-kindled rats. Male rats were implanted with a combination infusion cannula–stimulating electrode assembly into the right basolateral amygdala. Fully kindled animals received a single infusion of vehicle or BoNT/A or BoNT/B at doses of 1, 3.2, or 10 ng over a 20-minute period by convection-enhanced delivery. Electrographic (EEG) and behavioral kindling measures were determined at selected times during the 3- to 64-day period after the infusion. BoNT/B produced a dose-dependent elevation in after-discharge threshold and duration and a reduction in the seizure stage and duration of behavioral seizures that lasted for up to 50 days after infusion. BoNT/A had similar effects on EEG measures; behavioral seizure measures were also reduced, but the effect did not reach statistical significance. The effects of both toxins on EEG and behavioral measures progressively resolved during the latter half of the observation period. Animals gained weight normally, maintained normal body temperature, and did not show altered behavior. This study demonstrates for the first time that locally delivered BoNTs can produce prolonged inhibition of brain excitability, indicating that they could be useful for the treatment of brain disorders, including epilepsy, that would benefit from long-lasting suppression of neurotransmission within a circumscribed brain region. PMID:23772062

  11. QS-21: a potent vaccine adjuvant

    Technology Transfer Automated Retrieval System (TEKTRAN)

    QS-21 is an potent adjuvant derived from the bark of a Chilean tree, Quillaja saponaria. One of the advantages of this adjuvant is that it promotes a balanced humoral and cell-mediaed immune response and can be widely applicable to a variety of vaccines. This adjuvant has used for some veterinary va...

  12. Acetylcholinesterase-reduced graphene oxide hybrid films for organophosphorus neurotoxin sensing via quartz crystal microbalance

    NASA Astrophysics Data System (ADS)

    Tang, Shi; Ma, Wenying; Xie, Guangzhong; Su, Yuanjie; Jiang, Yadong

    2016-09-01

    An acetylcholinesterase (AChE)-reduced graphene oxide (RGO) hybrid films based biosensor enabled by quartz crystal microbalance (QCM) has been developed for the detection of organophosphorus neurotoxin in gas phase at room temperature. To improve the sensing performance, RGO was used to immobilize large quantities of enzyme and provide a favorable microenvironment to maintain the enzyme activity. The experimental results reveal that the response of AChE-RGO/glutaraldehyde based sensors is about 8 times larger than that of the AChE with the sensitivity of 1.583 Hz/mg/m3. 1.0 mg amount of RGO, 5% concentration of glutaraldehyde and pH 6.8 is the optimal condition of this biosensor.

  13. Mass Spectrometric Detection of Botulinum Neurotoxin by Measuring its Activity in Serum and Milk

    NASA Astrophysics Data System (ADS)

    Kalb, Suzanne R.; Pirkle, James L.; Barr, John R.

    Botulinum neurotoxins (BoNTs) are bacterial protein toxins which are considered likely agents for bioterrorism due to their extreme toxicity and high availability. A new mass spectrometry based assay called Endopep MS detects and defines the toxin serotype in clinical and food matrices via toxin activity upon a peptide substrate which mimics the toxin's natural target. Furthermore, the subtype of the toxin is differentiated by employing mass spectrometry based proteomic techniques on the same sample. The Endopep-MS assay selectively detects active BoNT and defines the serotype faster and with sensitivity greater than the mouse bioassay. One 96-well plate can be analyzed in under 7 h. On higher level or "hot" samples, the subtype can then be differentiated in less than 2 h with no need for DNA.

  14. Three Dimensional Solution Structure of Neurotoxin CM-11 from the Ophiophagus hannah.

    PubMed

    Liu, Wei-Dong; Pei, Feng-Kui; Liu, Ai-Zhuo; Pang, Yu-Xi

    1998-01-01

    The Ophiophagus hannah (King Cobra) neurotoxin CM-11 is a small protein with 72 amino acid residues. Based on complete assignments of (1)H-NMR resonances and determination of secondary structures of CM-11, 349 distance and 27 dihedral angle constraints including 19 psi's and 8 Xi (1)'s were collected from NOESY and DQF-COSY, and the chemical stereospecific assignment of Beta(1)H was partially achieved. Twelve structures with lower energy were obtained via metric matrix distance geometry and refinement with simulated annealing. These structures have a low RMSD of 0.14 nm for backbone atoms and 0.20 nm for heavy atoms, with no distance constraint violation more than 0.05 nm, and no dihedral angle violation more than 3deg;. PMID:12174257

  15. Determination of the Secondary Structure of the king Cobra Neurotoxin CM-11.

    PubMed

    Pang, Yu-Xi; Liu, Wei-Dong; Liu, Ai-Zhuo; Pei, Feng-Kui

    1997-01-01

    The king cobra neurotoxin CM-11 is a small protein with 72 amino acid residues. After its complete assignments of (1)H-NMR resonance's were obtained using various 2D-NMR technologies, including DQF-COSY, clean-TOCSY and NOESY, the secondary structure was analysed by studying the various NOEs extracted from the NOESY spectra and the distribution of chemical shifts. The secondary structure was finally determined by MCD as follows: a triple-strand antiparallel beta sheet with I20-W26, R37-A43 and V53-S59 as its beta strands, a short alpha helix formed by W30-G35 and four turns formed by P7-K1O, C14-G17, K50-V53 and D61-N64.

  16. Immunochemical and Pharmacological Distinctions between Curaremimetic Neurotoxin Binding Sites of Central, Autonomic, and Peripheral Origin

    NASA Astrophysics Data System (ADS)

    Lukas, Ronald J.

    1986-08-01

    Comparative pharmacological and immunochemical studies were conducted on α -bungarotoxin binding sites from rat brain or muscle, Torpedo electric tissue, or the TE671 or PC12 clonal cell lines. Characteristic distinctions were observed in the pharmacological profile of drugs competing for toxin binding to different tissues. Differences also were found in the proportion of toxin binding sites (membrane-bound or detergent-solubilized) that are immunologically reactive with either monoclonal antibodies directed against nicotinic acetylcholine receptors from the electric organ of Torpedo or polyclonal antisera raised against nicotinic receptors from the electric organ of Electrophorus. These results suggest that toxin binding sites are structurally heterogeneous. Structural heterogeneity of nicotinic acetylcholine receptors, neurotoxin binding sites, or both, may contribute to the manifestation of nicotinic receptor functional heterogeneity and may explain the apparent discrepancy at some sites between toxin binding activity and toxin functional potency.

  17. Calcium influx and mitochondrial alterations at synapses exposed to snake neurotoxins or their phospholipid hydrolysis products.

    PubMed

    Rigoni, Michela; Pizzo, Paola; Schiavo, Giampietro; Weston, Anne E; Zatti, Giancarlo; Caccin, Paola; Rossetto, Ornella; Pozzan, Tullio; Montecucco, Cesare

    2007-04-13

    Snake presynaptic phospholipase A2 neurotoxins (SPANs) bind to the presynaptic membrane and hydrolyze phosphatidylcholine with generation of lysophosphatidylcholine (LysoPC) and fatty acid (FA). The LysoPC+FA mixture promotes membrane fusion, inducing the exocytosis of the ready-to-release synaptic vesicles. However, also the reserve pool of synaptic vesicles disappears from nerve terminals intoxicated with SPAN or LysoPC+FA. Here, we show that LysoPC+FA and SPANs cause a large influx of extracellular calcium into swollen nerve terminals, which accounts for the extensive synaptic vesicle release. This is paralleled by the change of morphology and the collapse of membrane potential of mitochondria within nerve bulges. These results complete the picture of events occurring at nerve terminals intoxicated by SPANs and define the LysoPC+FA lipid mixture as a novel and effective agonist of synaptic vesicle release.

  18. Differential Response of Pig Masseter to Botulinum Neurotoxin Serotypes A and B

    PubMed Central

    Liu, Zi-Jun; Rafferty, Katherine L.; Ye, Wenmin; Herring, Susan W.

    2014-01-01

    Introduction Pigs respond to direct administration of botulinum neurotoxins (BoNTs), although they are resistant to botulism. The human masseter is frequently targeted for BoNT therapy. We aimed to understand how BoNT affects chewing by injecting porcine masseters. Methods One masseter of minipigs was injected with BoNT serotype A or B at doses comparable to those used in humans. Masticatory function was evaluated electromyographically. Muscle force was measured during tetany. Four weeks after injection, strain gauges affixed to the mandible assessed bone strain during chewing. Masseter mass and fiber diameter were measured after euthanasia. Results BoNT-A had no measurable effect. In contrast, BoNT-B reduced electrical activity and muscle force, producing substantial asymmetry between injected and uninjected muscles. Discussion The pig masseter is highly resistant to direct injection of BoNT-A, but it is affected by BoNT-B. PMID:26039454

  19. [Expression, antiserum preparation and bioactivity assays of insect neurotoxin LqhIT2].

    PubMed

    Li, Hongbo; Xia, Yuxian

    2008-10-01

    According to the codon bias of Pichia pastoris, the mature insect neurotoxin gene LqhIT2 was synthesized based on its amino acid sequence and was cloned to vector of PET-30a (+) and pPIC9K respectively. The fusion protein expressed in Escherichia. coli was induced with IPTG and purified with Ni-NTA His Bind Column. The purified fusion protein was used to immunize BALB/c mice, and antiserum obtained was highly specific with the titer of over 1:128 000. Using the antiserum, high-level expression transformants of P. pastoris were screened by dot blotting. The highest expression of recombinant LqhIT2 was about 9 mg/L in baffled flasks. The fusion protein of LqhIT2 expressed in E. coli was not toxic to locust, but the recombinant LqhIT2 expressed in P. pastoris had insecticidal activity against locust through injection. PMID:19149189

  20. Recombinant botulinum neurotoxin A heavy chain-based delivery vehicles for neuronal cell targeting

    PubMed Central

    Ho, Mengfei; Chang, Li-Hsin; Pires-Alves, Melissa; Thyagarajan, Baskaran; Bloom, Jordan E.; Gu, Zhengrong; Aberle, Karla K.; Teymorian, Sasha A.; Bannai, Yuka; Johnson, Steven C.; McArdle, Joseph J.; Wilson, Brenda A.

    2011-01-01

    The long half-life of botulinum neurotoxin serotype A (BoNT/A) in cells poses a challenge in developing post-exposure therapeutics complementary to existing antitoxin strategies. Delivery vehicles consisting of the toxin heavy chain (HC), including the receptor-binding domain and translocation domain, connected to an inhibitory cargo offer a possible solution for rescuing intoxicated neurons in victims paralyzed from botulism. Here, we report the expression and purification of soluble recombinant prototype green fluorescent protein (GFP) cargo proteins fused to the entire BoNT/A-HC (residues 544–1295) in Escherichia coli with up to a 40 amino acid linker inserted between the cargo and BoNT/A-HC vehicle. We show that these GFP-HC fusion proteins are functionally active and readily taken up by cultured neuronal cells as well as by neuronal cells in mouse motor nerve endings. PMID:21051321

  1. Effects of the noradrenergic neurotoxin DSP4 on spatial memory in the rat.

    PubMed

    Sontag, T A; Hauser, J; Kaunzinger, I; Gerlach, M; Tucha, O; Lange, K W

    2008-01-01

    Patients with attention-deficit/hyperactivity disorder (ADHD) show various cognitive impairments such as deficits in attention or working memory. Most symptoms of ADHD are thought to be associated with a dysbalance between the neurotransmitters noradrenaline and dopamine in the brain. In order to investigate the role of noradrenaline in this context we have produced a central depletion of noradrenaline in rats by administering different doses (10, 20 or 50 mg/kg body weight) of the neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) and assessing the behavioral consequences with a modified hole board task. The administration of DSP4 affected the working memory error, while reference memory and motor functions were not affected. The use of different doses of DSP4 to influence prefrontal functions and to understand the dysbalance of dopamine and noradrenaline in ADHD appears to be a promising approach.

  2. Discovery and characterization of a family of insecticidal neurotoxins with a rare vicinal disulfide bridge.

    PubMed

    Wang, X; Connor, M; Smith, R; Maciejewski, M W; Howden, M E; Nicholson, G M; Christie, M J; King, G F

    2000-06-01

    We have isolated a family of insect-selective neurotoxins from the venom of the Australian funnel-web spider that appear to be good candidates for biopesticide engineering. These peptides, which we have named the Janus-faced atracotoxins (J-ACTXs), each contain 36 or 37 residues, with four disulfide bridges, and they show no homology to any sequences in the protein/DNA databases. The three-dimensional structure of one of these toxins reveals an extremely rare vicinal disulfide bridge that we demonstrate to be critical for insecticidal activity. We propose that J-ACTX comprises an ancestral protein fold that we refer to as the disulfide-directed beta-hairpin.

  3. A protein chip membrane-capture assay for botulinum neurotoxin activity

    SciTech Connect

    Marconi, Severine; Ferracci, Geraldine; Berthomieu, Maelys; Kozaki, Shunji; Miquelis, Raymond; Boucraut, Jose; Seagar, Michael

    2008-12-15

    Botulinum neurotoxins A and B (BoNT/A and B) are neuromuscular blocking agents which inhibit neurotransmission by cleaving the intra-cellular presynaptic SNARE proteins SNAP-25 and VAMP2, localized respectively in plasma membrane and synaptic vesicles. These neurotoxins are both dangerous pathogens and powerful therapeutic agents with numerous clinical and cosmetic applications. Consequently there is a need for in vitro assays of their biological activity to screen for potential inhibitors and to replace the widely used in vivo mouse assay. Surface plasmon resonance (SPR) was used to measure membrane vesicle capture by antibodies against SNAP-25 and VAMP2. Substrate cleavage by BoNTs modified capture providing a method to assay toxin activity. Firstly using synaptic vesicles as a substrate, a comparison of the EC{sub 50}s for BoNT/B obtained by SPR, ELISA or flow cytometry indicated similar sensitivity although SPR assays were more rapid. Sonication of brain or neuronal cultures generated plasma membrane fragments with accessible intra-cellular epitopes adapted to measurement of BoNT/A activity. SPR responses were proportional to antigen concentration permitting detection of as little as 4 pM SNAP-25 in crude lysates. BoNT/A activity was assayed using monoclonal antibodies that specifically recognize a SNAP-25 epitope generated by the proteolytic action of the toxin. Incubation of intact primary cultured neurons with BoNT/A yielded an EC{sub 50} of 0.5 pM. The SPR biosensor method was sensitive enough to monitor BoNT/A and B activity in cells cultured in a 96-well format providing an alternative to experimental animals for toxicological assays.

  4. Novel Ganglioside-mediated Entry of Botulinum Neurotoxin Serotype D into Neurons

    SciTech Connect

    Kroken, Abby R.; Karalewitz, Andrew P.-A.; Fu, Zhuji; Kim, Jung-Ja P.; Barbieri, Joseph T.

    2012-02-07

    Botulinum Neurotoxins (BoNTs) are organized into seven serotypes, A-G. Although several BoNT serotypes enter neurons through synaptic vesicle cycling utilizing dual receptors (a ganglioside and a synaptic vesicle-associated protein), the entry pathway of BoNT/D is less well understood. Although BoNT/D entry is ganglioside-dependent, alignment and structural studies show that BoNT/D lacks key residues within a conserved ganglioside binding pocket that are present in BoNT serotypes A, B, E, F, and G, which indicate that BoNT/D-ganglioside interactions may be unique. In this study BoNT/D is shown to have a unique association with ganglioside relative to the other BoNT serotypes, utilizing a ganglioside binding loop (GBL, residues Tyr-1235-Ala-1245) within the receptor binding domain of BoNT/D (HCR/D) via b-series gangliosides, including GT1b, GD1b, and GD2. HCR/D bound gangliosides and entered neurons dependent upon the aromatic ring of Phe-1240 within the GBL. This is the first BoNT-ganglioside interaction that is mediated by a phenylalanine. In contrast, Trp-1238, located near the N terminus of the ganglioside binding loop, was mostly solvent-inaccessible and appeared to contribute to maintaining the loop structure. BoNT/D entry and intoxication were enhanced by membrane depolarization via synaptic vesicle cycling, where HCR/D colocalized with synaptophysin, a synaptic vesicle marker, but immunoprecipitation experiments did not detect direct association with synaptic vesicle protein 2. Thus, BoNT/D utilizes unique associations with gangliosides and synaptic vesicles to enter neurons, which may facilitate new neurotoxin therapies.

  5. Botulinum neurotoxin type A inhibits synaptic vesicle 2 expression in breast cancer cell lines

    PubMed Central

    Bandala, C; Cortés-Algara, AL; Mejía-Barradas, CM; Ilizaliturri-Flores, I; Dominguez-Rubio, R; Bazán-Méndez, CI; Floriano-Sánchez, E; Luna-Arias, JP; Anaya-Ruiz, M; Lara-Padilla, E

    2015-01-01

    Aim: It is known that botulinum neurotoxin type A (BoNTA) improves some kinds of cancer (e.g. prostate) and that synaptic vesicle glycoprotein 2 (SV2) is the molecular target of this neurotoxin. Besides having potential therapeutic value, this glycoprotein has recently been proposed as a molecular marker for several types of cancer. Although the mechanisms of cancer development and the improvement found with botulinum treatment are not well understood, the formation of the botulinum-SV2 complex may influence the presence and distribution of SV2 and the function of vesicles. To date, there are no reports on the possible effect of botulinum on breast cancer of unknown causes, which have a great impact on women’s health. Thus we determined the presence of SV2 in three breast cancer cell lines and the alterations found with botulinum application. Materials and methods: With and without adding 10 units of botulinum, SV2 protein expression was determined by optical densitometry in T47D, MDA-MB-231 and MDA-MB-453 cell lines and the distribution of SV2 was observed with immunochemistry (hematoxylin staining). Results: The SV2 protein was abundant in the cancer cells herein tested, and maximally so in T47D. In all three cancer cell lines botulinum diminished SV2 expression, which was found mostly in the cell periphery. Conclusion: SV2 could be a molecular marker in breast cancer. Its expression and distribution is regulated by botulinum, suggesting an interesting control mechanism for SV2 expression and a possible alternative therapy. Further studies are needed in this sense. PMID:26339411

  6. Environmental neurotoxins β-N-methylamino-l-alanine (BMAA) and mercury in shark cartilage dietary supplements.

    PubMed

    Mondo, Kiyo; Broc Glover, W; Murch, Susan J; Liu, Guangliang; Cai, Yong; Davis, David A; Mash, Deborah C

    2014-08-01

    Shark cartilage products are marketed as dietary supplements with claimed health benefits for animal and human use. Shark fin and cartilage products sold as extracts, dry powders and in capsules are marketed based on traditional Chinese medicine claims that it nourishes the blood, enhances appetite, and energizes multiple internal organs. Shark cartilage contains a mixture of chondroitin and glucosamine, a popular nutritional supplement ingested to improve cartilage function. Sharks are long-lived apex predators, that bioaccumulate environmental marine toxins and methylmercury from dietary exposures. We recently reported detection of the cyanobacterial toxin β-N-methylamino-l-alanine (BMAA) in the fins of seven different species of sharks from South Florida coastal waters. Since BMAA has been linked to degenerative brain diseases, the consumption of shark products may pose a human risk for BMAA exposures. In this report, we tested sixteen commercial shark cartilage supplements for BMAA by high performance liquid chromatography (HPLC-FD) with fluorescence detection and ultra performance liquid chromatography/mass spectrometry/mass spectrometry (UPLC-MS/MS). Total mercury (Hg) levels were measured in the same shark cartilage products by cold vapor atomic fluorescence spectrometry (CVAFS). We report here that BMAA was detected in fifteen out of sixteen products with concentrations ranging from 86 to 265μg/g (dry weight). All of the shark fin products contained low concentrations of Hg. While Hg contamination is a known risk, the results of the present study demonstrate that shark cartilage products also may contain the neurotoxin BMAA. Although the neurotoxic potential of dietary exposure to BMAA is currently unknown, the results demonstrate that shark cartilage products may contain two environmental neurotoxins that have synergistic toxicities.

  7. Protective potential of recombinant non-purified botulinum neurotoxin serotypes C and D.

    PubMed

    Moreira, Clóvis; da Cunha, Carlos Eduardo Pouey; Moreira, Gustavo Marçal Schmidt Garcia; Mendonça, Marcelo; Salvarani, Felipe Masiero; Moreira, Ângela Nunes; Conceição, Fabricio Rochedo

    2016-08-01

    Botulinum neurotoxin (BoNT) serotypes C and D are responsible for cattle botulism, a fatal paralytic disease that results in great economic losses in livestock production. Vaccination is the main approach to prevent cattle botulism. However, production of commercially available vaccines (toxoids) involves high risk and presents variation of BoNT production between batches. Such limitations can be attenuated by the development of novel nontoxic recombinant vaccines through a simple and reproducible process. The aim of this study was to evaluate the protective potential of recombinant non-purified botulinum neurotoxin serotypes C and D. Bivalent vaccines containing 200 μg rHCC and rHCD each were formulated in three different ways: (1) purified antigens; (2) recombinant Escherichia coli bacterins; (3) recombinant E. coli cell lysates (supernatant and inclusion bodies). Guinea pigs immunized subcutaneously with recombinant formulations developed a protective immune response against the respective BoNTs as determined by a mouse neutralization bioassay with pooled sera. Purified recombinant antigens were capable of inducing 13 IU/mL antitoxin C and 21 IU/mL antitoxin D. Similarly, both the recombinant bacterins and the cell lysate formulations were capable of inducing 12 IU/mL antitoxin C and 20 IU/mL antitoxin D. These values are two times as high as compared to values induced by the commercial toxoid used as control, and two to ten times as high as the minimum amount required by the Brazilian Ministry of Agriculture, Livestock and Food Supply (MAPA), respectively. Therefore, we used a practical, industry-friendly, and efficient vaccine production process that resulted in formulations capable of inducing protective immune response (neutralizing antitoxins) against botulism serotypes C and D. PMID:27236078

  8. [The in vitro production of three-finger neurotoxins from snake venoms with a high abundance of disulfide bonds. Problems and their solutions].

    PubMed

    Liukmanova, E N; Shulepko, M A; Shenkarev, Z O; Dolgikh, D A; Kirpichnikov, M P

    2010-01-01

    alpha-Neurotoxins from snake venom are highly efficient inhibitors of nicotinic acetylcholine receptors (nAChR). These small proteins that have a beta-structural organization attract much interest as a tool for studies of nACh R and as prototypes for the development of new Pharmaceuticals for the treatment of diseases of the nervous system. However, the in vitro production of "three-finger" neurotoxins is complicated by the presence of four or five disulfide bonds that are closely located in their molecules. The present review contains a description of the most frequently used modern approaches for the E. coli expression of recombinant proteins (direct expression, expression as fusions, and secretion) with an emphasis placed on the recombinant production of snake alpha-neurotoxins. The methods of E. coli expression of isotopically labeled neurotoxins are described. The proposed solutions will be of broad interest for the bacterial production of other disulfide-abundant proteins.

  9. Reinterpretation of luminiscence properties of neurotoxins from the venom of Middle-Asian corba Naja oxiana eichw.

    PubMed

    Bukolova-Orlova, T G; Permyakov, E A; Burstein, E A; Yukelson, L Y

    1976-08-01

    A new interpretation of previous work (Bukolova-Orlova, T. G., Burstein, E.A. and Yukelson, L. Ya (1974) Biochim. Biophys. Acta 342, 272-280) and some new data on the luminescence of neurotoxins I and II from Naja oxiana venom is given, based on the newer data on their complete amino acid sequences. Very effective excitation energy exchange exists between Trp-27 and Trp-33 in neurotoxin I and between Trp-27 and Trp-28 in neurotoxin II, Which results in the tryptophanyl fluorescence spectra of each of the proteins seeming to be monocomponent ones. The lowered fluorescence quantium yield value, the shortened phosphorescence lifetime (80% of the emission has tau p less than 0.5 s, 20% has tau p = 4.8 s, comparing with usual tau p = 5.5-5.9 s) and decreased phosphorescence to fluorescence ratio (0.042, as compared to the usual 0.4-0.7) for neurotoxin I suggest that the indole chromophore of Trp-27 and/or Trp-33 are in contact with heavy sulfur atoms of disulfide, most probably of Cys(28)-Cys(32). Tryptophanyls in neurotoxin II are exposed to the solvent, however their accessibility in relation to that of the free tryptophan to the negatively charged quencher I- (0.455) is much higher than that for the positively charged Cs+ (0.08), which is probably due to the proximity of cationic Lys-25, Lys-26 and His-31. The difference of accessibility to the negative and positive quenchers is even more pronounced in the case of the neurotoxin I (1.04 and 0 +/- 0.02, respectively), though in its chromophore vicinity along the primary structure there is only one cationic group, Lys-25. This fact together with the analysis of the amino acid sequence, suggest that the space folding of this polypeptide results in the close proximity of Trp-27 and/or Trp-33 with the C-terminal peptide segment 67-73, which contains four cationic groups (His-67, Lys-69, Lys-71 and Arg-72).

  10. Dmt and opioid peptides: a potent alliance.

    PubMed

    Bryant, Sharon D; Jinsmaa, Yunden; Salvadori, Severo; Okada, Yoshio; Lazarus, Lawrence H

    2003-01-01

    The introduction of the Dmt (2',6'-dimethyl-L-tyrosine)-Tic pharmacophore into the design of opioid ligands produced an extraordinary family of potent delta-opioid receptor antagonists and heralded a new phase in opioid research. First reviewed extensively in 1998, the incorporation of Dmt into a diverse group of opioid molecules stimulated the opioid field leading to the development of unique analogues with remarkable properties. This overview will document the crucial role played by this residue in the proliferation of opioid peptides with high receptor affinity (K(i) equal to or less than 1 nM) and potent bioactivity. The discussion will include the metamorphosis between delta-opioid receptor antagonists to delta-agonists based solely on subtle structural changes at the C-terminal region of the Dmt-Tic pharmacophore as well as their behavior in vivo. Dmt may be considered promiscuous due to the acquisition of potent mu-agonism by dermorphin and endomorphin derivatives as well as by a unique class of opioidmimetics containing two Dmt residues separated by alkyl or pyrazinone linkers. Structural studies on the Dmt-Tic compounds were enhanced tremendously by x-ray diffraction data for three potent and biologically diverse Dmt-Tic opioidmimetics that led to the development of pharmacophores for both delta-opioid receptor agonists and antagonists. Molecular modeling studies of other unique Dmt opioid analogues illuminated structural differences between delta- and mu-receptor ligand interactions. The future of these compounds as therapeutic applications for various medical syndromes including the control of cancer-associated pain is only a matter of time and perseverance.

  11. Structure-function relationships of curaremimetic neurotoxin loop 2 and of a structurally similar segment of rabies virus glycoprotein in their interaction with the nicotinic acetylcholine receptor

    SciTech Connect

    Lentz, T.L. )

    1991-11-12

    Peptides corresponding to portions of curaremimetic neurotoxin loop 2 and to a structurally similar segment of rabies virus glycoprotein were synthetically modified in order to gain information on structure-function relationships of neurotoxin loop 2 interactions with the acetylcholine receptor. Binding of synthetic peptides to the acetylcholine receptor of Torpedo electric organ membranes was assessed by measuring their ability to inhibit the binding of {sup 125}I-{alpha}-bungarotoxin to the receptor. The peptides showing the highest affinity for the receptor were a peptide corresponding to the sequence of loop 2 (residues 25-44) of Ophiophagus hannah (king cobra) toxin b and the structurally similar segment of CVS rabies virus glycoprotein. These affinities were comparable to those of d-tubocurarine and suberyldicholine. These results demonstrate the importance of loop 2 in the neurotoxin interaction with the receptor. N- and C-terminal deletions of the loop 2 peptides and substitution of residues invariant or highly conserved among neurotoxins were performed in order to determine the role of individual residues in binding. Residues 25-40 are the most crucial in the interaction with the acetylcholine receptor. Since this region of the glycoprotein contains residues corresponding to all of the functionally invariant neurotoxin residues, it may interact with the acetylcholine receptor through a mechanism similar to that of the neurotoxins.

  12. Optimizing facial rejuvenation outcomes by combining poly-L-lactic acid, hyaluronic acid, calcium hydroxylapatite, and neurotoxins: two case studies.

    PubMed

    Lorenc, Z Paul; Daro-Kaftan, Elizabeth

    2014-02-01

    Reversal of the visible signs of facial aging with the use of injectable products as an alternative to surgery has become more popular, with nearly 5 million procedures performed in the United States in 2012. Volume augmentation products, such as hyaluronic acid (HA), calcium hydroxylapatite (CaHA), and poly-L-lactic acid (PLLA), are often used in combination with one another and with neurotoxins for facial rejuvenation because of the complementary modes of action. This article presents 2 case reports involving patientspecific combinations of 2 different HA products, injectable PLLA, and CaHA with incobotulinumtoxinA or abobotulinumtoxinA. The combination of HA, CaHA, PLLA, and neurotoxins has resulted in outstanding outcomes for many patients, with no clinical evidence of increased adverse events secondary to combination therapy. PMID:24509971

  13. Proton nuclear magnetic resonance studies on the variant-3 neurotoxin from Centruroides sculpturatus Ewing: Sequential assignment of resonances

    SciTech Connect

    Nettesheim, D.G.; Klevit, R.E.; Drobny, G.; Watt, D.D.; Krishna, N.R. )

    1989-02-21

    The authors report the sequential assignment of resonances to specific residues in the proton nuclear magnetic resonance spectrum of the variant-3 neurotoxin from the scorpion Centruroides sculpturatus Ewing (range southwestern U.S.A.). A combination of two-dimensional NMR experiments such as 2D-COSY, 2D-NOESY, and single- and double-RELAY coherence transfer spectroscopy has been employed on samples of the protein dissolved in D{sub 2}O and in H{sub 2}O for assignment purposes. These studies provide a basis for the determination of the solution-phase conformation of this protein and for undertaking detailed structure-function studies of these neurotoxins that modulate the flow of sodium current by binding to the sodium channels of excitable membranes.

  14. Hyaluronic acid filler and botulinum Neurotoxin delivered simultaneously in the same syringe for effective and convenient combination aesthetic rejuvenation therapy.

    PubMed

    Kenner, Julie R

    2010-09-01

    Facial aesthetics and rejuvenation techniques have been evolving, with the most commonly applied techniques being the use of hyaluronic acid fillers and botulinum neurotoxins. Because of complementary actions, it is common for both products to be used in the same anatomical sites to optimize outcomes, either administered consecutively at one visit or at two separate visits. The author shows for the first time that hyaluronic acid (HA) and botulinum neurotoxin (BNT) can be delivered in combination in the same syringe--at the same time--to rejuvenate the upper face. Not only does concomitant administration result in excellent clinical outcome, without apparently compromising the attributes of either product alone, but this technique enhances the patient experience by allowing the use of small-gauge needles and inherently decreasing, by half or more, the number of needle sticks incurred. Larger studies are underway to study optimal techniques for administering HA and BNT combined in a single syringe.

  15. Optimizing facial rejuvenation outcomes by combining poly-L-lactic acid, hyaluronic acid, calcium hydroxylapatite, and neurotoxins: two case studies.

    PubMed

    Lorenc, Z Paul; Daro-Kaftan, Elizabeth

    2014-02-01

    Reversal of the visible signs of facial aging with the use of injectable products as an alternative to surgery has become more popular, with nearly 5 million procedures performed in the United States in 2012. Volume augmentation products, such as hyaluronic acid (HA), calcium hydroxylapatite (CaHA), and poly-L-lactic acid (PLLA), are often used in combination with one another and with neurotoxins for facial rejuvenation because of the complementary modes of action. This article presents 2 case reports involving patientspecific combinations of 2 different HA products, injectable PLLA, and CaHA with incobotulinumtoxinA or abobotulinumtoxinA. The combination of HA, CaHA, PLLA, and neurotoxins has resulted in outstanding outcomes for many patients, with no clinical evidence of increased adverse events secondary to combination therapy.

  16. Structural And Biochemical Studies of Botulinum Neurotoxin Serotype C1 Light Chain Protease: Implications for Dual Substrate Specificity

    SciTech Connect

    Jin, R.; Sikorra, S.; Stegmann, C.M.; Pich, A.; Binz, T.; Brunger, A.T.

    2009-06-01

    Clostridial neurotoxins are the causative agents of the neuroparalytic disease botulism and tetanus. They block neurotransmitter release through specific proteolysis of one of the three soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) SNAP-25, syntaxin, and synaptobrevin, which constitute part of the synaptic vesicle fusion machinery. The catalytic component of the clostridial neurotoxins is their light chain (LC), a Zn2+ endopeptidase. There are seven structurally and functionally related botulinum neurotoxins (BoNTs), termed serotype A to G, and tetanus neurotoxin (TeNT). Each of them exhibits unique specificity for their target SNAREs and peptide bond(s) they cleave. The mechanisms of action for substrate recognition and target cleavage are largely unknown. Here, we report structural and biochemical studies of BoNT/C1-LC, which is unique among BoNTs in that it exhibits dual specificity toward both syntaxin and SNAP-25. A distinct pocket (S1') near the active site likely achieves the correct register for the cleavage site by only allowing Ala as the P1' residue for both SNAP-25 and syntaxin. Mutations of this SNAP-25 residue dramatically reduce enzymatic activity. The remote a-exosite that was previously identified in the complex of BoNT/A-LC and SNAP-25 is structurally conserved in BoNT/C1. However, mutagenesis experiments show that the a-exosite of BoNT/C1 plays a less stringent role in substrate discrimination in comparison to that of BoNT/A, which could account for its dual substrate specificity.

  17. Lycopodium clavatum exine microcapsules enable safe oral delivery of 3,4-diaminopyridine for treatment of botulinum neurotoxin A intoxication.

    PubMed

    Harris, T L; Wenthur, C J; Diego-Taboada, A; Mackenzie, G; Corbitt, T S; Janda, K D

    2016-03-18

    3,4-Diaminopyridine has shown promise in reversing botulinum intoxication, but poor pharmacokinetics and a narrow therapeutic window limit its clinical utility. Thus, we developed a pH-dependent oral delivery platform using club moss spore exines. These exine microcapsules slowed 3,4-diaminopyridine absorption, limited its seizure activity, and enabled delivery of doses which prolonged mouse survival after botulism neurotoxin A intoxication. PMID:26906286

  18. In vitro detection of cardiotoxins or neurotoxins affecting ion channels or pumps using beating cardiomyocytes as alternative for animal testing.

    PubMed

    Nicolas, Jonathan; Hendriksen, Peter J M; de Haan, Laura H J; Koning, Rosella; Rietjens, Ivonne M C M; Bovee, Toine F H

    2015-03-01

    The present study investigated if and to what extent murine stem cell-derived beating cardiomyocytes within embryoid bodies can be used as a broad screening in vitro assay for neurotoxicity testing, replacing for example in vivo tests for marine neurotoxins. Effect of nine model compounds, acting on either the Na(+), K(+), or Ca(2+) channels or the Na(+)/K(+) ATP-ase pump, on the beating was assessed. Diphenhydramine, veratridine, isradipine, verapamil and ouabain induced specific beating arrests that were reversible and none of the concentrations tested induced cytotoxicity. Three K(+) channel blockers, amiodarone, clofilium and sematilide, and the Na(+)/K(+) ATPase pump inhibitor digoxin had no specific effect on the beating. In addition, two marine neurotoxins i.e. saxitoxin and tetrodotoxin elicited specific beating arrests in cardiomyocytes. Comparison of the results obtained with cardiomyocytes to those obtained with the neuroblastoma neuro-2a assay revealed that the cardiomyocytes were generally somewhat more sensitive for the model compounds affecting Na(+) and Ca(2+) channels, but less sensitive for the compounds affecting K(+) channels. The stem cell-derived cardiomyocytes were not as sensitive as the neuroblastoma neuro-2a assay for saxitoxin and tetrodotoxin. It is concluded that the murine stem cell-derived beating cardiomyocytes provide a sensitive model for detection of specific neurotoxins and that the neuroblastoma neuro-2a assay may be a more promising cell-based assay for the screening of marine biotoxins.

  19. Resveratrol preconditioning increases methionine sulfoxide reductases A expression and enhances resistance of human neuroblastoma cells to neurotoxins.

    PubMed

    Wu, Peng-Fei; Xie, Na; Zhang, Juan-Juan; Guan, Xin-Lei; Zhou, Jun; Long, Li-Hong; Li, Yuan-Long; Xiong, Qiu-Ju; Zeng, Jian-Hua; Wang, Fang; Chen, Jian-Guo

    2013-06-01

    Methionine sulfoxide reductases A (MsrA) has been postulated to act as a catalytic antioxidant system involved in the protection of oxidative stress-induced cell injury. Recently, attention has turned to MsrA in coupling with the pathology of Parkinson's disease, which is closely related to neurotoxins that cause dopaminergic neuron degeneration. Here, we firstly provided evidence that pretreatment with a natural polyphenol resveratrol (RSV) up-regulated the expression of MsrA in human neuroblastoma SH-SY5Y cells. It was also observed that the expression and nuclear translocation of forkhead box group O 3a (FOXO3a), a transcription factor that activates the human MsrA promoter, increased after RSV pretreatment. Nicotinamide , an inhibitor of silent information regulator 1 (SIRT1), prevented RSV-induced elevation of FOXO3a and MsrA expression, indicating that the effect of RSV was mediated by a SIRT1-dependent pathway. RSV preconditioning increased methionine sulfoxide(MetO)-reducing activity in SH-SY5Y cells and enhanced their resistance to neurotoxins, including chloramine-T and 1-methyl-4-phenyl-pyridinium. In addition, the enhancement of cell resistance to neurotoxins caused by RSV preconditioning can be largely prevented by MsrA inhibitor dimethyl sulfoxide. Our findings suggest that treatment with polyphenols such as RSV can be used as a potential regulatory strategy for MsrA expression and function.

  20. Induction of protective neutralizing antibody responses against botulinum neurotoxin serotype C using plasmid carried by PLGA nanoparticles.

    PubMed

    Ruwona, Tinashe B; Xu, Haiyue; Li, Junwei; Diaz-Arévalo, Diana; Kumar, Amit; Zeng, Mingtao; Cui, Zhengrong

    2016-05-01

    Botulinum neurotoxin (BoNT) is a lethal neurotoxin, for which there is currently not an approved vaccine. Recent efforts in developing vaccine candidates against botulism have been directed at the heavy chain fragment of BoNT, because antibodies against this region have been shown to prevent BoNT from binding to its receptor and thus to nerve cell surface, offering protection against BoNT intoxication. In the present study, it was shown that immunization with plasmid DNA that encodes the 50 KDa C-terminal fragment of the heavy chain of BoNT serotype C (i.e., BoNT/C-Hc50) and is carried by cationic poly (lactic-co-glycolic) acid (PLGA) nanoparticles induces stronger BoNT/C-specific antibody responses, as compared to immunization with the plasmid alone. Importantly, the antibodies have BoNT/C-neutralizing activity, protecting the immunized mice from a lethal dose of BoNT/C challenge. A plasmid DNA vaccine encoding the Hc50 fragments of BoNT serotypes that cause human botulism may represent a viable vaccine candidate for protecting against botulinum neurotoxin intoxication. PMID:26837242

  1. Specific neurotoxin lesions of median raphe serotonergic neurons disrupt maternal behavior in the lactating rat.

    PubMed

    Barofsky, A L; Taylor, J; Tizabi, Y; Kumar, R; Jones-Quartey, K

    1983-11-01

    Impairments in lactation after electrolytic lesions of the median raphe (MR) nucleus have been corrected by treatment with PRL. Specific serotonin neurotoxin lesions were used in the present study to determine whether decrements in litter growth after electrolytic lesions could be attributed to serotonergic neuron damage at the MR locus, and whether MR lesions (MRL) disrupted suckling-induced PRL release. Intracerebral microinjection of 5,7-dihydroxytryptamine (5,7-DHT) into the MR nucleus produced dose-related decrements in litter growth after either 4 micrograms (sham, 1.35 +/- 0.05; MRL, 1.04 +/- 0.05 g/pup X day; P less than 0.001) or 8 micrograms 5,7-DHT (sham, 1.35 +/- 0.06; MRL, 0.87 +/- 0.11 g/pup X day; P less than 0.001). Despite hypothalamic serotonin depletions of 15% and 55%, respectively, for the two doses of 5,7-DHT, there was no difference between sham and MRL animals in either basal or suckling-induced PRL release. When lesions were placed on day 1 of lactation (L) so that killing on day 7-L corresponded to the early maximal neurotoxin effect, MRL mothers still showed litter growth decrements (0.37 +/- 0.07; sham, 0.98 +/- 0.08 g/pup X day; P less than 0.001) and normal PRL values. When maternal behavior was examined, MRL animals exhibited a higher incidence of abnormal behaviors (failure to retrieve pups, cannibalism, and failure to initiate suckling during a 1-h test period; Fisher's exact P, Sham vs. MRL, less than 0.01, less than 0.05, and 0.15, respectively) than sham animals or animals with 5,7-DHT lesions in the dorsal raphe nucleus or superior colliculus. In addition, suckling behavior scores, determined from daily suckling behavior observations, were lowest in the MRL group and correlated with litter growth only in this group (r = 0.789; P less than 0.01). These data suggest that serotonergic elements in the MR nucleus play an obligatory role in maintaining normal maternal behavior during lactation, but they are not involved in suckling

  2. Immunoprecipitation of Native Botulinum Neurotoxin Complexes from Clostridium botulinum Subtype A Strains

    PubMed Central

    Lin, Guangyun; Tepp, William H.; Bradshaw, Marite; Fredrick, Chase M.

    2014-01-01

    Botulinum neurotoxins (BoNTs) naturally exist as components of protein complexes containing nontoxic proteins. The nontoxic proteins impart stability of BoNTs in the gastrointestinal tract and during purification and handling. The two primary neurotoxin complexes (TCs) are (i) TC1, consisting of BoNT, nontoxin-nonhemagglutinin (NTNH), and hemagglutinins (HAs), and (ii) TC2, consisting of BoNT and NTNH (and possibly OrfX proteins). In this study, BoNT/A subtypes A1, A2, A3, and A5 were examined for the compositions of their TCs in culture extracts using immunoprecipitation (IP). IP analyses showed that BoNT/A1 and BoNT/A5 form TC1s, while BoNT/A2 and BoNT/A3 form TC2s. A Clostridium botulinum host strain expressing recombinant BoNT/A4 (normally present as a TC2) from an extrachromosomal plasmid formed a TC1 with complexing proteins from the host strain, indicating that the HAs and NTNH encoded on the chromosome associated with the plasmid-encoded BoNT/A4. Strain NCTC 2916 (A1/silent B1), which carries both an ha silent bont/b cluster and an orfX bont/a1 cluster, was also examined. IP analysis revealed that NCTC 2916 formed only a TC2 containing BoNT/A1 and its associated NTNH. No association between BoNT/A1 and the nontoxic proteins from the silent bont/b cluster was detected, although the HAs were expressed as determined by Western blotting analysis. Additionally, NTNH and HAs from the silent bont/b cluster did not form a complex in NCTC 2916. The stabilities of the two types of TC differed at various pHs and with addition of KCl and NaCl. TC1 complexes were more stable than TC2 complexes. Mouse serum stabilized TC2, while TC1 was unaffected. PMID:25362065

  3. Hinge peptide combinatorial libraries for inhilbitors of botulinum neurotoxins and saxitoxin: deconvolution strategy.

    PubMed

    Moore, Graham J; Moore, Diana M; Roy, Samir S; Hayden, Lawrence J; Hamilton, Murray G; Chan, Nora W C; Lee, William E

    2006-02-01

    Abstract Combinatorial library screening offers a rapid process for identifying potential therapies to toxins. Hinge peptide libraries, which rely on conformational diversity rather than traditional molecular diversity, reduce the need for huge numbers of syntheses and screening steps and greatly expedite the discovery process of active molecules. Hinge peptide libraries having the structures: Acetyl-X1-X2-hinge-X3-X4-NH2 (capped) and X1-hinge-X2-X3 (uncapped), where X1 through X4 are near-equimolar mixtures of twelve L-amino acids and hinge = 4-aminobutyric acid, were screened for inhibitory activity in bioassays for botulinum neurotoxins A and B (BoNT/A, BoNT/B) and saxitoxin. The zinc protease activity of the reduced light chains of BoNT/A and /B was assayed by measuring the cleavage of synthetic substrates. Saxitoxin activity was measured by the restoration of the viability of neuroblastoma cells treated with ouabain and veratridine. Deconvolution of libraries was accomplished by fixing one position at a time beginning with the C-terminus. Primary library subsets in which position 4 was fixed showed moderate levels of inhibition for BoNT/A. Secondary library subsets showed stronger inhibition in the bioassays. In each of the bioassays, inhibitory potency was stronger when the second position to be fixed was on the opposite side of the hinge, rather than on the same side with respect to the C-terminus, suggesting that the hinge facilitates the interaction of side chains. Inhibitors for all three of the toxins studied were discovered within library subsets, although not necessarily in primary subsets. These studies demonstrate that (1) the best strategy for deconvoluting hinge peptide libraries is by fixing residues alternately on each side of the hinge moiety, and (2) it is essential to investigate secondary subsets even when primary subsets are inactive. The present findings support the concept that the increased flexibility imposed by the inclusion of a

  4. Molecular components required for resting and stimulated endocytosis of botulinum neurotoxins by glutamatergic and peptidergic neurons.

    PubMed

    Meng, Jianghui; Wang, Jiafu; Lawrence, Gary W; Dolly, J Oliver

    2013-08-01

    Proteins responsible for basal and stimulated endocytosis in nerves containing small clear synaptic vesicles (SCSVs) or large dense-core vesicles (LDCVs) are revealed herein, using probes that exploit surface-exposed vesicle proteins as acceptors for internalization. Basal uptake of botulinum neurotoxins (BoNTs) by both SCSV-releasing cerebellar granule neurons (CGNs) and LDCV-enriched trigeminal ganglionic neurons (TGNs) was found to require protein acceptors and acidic compartments. In addition, dynamin, clathrin, adaptor protein complex-2 (AP2), and amphiphysin contribute to the depolarization-evoked entry. For fast recycling of SCSVs, knockdown and knockout strategies demonstrated that CGNs use predominantly dynamin 1, whereas isoform 2 and, to a smaller extent, isoform 3 support a less rapid mode of stimulated endocytosis. Accordingly, proximity ligation assay confirmed that dynamin 1 and 2 colocalize with amphiphysin 1 in CGNs, and the latter copurified with both dynamins from cell extracts. In contrast, LDCV-releasing TGNs preferentially employ dynamins 2 and 3 and amphiphysin 1 for evoked endocytosis and lack the fast phase. Hence, stimulation recruits dynamin, clathrin, AP2, and amphiphysin to augment BoNT internalization, and neurons match endocytosis mediators to the different demands for locally recycling SCSVs or replenishing distally synthesized LDCVs. PMID:23640057

  5. Clinical Uses of Botulinum Neurotoxins: Current Indications, Limitations and Future Developments

    PubMed Central

    Chen, Sheng

    2012-01-01

    Botulinum neurotoxins (BoNTs) cause flaccid paralysis by interfering with vesicle fusion and neurotransmitter release in the neuronal cells. BoNTs are the most widely used therapeutic proteins. BoNT/A was approved by the U.S. FDA to treat strabismus, blepharospam, and hemificial spasm as early as 1989 and then for treatment of cervical dystonia, glabellar facial lines, axillary hyperhidrosis, chronic migraine and for cosmetic use. Due to its high efficacy, longevity of action and satisfactory safety profile, it has been used empirically in a variety of ophthalmological, gastrointestinal, urological, orthopedic, dermatological, secretory, and painful disorders. Currently available BoNT therapies are limited to neuronal indications with the requirement of periodic injections resulting in immune-resistance for some indications. Recent understanding of the structure-function relationship of BoNTs prompted the engineering of novel BoNTs to extend therapeutic interventions in non-neuronal systems and to overcome the immune-resistance issue. Much research still needs to be done to improve and extend the medical uses of BoNTs. PMID:23162705

  6. In vitro detection and quantification of botulinum neurotoxin type E activity in avian blood

    USGS Publications Warehouse

    Piazza, T.M.; Blehert, D.S.; Dunning, F.M.; Berlowski-Zier, B. M.; Zeytin, F.N.; Samuel, M.D.; Tucker, W.C.

    2011-01-01

    Botulinum neurotoxin serotype E (BoNT/E) outbreaks in the Great Lakes region cause large annual avian mortality events, with an estimated 17,000 bird deaths reported in 2007 alone. During an outbreak investigation, blood collected from bird carcasses is tested for the presence of BoNT/E using the mouse lethality assay. While sensitive, this method is labor-intensive and low throughput and can take up to 7 days to complete. We developed a rapid and sensitive in vitro assay, the BoTest Matrix E assay, that combines immunoprecipitation with high-affinity endopeptidase activity detection by F??rster resonance energy transfer (FRET) to rapidly quantify BoNT/E activity in avian blood with detection limits comparable to those of the mouse lethality assay. On the basis of the analysis of archived blood samples (n = 87) collected from bird carcasses during avian mortality investigations, BoTest Matrix E detected picomolar quantities of BoNT/E following a 2-h incubation and femtomolar quantities of BoNT/E following extended incubation (24 h) with 100% diagnostic specificity and 91% diagnostic sensitivity. ?? 2011, American Society for Microbiology.

  7. In vitro detection and quantification of botulinum neurotoxin type E activity in avian blood

    USGS Publications Warehouse

    Piazza, Timothy M.; Blehert, David S.; Dunning, F. Mark; Berlowski-Zier, Brenda M.; Zeytin, Fusun N.; Samuel, Michael D.; Tucker, Ward C.

    2011-01-01

    Botulinum neurotoxin serotype E (BoNT/E) outbreaks in the Great Lakes region cause large annual avian mortality events, with an estimated 17,000 bird deaths reported in 2007 alone. During an outbreak investigation, blood collected from bird carcasses is tested for the presence of BoNT/E using the mouse lethality assay. While sensitive, this method is labor-intensive and low throughput and can take up to 7 days to complete. We developed a rapid and sensitive in vitro assay, the BoTest Matrix E assay, that combines immunoprecipitation with high-affinity endopeptidase activity detection by Förster resonance energy transfer (FRET) to rapidly quantify BoNT/E activity in avian blood with detection limits comparable to those of the mouse lethality assay. On the basis of the analysis of archived blood samples (n = 87) collected from bird carcasses during avian mortality investigations, BoTest Matrix E detected picomolar quantities of BoNT/E following a 2-h incubation and femtomolar quantities of BoNT/E following extended incubation (24 h) with 100% diagnostic specificity and 91% diagnostic sensitivity.

  8. Recommended Immunological Strategies to Screen for Botulinum Neurotoxin-Containing Samples

    PubMed Central

    Simon, Stéphanie; Fiebig, Uwe; Liu, Yvonne; Tierney, Rob; Dano, Julie; Worbs, Sylvia; Endermann, Tanja; Nevers, Marie-Claire; Volland, Hervé; Sesardic, Dorothea; Dorner, Martin B.

    2015-01-01

    Botulinum neurotoxins (BoNTs) cause the life-threatening neurological illness botulism in humans and animals and are divided into seven serotypes (BoNT/A–G), of which serotypes A, B, E, and F cause the disease in humans. BoNTs are classified as “category A” bioterrorism threat agents and are relevant in the context of the Biological Weapons Convention. An international proficiency test (PT) was conducted to evaluate detection, quantification and discrimination capabilities of 23 expert laboratories from the health, food and security areas. Here we describe three immunological strategies that proved to be successful for the detection and quantification of BoNT/A, B, and E considering the restricted sample volume (1 mL) distributed. To analyze the samples qualitatively and quantitatively, the first strategy was based on sensitive immunoenzymatic and immunochromatographic assays for fast qualitative and quantitative analyses. In the second approach, a bead-based suspension array was used for screening followed by conventional ELISA for quantification. In the third approach, an ELISA plate format assay was used for serotype specific immunodetection of BoNT-cleaved substrates, detecting the activity of the light chain, rather than the toxin protein. The results provide guidance for further steps in quality assurance and highlight problems to address in the future. PMID:26703727

  9. A high content imaging assay for identification of Botulinum neurotoxin inhibitors.

    PubMed

    Kota, Krishna P; Soloveva, Veronica; Wanner, Laura M; Gomba, Glenn; Kiris, Erkan; Panchal, Rekha G; Kane, Christopher D; Bavari, Sina

    2014-01-01

    Synaptosomal-associated protein-25 (SNAP-25) is a component of the soluble NSF attachment protein receptor (SNARE) complex that is essential for synaptic neurotransmitter release. Botulinum neurotoxin serotype A (BoNT/A) is a zinc metalloprotease that blocks exocytosis of neurotransmitter by cleaving the SNAP-25 component of the SNARE complex. Currently there are no licensed medicines to treat BoNT/A poisoning after internalization of the toxin by motor neurons. The development of effective therapeutic measures to counter BoNT/A intoxication has been limited, due in part to the lack of robust high-throughput assays for screening small molecule libraries. Here we describe a high content imaging (HCI) assay with utility for identification of BoNT/A inhibitors. Initial optimization efforts focused on improving the reproducibility of inter-plate results across multiple, independent experiments. Automation of immunostaining, image acquisition, and image analysis were found to increase assay consistency and minimize variability while enabling the multiparameter evaluation of experimental compounds in a murine motor neuron system. PMID:25489815

  10. Inhibiting oral intoxication of botulinum neurotoxin A complex by carbohydrate receptor mimics.

    PubMed

    Lee, Kwangkook; Lam, Kwok-Ho; Kruel, Anna-Magdalena; Mahrhold, Stefan; Perry, Kay; Cheng, Luisa W; Rummel, Andreas; Jin, Rongsheng

    2015-12-01

    Botulinum neurotoxins (BoNTs) cause the disease botulism manifested by flaccid paralysis that could be fatal to humans and animals. Oral ingestion of the toxin with contaminated food is one of the most common routes for botulism. BoNT assembles with several auxiliary proteins to survive in the gastrointestinal tract and is subsequently transported through the intestinal epithelium into the general circulation. Several hemagglutinin proteins form a multi-protein complex (HA complex) that recognizes host glycans on the intestinal epithelial cell surface to facilitate BoNT absorption. Blocking carbohydrate binding to the HA complex could significantly inhibit the oral toxicity of BoNT. Here, we identify lactulose, a galactose-containing non-digestible sugar commonly used to treat constipation, as a prototype inhibitor against oral BoNT/A intoxication. As revealed by a crystal structure, lactulose binds to the HA complex at the same site where the host galactose-containing carbohydrate receptors bind. In vitro assays using intestinal Caco-2 cells demonstrated that lactulose inhibits HA from compromising the integrity of the epithelial cell monolayers and blocks the internalization of HA. Furthermore, co-administration of lactulose significantly protected mice against BoNT/A oral intoxication in vivo. Taken together, these data encourage the development of carbohydrate receptor mimics as a therapeutic intervention to prevent BoNT oral intoxication.

  11. Biodistribution and Lymphatic Tracking of the Main Neurotoxin of Micrurus fulvius Venom by Molecular Imaging.

    PubMed

    Vergara, Irene; Castillo, Erick Y; Romero-Piña, Mario E; Torres-Viquez, Itzel; Paniagua, Dayanira; Boyer, Leslie V; Alagón, Alejandro; Medina, Luis Alberto

    2016-04-01

    The venom of the Eastern coral snake Micrurus fulvius can cause respiratory paralysis in the bitten patient, which is attributable to β-neurotoxins (β-NTx). The aim of this work was to study the biodistribution and lymphatic tracking by molecular imaging of the main β-NTx of M. fulvius venom. β-NTx was bioconjugated with the chelator diethylenetriaminepenta-acetic acid (DTPA) and radiolabeled with the radionuclide Gallium-67. Radiolabeling efficiency was 60%-78%; radiochemical purity ≥92%; and stability at 48 h ≥ 85%. The median lethal dose (LD50) and PLA₂ activity of bioconjugated β-NTx decreased 3 and 2.5 times, respectively, in comparison with native β-NTx. The immune recognition by polyclonal antibodies decreased 10 times. Biodistribution of β-NTx-DTPA-(67)Ga in rats showed increased uptake in popliteal, lumbar nodes and kidneys that was not observed with (67)Ga-free. Accumulation in organs at 24 h was less than 1%, except for kidneys, where the average was 3.7%. The inoculation site works as a depot, since 10% of the initial dose of β-NTx-DTPA-(67)Ga remains there for up to 48 h. This work clearly demonstrates the lymphatic system participation in the biodistribution of β-NTx-DTPA-(67)Ga. Our approach could be applied to analyze the role of the lymphatic system in snakebite for a better understanding of envenoming. PMID:27023607

  12. Mode of VAMP Substrate Recognition and Inhibition of Clostridium botulinum Neurotoxin F

    SciTech Connect

    Agarwal, R.; Schmidt, J; Stafford, R; Swaminathan, S

    2009-01-01

    Clostridium botulinum neurotoxins (BoNTs) cleave neuronal proteins responsible for neurotransmitter release, causing the neuroparalytic disease botulism. BoNT serotypes B, D, F and G cleave and inactivate vesicle-associated membrane protein (VAMP), each at a unique peptide bond. The specificity of BoNTs depends on the mode of substrate recognition. We have investigated the mechanism of substrate recognition of BoNT F by determining the crystal structures of its complex with two substrate-based inhibitors, VAMP 22-58/Gln58D-cysteine and 27-58/Gln58D-cysteine. The inhibitors bind to BoNT F in the canonical direction (as seen for BoNTs A and E substrates) but are positioned specifically via three major exosites away from the active site. The cysteine sulfur of the inhibitors interacts with the zinc and exists as sulfinic acid in the inhibitor VAMP 27-58/Gln58D-cysteine. Arg133 and Arg171, which form part of two separate exosites, are crucial for substrate binding and catalysis.

  13. The Receptor Binding Domain of Botulinum Neurotoxin Stereotype C Binds Phosphoinositides

    SciTech Connect

    Zhang, Yanfeng; Varnum, Susan M.

    2012-03-01

    Botulinum neurotoxins (BoNTs) are the most toxic proteins known for humans and animals with an extremely low LD50 of {approx} 1 ng/kg. BoNTs generally require a protein and a ganglioside on the cell membrane surface for binding, which is known as a 'dual receptor' mechanism for host intoxication. Recent studies have suggested that in addition to gangliosides, other membrane lipids such as phosphoinositides may be involved in the interactions with the receptor binding domain (HCR) of BoNTs for better membrane penetration. Here, using two independent lipid-binding assays, we tested the interactions of BoNT/C-HCR with lipids in vitro. BoNT/C-HCR was found to bind negatively charged phospholipids, preferentially phosphoinositides. Additional interactions to phosphoinositides may help BoNT/C bind membrane more tightly and transduct signals for subsequent steps of intoxication. Our results provide new insights into the mechanisms of host cell membrane recognition by BoNTs.

  14. High sensitivity detection of active botulinum neurotoxin by glyco-quantitative polymerase chain-reaction.

    PubMed

    Kwon, Seok Joon; Jeong, Eun Ji; Yoo, Yung Choon; Cai, Chao; Yang, Gi-Hyeok; Lee, Jae Chul; Dordick, Jonathan S; Linhardt, Robert J; Lee, Kyung Bok

    2014-03-01

    The sensitive detection of highly toxic botulinum neurotoxin (BoNT) from Clostridium botulinum is of critical importance because it causes human illnesses if foodborne or introduced in wounds and as an iatrogenic substance. Moreover, it has been recently considered a possible biological warfare agent. Over the past decade, significant progress has been made in BoNT detection technologies, including mouse lethality assays, enzyme-linked immunosorbent assays, and endopeptidase assays and by mass spectrometry. Critical assay requirements, including rapid assay, active toxin detection, sensitive and accurate detection, still remain challenging. Here, we present a novel method to detect active BoNTs using a Glyco-quantitative polymerase chain-reaction (qPCR) approach. Sialyllactose, which interacts with the binding-domain of BoNTs, is incorporated into a sialyllactose-DNA conjugate as a binding-probe for active BoNT and recovered through BoNT-immunoprecipitation. Glyco-qPCR analysis of the bound sialyllactose-DNA is then used to detect low attomolar concentrations of BoNT and attomolar to femtomolar concentrations of BoNT in honey, the most common foodborne source of infant botulism.

  15. Activity of botulinum neurotoxin type D (strain 1873) in human neurons

    PubMed Central

    Pellett, Sabine; Tepp, William H.; Scherf, Jacob M.; Pier, Christina L.; Johnson, Eric A.

    2015-01-01

    Botulinum Neurotoxin type D (BoNT/D) causes periodic outbreaks of botulism in cattle and horses, but is rarely associated with human botulism. Previous studies have shown that humans responded poorly to peripheral injection of up to 10 U of BoNT/D. Isolated human pyramidalis muscle preparations were resistant to BoNT/D, whereas isolated human intercostal muscle preparations responded to BoNT/D similarly as to other BoNT serotypes. In vitro data indicate that BoNT/D does not cleave human VAMP1 efficiently, and differential expression of the VAMP 1 and 2 isoforms may be responsible for the above observations. Here we examined sensitivity of cultured human neurons derived from human induced pluripotent stem cells to BoNT/D. Our data indicate that BoNT/D can enter and cleave VAMP 2 in human neurons, but at significantly lower efficiency than other BoNT serotypes. In addition, BoNT/D had a short duration of action in the cultured neurons, similar to that of BoNT/E. In vivo analyses indicated a slower time to death in mice, as well as a later onset and shorter duration of action than BoNT/A1. Finally, examination of BoNT/D activity in various rodent and human cell models resulted in dramatic differences in sensitivity, indicating a unique cell entry mechanism of BoNT/D. PMID:25937339

  16. Quantification of neurotoxin BMAA (β-N-methylamino-L-alanine) in seafood from Swedish markets

    NASA Astrophysics Data System (ADS)

    Jiang, Liying; Kiselova, Nadezda; Rosén, Johan; Ilag, Leopold L.

    2014-11-01

    The neurotoxin β-N-methylamino-L-alanine (BMAA) produced naturally by cyanobacteria, diatoms and dinoflagellates can be transferred and accumulated up the food chain, and may be a risk factor for neurodegenerative diseases. This study provides the first systematic screening of BMAA exposure of a large population through the consumption of seafood sold in metropolitan markets. BMAA was distinguished from known isomers by liquid chromatography tandem mass spectrometry after acidic hydrolysis and derivatization. Using deuterium-labeled internal standard, BMAA was quantified as 0.01-0.90 μg/g wet weight of tissues in blue mussel, oyster, shrimp, plaice, char and herring, but was undetectable (<0.01 μg/g) in other samples (salmon, cod, perch and crayfish). Provided that the content of BMAA detected is relevant for intake calculations, the data presented may be used for a first estimation of BMAA exposure through seafood from Swedish markets, and to refine the design of future toxicological experiments and assessments.

  17. A mechanism for slow release of biomagnified cyanobacterial neurotoxins and neurodegenerative disease in Guam

    PubMed Central

    Murch, Susan J.; Cox, Paul Alan; Banack, Sandra Anne

    2004-01-01

    As root symbionts of cycad trees, cyanobacteria of the genus Nostoc produce β-methylamino-l-alanine (BMAA), a neurotoxic nonprotein amino acid. The biomagnification of BMAA through the Guam ecosystem fits a classic triangle of increasing concentrations of toxic compounds up the food chain. However, because BMAA is polar and nonlipophilic, a mechanism for its biomagnification through increasing trophic levels has been unclear. We report that BMAA occurs not only as a free amino acid in the Guam ecosystem but also can be released from a bound form by acid hydrolysis. After first removing free amino acids from tissue samples of various trophic levels (cyanobacteria, root symbioses, cycad seeds, cycad flour, flying foxes eaten by the Chamorro people, and brain tissues of Chamorros who died from amyotrophic lateral sclerosis/Parkinsonism dementia complex), we then hydrolyzed the remaining fraction and found BMAA concentrations increased 10- to 240-fold. This bound form of BMAA may function as an endogenous neurotoxic reservoir, accumulating and being transported between trophic levels and subsequently being released during digestion and protein metabolism. Within brain tissues, the endogenous neurotoxic reservoir can slowly release free BMAA, thereby causing incipient and recurrent neurological damage over years or even decades, which may explain the observed long latency period for neurological disease onset among the Chamorro people. The presence of BMAA in brain tissues from Canadian patients who died of Alzheimer's disease suggests that exposure to cyanobacterial neurotoxins occurs outside of Guam. PMID:15295100

  18. Disulfide isomers of alpha-neurotoxins from King cobra (Ophiophagus hannah) venom.

    PubMed

    Lin, S R; Chang, L S; Chang, C C

    1999-01-01

    Two novel alpha-neurotoxins, Oh-6A and Oh-6B, isolated from the king cobra (Ophiophagus hannah) venom, consist of 70 amino acid residues with 10 cysteine residues and share the same amino acid sequences as determined by Edman degradation on the peptide fragments generated from the proteolytic hydrolysates. Their sequences share 46-53% homology with Oh-4, Oh-5, Toxin a, and Toxin b from the same venom. The finding that Oh-6A and Oh-6B had different retention times in the reversed-phase column suggested that the two toxin molecules should not have the same conformation. Selective reduction on the disulfide bond, Cys26--Cys30, at the tip of their loop II structures resulted in the production of the partially reduced derivatives eluted at the same position. Under redox conditions, the partially reduced Oh-6A and 6B exclusively converted into native Oh-6A as evidenced by HPLC analyses. This suggests that Oh-6A and Oh-6B are disulfide isomers which probably arise from cis-trans isomerization of the Cys26--Cys30 disulfide bond. Alternatively, the two toxins exhibited binding activity toward nAChR and lethal toxicity equally. It reflects that the diversity around the extra loop at the loop II structure does not exert a significant effect on the manifestation of the neurotoxicity of Oh-6A and Oh-6B.

  19. The destructive effect of botulinum neurotoxins on the SNARE protein: SNAP-25 and synaptic membrane fusion.

    PubMed

    Lu, Bin

    2015-01-01

    Synaptic exocytosis requires the assembly of syntaxin 1A and SNAP-25 on the plasma membrane and synaptobrevin 2 (VAMP2) on the vesicular membrane to bridge the two opposite membranes. It is believed that the three SNARE proteins assemble in steps along the dynamic assembly pathway. The C-terminus of SNAP-25 is known to be the target of botulinum neurotoxins (BoNT/A and BoNT/E) that block neurotransmitters release in vivo. In this study, we employed electron paramagnetic resonance (EPR) spectroscopy to investigate the conformation of the SNAP-25 C-terminus in binary and ternary SNARE complexes. The fluorescence lipid mixing assay shows that the C-terminal of SNAP-25 is essential for membrane fusion, and that the truncated SNAP-25 mutants cleaved by BoNT/A and BoNT/E display different inhibition effects on membrane fusion: SNAP-25E (Δ26) abolishes the fusion activity of the SNARE complex, while SNAP-25A (Δ9) loses most of its function, although it can still form a SDS-resistant SNARE complex as the wild-type SNAP-25. CW-EPR spectra validate the unstable structures of the SNARE complex formed by SNAP-25 mutants. We propose that the truncated SNAP-25 mutants will disrupt the assembly of the SNARE core complex, and then inhibit the synaptic membrane fusion accordingly.

  20. Molecular Evolution of Antibody Cross-Reactivity for Two Subtypes of Type a Botulinum Neurotoxin

    SciTech Connect

    Garcia-Rodriguez, C.; Levy, R.; Arndt, J.W.; Forsyth, C.M.; Razai, A.; Lou, J.; Geren, I.; Stevens, R.C.; Marks, J.D.; /UC, San Francisco /Scripps Res. Inst.

    2007-07-09

    Broadening antibody specificity without compromising affinity should facilitate detection and neutralization of toxin and viral subtypes. We used yeast display and a co-selection strategy to increase cross-reactivity of a single chain (sc) Fv antibody to botulinum neurotoxin type A (BoNT/A). Starting with a scFv that binds the BoNT/A1 subtype with high affinity (136 pM) and the BoNT/A2 subtype with low affinity (109 nM), we increased its affinity for BoNT/A2 1,250-fold, to 87 pM, while maintaining high-affinity binding to BoNT/A1 (115 pM). To find the molecular basis for improved cross-reactivity, we determined the X-ray co-crystal structures of wild-type and cross-reactive antibodies complexed to BoNT/A1 at resolutions up to 2.6 A, and measured the thermodynamic contribution of BoNT/A1 and A2 amino acids to wild-type and cross-reactive antibody binding. The results show how an antibody can be engineered to bind two different antigens despite structural differences in the antigen-antibody interface and may provide a general strategy for tuning antibody specificity and cross-reactivity.

  1. Cannabidiol Exposure During Neuronal Differentiation Sensitizes Cells Against Redox-Active Neurotoxins.

    PubMed

    Schönhofen, Patrícia; de Medeiros, Liana M; Bristot, Ivi Juliana; Lopes, Fernanda M; De Bastiani, Marco A; Kapczinski, Flávio; Crippa, José Alexandre S; Castro, Mauro Antônio A; Parsons, Richard B; Klamt, Fábio

    2015-08-01

    Cannabidiol (CBD), one of the most abundant Cannabis sativa-derived compounds, has been implicated with neuroprotective effect in several human pathologies. Until now, no undesired side effects have been associated with CBD. In this study, we evaluated CBD's neuroprotective effect in terminal differentiation (mature) and during neuronal differentiation (neuronal developmental toxicity model) of the human neuroblastoma SH-SY5Y cell line. A dose-response curve was performed to establish a sublethal dose of CBD with antioxidant activity (2.5 μM). In terminally differentiated SH-SY5Y cells, incubation with 2.5 μM CBD was unable to protect cells against the neurotoxic effect of glycolaldehyde, methylglyoxal, 6-hydroxydopamine, and hydrogen peroxide (H2O2). Moreover, no difference in antioxidant potential and neurite density was observed. When SH-SY5Y cells undergoing neuronal differentiation were exposed to CBD, no differences in antioxidant potential and neurite density were observed. However, CBD potentiated the neurotoxicity induced by all redox-active drugs tested. Our data indicate that 2.5 μM of CBD, the higher dose tolerated by differentiated SH-SY5Y neuronal cells, does not provide neuroprotection for terminally differentiated cells and shows, for the first time, that exposure of CBD during neuronal differentiation could sensitize immature cells to future challenges with neurotoxins.

  2. The destructive effect of botulinum neurotoxins on the SNARE protein: SNAP-25 and synaptic membrane fusion

    PubMed Central

    2015-01-01

    Synaptic exocytosis requires the assembly of syntaxin 1A and SNAP-25 on the plasma membrane and synaptobrevin 2 (VAMP2) on the vesicular membrane to bridge the two opposite membranes. It is believed that the three SNARE proteins assemble in steps along the dynamic assembly pathway. The C-terminus of SNAP-25 is known to be the target of botulinum neurotoxins (BoNT/A and BoNT/E) that block neurotransmitters release in vivo. In this study, we employed electron paramagnetic resonance (EPR) spectroscopy to investigate the conformation of the SNAP-25 C-terminus in binary and ternary SNARE complexes. The fluorescence lipid mixing assay shows that the C-terminal of SNAP-25 is essential for membrane fusion, and that the truncated SNAP-25 mutants cleaved by BoNT/A and BoNT/E display different inhibition effects on membrane fusion: SNAP-25E (Δ26) abolishes the fusion activity of the SNARE complex, while SNAP-25A (Δ9) loses most of its function, although it can still form a SDS-resistant SNARE complex as the wild-type SNAP-25. CW-EPR spectra validate the unstable structures of the SNARE complex formed by SNAP-25 mutants. We propose that the truncated SNAP-25 mutants will disrupt the assembly of the SNARE core complex, and then inhibit the synaptic membrane fusion accordingly. PMID:26157630

  3. Camelid-derived heavy-chain nanobody against Clostridium botulinum neurotoxin E in Pichia pastoris.

    PubMed

    Baghban, Roghayyeh; Gargari, Seyed Latif Mousavi; Rajabibazl, Masoumeh; Nazarian, Shahram; Bakherad, Hamid

    2016-01-01

    Botulinum neurotoxins (BoNTs) result in severe and often fatal disease, botulism. Common remedial measures such as equine antitoxin and human botulism immunoglobulin in turn are problematic and time-consuming. Therefore, diagnosis and therapy of BoNTs are vital. The variable domain of heavy-chain antibodies (VHH) has unique features, such as the ability to identify and bind specifically to target epitopes and ease of production in bacteria and yeast. The Pichia pastoris is suitable for expression of recombinant antibody fragments. Disulfide bond formation and correct folds of protein with a high yield are some of the advantages of this eukaryotic host. In this study, we have expressed and purified the camelid VHH against BoNT/E in P. pastoris. The final yield of P. pastoris-expressed antibody was estimated to be 16 mg/l, which is higher than that expressed by Escherichia coli. The nanobody expressed in P. pastoris neutralized 4LD50 of the BoNT/E upon i.p. injection in 25% of mice. The nanobody expressed in E. coli extended the mice's survival to 1.5-fold compared to the control. This experiment indicated that the quality of expressed protein in the yeast is superior to that of the bacterial expression. Favorable protein folding by P. pastoris seems to play a role in its better toxin-binding property. PMID:24673401

  4. Neurotoxin-induced DNA damage is persistentin SH-SY5Y cells and LC neurons

    PubMed Central

    Wang, Yan; Musich, Phillip R.; Cui, Kui; Zou, Yue; Zhu, Meng-Yang

    2015-01-01

    Degeneration of the noradrenergic neurons has been reported in the brain of patients suffering from neurodegenerative diseases. However, their pathologic characteristics during the neurodegenerative course and underlying mechanisms remain to be elucidated. In the present study, we used the neurotoxincamptothecin (CPT)to induce the DNA damage response in neuroblastoma SH-SY5Y cells, normal fibroblast cells, and primarily cultured LC and raphe neurons to examine cellular responses and repair capabilities after neurotoxin exposure. To our knowledge, the present study is the first to show that noradrenergic SH-SY5Y cells are more sensitive to CPT-induced DNA damage and deficientin DNA repair, as compared to fibroblast cells. Furthermore, similar to SH-SY5Y cells, primarily cultured LC neurons are more sensitive to CPT-induced DNA damage and show a deficiency in repairing this damage. Moreover, while N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) exposure also results in DNA damage in cultured LC neurons, neither CPT nor DSP4 induce DNA damage in neuronal cultures from the raphe nuclei. Taken together, noradrenergic SH-SY5Y cells and LC neurons are sensitive to CPT-induced DNA damage and exhibit a repair deficiency, providing a mechanistic explanation for the pathologic characteristics of LC degeneration when facing endogenous and environmental DNA-damaging insultsin vivo. PMID:25724887

  5. SLEEPLESS, a Ly–6/Neurotoxin Family Member, Regulates Levels, Localization, and Activity of Shaker

    PubMed Central

    Wu, Mark N.; Joiner, William J.; Dean, Terry; Yue, Zhifeng; Smith, Corinne J.; Chen, Dechun; Hoshi, Toshinori; Sehgal, Amita; Koh, Kyunghee

    2009-01-01

    Sleep is a whole–organism phenomenon accompanied by global changes in neural activity. We previously identified SLEEPLESS (SSS) as a novel glycosylphosphatidyl–inositol–anchored protein required for sleep in Drosophila. Here, we demonstrate a critical role for SSS in regulating the sleep–modulating potassium channel, Shaker. SSS and Shaker exhibit similar expression patterns in the brain and specifically affect each other’s expression levels. sss mutants exhibit altered Shaker localization, reduced Shaker current density, and slower Shaker current kinetics. Transgenic expression of sss in sss mutants rescues defects in Shaker expression and activity cell–autonomously and also suggests that SSS functions in wake–promoting, cholinergic neurons. Importantly, in heterologous cells, SSS accelerates kinetics of Shaker currents and can be co–immunoprecipitated with Shaker, suggesting that SSS interacts with Shaker and modulates its activity. SSS is predicted to belong to the Ly–6/neurotoxin superfamily, suggesting a novel mechanism for regulation of neuronal excitability by endogenous toxin–like molecules. PMID:20010822

  6. Analysis of the neurotoxin anisatin in star anise by LC-MS/MS.

    PubMed

    Mathon, Caroline; Bongard, Benjamin; Duret, Monique; Ortelli, Didier; Christen, Philippe; Bieri, Stefan

    2013-01-01

    The aim of this work was to develop an analytical method capable of determining the presence of anisatin in star anise. This neurotoxin may induce severe side effects such as epileptic convulsions. It is therefore of prime importance to have rapid and accurate analytical methods able to detect and quantify anisatin in samples that are purportedly edible star anise. The sample preparation combined an automated accelerated solvent extraction with a solid-supported liquid-liquid purification step on EXtrelut®. Samples were analysed on a porous graphitic carbon HPLC column and quantified by tandem mass spectrometry operating in the negative ionisation mode. The quantification range of anisatin was between 0.2 and 8 mg kg⁻¹. The applicability of this validated method was demonstrated by the analysis of several Illicium species and star anise samples purchased on the Swiss market. High levels of anisatin were measured in Illicium lanceolatum, I. majus and I. anisatum, which may cause health concerns if they are misidentified or mixed with edible Illicium verum.

  7. Specificity and efficacy of noradrenaline, serotonin depletion in discrete brain areas of Swiss mice by neurotoxins.

    PubMed

    Dailly, Eric; Chenu, Franck; Petit-Demoulière, Benoit; Bourin, Michel

    2006-01-15

    The aim of this work is to define neurotoxins doses to have efficient and specific depletion of noradrenaline (NA), serotonin (5-HT) neurotransmission in cortex, striatum, hippocampus and hypothalamus of Swiss mice after intraperitoneal administration of, respectively, N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP-4) and para-chlorophenylalanine methyl ester hydrochloride (PCPA). The neurotransmitters concentrations were determined by high performance liquid chromatography with amperometric detection. The minimal single dose necessary to produce a highly significant decrease of NA levels (p<0.01 in comparison with control group) in hypothalamus (-44%), hippocampus (-91%), striatum (-40%) and cortex (-68%) was 50mg/kg but DA and 5-HT levels were modified, respectively, in hypothalamus and striatum. Three doses of PCPA 300 mg/kg over 3 consecutive days involve a profound depletion of 5-HT transmission in all discrete brain areas but NA and DA levels were also significantly reduced. In conclusion, DSP-4 has a different efficacy in discrete brain areas with a noradrenergic specificity which is not absolute, PCPA has a similar efficacy in all brain areas but is unspecific of 5-HT transmission.

  8. Botulinum neurotoxin: unique folding of enzyme domain of the most-poisonous poison.

    PubMed

    Kumar, Raj; Kukreja, Roshan V; Li, Li; Zhmurov, Artem; Kononova, Olga; Cai, Shuowei; Ahmed, Syed A; Barsegov, Valeri; Singh, Bal Ram

    2014-01-01

    Botulinum neurotoxin (BoNT), the most toxic substance known to mankind, is the first example of the fully active molten globule state. To understand its folding mechanism, we performed urea denaturation experiments and theoretical modeling using BoNT serotype A (BoNT/A). We found that the extent of BoNT/A denaturation from the native state (N) shows a nonmonotonic dependence on urea concentration indicating a unique multistep denaturation process, N → I1 [Formula: see text] I2 [Formula: see text] U, with two intermediate states I1 and I2. BoNT/A loses almost all its secondary structure in 3.75 M urea (I1), yet it displays a native-like secondary structure in 5 M urea (I2). This agrees with the results of theoretical modeling, which helped to determine the molecular basis of unique behavior of BoNT/A in solution. Except for I2, all the states revert back to full enzymatic activity for SNAP-25 including the unfolded state U stable in 7 M urea. Our results stress the importance of structural flexibility in the toxin's mechanism of survival and action, an unmatched evolutionary trait from billion-year-old bacteria, which also correlates with the long-lasting enzymatic activity of BoNT inside neuronal cells. BoNT/A provides a rich model to explore protein folding in relation to functional activity.

  9. Recommended Mass Spectrometry-Based Strategies to Identify Botulinum Neurotoxin-Containing Samples

    PubMed Central

    Kalb, Suzanne R.; Baudys, Jakub; Wang, Dongxia; Barr, John R.

    2015-01-01

    Botulinum neurotoxins (BoNTs) cause the disease called botulism, which can be lethal. BoNTs are proteins secreted by some species of clostridia and are known to cause paralysis by interfering with nerve impulse transmission. Although the human lethal dose of BoNT is not accurately known, it is estimated to be between 0.1 μg to 70 μg, so it is important to enable detection of small amounts of these toxins. Our laboratory previously reported on the development of Endopep-MS, a mass-spectrometric‑based endopeptidase method to detect, differentiate, and quantify BoNT immunoaffinity purified from complex matrices. In this work, we describe the application of Endopep-MS for the analysis of thirteen blinded samples supplied as part of the EQuATox proficiency test. This method successfully identified the presence or absence of BoNT in all thirteen samples and was able to successfully differentiate the serotype of BoNT present in the samples, which included matrices such as buffer, milk, meat extract, and serum. Furthermore, the method yielded quantitative results which had z-scores in the range of −3 to +3 for quantification of BoNT/A containing samples. These results indicate that Endopep-MS is an excellent technique for detection, differentiation, and quantification of BoNT in complex matrices. PMID:25996606

  10. Intraprostatic Botulinum Neurotoxin Type A Injection for Benign Prostatic Hyperplasia—A Spotlight in Reality

    PubMed Central

    Hsu, Yu-Chao; Wang, Hung-Jen; Chuang, Yao-Chi

    2016-01-01

    Botulinum toxin is a neurotoxin produced by the bacterium Clostridium botulinum. It inhibits the release of acetylcholine and other neurotransmitters from the nerve terminal. Botulinum toxin, specifically toxin type A (BoNT-A) has been used since the 1970s to reduce the muscular hypercontraction disorders. The application of BoNT-A in urology field started from intra-bladder injection for overactive bladder, which has been recognized as third line therapy in many countries. Since prostate gland as well as bladder is under the influence of autonomic innervation, theorectically, injection of BoNT-A into the prostate induces chemo-denervation and modulation of prostate function, and reduces lower urinary tract symptoms (LUTS). This article reviews the application of BoNT-A in patients with LUTS/ benign prostatic hyperplasia (BPH) from mechanisms of action to clinical results. BoNT-A has been shown to induce prostate apoptosis, downregulation of alpha 1A receptors, and reduce contractile function of prostate in animal studies. Open studies of intraprostate BoNT-A injection have demonstrated promising results of reducing LUTS and improvement of voiding function in human LUTS/BPH, however, intraprostatic BoNT-A injection did not perform better than the placebo group in recent publications of placebo controlled studies. We suggested that BoNT-A prostate injection might benefit selected population of BPH/LUTS, but it is unlikely to be an effective therapy for general population of male LUTS/BPH. PMID:27128942

  11. Expanding the use of neurotoxins in facial aesthetics: a consensus panel's assessment and recommendations.

    PubMed

    Kane, Michael; Donofrio, Lisa; Ascher, Benjamin; Hexsel, Doris; Monheit, Gary; Rzany, Berthold; Weiss, Robert

    2010-01-01

    Injection of botulinum toxin type A (BoNTA) is the most common nonsurgical aesthetic procedure undertaken in the United States (U.S.). A new formulation of BoNTA (abobotulinumtoxinA, Dysport™) has recently been approved in the U.S. for the treatment of glabellar lines. This product has been used for facial aesthetics in other parts of the world for more than 15 years, whereas in the U.S. a different formulation (onabotulinumtoxinA, Botox® Cosmetic) has been used for many years. The various formulations of neurotoxins are unique and are not interchangeable nor are doses convertible from one product to another, so it is important that recommendations be developed to assist U.S. clinicians in understanding the differences between the two available formulations of BoNTA, which should ensure successful outcomes with these products. A group of worldwide experts on the aesthetic use of BoNTA convened in February 2009 in New York, NY, to review the use of BoNTA and to develop consensus recommendations for the use of the new formulation, since such guidelines previously had only been published in German. This publication summarizes key discussions from the meeting as well as recommendations and suggestions regarding the use of abobotulinumtoxinA in the areas of the face most commonly treated with BoNTA. PMID:20919448

  12. A chip-based assay for botulinum neurotoxin A activity in pharmaceutical preparations.

    PubMed

    Lévêque, Christian; Ferracci, Géraldine; Maulet, Yves; Grand-Masson, Chloé; Seagar, Michael; El Far, Oussama

    2015-05-01

    The production of botulinum neurotoxin A (BoNT/A) for therapeutic and cosmetic applications requires precise determination of batch potency, and the enzymatic activity of BoNT/A light chain is a crucial index that can be measured in vitro. We previously established a SNAP-25 chip-based assay using surface plasmon resonance (SPR) that is more sensitive than the standard mouse bioassay for the quantification of BoNT/A activity. We have now adapted this procedure for pharmaceutical preparations. The optimized SPR assay allowed multiple measurements on a single chip, including the kinetics of substrate cleavage. The activity of five different batches of a pharmaceutical BoNT/A preparation was determined in a blind study by SPR and found to be in agreement with data from the in vivo mouse lethality assay. Biosensor detection of specific proteolytic products has the potential to accurately monitor the activity of pharmaceutical BoNT/A preparations, and a single chip can be used to assay more than 100 samples.

  13. Eosinophil-derived neurotoxin: a novel biomarker for diagnosis and monitoring of asthma

    PubMed Central

    2013-01-01

    Asthma is associated with increased levels of eosinophils in tissues, body fluids, and bone marrow. Elevated levels of eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP) have been noted in asthma patients. Higher levels of EDN and ECP are also associated with exacerbated asthmatic conditions. Thus, EDN, along with ECP, may aid the diagnosis and monitoring of asthma. Several groups have suggested that EDN is more useful than ECP in evaluating disease severity. This may partially be because of the recoverability of EDN (not sticky, 100% recovery rate), as ECP is a sticky and more highly charged protein. In terms of clinical utility, EDN level is a more accurate biomarker than ECP when analyzing the underlying pathophysiology of asthma. As a monitoring tool, EDN has shown good results in children with asthma as well as other allergic diseases. In children too young to fully participate in lung function tests, EDN levels may be useful as an alter native measurement of eosinophilic inflammation. EDN can also be used in adult patients and in multiple specimen types (e.g., serum, sputum, bronchoalveolar lavage fluid, and nasal lavage fluid). These results are repeatable and reproducible. In conclusion, EDN may be a novel biomarker for the diagnosis, treatment, and monitoring of asthma/allergic disease. PMID:23390439

  14. Eosinophil-Derived Neurotoxin Is Elevated in Patients with Amyotrophic Lateral Sclerosis

    PubMed Central

    Liu, Guan-Ting; Hwang, Chi-Shin; Hsieh, Chia-Hung; Lu, Chih-Hao; Chang, Sunny Li-Yun; Lee, Jin-Ching; Huang, Chien-Fu

    2013-01-01

    Background and Objectives. Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by loss of motor neurons in the brainstem, motor cortex, and spinal cord. Oxidative stress and neuroinflammation have been implicated in the pathophysiology of ALS. Members of the family of damage-associated molecular patterns, including reactive oxygen species, high-mobility group box 1, and eosinophil-derived neurotoxin (EDN), may participate in pathological conditions. In this study, we aim to discover new biomarker for detecting ALS. Materials and Methods. We examined 44 patients with ALS, 41 patients with Alzheimer's disease, 41 patients with Parkinson's disease, and 44 healthy controls. The concentration of serum EDN was measured using an enzyme-linked immunosorbent assay. Results. EDN levels were significantly increased 2.17-fold in the serum of patients with ALS as compared with healthy controls (P < 0.05). No correlation between the levels of serum EDN and various clinical parameters of ALS was found. Moreover, the levels of serum EDN in patients with Parkinson's disease and Alzheimer's disease and healthy controls were similar. Conclusion. A higher level of serum EDN was found specifically in patients with ALS, indicating that EDN may participate in the pathophysiology of ALS. PMID:23533305

  15. [Monoclonal antibodies to type A, B, E and F botulinum neurotoxins].

    PubMed

    Abbasova, S G; Ruddenko, N V; Gorokhovatskiĭ, A Iu; Kapralova, M V; Vinogradova, I D; Vertiev, Iu V; Nesmeianov, V A; Grishin, E V

    2011-01-01

    Mouse monoclonal antibodies against the most acutely toxic substances, botulinum neurotoxins (BoNTs) of types A, B, E, and F, was generated and characterized, that recognize their respective toxins in natural toxin complex. Based on these antibodies, we developed sandwich-ELISA for quantitative detection of these toxins. For each respective toxin the detection limit of the assay was: BoNT/A - 0.4 ng/ml, BoNT/B - 0.5 ng/ml; BoNT/E - 0.1 ng/ml; and for BoNT/F - 2.4 ng/ml. The developed assays permitted quantitative identification of the BoNTs in canned meat and vegetables. The BNTA-4.1 and BNTA-9.1 antibodies possessed neutralizing activity against natural complex of the botulinium toxin type A in vivo, both individually and in mixture, the mixture of the antibodies neutralized the higher dose of the toxin. The BNTA-4.1 antibody binds specifically the light chain (the chain with protease activity) of the toxin, whereas BNTA-9.1 interacts with the heavy chain. We believe that the BNTA-4.1 and BNTA-9.1 monoclonal antibodies are prospective candidates for development of humanized therapeutic antibodies for treatment of BoNT/A-caused botulism.

  16. Early cerebrovascular and parenchymal events following prenatal exposure to the putative neurotoxin methylazoxymethanol.

    PubMed

    Bassanini, Stefania; Hallene, Kerri; Battaglia, Giorgio; Finardi, Adele; Santaguida, Stefano; Cipolla, Marilyn; Janigro, Damir

    2007-05-01

    One of the most common causes of neurological disabilities are malformations of cortical development (MCD). A useful animal model of MCD consists of prenatal exposure to methylazoxymethanol (MAM), resulting in a postnatal phenotype characterized by cytological aberrations reminiscent of human MCD. Although postnatal effects of MAM are likely a consequence of prenatal events, little is known on how the developing brain reacts to MAM. General assumption is the effects of prenatally administered MAM are short lived (24 h) and neuroblast-specific. MAM persisted for several days after exposure in utero in both maternal serum and fetal brain, but at levels lower than predicted by a neurotoxic action. MAM levels and time course were consistent with a different mechanism of indirect neuronal toxicity. The most prominent acute effects of MAM were cortical swelling associated with mild cortical disorganization and neurodegeneration occurring in absence of massive neuronal cell death. Delayed or aborted vasculogenesis was demonstrated by MAM's ability to hinder vessel formation. In vitro, MAM reduced synthesis and release of VEGF by endothelial cells. Decreased expression of VEGF, AQP1, and lectin-B was consistent with a vascular target in prenatal brain. The effects of MAM on cerebral blood vessels persisted postnatally, as indicated by capillary hypodensity in heterotopic areas of adult rat brain. In conclusion, these results show that MAM does not act only as a neurotoxin per se, but may additionally cause a short-lived toxic effect secondary to cerebrovascular dysfunction, possibly due to a direct anti-angiogenic effect of MAM itself.

  17. A Monoclonal Antibody Based Capture ELISA for Botulinum Neurotoxin Serotype B: Toxin Detection in Food

    PubMed Central

    Stanker, Larry H.; Scotcher, Miles C.; Cheng, Luisa; Ching, Kathryn; McGarvey, Jeffery; Hodge, David; Hnasko, Robert

    2013-01-01

    Botulism is a serious foodborne neuroparalytic disease, caused by botulinum neurotoxin (BoNT), produced by the anaerobic bacterium Clostridium botulinum. Seven toxin serotypes (A – H) have been described. The majority of human cases of botulism are caused by serotypes A and B followed by E and F. We report here a group of serotype B specific monoclonal antibodies (mAbs) capable of binding toxin under physiological conditions. Thus, they serve as capture antibodies for a sandwich (capture) ELISA. The antibodies were generated using recombinant peptide fragments corresponding to the receptor-binding domain of the toxin heavy chain as immunogen. Their binding properties suggest that they bind a complex epitope with dissociation constants (KD’s) for individual antibodies ranging from 10 to 48 × 10−11 M. Assay performance for all possible combinations of capture-detector antibody pairs was evaluated and the antibody pair resulting in the lowest level of detection (L.O.D.), ~20 pg/mL was determined. Toxin was detected in spiked dairy samples with good recoveries at concentrations as low as 0.5 pg/mL and in ground beef samples at levels as low as 2 ng/g. Thus, the sandwich ELISA described here uses mAb for both the capture and detector antibodies (binding different epitopes on the toxin molecule) and readily detects toxin in those food samples tested. PMID:24253240

  18. Oxidative modification of human ceruloplasmin induced by a catechol neurotoxin, salsolinol

    PubMed Central

    Kim, Seung-Sub; Kang, Jae Yoon; Kang, Jung Hoon

    2016-01-01

    Salsolinol (SAL), a compound derived from dopamine metabolism, is the most probable neurotoxin involved in the pathogenesis of Parkinson’s disease (PD). In this study, we investigated the modification and inactivation of human ceruloplasmin (hCP) induced by SAL. Incubation of hCP with SAL increased the protein aggregation and enzyme inactivation in a dose-dependent manner. Reactive oxygen species scavengers and copper chelators inhibited the SAL-mediated hCP modification and inactivation. The formation of dityrosine was detected in SAL-mediated hCP aggregates. Amino acid analysis post the exposure of hCP to SAL revealed that aspartate, histidine, lysine, threonine and tyrosine residues were particularly sensitive. Since hCP is a major copper transport protein, oxidative damage of hCP by SAL may induce perturbation of the copper transport system, which subsequently leads to deleterious conditions in cells. This study of the mechanism by which ceruloplasmin is modified by salsolinol may provide an explanation for the deterioration of organs under neurodegenerative disorders such as PD. [BMB Reports 2016; 49(1): 45-50] PMID:26077029

  19. Recommended Immunological Strategies to Screen for Botulinum Neurotoxin-Containing Samples.

    PubMed

    Simon, Stéphanie; Fiebig, Uwe; Liu, Yvonne; Tierney, Rob; Dano, Julie; Worbs, Sylvia; Endermann, Tanja; Nevers, Marie-Claire; Volland, Hervé; Sesardic, Dorothea; Dorner, Martin B

    2015-11-26

    Botulinum neurotoxins (BoNTs) cause the life-threatening neurological illness botulism in humans and animals and are divided into seven serotypes (BoNT/A-G), of which serotypes A, B, E, and F cause the disease in humans. BoNTs are classified as "category A" bioterrorism threat agents and are relevant in the context of the Biological Weapons Convention. An international proficiency test (PT) was conducted to evaluate detection, quantification and discrimination capabilities of 23 expert laboratories from the health, food and security areas. Here we describe three immunological strategies that proved to be successful for the detection and quantification of BoNT/A, B, and E considering the restricted sample volume (1 mL) distributed. To analyze the samples qualitatively and quantitatively, the first strategy was based on sensitive immunoenzymatic and immunochromatographic assays for fast qualitative and quantitative analyses. In the second approach, a bead-based suspension array was used for screening followed by conventional ELISA for quantification. In the third approach, an ELISA plate format assay was used for serotype specific immunodetection of BoNT-cleaved substrates, detecting the activity of the light chain, rather than the toxin protein. The results provide guidance for further steps in quality assurance and highlight problems to address in the future.

  20. Presence of the Neurotoxin BMAA in Aquatic Ecosystems: What Do We Really Know?

    PubMed Central

    Faassen, Elisabeth J.

    2014-01-01

    The neurotoxin β-N-methylamino-l-alanine (BMAA) is suspected to play a role in the neurological diseases amyotrophic lateral sclerosis, Alzheimer’s disease, and Parkinson’s disease. BMAA production by cyanobacteria has been reported and contact with cyanobacteria infested waters or consumption of aquatic organisms are possible pathways to human exposure. However, there is little consensus regarding whether BMAA is present in cyanobacteria or not, and if so, at what concentrations. The aim of this review is to indicate the current state of knowledge on the presence of BMAA in aquatic ecosystems. Some studies have convincingly shown that BMAA can be present in aquatic samples at the µg/g dry weight level, which is around the detection limit of some equally credible studies in which no BMAA was detected. However, for the majority of the reviewed articles, it was unclear whether BMAA was correctly identified, either because inadequate analytical methods were used, or because poor reporting of analyses made it impossible to verify the results. Poor analysis, reporting and prolific errors have shaken the foundations of BMAA research. First steps towards estimation of human BMAA exposure are to develop and use selective, inter-laboratory validated methods and to correctly report the analytical work. PMID:24662480

  1. Mode of VAMP substrate recognition and inhibition of Clostridium botulinum neurotoxin F.

    PubMed

    Agarwal, Rakhi; Schmidt, James J; Stafford, Robert G; Swaminathan, Subramanyam

    2009-07-01

    Clostridium botulinum neurotoxins (BoNTs) cleave neuronal proteins responsible for neurotransmitter release, causing the neuroparalytic disease botulism. BoNT serotypes B, D, F and G cleave and inactivate vesicle-associated membrane protein (VAMP), each at a unique peptide bond. The specificity of BoNTs depends on the mode of substrate recognition. We have investigated the mechanism of substrate recognition of BoNT F by determining the crystal structures of its complex with two substrate-based inhibitors, VAMP 22-58/Gln58D-cysteine and 27-58/Gln58D-cysteine. The inhibitors bind to BoNT F in the canonical direction (as seen for BoNTs A and E substrates) but are positioned specifically via three major exosites away from the active site. The cysteine sulfur of the inhibitors interacts with the zinc and exists as sulfinic acid in the inhibitor VAMP 27-58/Gln58D-cysteine. Arg133 and Arg171, which form part of two separate exosites, are crucial for substrate binding and catalysis.

  2. Plastic enzyme-linked immunosorbent assays (ELISA)-on-a-chip biosensor for botulinum neurotoxin A.

    PubMed

    Han, Seung-Mok; Cho, Joung-Hwan; Cho, Il-Hoon; Paek, Eui-Hwan; Oh, Hee-Bok; Kim, Bong-Su; Ryu, Chunsun; Lee, Kyunghee; Kim, Young-Kee; Paek, Se-Hwan

    2007-03-21

    A plastic ELISA-on-a-chip (EOC) employing the concept of cross-flow immuno-chromatographic analysis was applied to the measurement of botulinum neurotoxin A (BoNT/A) as agent for bio-terrorism. Two monoclonal antibodies specific to the heavy chain of the toxin were raised and identified to form sandwich binding complexes as the pair with the analyte. For the construction of an immuno-strip, one was utilized as the capture antibody immobilized onto nitrocellulose membrane and the other as the detection coupled to an enzyme, horseradish peroxidase. The two plates of EOC used in this study were fabricated by injection molding of polycarbonate to improve the reproducibility of manufacture and, after inclusion of the immuno-strip, bonded using a UV-sensitive adhesive. Under optimal conditions of analysis, the chip produced a color signal in proportion to the analyte dose and the signal was quantified using a detector equipped with a digital camera. From the dose-response curve, the detection limit of BoNT/A was 2.0 ng mL(-1), approximately five times more sensitive than a commercial-version detection kit employing colloidal gold tracer.

  3. Biodistribution and Lymphatic Tracking of the Main Neurotoxin of Micrurus fulvius Venom by Molecular Imaging

    PubMed Central

    Vergara, Irene; Castillo, Erick Y.; Romero-Piña, Mario E.; Torres-Viquez, Itzel; Paniagua, Dayanira; Boyer, Leslie V.; Alagón, Alejandro; Medina, Luis Alberto

    2016-01-01

    The venom of the Eastern coral snake Micrurus fulvius can cause respiratory paralysis in the bitten patient, which is attributable to β-neurotoxins (β-NTx). The aim of this work was to study the biodistribution and lymphatic tracking by molecular imaging of the main β-NTx of M. fulvius venom. β-NTx was bioconjugated with the chelator diethylenetriaminepenta-acetic acid (DTPA) and radiolabeled with the radionuclide Gallium-67. Radiolabeling efficiency was 60%–78%; radiochemical purity ≥92%; and stability at 48 h ≥ 85%. The median lethal dose (LD50) and PLA2 activity of bioconjugated β-NTx decreased 3 and 2.5 times, respectively, in comparison with native β-NTx. The immune recognition by polyclonal antibodies decreased 10 times. Biodistribution of β-NTx-DTPA-67Ga in rats showed increased uptake in popliteal, lumbar nodes and kidneys that was not observed with 67Ga-free. Accumulation in organs at 24 h was less than 1%, except for kidneys, where the average was 3.7%. The inoculation site works as a depot, since 10% of the initial dose of β-NTx-DTPA-67Ga remains there for up to 48 h. This work clearly demonstrates the lymphatic system participation in the biodistribution of β-NTx-DTPA-67Ga. Our approach could be applied to analyze the role of the lymphatic system in snakebite for a better understanding of envenoming. PMID:27023607

  4. Proteomic Changes in Rat Thyroarytenoid Muscle Induced by Botulinum Neurotoxin Injection

    PubMed Central

    Welham, Nathan V.; Marriott, Gerard; Tateya, Ichiro; Bless, Diane M.

    2009-01-01

    Botulinum neurotoxin (BoNT) injection into the thyroarytenoid (TA) muscle is a commonly performed medical intervention for adductor spasmodic dysphonia. The mechanism of action of BoNT at the neuromuscular junction is well understood, however, aside from reports focused on myosin heavy chain isoform abundance, there is a paucity of data addressing the effects of therapeutic BoNT injection on the TA muscle proteome. In this study, 12 adult Sprague Dawley rats underwent unilateral TA muscle BoNT serotype A injection followed by tissue harvest at 72 hrs, 7 days, 14 days, and 56 days post-injection. Three additional rats were reserved as controls. Proteomic analysis was performed using 2D SDS-PAGE followed by MALDI-MS. Vocal fold movement was significantly reduced by 72 hrs, with complete return of function by 56 days. Twenty-five protein spots demonstrated significant protein abundance changes following BoNT injection, and were associated with alterations in energy metabolism, muscle contractile function, cellular stress response, transcription, translation, and cell proliferation. A number of protein abundance changes persisted beyond the return of gross physiologic TA function. These findings represent the first report of BoNT induced changes in any skeletal muscle proteome, and reinforce the utility of applying proteomic tools to the study of system-wide biological processes in normal and perturbed TA muscle function. PMID:18442174

  5. Chapter 3: Molecular basis for the therapeutic effectiveness of botulinum neurotoxin type A.

    PubMed

    Dolly, J Oliver; Lawrence, Gary W

    2014-07-01

    The utility of botulinum neurotoxin type A (BoNT/A) for treating overactive muscles and endocrine glands is attributable to a unique conflation of properties honed to exploit and inactivate synaptic transmission. Specific, high-affinity coincident binding to gangliosides plus an intraluminal loop of synaptic vesicle protein 2 (SV2) by the heavy chain (HC) of BoNT/A confers selectivity for presynaptic nerve terminals and subsequent uptake by endocytosis. Upon vesicle acidification, the HC forms a channel for transmembrane transfer of the light chain to the cytosol, as observed by single channel recordings. The light chain is a Zn(2+) -dependent endoprotease that cleaves and inactivates SNAP-25, thereby blocking exocytotic release of transmitters, a discovery that revealed the pivotal role of the latter in synaptic vesicle fusion. A di-leucine motif in BoNT/A light chain stabilizes this protease, contributing to its longevity inside nerves. The ubiquity of SV2 and SNAP-25 has prompted re-evaluation of the nerve types susceptible to BoNT/A. In urology, there is emerging evidence that BoNT/A blocks neuropeptide release from afferent nerves, exocytosis of acetylcholine and purines from efferent nerves, and possibly ATP release from the urothelium. Suppression by BoNT/A of the surface expression of nociceptor channels on bladder afferents might also contribute to its improvement of urological sensory symptoms.

  6. Molecular topography and secondary structure comparisons of botulinum neurotoxin types A, B and E.

    PubMed

    Singh, B R; DasGupta, B R

    1989-03-16

    Botulinum neurotoxin (NT) serotypes A, B and E differ in microstructure and biological activities. The three NTs were examined for secondary structure parameters (alpha-helix, beta-sheet, beta-turn and random coil content) on the basis of circular dichroism; degree of exposed Tyr residues (second derivative spectroscopy) and state of the Trp residues (fluorescence and fluorescence quantum yield). The proteins are high in beta-pleated sheet content (41-44%) and low in alpha-helical content (21-28%). About 30-36% of the amino acids are in random coils. The beta-sheet contents in the NTs are similar irrespective of their structural forms (i.e. single or dichain forms) or level of toxicity. About 84%, 58% and 61% of Tyr residues of types A, B, and E NT, respectively, were exposed to the solvent (pH 7.2 phosphate buffer). Although the fluorescence emission maximum of Trp residues of type B NT was most blue shifted (331 nm compared to 334 for types A and E NT, and 346 nm for free tryptophan) the fluorescence quantum yields of types A and B were similar and higher than type E. In general the NTs have similar secondary (low alpha-helix and high beta-sheets) and tertiary (exposed tyrosine residues and tryptophan fluorescence quantum yield) structures. Within this generalized picture there are significant differences which might be related to the differences in their biological activities.

  7. Resolution of sub-nanosecond motions in botulinum neurotoxin endopeptidase: An evidence of internal flexibility.

    PubMed

    Kumar, Raj; Cai, Shuowei; Ojadi, Emmanuel; Singh, Bal R

    2015-05-01

    Botulinum neurotoxins (BoNTs) are the most poisonous substances known to mankind, which act on the peripheral nervous system leading to flaccid paralysis. Although co-crystal structure of BoNT/A light chain (LC) reveals some unique features of the biological function of this molecule, structural characteristics in solution reveal its dynamic features, not available through the published crystal structures. In this study, we have examined internal flexibility of this molecule by measuring rotational correlation time as a function of viscosity, using frequency domain fluorescence anisotropy decay technique. Fluorescence anisotropy decay of BoNT/A LC resolved sub-nanosecond local motion (faster component), interpreted as internal flexibility of the molecule was affected significantly with viscosity. Both local and global movements were affected by viscosity, which indicates the accessibility of protein core and flexibility of overall structure. In conclusion, this work demonstrates the presence of flexibility in the internal peptide segments, which appears to play a significant role in BoNT/A LC biological function. PMID:25578806

  8. Botulinum neurotoxin: unique folding of enzyme domain of the most-poisonous poison.

    PubMed

    Kumar, Raj; Kukreja, Roshan V; Li, Li; Zhmurov, Artem; Kononova, Olga; Cai, Shuowei; Ahmed, Syed A; Barsegov, Valeri; Singh, Bal Ram

    2014-01-01

    Botulinum neurotoxin (BoNT), the most toxic substance known to mankind, is the first example of the fully active molten globule state. To understand its folding mechanism, we performed urea denaturation experiments and theoretical modeling using BoNT serotype A (BoNT/A). We found that the extent of BoNT/A denaturation from the native state (N) shows a nonmonotonic dependence on urea concentration indicating a unique multistep denaturation process, N → I1 [Formula: see text] I2 [Formula: see text] U, with two intermediate states I1 and I2. BoNT/A loses almost all its secondary structure in 3.75 M urea (I1), yet it displays a native-like secondary structure in 5 M urea (I2). This agrees with the results of theoretical modeling, which helped to determine the molecular basis of unique behavior of BoNT/A in solution. Except for I2, all the states revert back to full enzymatic activity for SNAP-25 including the unfolded state U stable in 7 M urea. Our results stress the importance of structural flexibility in the toxin's mechanism of survival and action, an unmatched evolutionary trait from billion-year-old bacteria, which also correlates with the long-lasting enzymatic activity of BoNT inside neuronal cells. BoNT/A provides a rich model to explore protein folding in relation to functional activity. PMID:23746226

  9. Amino acid neurotoxins in feathers of the Lesser Flamingo, Phoeniconaias minor.

    PubMed

    Metcalf, J S; Banack, S A; Kotut, K; Krienitz, L; Codd, G A

    2013-01-01

    The Lesser Flamingo (Phoeniconaias minor) is known to use cyanobacteria (primarily Arthrospira) as a major food source in the East African Rift Valley lakes. Periodically, mass mortalities have occurred, associated with the cyanobacterial toxins (cyanotoxins), microcystins and anatoxin-a. Deposition of these cyanotoxins into P. minor feathers has been shown to occur, consistent with the presence of cyanotoxins in the livers, stomach and faecal contents after dietary intake. As cyanobacteria have been shown to also produce the neurotoxins β-N-methylamino-L-alanine (BMAA) and 2,4-diaminobutyric acid (DAB), stored wing feathers, previously recovered from flamingos which had been exposed to microcystins and anatoxin-a and had subsequently died, were analysed for these neurotoxic amino acids. Trace amounts of BMAA were detected in extracts from Lake Nakuru flamingo feathers, with DAB also present at concentrations between 3.5 and 8.5 μg g(-1) dry weight in feathers from both lakes. Toxin recovery by solid-phase extraction of feather digests was tested with spiked deuterated BMAA and showed good recovery when analysed by LC-MS/MS (80-94%). This is the first report of these neurotoxic amino acids in birds. We discuss the origin and significance of DAB, alongside other cyanotoxins of dietary origin, in the feathers of the Lesser Flamingo.

  10. Novel chimeras of botulinum and tetanus neurotoxins yield insights into their distinct sites of neuroparalysis.

    PubMed

    Wang, Jiafu; Zurawski, Tomas H; Meng, Jianghui; Lawrence, Gary W; Aoki, K Roger; Wheeler, Larry; Dolly, J Oliver

    2012-12-01

    Botulinum neurotoxin (BoNT) A or E and tetanus toxin (TeTx) bind to motor-nerve endings and undergo distinct trafficking; their light-chain (LC) proteases cleave soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) peripherally or centrally and cause flaccid or spastic paralysis, respectively. To seek protein domains responsible for local blockade of transmitter release (BoNTs) rather than retroaxonal transport to spinal neurons (TeTx), their acceptor-binding moieties (H(C))--or in one case, heavy chain (HC)--were exchanged by gene recombination. Each chimera, expressed and purified from Escherichia coli, entered rat cerebellar neurons to cleave their substrates, blocked in vitro nerve-induced muscle contractions, and produced only flaccid paralysis in mice. Thus, the local cytosolic delivery of BoNT/A or BoNT/E proteases and the contrasting retrograde transport of TeTx are not specified solely by their HC or H(C); BoNT/A LC translocated locally irrespective of being targeted by either of the latter TeTx domains. In contrast, BoNT/E protease fused to a TeTx enzymatically inactive mutant (TeTIM) caused spastic paralysis and cleaved SNAP-25 in spinal cord but not the injected muscle. Apparently, TeTIM precludes cytosolic release of BoNT/E protease at motor nerve endings. It is deduced that the LCs of the toxins, acting in conjunction with HC domains, dictate their local or distant destinations.

  11. The voltage-gated sodium channel: a major target of marine neurotoxins.

    PubMed

    Mattei, César; Legros, Christian

    2014-12-01

    Voltage-gated sodium channels (Nav) are key components for nerve excitability. They initiate and propagate the action potential in excitable cells, throughout the central and peripheral nervous system, thus enabling a variety of physiological functions to be achieved. The rising phase of the action potential is driven by the opening of Nav channels which activate rapidly and carry Na(+) ions in the intracellular medium, and ends with the Na(+) current inactivation. The biophysical properties of these channels have been elucidated, through the use of pharmacological agents that disrupt the molecular mechanism of the channel functioning. Among them, marine toxins produced by venomous animals or microorganisms have been crucial to map the different allosteric binding sites of the channels, understand their mode of action and represent an emerging source of therapeutic agents to alleviate or cure Na(+) channels-linked human diseases. In this article, we review recent discoveries on the molecular and biophysical properties of the Na(+) channel as a target for marine neurotoxins, and present the ongoing developments of pharmacological agents as therapeutic tools.

  12. Molecular components required for resting and stimulated endocytosis of botulinum neurotoxins by glutamatergic and peptidergic neurons.

    PubMed

    Meng, Jianghui; Wang, Jiafu; Lawrence, Gary W; Dolly, J Oliver

    2013-08-01

    Proteins responsible for basal and stimulated endocytosis in nerves containing small clear synaptic vesicles (SCSVs) or large dense-core vesicles (LDCVs) are revealed herein, using probes that exploit surface-exposed vesicle proteins as acceptors for internalization. Basal uptake of botulinum neurotoxins (BoNTs) by both SCSV-releasing cerebellar granule neurons (CGNs) and LDCV-enriched trigeminal ganglionic neurons (TGNs) was found to require protein acceptors and acidic compartments. In addition, dynamin, clathrin, adaptor protein complex-2 (AP2), and amphiphysin contribute to the depolarization-evoked entry. For fast recycling of SCSVs, knockdown and knockout strategies demonstrated that CGNs use predominantly dynamin 1, whereas isoform 2 and, to a smaller extent, isoform 3 support a less rapid mode of stimulated endocytosis. Accordingly, proximity ligation assay confirmed that dynamin 1 and 2 colocalize with amphiphysin 1 in CGNs, and the latter copurified with both dynamins from cell extracts. In contrast, LDCV-releasing TGNs preferentially employ dynamins 2 and 3 and amphiphysin 1 for evoked endocytosis and lack the fast phase. Hence, stimulation recruits dynamin, clathrin, AP2, and amphiphysin to augment BoNT internalization, and neurons match endocytosis mediators to the different demands for locally recycling SCSVs or replenishing distally synthesized LDCVs.

  13. Clinical uses of botulinum neurotoxins: current indications, limitations and future developments.

    PubMed

    Chen, Sheng

    2012-10-01

    Botulinum neurotoxins (BoNTs) cause flaccid paralysis by interfering with vesicle fusion and neurotransmitter release in the neuronal cells. BoNTs are the most widely used therapeutic proteins. BoNT/A was approved by the U.S. FDA to treat strabismus, blepharospam, and hemificial spasm as early as 1989 and then for treatment of cervical dystonia, glabellar facial lines, axillary hyperhidrosis, chronic migraine and for cosmetic use. Due to its high efficacy, longevity of action and satisfactory safety profile, it has been used empirically in a variety of ophthalmological, gastrointestinal, urological, orthopedic, dermatological, secretory, and painful disorders. Currently available BoNT therapies are limited to neuronal indications with the requirement of periodic injections resulting in immune-resistance for some indications. Recent understanding of the structure-function relationship of BoNTs prompted the engineering of novel BoNTs to extend therapeutic interventions in non-neuronal systems and to overcome the immune-resistance issue. Much research still needs to be done to improve and extend the medical uses of BoNTs.

  14. An optical biosensor assay for rapid dual detection of Botulinum neurotoxins A and E

    PubMed Central

    Lévêque, Christian; Ferracci, Géraldine; Maulet, Yves; Mazuet, Christelle; Popoff, Michel R.; Blanchard, Marie-Pierre; Seagar, Michael; El Far, Oussama

    2015-01-01

    The enzymatic activity of the pathogenic botulinum neurotoxins type A and E (BoNT/A and E) leads to potentially lethal paralytic symptoms in humans and their prompt detection is of crucial importance. A chip assay based on Surface Plasmon Resonance monitoring of the cleavage products is a simple method that we have previously established to detect BoNT/A activity. We have now developed a similar format assay to measure BoNT/E activity. A monoclonal antibody specifically recognizing SNAP25 cleaved by BoNT/E was generated and used to measure the appearance of the neo-epitope following injection of BoNT/E over SNAP-25 immobilized on a chip. This assay detects BoNT/E activity at 1 LD50/ml within minutes and linear dose-responses curves were obtained using a multiplexed biosensor. A threshold of 0.01 LD50/ml was achieved after 5 h of cleavage. This assay is 10-fold more sensitive than the in vivo assay for direct detection of BoNT/E in serum samples. The SNAP25 chip assay is able to discriminate in an automated manner the presence of BoNT/E, BoNT/A or a combination of both toxins. PMID:26648139

  15. Recommended Immunological Strategies to Screen for Botulinum Neurotoxin-Containing Samples.

    PubMed

    Simon, Stéphanie; Fiebig, Uwe; Liu, Yvonne; Tierney, Rob; Dano, Julie; Worbs, Sylvia; Endermann, Tanja; Nevers, Marie-Claire; Volland, Hervé; Sesardic, Dorothea; Dorner, Martin B

    2015-12-01

    Botulinum neurotoxins (BoNTs) cause the life-threatening neurological illness botulism in humans and animals and are divided into seven serotypes (BoNT/A-G), of which serotypes A, B, E, and F cause the disease in humans. BoNTs are classified as "category A" bioterrorism threat agents and are relevant in the context of the Biological Weapons Convention. An international proficiency test (PT) was conducted to evaluate detection, quantification and discrimination capabilities of 23 expert laboratories from the health, food and security areas. Here we describe three immunological strategies that proved to be successful for the detection and quantification of BoNT/A, B, and E considering the restricted sample volume (1 mL) distributed. To analyze the samples qualitatively and quantitatively, the first strategy was based on sensitive immunoenzymatic and immunochromatographic assays for fast qualitative and quantitative analyses. In the second approach, a bead-based suspension array was used for screening followed by conventional ELISA for quantification. In the third approach, an ELISA plate format assay was used for serotype specific immunodetection of BoNT-cleaved substrates, detecting the activity of the light chain, rather than the toxin protein. The results provide guidance for further steps in quality assurance and highlight problems to address in the future. PMID:26703727

  16. Rapid downward transport of the neurotoxin domoic acid in coastal waters

    NASA Astrophysics Data System (ADS)

    Sekula-Wood, Emily; Schnetzer, Astrid; Benitez-Nelson, Claudia R.; Anderson, Clarissa; Berelson, William M.; Brzezinski, Mark A.; Burns, Justina M.; Caron, David A.; Cetinic, Ivona; Ferry, John L.; Fitzpatrick, Elizabeth; Jones, Burton H.; Miller, Peter E.; Morton, Steve L.; Schaffner, Rebecca A.; Siegel, David A.; Thunell, Robert

    2009-04-01

    Toxic phytoplankton blooms threaten coastlines worldwide by diminishing beach quality and adversely affecting marine ecosystems and human health. The common diatom genus Pseudo-nitzschia consists of several species known to produce the neurotoxin domoic acid. Recent studies suggest that algal blooms dominated by Pseudo-nitzschia are increasing in frequency and duration owing to changes in coastal nutrient regimes. However, few studies have examined the persistence or long-term biogeochemical cycling of domoic acid in marine waters. Here, we measure the concentration of domoic acid in surface waters and sediment traps-up to 800m in depth-off the coast of Southern California. We show that peaks in Pseudo-nitzschia abundance and domoic acid concentrations in surface waters coincide with peaks in diatom and toxin abundance at depth, suggesting rapid downward transport of the toxin. In some cases, the sinking particles contain over five times the United States federal limit of domoic acid. Detection of domoic acid in bottom sediments indicates that the toxin may persist long after the Pseudo-nitzschia blooms. Our results indicate that vertical fluxes of domoic acid are a substantial source of the toxin to deep-ocean food webs, and could explain high levels of domoic acid previously observed in benthic organisms.

  17. Orally Bioavailable Potent Soluble Epoxide Hydrolase Inhibitors

    PubMed Central

    Hwang, Sung Hee; Tsai, Hsing-Ju; Liu, Jun-Yan; Morisseau, Christophe; Hammock, Bruce D.

    2008-01-01

    A series of N,N′-disubstituted ureas having a conformationally restricted cis- or trans-1,4-cyclohexane α to the urea were prepared and tested as soluble epoxide hydrolase (sEH) inhibitors. This series of compounds showed low nanomolar to picomolar activities against recombinant human sEH. Both isomers showed similar potencies, but the trans isomers were more metabolically stable in human hepatic microsomes. Furthermore, these new potent inhibitors show a greater metabolic stability in vivo than previously described sEH inhibitors. We demonstrated that trans-4-[4-(3-adamantan-1-ylureido)cyclohexyloxy]benzoic acid 13g (t-AUCB, IC50 = 1.3 ± 0.05 nM) had excellent oral bioavailability (98%, n = 2) and blood area under the curve in dogs and was effective in vivo to treat hypotension in lipopolysaccharide challenged murine models. PMID:17616115

  18. Highly potent fibrinolytic serine protease from Streptomyces.

    PubMed

    Uesugi, Yoshiko; Usuki, Hirokazu; Iwabuchi, Masaki; Hatanaka, Tadashi

    2011-01-01

    We introduce a highly potent fibrinolytic serine protease from Streptomyces omiyaensis (SOT), which belongs to the trypsin family. The fibrinolytic activity of SOT was examined using in vitro assays and was compared with those of known fibrinolytic enzymes such as plasmin, tissue-type plasminogen activator (t-PA), urokinase, and nattokinase. Compared to other enzymes, SOT showed remarkably higher hydrolytic activity toward mimic peptides of fibrin and plasminogen. The fibrinolytic activity of SOT is about 18-fold higher than that of plasmin, and is comparable to that of t-PA by fibrin plate assays. Furthermore, SOT had some plasminogen activator-like activity. Results show that SOT and nattokinase have very different fibrinolytic and fibrinogenolytic modes, engendering significant synergetic effects of SOT and nattokinase on fibrinolysis. These results suggest that SOT presents important possibilities for application in the therapy of thrombosis.

  19. Potent antitrypanosomal triterpenoid saponins from Mussaenda luteola

    PubMed Central

    Mohamed, Shaymaa M.; Bachkeet, Enaam Y.; Bayoumi, Soad A.; Jain, Surendra; Cutler, Stephen J.; Tekwani, Babu L.; Ross, Samir A.

    2016-01-01

    Five new triterpenoid saponins, heinsiagenin A 3-O-[α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl-(1→2)]-β-d-glucopyranoside (1), heinsiagenin A 3-O-[α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl-(1→2)]-[β-d-glucopyranosyl-(1→4)]-β-d-glucopyranoside (2), 2α-hydroxyheinsiagenin A 3-O-[α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl-(1→2)]-β-d-glucopyranoside (3), 2α-hydroxyheinsiagenin A 3-O-[β-d-glucopyranosyl-(1→2)]-[β-d-glucopyranosyl-(1→4)]-β-d-glucopyranoside (4) and N-(2S, 3R, 4R-3-methyl-4-pentanolid-2-yl)-18-hydroxylanosta-8 (9), 22E, 24E-trien-27-amide-3-O-[α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl-(1→2)]-[β-d-glucopyranosyl-(1→4)]-β-d-glucopyranoside (5) were isolated from the aerial parts of Mussaenda luteola Delile (Rubiaceae). Structural elucidation was based on the analysis of spectroscopic data (1D and 2D NMR) and HR-ESI-MS. Compound 1 showed potent antitrypanosomal activity with an IC50 value of 8.80 μM. Compounds 2–4 showed highly potent antitrypanosomal activity with IC50 values ranging between (2.57–2.84 μM) and IC90 values ranging between (3.36–4.35 μM), which are 5 fold greater than the positive control DFMO (IC50 and IC90 values of 13.06 and 28.99 μM, respectively). Compounds 1 and 2 showed moderate affinity to μ-opioid receptors with Ki values of 9.936 μM and 0.872 μM, respectively compared to a Ki value of 1.958 nM for the positive control, naloxone HCl. PMID:26524249

  20. Potent antitrypanosomal triterpenoid saponins from Mussaenda luteola.

    PubMed

    Mohamed, Shaymaa M; Bachkeet, Enaam Y; Bayoumi, Soad A; Jain, Surendra; Cutler, Stephen J; Tekwani, Babu L; Ross, Samir A

    2015-12-01

    Five new triterpenoid saponins, heinsiagenin A 3-O-[α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl-(1→2)]-β-d-glucopyranoside (1), heinsiagenin A 3-O-[α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl-(1→2)]-[β-d-glucopyranosyl-(1→4)]-β-d-glucopyranoside (2), 2α-hydroxyheinsiagenin A 3-O-[α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl-(1→2)]-β-d-glucopyranoside (3), 2α-hydroxyheinsiagenin A 3-O-[β-d-glucopyranosyl-(1→2)]-[β-d-glucopyranosyl-(1→4)]-β-d-glucopyranoside (4) and N-(2S, 3R, 4R-3-methyl-4-pentanolid-2-yl)-18-hydroxylanosta-8 (9), 22E, 24E-trien-27-amide-3-O-[α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl-(1→2)]-[β-d-glucopyranosyl-(1→4)]-β-d-glucopyranoside (5) were isolated from the aerial parts of Mussaenda luteola Delile (Rubiaceae). Structural elucidation was based on the analysis of spectroscopic data (1D and 2D NMR) and HR-ESI-MS. Compound 1 showed potent antitrypanosomal activity with an IC50 value of 8.80μM. Compounds 2-4 showed highly potent antitrypanosomal activity with IC50 values ranging between (2.57-2.84μM) and IC90 values ranging between (3.36-4.35μM), which are 5 fold greater than the positive control DFMO (IC50 and IC90 values of 13.06 and 28.99μM, respectively). Compounds 1 and 2 showed moderate affinity to μ-opioid receptors with Ki values of 9.936μM and 0.872μM, respectively compared to a Ki value of 1.958nM for the positive control, naloxone HCl. PMID:26524249

  1. Predicting Improvement in Writer’s Cramp Symptoms following Botulinum Neurotoxin Injection Therapy

    PubMed Central

    Jackman, Mallory; Delrobaei, Mehdi; Rahimi, Fariborz; Atashzar, S. Farokh; Shahbazi, Mahya; Patel, Rajni; Jog, Mandar

    2016-01-01

    Introduction Writer’s cramp is a specific focal hand dystonia causing abnormal posturing and tremor in the upper limb. The most popular medical intervention, botulinum neurotoxin type A (BoNT-A) therapy, is variably effective for 50–70% of patients. BoNT-A non-responders undergo ineffective treatment and may experience significant side effects. Various assessments have been used to determine response prediction to BoNT-A, but not in the same population of patients. Methods A comprehensive assessment was employed to measure various symptom aspects. Clinical scales, full upper-limb kinematic measures, self-report, and task performance measures were assessed for nine writer’s cramp patients at baseline. Patients received two BoNT-A injections then were classified as responders or non-responders based on a quantified self-report measure. Baseline scores were compared between groups, across all measures, to determine which scores predicted a positive BoNT-A response. Results Five of nine patients were responders. No kinematic measures were predictably different between groups. Analyses revealed three features that predicted a favorable response and separated the two groups: higher than average cramp severity and cramp frequency, and below average cramp latency. Discussion Non-kinematic measures appear to be superior in making such predictions. Specifically, measures of cramp severity, frequency, and latency during performance of a specific set of writing and drawing tasks were predictive factors. Since kinematic was not used to determine the injection pattern and the injections were visually guided, it may still be possible to use individual patient kinematics for better outcomes. PMID:27625900

  2. Botulinum Neurotoxins Can Enter Cultured Neurons Independent of Synaptic Vesicle Recycling

    PubMed Central

    Pellett, Sabine; Tepp, William H.; Scherf, Jacob M.; Johnson, Eric A.

    2015-01-01

    Botulinum neurotoxins (BoNTs) are the causative agent of the severe and long-lasting disease botulism. At least seven different serotypes of BoNTs (denoted A-G) have been described. All BoNTs enter human or animal neuronal cells via receptor mediated endocytosis and cleave cytosolic SNARE proteins, resulting in a block of synaptic vesicle exocytosis, leading to the flaccid paralysis characteristic of botulism. Previous data have indicated that once a neuronal cell has been intoxicated by a BoNT, further entry of the same or other BoNTs is prevented due to disruption of synaptic vesicle recycling. However, it has also been shown that cultured neurons exposed to BoNT/A are still capable of taking up BoNT/E. In this report we show that in general BoNTs can enter cultured human or mouse neuronal cells that have previously been intoxicated with another BoNT serotype. Quantitative analysis of cell entry by assessing SNARE cleavage revealed none or only a minor difference in the efficiency of uptake of BoNTs into previously intoxicated neurons. Examination of the endocytic entry pathway by specific endocytosis inhibitors indicated that BoNTs are taken up by clathrin coated pits in both non pre-exposed and pre-exposed neurons. LDH release assays indicated that hiPSC derived neurons exposed consecutively to two different BoNT serotypes remained viable and healthy except in the case of BoNT/E or combinations of BoNT/E with BoNT/B, /D, or /F. Overall, our data indicate that previous intoxication of neuronal cells with BoNT does not inhibit further uptake of BoNTs. PMID:26207366

  3. NRSF/REST neuronal deficient mice are more vulnerable to the neurotoxin MPTP.

    PubMed

    Yu, Mei; Suo, Haiyun; Liu, Ming; Cai, Lei; Liu, Jie; Huang, Yufang; Xu, Jing; Wang, Yancong; Zhu, Cuiqing; Fei, Jian; Huang, Fang

    2013-03-01

    Parkinson's disease (PD) is characterized by progressing loss of dopaminergic neurons in the midbrain. Abnormal gene expression plays a critical role in its pathogenesis. Neuron-restrictive silencer factor (NRSF)/neuronal repressor element-1 silencing transcription factor (REST), a member of the zinc finger transcription factors, inhibits the expression of neuron-specific genes in nonneuronal cells, and regulates neurogenesis. Our previous work showed that 1-methyl-4-phenyl-pyridinium ion triggers dynamic changes of messenger RNA and protein expression of NRSF in human dopaminergic SH-SY5Y cells, and alteration of NRSF expression exacerbates 1-methyl-4-phenyl-pyridinium ion-induced cell death. The purpose of this study was to explore the in vivo role of NRSF in the progress of PD by using NRSF/REST neuron-specific conditional knockout mice (cKO). 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was adopted to generate PD models in the cKO mice and wild type littermates. At 1, 3, 7, 14, 21, and 28 days after MPTP injection, behavioral tests were performed, and cKO mice displayed some impairments in locomotor activities. Also, the reduction of tyrosine hydroxylase protein in the striatum and the loss of dopaminergic neurons in the substantia nigra were more severe in the cKO mice. Meanwhile, the cKO mice exhibited a more dramatic depletion of striatal dopamine, accompanied by an increase in glial fibrillary acidic protein (GFAP) expression and sustained interleukin-1β transcription. These results suggested that NRSF/REST neuronal cKO mice are more vulnerable to the dopaminergic neurotoxin MPTP. Disturbance of the homeostasis of NRSF and its target genes, gliogenesis, and inflammation may contribute to the higher MPTP sensitivity in NRSF/REST neuronal cKO mice. PMID:22766071

  4. Development of Human-Like scFv-Fc Neutralizing Botulinum Neurotoxin E

    PubMed Central

    Miethe, Sebastian; Rasetti-Escargueil, Christine; Avril, Arnaud; Liu, Yvonne; Chahboun, Siham; Korkeala, Hannu; Mazuet, Christelle; Popoff, Michel-Robert; Pelat, Thibaut; Thullier, Philippe; Sesardic, Dorothea; Hust, Michael

    2015-01-01

    Background Botulinum neurotoxins (BoNTs) are considered to be the most toxic substances known on earth and are responsible for human botulism, a life-threatening disease characterized by flaccid muscle paralysis that occurs naturally by food-poisoning or colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNTs have been classified as category A agent by the Centers of Disease Control and Prevention (CDC) and are listed among the six agents with the highest risk to be used as bioweapons. Neutralizing antibodies are required for the development of effective anti-botulism therapies to deal with the potential risk of exposure. Results In this study, a macaque (Macaca fascicularis) was immunized with recombinant light chain of BoNT/E3 and an immune phage display library was constructed. After a multi-step panning, several antibody fragments (scFv, single chain fragment variable) with nanomolar affinities were isolated, that inhibited the endopeptidase activity of pure BoNT/E3 in vitro by targeting its light chain. Furthermore, three scFv were confirmed to neutralize BoNT/E3 induced paralysis in an ex vivo mouse phrenic nerve-hemidiaphragm assay. The most effective neutralization (20LD50/mL, BoNT/E3) was observed with scFv ELC18, with a minimum neutralizing concentration at 0.3 nM. Furthermore, ELC18 was highly effective in vivo when administered as an scFv-Fc construct. Complete protection of 1LD50 BoNT/E3 was observed with 1.6 ng/dose in the mouse flaccid paralysis assay. Conclusion These scFv-Fcs antibodies are the first recombinant antibodies neutralizing BoNT/E by targeting its light chain. The human-like nature of the isolated antibodies is predicting a good tolerance for further clinical development. PMID:26440796

  5. Pre-Clinical Study of a Novel Recombinant Botulinum Neurotoxin Derivative Engineered for Improved Safety

    PubMed Central

    Vazquez-Cintron, Edwin; Tenezaca, Luis; Angeles, Christopher; Syngkon, Aurelia; Liublinska, Victoria; Ichtchenko, Konstantin; Band, Philip

    2016-01-01

    Cyto-012 is a recombinant derivative of Botulinum neurotoxin Type A (BoNT/A). It primarily differs from wild type (wt) BoNT/A1 in that it incorporates two amino acid substitutions in the catalytic domain of the light chain (LC) metalloprotease (E224 > A and Y366 > A), designed to provide a safer clinical profile. Cyto-012 is specifically internalized into rat cortical and hippocampal neurons, and cleaves Synaptosomal-Associated Protein 25 (SNAP-25), the substrate of wt BoNT/A, but exhibits slower cleavage kinetics and therefore requires a higher absolute dose to exhibit pharmacologic activity. The pharmacodynamics of Cyto-012 and wt BoNT/A have similar onset and duration of action using the Digital Abduction Assay (DAS). Intramuscular LD50 values for Cyto-012 and wt BoNT/A respectively, were 0.63 ug (95% CI = 0.61, 0.66) and 6.22 pg (95% CI = 5.42, 7.02). ED50 values for Cyto-012 and wt BoNT/A were respectively, 0.030 ug (95% CI = 0.026, 0.034) and 0.592 pg (95% CI = 0.488, 0.696). The safety margin (intramuscular LD50/ED50 ratio) for Cyto-012 was found to be improved 2-fold relative to wt BoNT/A (p < 0.001). The DAS response to Cyto-012 was diminished when a second injection was administered 32 days after the first. These data suggest that the safety margin of BoNT/A can be improved by modulating their activity towards SNAP-25. PMID:27484492

  6. Direct biosensor detection of botulinum neurotoxin endopeptidase activity in sera from patients with type A botulism.

    PubMed

    Lévêque, Christian; Ferracci, Géraldine; Maulet, Yves; Mazuet, Christelle; Popoff, Michel; Seagar, Michael; El Far, Oussama

    2014-07-15

    Botulinum neurotoxin A (BoNT/A) has intrinsic endoprotease activity specific for SNAP-25, a key protein for presynaptic neurotransmitter release. The inactivation of SNAP-25 by BoNT/A underlies botulism, a rare but potentially fatal disease. There is a crucial need for a rapid and sensitive in vitro serological test for BoNT/A to replace the current in vivo mouse bioassay. Cleavage of SNAP-25 by BoNT/A generates neo-epitopes which can be detected by binding of a monoclonal antibody (mAb10F12) and thus measured by surface plasmon resonance (SPR). We have explored two SPR assay formats, with either mAb10F12 or His6-SNAP-25 coupled to the biosensor chip. When BoNT/A was incubated with SNAP-25 in solution and the reaction products were captured on a mAb-coated chip, a sensitivity of 5 fM (0.1LD50/ml serum) was obtained. However, this configuration required prior immunoprecipitation of BoNT/A. A sensitivity of 0.5 fM in 10% serum (0.1 LD50/ml serum) was attained when SNAP-25 was coupled directly to the chip, followed by sequential injection of BoNT/A samples and mAb10F12 into the flow system to achieve on-chip cleavage and detection, respectively. This latter format detected BoNT/A endoprotease activity in 50-100 µl serum samples from all patients (11/11) with type A botulism within 5h. No false positives occurred in sera from healthy subjects or patients with other neurological diseases. The automated chip-based procedure has excellent specificity and sensitivity, with significant advantages over the mouse bioassay in terms of rapidity, required sample volume and animal ethics.

  7. Facile electrochemical detection of botulinum neurotoxin type E using a two-step proteolytic cleavage.

    PubMed

    Park, Seonhwa; Shin, Yu Mi; Song, Ji-Joon; Yang, Haesik

    2015-10-15

    Facile electrochemical methods for measuring protease concentration or protease activity are essential for point-of-care testing of toxic proteases. However, electrochemical detection of proteases, such as botulinum neurotoxin type E (BoNT/E), that cleave a peptide bond between two specific amino acid residues is challenging. This study reports a facile and sensitive electrochemical method for BoNT/E detection. The method is based on a two-step proteolytic cleavage using a target BoNT/E light chain (BoNT/E-LC) and an externally supplemented exopeptidase, L-leucine-aminopeptidase (LAP). BoNT/E-LC cleaves a peptide bond between arginine and isoleucine in IDTQNRQIDRI-4-amino-1-naphthol (oligopeptide-AN) to generate isoleucine-AN. Subsequently, LAP cleaves a bond between isoleucine and AN to liberate a free electroactive AN species. The liberated AN participates in electrochemical-chemical-chemical (ECC) redox cycling involving Ru(NH3)6(3+), AN, and a reducing agent, which allows a high signal amplification. Electrochemical detection is carried out without surface modification of indium-tin oxide electrodes. We show that dithiothreitol is beneficial for enhancing the enzymatic activity of BoNT/E-LC and also for achieving a fast ECC redox cycling. An incubation temperature of 37°C and the use of phosphate buffered saline (PBS) buffer resulted in optimal signal-to-background ratios for efficient BoNT/E detection. BoNT/E-LC could be detected at concentrations of approximately 2.0 pg/mL, 0.2, and 3 ng/mL after 4h, 2h, and 15 min incubation in PBS buffer, respectively, and approximately 0.3 ng/mL after 2-h incubation in bottled water. The method developed could be applied in fast, sensitive, and selective detection of any protease that cleaves a peptide bond between two specific amino acid residues.

  8. Pre-Clinical Study of a Novel Recombinant Botulinum Neurotoxin Derivative Engineered for Improved Safety.

    PubMed

    Vazquez-Cintron, Edwin; Tenezaca, Luis; Angeles, Christopher; Syngkon, Aurelia; Liublinska, Victoria; Ichtchenko, Konstantin; Band, Philip

    2016-01-01

    Cyto-012 is a recombinant derivative of Botulinum neurotoxin Type A (BoNT/A). It primarily differs from wild type (wt) BoNT/A1 in that it incorporates two amino acid substitutions in the catalytic domain of the light chain (LC) metalloprotease (E224 > A and Y366 > A), designed to provide a safer clinical profile. Cyto-012 is specifically internalized into rat cortical and hippocampal neurons, and cleaves Synaptosomal-Associated Protein 25 (SNAP-25), the substrate of wt BoNT/A, but exhibits slower cleavage kinetics and therefore requires a higher absolute dose to exhibit pharmacologic activity. The pharmacodynamics of Cyto-012 and wt BoNT/A have similar onset and duration of action using the Digital Abduction Assay (DAS). Intramuscular LD50 values for Cyto-012 and wt BoNT/A respectively, were 0.63 ug (95% CI = 0.61, 0.66) and 6.22 pg (95% CI = 5.42, 7.02). ED50 values for Cyto-012 and wt BoNT/A were respectively, 0.030 ug (95% CI = 0.026, 0.034) and 0.592 pg (95% CI = 0.488, 0.696). The safety margin (intramuscular LD50/ED50 ratio) for Cyto-012 was found to be improved 2-fold relative to wt BoNT/A (p < 0.001). The DAS response to Cyto-012 was diminished when a second injection was administered 32 days after the first. These data suggest that the safety margin of BoNT/A can be improved by modulating their activity towards SNAP-25.

  9. Trans generational effects of the neurotoxin BMAA on the aquatic grazer Daphnia magna.

    PubMed

    Faassen, Elisabeth J; García-Altares, María; Mendes e Mello, Mariana; Lürling, Miquel

    2015-11-01

    β-N-Methylamino-l-alanine (BMAA) is a neurotoxin that is suspected to play a role in the neurological diseases amyotrophic lateral sclerosis, Alzheimer's disease and Parkinson's disease. BMAA has been detected in phytoplankton and globally, the main exposure routes for humans to BMAA are through direct contact with phytoplankton-infested waters and consumption of BMAA-contaminated fish and invertebrates. As BMAA can be transferred from mothers to offspring in mammals, BMAA exposure is expected to have transgenerational effects. The aim of our study was to determine whether maternal exposure to BMAA affects offspring fitness in zooplankton. We performed a multigenerational life history experiment and a multigenerational uptake experiment with the water flea Daphnia magna as a model species. In both experiments, offspring from nonexposed and exposed mothers were raised in clean and BMAA-containing medium. Direct exposure to 110μg/l BMAA reduced survival, somatic growth, reproduction and population growth. Maternal exposure did not affect D. magna fitness: animals from exposed mothers that were raised in clean medium had a higher mortality and produced lighter neonates than the controls, but this did not result in lower population growth rates. No evidence of adaptation was found. Instead, multigeneration exposure to BMAA had a negative effect: animals that were exposed during two generations had a lower brood viability and neonate weight than animals born from unexposed mothers, but raised in BMAA-containing medium. Maternal transfer of BMAA was observed, but BMAA concentrations in neonates raised in BMAA containing medium were similar for animals born from exposed and unexposed mothers. Our results indicate that zooplankton might be an important vector for the transfer of BMAA along the pelagic food chain, but whether BMAA plays a role in preventing zooplankton from controlling cyanobacterial blooms needs further investigation.

  10. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library.

    PubMed

    Wang, Han; Yu, Rui; Fang, Ting; Yu, Ting; Chi, Xiangyang; Zhang, Xiaopeng; Liu, Shuling; Fu, Ling; Yu, Changming; Chen, Wei

    2016-01-01

    Tetanus neurotoxin (TeNT) produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective. PMID:27626445

  11. Recovery of mouse neuromuscular junctions from single and repeated injections of botulinum neurotoxin A

    PubMed Central

    Rogozhin, A A; Pang, K K; Bukharaeva, E; Young, C; Slater, C R

    2008-01-01

    Botulinum neurotoxin type A (BoNT/A) paralyses muscles by blocking acetylcholine (ACh) release from motor nerve terminals. Although highly toxic, it is used clinically to weaken muscles whose contraction is undesirable, as in dystonias. The effects of an injection of BoNT/A wear off after 3–4 months so repeated injections are often used. Recovery of neuromuscular transmission is accompanied by the formation of motor axon sprouts, some of which form new synaptic contacts. However, the functional importance of these new contacts is unknown. Using intracellular and focal extracellular recording we show that in the mouse epitrochleoanconeus (ETA), quantal release from the region of the original neuromuscular junction (NMJ) can be detected as soon as from new synaptic contacts, and generally accounts for > 80% of total release. During recovery the synaptic delay and the rise and decay times of endplate potentials (EPPs) become prolonged approximately 3-fold, but return to normal after 2–3 months. When studied after 3–4 months, the response to repetitive stimulation at frequencies up to 100 Hz is normal. When two or three injections of BoNT/A are given at intervals of 3–4 months, quantal release returns to normal values more slowly than after a single injection (11 and 15 weeks to reach 50% of control values versus 6 weeks after a single injection). In addition, branching of the intramuscular muscular motor axons, the distribution of the NMJs and the structure of many individual NMJs remain abnormal. These findings highlight the plasticity of the mammalian NMJ but also suggest important limits to it. PMID:18467364

  12. Chemical and enzyme-mediated oxidation of the serotonergic neurotoxin 5,7-dihydroxytryptamine: mechanistic insights.

    PubMed

    Tabatabaie, T; Dryhurst, G

    1992-06-12

    The oxidation chemistry and biochemistry of the serotonergic neurotoxin 5,7-dihydroxytryptamine (1) has been studied under anaerobic and aerobic conditions in aqueous solution at physiological pH. Under anaerobic conditions, one-electron oxidants (ferricytochrome c, peroxidase/H2O2, ceruloplasmin, Cu2+) generate a radical intermediate. Dimerization of the C(6)-centered resonance form of this radical followed by secondary oxidations yields 3-(2-aminoethyl)-6-[3-(2-aminoethyl)-1,7-dihydro- 5-hydroxy-7-oxo-6H-indol-6-ylidene]-1-H-indole-5,7(4H,6H)-dione. Under aerobic conditions, molecular O2 attacks the C(4)-centered 1 radical to yield a hydroperoxy radical which decomposes to 5-hydroxytryptamine-4,7-dione (2). Autoxidation of 1 proceeds by primary attack by molecular O2 on a C(4)-centered carbanion to form a superoxide-radical complex. This rearranges to a C(4)-centered hydroperoxide which decomposes to 2. A C(6)-centered carbanion of 1 combines with 2 to give, ultimately, 6,6'-bi-5-hydroxytryptamine-4,7-dione (3). Trace concentrations of transition metal ions (Fe3+, Fe2+, Cu2+, Mn2+) catalyze the autoxidation of 1 by catalytic cycles in which a hydroperoxide intermediate plays key roles. A byproduct of the transition metal-catalyzed oxidation of 1 is superoxide, O2-. Because of its enormous basicity O2- facilitates deprotonation of 1. The C(4)-centered carbanion so produced is oxidized by molecular O2 or by the hydroperoxy radical (HO2) to give radical intermediates and thence 2 and 3. Mechanistic pathways leading to the various products of oxidation of 1 are proposed and the potential roles of oxidation reactions of the indolamine are related to its neurodegenerative properties. PMID:1319496

  13. Blockade of glutamate release by botulinum neurotoxin type A in humans: A dermal microdialysis study

    PubMed Central

    da Silva, Larissa Bittencourt; Karshenas, Ali; Bach, Flemming W; Rasmussen, Sten; Arendt-Nielsen, Lars; Gazerani, Parisa

    2014-01-01

    BACKGROUND: The analgesic action of botulinum neurotoxin type A (BoNTA) has been linked to the blockade of peripheral release of neuropeptides and neurotransmitters in animal models; however, there is no direct evidence of this in humans. OBJECTIVES: To investigate the effect of BoNTA on glutamate release in humans, using an experimental model of pain and sensitization provoked by capsaicin plus mild heat. METHODS: Twelve healthy volunteers (six men, six women) were pretreated with BoNTA (10 U) on the volar forearm and with a saline control on the contralateral side. Dermal microdialysis was applied one week later to collect interstitial samples before and after the application of a capsaicin patch (8%) plus mild heat (40°C/60 min) to provoke glutamate release, pain and vasodilation. Samples were collected every hour for 3 h using linear microdialysis probes (10 mm, 100 kD). Dialysate was analyzed for glutamate concentration. Pain intensity and skin vasomotor reactions (temperature and blood flow changes) were also recorded. RESULTS: BoNTA significantly reduced glutamate release compared with saline (P<0.05). The provoked pain intensity was lower in the BoNTA-pretreated arm (P<0.01). The reduction in pain scores was not correlated with glutamate level. Cutaneous blood flow (P<0.05), but not cutaneous temperature (P≥0.05), was significantly reduced by BoNTA. There was a correlation between glutamate level and skin blood flow (r=0.58/P<0.05) but not skin temperature (P≥0.05). No differences according to sex were observed in any response. CONCLUSIONS: The present study provided the first direct evidence supporting the inhibitory effect of BoNTA on glutamate release in human skin, which is potentially responsible for some of the analgesic action of BoNTA. PMID:24851237

  14. Enhanced insecticidal activity of Chilo iridescent virus expressing an insect specific neurotoxin.

    PubMed

    Nalcacioglu, Remziye; Muratoglu, Hacer; Yesilyurt, Aydın; van Oers, Monique M; Vlak, Just M; Demirbag, Zihni

    2016-07-01

    Previously we have generated a recombinant Chilo iridescent virus (CIV) by inserting the green fluorescent protein gene (gfp) into the CIV 157L open reading frame (ORF) locus and showed that this recombinant (rCIV-Δ157L-gfp) was fully infectious both in cell culture as well as in insect larvae. This study opened up a new avenue for increasing the speed of kill of CIV and other iridoviruses by inserting virulence or toxin genes into the viral genome. In the current study we constructed a recombinant CIV (rCIV-Δ157L/gfp-AaIT) where the 157L ORF was replaced with both the AaIT neurotoxin gene from the scorpion Androctonus australis and the gfp gene, each under control of the viral major capsid protein (mcp) gene promoter. Recombinant virus was purified by successive rounds of plaque purification using Spodoptera frugiperda (Sf-9) cells. One-step growth curves for the recombinant viruses, rCIV-Δ157L/gfp-AaIT and rCIV-Δ157L-gfp, and wild-type CIVs in Sf-9 cells showed similar profiles. AaIT toxin expression in infected third instar Galleria mellonella larvae was confirmed by western blot analysis using an antibody against the AaIT protein. rCIV-Δ157L/gfp-AaIT infection at a concentration that kills 100% of the larvae caused paralysis in infected third instar G. mellonella larvae from two days after injection, whereas infection with non-AaIT containing viruses showed mortality starting much later (>10days). Bioassays on these larvae demonstrated that the speed of kill of CIV carrying AaIT was strikingly enhanced as compared to wild-type CIV. These results suggest that insertion of a toxin gene into CIV provides further opportunities to control a wide range of pest insects, such as weevils, using an iridovirus. PMID:27369385

  15. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library

    PubMed Central

    Wang, Han; Yu, Rui; Fang, Ting; Yu, Ting; Chi, Xiangyang; Zhang, Xiaopeng; Liu, Shuling; Fu, Ling; Yu, Changming; Chen, Wei

    2016-01-01

    Tetanus neurotoxin (TeNT) produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective. PMID:27626445

  16. Tetanus neurotoxin HCC protein commits T cells to IFN-γ producing cells.

    PubMed

    Torabi Goudarzi, S; Hajivalili, M; Hosseini, M; Ghafari Khamene, M; Yazdani, Y; Sadreddini, S; Miahipour, A; Younesi, V; Yousefi, M

    2016-01-01

    A protective response against tetanus toxin and toxoid demands efficient specific T cell and B cell responses. Tetanus neurotoxin (TeNT), a 150 kDa polypeptide, is the main cause of tetanus disease. TeNT consists of two structurally distinct chains, a 50 kDa N-terminal light (L) and a 100 kDa C-terminal heavy (H) chain. C-terminal heavy (H) chain (fragment C) has two sub-domains named as proximal HCN and carboxy sub-domain or HCC. Beside neural binding property, HCC has been recently found as an immunodominant module of TeNT. In the present study, we investigated the effects of recombinant HCC (rHCC) on the expression of lineage specific transcription factors and secretion of a panel of functional cytokines including IFN-γ, IL-4, and IL-17 from purified human T cells. Our results revealed that T-bet transcript level, as TH1 specific transcription factor, was significantly increased in the cells treated with 10 and 20 µg/ml of rHCC following 48 h treatment(p<0.05). Treated purified human T cells with rHCC showed significant increase in IFN-γ mRNA level and cytokine secretion, but not IL-4 and IL-17, following 48 h treatment. In conclusion, our results showed that treatment of T cells with r HCC resulted in development of Th1 lineage phenotype, which might lead to a specific and protective antibody mediated response against tetanus toxin. PMID:27064869

  17. Synergistic capture of Clostridium botulinum Type A neurotoxin by scFv antibodies to novel epitopes

    SciTech Connect

    Gray, Sean A.; Barr, John R.; Kalb, Suzanne R.; Marks, James D.; Baird, Cheryl L.; Cangelosi, Gerard A.; Miller, Keith D.; Feldhaus, Michael J.

    2011-10-01

    A non-immune library of human single chain fragment variable (scFv) antibodies displayed on Saccharomyces cerevisiae was screened for binding to the Clostridium botulinum neurotoxin serotype A binding domain [BoNT/A (Hc)] with the goal of identifying scFv to novel epitopes. To do this, an antibody-mediated labeling strategy was used in which antigen-binding yeast clones were selected after labeling with previously characterized monoclonal antibodies (MAbs) specific to the Hc. Twenty unique scFv clones were isolated that bound Hc. Of these, three also bound to full-length BoNT/A toxin complex with affinities ranging from 5 nM to 170 nM. Epitope binning showed that the three unique clones recognized at least two epitopes that were distinct from one another and from the detection MAbs. After production in E. coli, the scFv were coupled to magnetic particles and tested for their ability to capture BoNT/A holotoxin using an Endopep-MS assay. In this assay, toxin captured by scFv coated magnetic particles was detected by incubation of the complex with a peptide containing a BoNT/A-specific cleavage sequence. Mass spectrometry was used to detect the ratio of intact peptide to cleavage products as evidence for toxin capture. When tested individually, each of the scFv showed a weak positive Endopep-MS result. However, when the particles were coated with all three scFv simultaneously, they exhibited significantly higher Endopep-MS activity, consistent with synergistic binding. These results demonstrate novel approaches toward the isolation and characterization of scFv antibodies specific to unlabeled antigen. They also provide evidence that distinct scFv antibodies can work synergistically to increase the efficiency of antigen capture onto a solid support.

  18. Monoclonal Antibodies Targeting the Alpha-Exosite of Botulinum Neurotoxin Serotype/A Inhibit Catalytic Activity.

    PubMed

    Fan, Yongfeng; Geren, Isin N; Dong, Jianbo; Lou, Jianlong; Wen, Weihua; Conrad, Fraser; Smith, Theresa J; Smith, Leonard A; Ho, Mengfei; Pires-Alves, Melissa; Wilson, Brenda A; Marks, James D

    2015-01-01

    The paralytic disease botulism is caused by botulinum neurotoxins (BoNT), multi-domain proteins containing a zinc endopeptidase that cleaves the cognate SNARE protein, thereby blocking acetylcholine neurotransmitter release. Antitoxins currently used to treat botulism neutralize circulating BoNT but cannot enter, bind to or neutralize BoNT that has already entered the neuron. The light chain endopeptidase domain (LC) of BoNT serotype A (BoNT/A) was targeted for generation of monoclonal antibodies (mAbs) that could reverse paralysis resulting from intoxication by BoNT/A. Single-chain variable fragment (scFv) libraries from immunized humans and mice were displayed on the surface of yeast, and 19 BoNT/A LC-specific mAbs were isolated by using fluorescence-activated cell sorting (FACS). Affinities of the mAbs for BoNT/A LC ranged from a KD value of 9.0×10-11 M to 3.53×10-8 M (mean KD 5.38×10-9 M and median KD 1.53×10-9 M), as determined by flow cytometry analysis. Eleven mAbs inhibited BoNT/A LC catalytic activity with IC50 values ranging from 8.3 ~73×10-9 M. The fine epitopes of selected mAbs were also mapped by alanine-scanning mutagenesis, revealing that the inhibitory mAbs bound the α-exosite region remote from the BoNT/A LC catalytic center. The results provide mAbs that could prove useful for intracellular reversal of paralysis post-intoxication and further define epitopes that could be targeted by small molecule inhibitors.

  19. Monoclonal Antibodies Targeting the Alpha-Exosite of Botulinum Neurotoxin Serotype/A Inhibit Catalytic Activity

    PubMed Central

    Fan, Yongfeng; Geren, Isin N.; Dong, Jianbo; Lou, Jianlong; Wen, Weihua; Conrad, Fraser; Smith, Theresa J.; Smith, Leonard A.; Ho, Mengfei; Pires-Alves, Melissa; Wilson, Brenda A.; Marks, James D.

    2015-01-01

    The paralytic disease botulism is caused by botulinum neurotoxins (BoNT), multi-domain proteins containing a zinc endopeptidase that cleaves the cognate SNARE protein, thereby blocking acetylcholine neurotransmitter release. Antitoxins currently used to treat botulism neutralize circulating BoNT but cannot enter, bind to or neutralize BoNT that has already entered the neuron. The light chain endopeptidase domain (LC) of BoNT serotype A (BoNT/A) was targeted for generation of monoclonal antibodies (mAbs) that could reverse paralysis resulting from intoxication by BoNT/A. Single-chain variable fragment (scFv) libraries from immunized humans and mice were displayed on the surface of yeast, and 19 BoNT/A LC-specific mAbs were isolated by using fluorescence-activated cell sorting (FACS). Affinities of the mAbs for BoNT/A LC ranged from a KD value of 9.0×10−11 M to 3.53×10−8 M (mean KD 5.38×10−9 M and median KD 1.53×10−9 M), as determined by flow cytometry analysis. Eleven mAbs inhibited BoNT/A LC catalytic activity with IC50 values ranging from 8.3 ~73×10−9 M. The fine epitopes of selected mAbs were also mapped by alanine-scanning mutagenesis, revealing that the inhibitory mAbs bound the α-exosite region remote from the BoNT/A LC catalytic center. The results provide mAbs that could prove useful for intracellular reversal of paralysis post-intoxication and further define epitopes that could be targeted by small molecule inhibitors. PMID:26275214

  20. Ultrafiltration improves ELISA and Endopep MS analysis of botulinum neurotoxin type A in drinking water.

    PubMed

    Raphael, Brian H; Lautenschlager, Matthew; Kahler, Amy; Pai, Suresh; Parks, Bryan A; Kalb, Suzanne R; Maslanka, Susan E; Shah, Sanjiv; Magnuson, Matthew; Hill, Vincent R

    2012-09-01

    The objective of this study was to adapt and evaluate two in vitro botulinum neurotoxin (BoNT) detection methods, including the Botulinum Toxin ELISA and the Endopep MS (a mass spectrometric-based endopeptidase method), for use with drinking water samples. The method detection limits (MDL) of the ELISA and Endopep MS were 260 pg/mL and 21 pg/mL of BoNT/A complex toxin, respectively. Since toxin could be present in water samples at highly dilute concentrations, large volume (100-L) samples of municipal tap water from five US municipalities having distinct water compositions were dechlorinated, spiked with 5 μg BoNT/A, and subjected to tangential-flow ultrafiltration (UF) using hollow fiber dialyzers. The recovery efficiency of BoNT/A using UF and quantified by ELISA ranged from 11% to 36% while efficiencies quantified by MS ranged from 26% to 55%. BoNT/A was shown to be stable in dechlorinated municipal tap water stored at 4°C for up to four weeks. In addition, toxin present in UF-concentrated water samples was also shown to be stable at 4°C for up to four weeks, allowing holding of samples prior to analysis. Finally, UF was used to concentrate a level of toxin (7 pg/mL) which is below the MDL for direct analysis by both ELISA and Endopep MS. Following UF, toxin was detectable in these samples using both in vitro analysis methods. These data demonstrate that UF-concentration of toxin from large volume water samples followed by use of existing analytical methods for detection of BoNT/A can be used in support of a monitoring program for contaminants in drinking water.

  1. "BINACLE" assay for in vitro detection of active tetanus neurotoxin in toxoids.

    PubMed

    Behrensdorf-Nicol, Heike A; Weisser, Karin; Krämer, Beate

    2015-01-01

    Tetanus neurotoxin (TeNT) consists of two protein chains connected by a disulfide linkage: The heavy chain mediates the toxin binding and uptake by neurons, whereas the light chain cleaves synaptobrevin and thus blocks neurotransmitter release.Chemically inactivated TeNT (tetanus toxoid) is utilized for the production of tetanus vaccines. For safety reasons, each toxoid bulk has to be tested for the "absence of toxin and irreversibility of toxoid". To date, these mandatory tests are performed as toxicity tests in guinea pigs. A replacement by an animal-free method for the detection of TeNT would be desirable. The BINACLE (BINding And CLEavage) assay takes into account the receptor-binding as well as the proteolytic characteristics of TeNT: The toxin is bound to immobilized receptor molecules, the light chains are then released by reduction and transferred to a microplate containing synaptobrevin, and the fragment resulting from TeNT-induced cleavage is finally detected. This assay offers a higher specificity for discriminating between toxic TeNT and inactivated toxoid molecules than other published assays. Validation studies have shown that the BINACLE assay allows the sensitive and robust detection of TeNT in toxoids, and thus may indeed represent a suitable alternative to the prescribed animal safety tests for toxoids from several European vaccine manufacturers. Product-specific validations (and possibly adaptations) of the assay protocol will be required. A European collaborative study is currently being initiated to further examine the applicability of the method for toxoid testing. The final aim is the inclusion of the method into the European Pharmacopoeia.

  2. Structural Analysis of the Receptor Binding Domain of Botulinum Neurotoxin Serotype D

    SciTech Connect

    Y Zhang; G Buchko; L Qin; H Robinson; S Varnum

    2011-12-31

    Botulinum neurotoxins (BoNTs) are the most toxic proteins known. The mechanism for entry into neuronal cells for serotypes A, B, E, F, and G involves a well understood dual receptor (protein and ganglioside) process, however, the mechanism of entry for serotypes C and D remains unclear. To provide structural insights into how BoNT/D enters neuronal cells, the crystal structure of the receptor binding domain (S863-E1276) for this serotype (BoNT/D-HCR) was determined at 1.65{angstrom} resolution. While BoNT/D-HCR adopts an overall fold similar to that observed in other known BoNT HCRs, several major structural differences are present. These structural differences are located at, or near, putative receptor binding sites and may be responsible for BoNT/D host preferences. Two loops, S1195-I1204 and K1236-N1244, located on both sides of the putative protein receptor binding pocket, are displaced >10{angstrom} relative to the corresponding residues in the crystal structures of BoNT/B and G. Obvious clashes were observed in the putative protein receptor binding site when the BoNT/B protein receptor synaptotagmin II was modeled into the BoNT/D-HCR structure. Although a ganglioside binding site has never been unambiguously identified in BoNT/D-HCR, a shallow cavity in an analogous location to the other BoNT serotypes HCR domains is observed in BoNT/D-HCR that has features compatible with membrane binding. A portion of a loop near the putative receptor binding site, K1236-N1244, is hydrophobic and solvent-exposed and may directly bind membrane lipids. Liposome-binding experiments with BoNT/D-HCR demonstrate that this membrane lipid may be phosphatidylethanolamine.

  3. Botulinum Neurotoxins: Qualitative and Quantitative Analysis Using the Mouse Phrenic Nerve Hemidiaphragm Assay (MPN).

    PubMed

    Bigalke, Hans; Rummel, Andreas

    2015-11-25

    The historical method for the detection of botulinum neurotoxin (BoNT) is represented by the mouse bioassay (MBA) measuring the animal survival rate. Since the endpoint of the MBA is the death of the mice due to paralysis of the respiratory muscle, an ex vivo animal replacement method, called mouse phrenic nerve (MPN) assay, employs the isolated N. phrenicus-hemidiaphragm tissue. Here, BoNT causes a dose-dependent characteristic decrease of the contraction amplitude of the indirectly stimulated muscle. Within the EQuATox BoNT proficiency 13 test samples were analysed using the MPN assay by serial dilution to a bath concentration resulting in a paralysis time within the range of calibration curves generated with BoNT/A, B and E standards, respectively. For serotype identification the diluted samples were pre-incubated with polyclonal anti-BoNT/A, B or E antitoxin or a combination of each. All 13 samples were qualitatively correctly identified thereby delivering superior results compared to single in vitro methods like LFA, ELISA and LC-MS/MS. Having characterized the BoNT serotype, the final bath concentrations were calculated using the calibration curves and then multiplied by the respective dilution factor to obtain the sample concentration. Depending on the source of the BoNT standards used, the quantitation of ten BoNT/A containing samples delivered a mean z-score of 7 and of three BoNT/B or BoNT/E containing samples z-scores <2, respectively.

  4. Structural analysis of the receptor binding domain of botulinum neurotoxin serotype D

    SciTech Connect

    Zhang, Yanfeng; Buchko, Garry W.; Qin, Lin; Robinson, Howard; Varnum, Susan M.

    2010-10-28

    Botulinum neurotoxins (BoNTs) are the most toxic proteins known. The mechanism for entry into neuronal cells for serotypes A, B, E, F, and G involves a well understood dual receptor (protein and ganglioside) process, however, the mechanism of entry for serotypes C and D remains unclear. To provide structural insights into how BoNT/D enters neuronal cells, the crystal structure of the receptor binding domain (S863-E1276) for this serotype (BoNT/D-HCR) was determined at 1.65 Å resolution. While BoNT/D-HCR adopts an overall fold similar to that observed in other known BoNT HCRs, several major structural differences are present. These structural differences are located at, or near, putative receptor binding sites and may be responsible for BoNT/D host preferences. Two loops, S1195-I1204 and K1236-N1244, located on both sides of the putative protein receptor binding pocket, are displaced >10 Å relative to the corresponding residues in the crystal structures of BoNT/B and G. Obvious clashes were observed in the putative protein receptor binding site when the BoNT/B protein receptor synaptotagmin II was modeled into the BoNT/D-HCR structure. Although a ganglioside binding site has never been unambiguously identified in BoNT/D-HCR, a shallow cavity in an analogous location to the other BoNT serotypes HCR domains is observed in BoNT/D-HCR that has features compatible with membrane binding. A portion of a loop near the putative receptor binding site, K1236-N1244, is hydrophobic and solvent-exposed and may directly bind membrane lipids. Liposome-binding experiments with BoNT/D-HCR demonstrate that this membrane lipid may be phosphatidylethanolamine.

  5. Predicting Improvement in Writer’s Cramp Symptoms following Botulinum Neurotoxin Injection Therapy

    PubMed Central

    Jackman, Mallory; Delrobaei, Mehdi; Rahimi, Fariborz; Atashzar, S. Farokh; Shahbazi, Mahya; Patel, Rajni; Jog, Mandar

    2016-01-01

    Introduction Writer’s cramp is a specific focal hand dystonia causing abnormal posturing and tremor in the upper limb. The most popular medical intervention, botulinum neurotoxin type A (BoNT-A) therapy, is variably effective for 50–70% of patients. BoNT-A non-responders undergo ineffective treatment and may experience significant side effects. Various assessments have been used to determine response prediction to BoNT-A, but not in the same population of patients. Methods A comprehensive assessment was employed to measure various symptom aspects. Clinical scales, full upper-limb kinematic measures, self-report, and task performance measures were assessed for nine writer’s cramp patients at baseline. Patients received two BoNT-A injections then were classified as responders or non-responders based on a quantified self-report measure. Baseline scores were compared between groups, across all measures, to determine which scores predicted a positive BoNT-A response. Results Five of nine patients were responders. No kinematic measures were predictably different between groups. Analyses revealed three features that predicted a favorable response and separated the two groups: higher than average cramp severity and cramp frequency, and below average cramp latency. Discussion Non-kinematic measures appear to be superior in making such predictions. Specifically, measures of cramp severity, frequency, and latency during performance of a specific set of writing and drawing tasks were predictive factors. Since kinematic was not used to determine the injection pattern and the injections were visually guided, it may still be possible to use individual patient kinematics for better outcomes.

  6. Botulinum and Tetanus Neurotoxin-Induced Blockade of Synaptic Transmission in Networked Cultures of Human and Rodent Neurons.

    PubMed

    Beske, Phillip H; Bradford, Aaron B; Grynovicki, Justin O; Glotfelty, Elliot J; Hoffman, Katie M; Hubbard, Kyle S; Tuznik, Kaylie M; McNutt, Patrick M

    2016-02-01

    Clinical manifestations of tetanus and botulism result from an intricate series of interactions between clostridial neurotoxins (CNTs) and nerve terminal proteins that ultimately cause proteolytic cleavage of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins and functional blockade of neurotransmitter release. Although detection of cleaved SNARE proteins is routinely used as a molecular readout of CNT intoxication in cultured cells, impaired synaptic function is the pathophysiological basis of clinical disease. Work in our laboratory has suggested that the blockade of synaptic neurotransmission in networked neuron cultures offers a phenotypic readout of CNT intoxication that more closely replicates the functional endpoint of clinical disease. Here, we explore the value of measuring spontaneous neurotransmission frequencies as novel and functionally relevant readouts of CNT intoxication. The generalizability of this approach was confirmed in primary neuron cultures as well as human and mouse stem cell-derived neurons exposed to botulinum neurotoxin serotypes A-G and tetanus neurotoxin. The sensitivity and specificity of synaptic activity as a reporter of intoxication was evaluated in assays representing the principal clinical and research purposes of in vivo studies. Our findings confirm that synaptic activity offers a novel and functionally relevant readout for the in vitro characterizations of CNTs. They further suggest that the analysis of synaptic activity in neuronal cell cultures can serve as a surrogate for neuromuscular paralysis in the mouse lethal assay, and therefore is expected to significantly reduce the need for terminal animal use in toxin studies and facilitate identification of candidate therapeutics in cell-based screening assays.

  7. Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

    PubMed Central

    Hubbard, Kyle; Beske, Phillip; Lyman, Megan; McNutt, Patrick

    2015-01-01

    Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, cell-based model that produces functioning synapses and undergoes physiological responses to intoxication. Here we describe a simple and robust method to efficiently differentiate murine embryonic stem cells (ESCs) into defined lineages of synaptically active, networked neurons. Following an 8 day differentiation protocol, mouse embryonic stem cell-derived neurons (ESNs) rapidly express and compartmentalize neurotypic proteins, form neuronal morphologies and develop intrinsic electrical responses. By 18 days after differentiation (DIV 18), ESNs exhibit active glutamatergic and γ-aminobutyric acid (GABA)ergic synapses and emergent network behaviors characterized by an excitatory:inhibitory balance. To determine whether intoxication with CNTs functionally antagonizes synaptic neurotransmission, thereby replicating the in vivo pathophysiology that is responsible for clinical manifestations of botulism or tetanus, whole-cell patch clamp electrophysiology was used to quantify spontaneous miniature excitatory post-synaptic currents (mEPSCs) in ESNs exposed to tetanus neurotoxin (TeNT) or botulinum neurotoxin (BoNT) serotypes /A-/G. In all cases, ESNs exhibited near-complete loss of synaptic activity within 20 hr. Intoxicated neurons remained viable, as demonstrated by unchanged resting membrane potentials and intrinsic electrical responses. To further characterize the sensitivity of this approach, dose-dependent effects of intoxication on synaptic activity were measured 20 hr after addition of BoNT/A. Intoxication with 0.005 pM BoNT/A resulted in a significant decrement in mEPSCs, with a median inhibitory concentration (IC50) of 0.013 pM. Comparisons of median doses indicate that functional measurements of synaptic inhibition are faster, more specific and more sensitive than SNARE cleavage assays or the mouse lethality assay

  8. Highly toxic Microcystis aeruginosa strain, isolated from São Paulo-Brazil, produce hepatotoxins and paralytic shellfish poison neurotoxins.

    PubMed

    Sant'Anna, Célia L; de Carvalho, Luciana R; Fiore, Marli F; Silva-Stenico, Maria Estela; Lorenzi, Adriana S; Rios, Fernanda R; Konno, Katsuhiro; Garcia, Carlos; Lagos, Nestor

    2011-04-01

    While evaluating several laboratory-cultured cyanobacteria strains for the presence of paralytic shellfish poison neurotoxins, the hydrophilic extract of Microcystis aeruginosa strain SPC777--isolated from Billings's reservoir, São Paulo, Brazil--was found to exhibit lethal neurotoxic effect in mouse bioassay. The in vivo test showed symptoms that unambiguously were those produced by PSP. In order to identify the presence of neurotoxins, cells were lyophilized, and the extracts were analyzed by HPLC-FLD and HPLC-MS. HPLC-FLD analysis revealed four main Gonyautoxins: GTX4(47.6%), GTX2(29.5%), GTX1(21.9%), and GTX3(1.0%). HPLC-MS analysis, on other hand, confirmed both epimers, with positive Zwitterions M(+) 395.9 m/z for GTX3/GTX2 and M(+) 411 m/z for GTX4/GTX1 epimers.The hepatotoxins (Microcystins) were also evaluated by ELISA and HPLC-MS analyses. Positive immunoreaction was observed by ELISA assay. Alongside, the HPLC-MS analyses revealed the presence of [L: -ser(7)] MCYST-RR. The N-methyltransferase (NMT) domain of the microcystin synthetase gene mcyA was chosen as the target sequence to detect the presence of the mcy gene cluster. PCR amplification of the NMT domain, using the genomic DNA of the SPC777 strain and the MSF/MSR primer set, resulted in the expected 1,369 bp product. The phylogenetic analyses grouped the NMT sequence with the NMT sequences of other known Microcystis with high bootstrap support. The taxonomical position of M. aeruginosa SPC777 was confirmed by a detailed morphological description and a phylogenetic analysis of 16S rRNA gene sequence. Therefore, co-production of PSP neurotoxins and microcystins by an isolated M. aeruginosa strain is hereby reported for the first time.

  9. Botulinum and Tetanus Neurotoxin-Induced Blockade of Synaptic Transmission in Networked Cultures of Human and Rodent Neurons.

    PubMed

    Beske, Phillip H; Bradford, Aaron B; Grynovicki, Justin O; Glotfelty, Elliot J; Hoffman, Katie M; Hubbard, Kyle S; Tuznik, Kaylie M; McNutt, Patrick M

    2016-02-01

    Clinical manifestations of tetanus and botulism result from an intricate series of interactions between clostridial neurotoxins (CNTs) and nerve terminal proteins that ultimately cause proteolytic cleavage of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins and functional blockade of neurotransmitter release. Although detection of cleaved SNARE proteins is routinely used as a molecular readout of CNT intoxication in cultured cells, impaired synaptic function is the pathophysiological basis of clinical disease. Work in our laboratory has suggested that the blockade of synaptic neurotransmission in networked neuron cultures offers a phenotypic readout of CNT intoxication that more closely replicates the functional endpoint of clinical disease. Here, we explore the value of measuring spontaneous neurotransmission frequencies as novel and functionally relevant readouts of CNT intoxication. The generalizability of this approach was confirmed in primary neuron cultures as well as human and mouse stem cell-derived neurons exposed to botulinum neurotoxin serotypes A-G and tetanus neurotoxin. The sensitivity and specificity of synaptic activity as a reporter of intoxication was evaluated in assays representing the principal clinical and research purposes of in vivo studies. Our findings confirm that synaptic activity offers a novel and functionally relevant readout for the in vitro characterizations of CNTs. They further suggest that the analysis of synaptic activity in neuronal cell cultures can serve as a surrogate for neuromuscular paralysis in the mouse lethal assay, and therefore is expected to significantly reduce the need for terminal animal use in toxin studies and facilitate identification of candidate therapeutics in cell-based screening assays. PMID:26615023

  10. [Botulinum neurotoxin type A in neurogenic detrusor overactivity: consensus paper of the Working Group Neuro-Urology of the DMGP].

    PubMed

    Böthig, R; Kaufmann, A; Bremer, J; Pannek, J; Domurath, B

    2014-04-01

    The use of botulinum neurotoxin (BoNT-A) for suppression of neurogenic detrusor overactivity was first reported in 2000. Since that time, this method has gained widespread use. A number of recommendations and consensus statements have already been published. The current practice-oriented consensus paper takes into account recent developments and the over 10-year experience of most members of the Working Group Neuro-Urology of the German-speaking Medical Society for Paraplegia (DMGP) with a focus on the use of BoNT-A in paraplegic patients and in patients with multiple sclerosis. PMID:24604016

  11. [Localization of binding sites in rat muscles and brain for cobra neurotoxin and serum immunoglobulins from patients with myasthenia].

    PubMed

    Smirnov, V A; Vladeeva, N V; Lobzin, V S; Paniukov, A N

    1986-09-01

    Using the immunohistochemical technique, it was revealed that serum immunoglobulins of patients with myasthenia gravis (Ig) were irreversibly attached to the myoneuronal connections of the rat intercostal muscles like the marker of the nicotin cholinireceptors--the cobra venom neurotoxin (CT). In addition, Ig differs from CT in the binding to nervous cells of the claustrum and diencephalon reticular formation and with certain cells of the nucleus caudatus and hemispheric cerebral cortex. It is suggested that the autoimmune processes in patients with myasthenia gravis do not only involve myoneuronal connection but also participate in central mechanisms of the disease genesis. PMID:3756323

  12. Detection of Type A, B, E, and F Clostridium botulinum Neurotoxins in Foods by Using an Amplified Enzyme-Linked Immunosorbent Assay with Digoxigenin-Labeled Antibodies

    PubMed Central

    Sharma, Shashi K.; Ferreira, Joseph. L.; Eblen, Brian S.; Whiting, Richard C.

    2006-01-01

    An amplified enzyme-linked immunosorbent assay (ELISA) for the detection of Clostridium botulinum complex neurotoxins was evaluated for its ability to detect these toxins in food. The assay was found to be suitable for detecting type A, B, E, and F botulinum neurotoxins in a variety of food matrices representing liquids, solid, and semisolid food. Specific foods included broccoli, orange juice, bottled water, cola soft drinks, vanilla extract, oregano, potato salad, apple juice, meat products, and dairy foods. The detection sensitivity of the test for these botulinum complex serotypes was found to be 60 pg/ml (1.9 50% lethal dose [LD50]) for botulinum neurotoxin type A (BoNT/A), 176 pg/ml (1.58 LD50) for BoNT/B, 163 pg/ml for BoNT/E (4.5 LD50), and 117 pg/ml for BoNT/F (less than 1 LD50) in casein buffer. The test could also readily detect 2 ng/ml of neurotoxins type A, B, E, and F in a variety of food samples. For specificity studies, the assay was also used to test a large panel of type A C. botulinum, a smaller panel of proteolytic and nonproteolytic type B, E, and F neurotoxin-producing Clostridia, and nontoxigenic organisms using an overnight incubation of toxin production medium. The assay appears to be an effective tool for large-scale screening of the food supply in the event of a botulinum neurotoxin contamination event. PMID:16461671

  13. POTENT Reconstruction from Mark III Velocities

    NASA Astrophysics Data System (ADS)

    Dekel, A.; Eldar, A.; Kolatt, T.; Yahil, A.; Willick, J. A.; Faber, S. M.; Courteau, S.; Burstein, D.

    1999-09-01

    We present an improved version of the POTENT method for reconstructing the cosmological velocity and mass density fields from radial peculiar velocities, test it with mock catalogs, and apply it to the Mark III Catalog of Galaxy Peculiar Velocities. The method is improved in several ways: (1) the inhomogeneous Malmquist bias is reduced by grouping and corrected statistically in either forward or inverse analyses of inferred distances, (2) the smoothing into a radial velocity field is optimized such that window and sampling biases are reduced, (3) the density field is derived from the velocity field using an improved weakly nonlinear approximation in Eulerian space, and (4) the computational errors are made negligible compared to the other errors. The method is carefully tested and optimized using realistic mock catalogs based on an N-body simulation that mimics our cosmological neighborhood, and the remaining systematic and random errors are evaluated quantitatively. The Mark III catalog, with ~3300 grouped galaxies, allows a reliable reconstruction with fixed Gaussian smoothing of 10-12 h-1 Mpc out to ~60 h-1 Mpc and beyond in some directions. We present maps of the three-dimensional velocity and mass-density fields and the corresponding errors. The typical systematic and random errors in the density fluctuations inside 40 h-1 Mpc are +/-0.13 and +/-0.18 (for Ω=1). In its gross features, the recovered mass distribution resembles the galaxy distribution in redshift surveys and the mass distribution in a similar POTENT analysis of a complementary velocity catalog (SFI), including such features as the Great Attractor, Perseus-Pisces, and the large void in between. The reconstruction inside ~40 h-1 Mpc is not affected much by a revised calibration of the distance indicators (VM2, tailored to match the velocities from the IRAS 1.2 Jy redshift survey). The volume-weighted bulk velocity within the sphere of radius 50 h-1 Mpc about the Local Group is V50=370+/-110 km s-1

  14. Potent effects of dioscin against liver fibrosis

    PubMed Central

    Zhang, Xiaoling; Han, Xu; Yin, Lianhong; Xu, Lina; Qi, Yan; Xu, Youwei; Sun, Huijun; Lin, Yuan; Liu, Kexin; Peng, Jinyong

    2015-01-01

    We previously reported the promising effects of dioscin against liver injury, but its effect on liver fibrosis remains unknown. The present work investigated the activities of dioscin against liver fibrosis and the underlying molecular mechanisms. Dioscin effectively inhibited the cell viabilities of HSC-T6, LX-2 and primary rat hepatic stellate cells (HSCs), but not hepatocytes. Furthermore, dioscin markedly increased peroxisome proliferator activated receptor-γ (PPAR-γ) expression and significantly reduced a-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1), collagen α1 (I) (COL1A1) and collagen α1 (III) (COL3A1) levels in vitro. Notably, dioscin inhibited HSCs activation and induced apoptosis in activated HSCs. In vivo, dioscin significantly improved body weight and hydroxylproline, laminin, α-SMA, TGF-β1, COL1A1 and COL3A1 levels, which were confirmed by histopathological assays. Dioscin facilitated matrix degradation, and exhibited hepatoprotective effects through the attenuation of oxidative stress and inflammation, in addition to exerting anti-fibrotic effects through the modulation of the TGF-β1/Smad, Wnt/β-catenin, mitogen-activated protein kinase (MAPK) and mitochondrial signaling pathways, which triggered the senescence of activated HSCs. In conclusion, dioscin exhibited potent effects against liver fibrosis through the modulation of multiple targets and signaling pathways and should be developed as a novel candidate for the treatment of liver fibrosis in the future. PMID:25853178

  15. Potent effects of dioscin against liver fibrosis.

    PubMed

    Zhang, Xiaoling; Han, Xu; Yin, Lianhong; Xu, Lina; Qi, Yan; Xu, Youwei; Sun, Huijun; Lin, Yuan; Liu, Kexin; Peng, Jinyong

    2015-01-01

    We previously reported the promising effects of dioscin against liver injury, but its effect on liver fibrosis remains unknown. The present work investigated the activities of dioscin against liver fibrosis and the underlying molecular mechanisms. Dioscin effectively inhibited the cell viabilities of HSC-T6, LX-2 and primary rat hepatic stellate cells (HSCs), but not hepatocytes. Furthermore, dioscin markedly increased peroxisome proliferator activated receptor-γ (PPAR-γ) expression and significantly reduced a-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1), collagen α1 (I) (COL1A1) and collagen α1 (III) (COL3A1) levels in vitro. Notably, dioscin inhibited HSCs activation and induced apoptosis in activated HSCs. In vivo, dioscin significantly improved body weight and hydroxylproline, laminin, α-SMA, TGF-β1, COL1A1 and COL3A1 levels, which were confirmed by histopathological assays. Dioscin facilitated matrix degradation, and exhibited hepatoprotective effects through the attenuation of oxidative stress and inflammation, in addition to exerting anti-fibrotic effects through the modulation of the TGF-β1/Smad, Wnt/β-catenin, mitogen-activated protein kinase (MAPK) and mitochondrial signaling pathways, which triggered the senescence of activated HSCs. In conclusion, dioscin exhibited potent effects against liver fibrosis through the modulation of multiple targets and signaling pathways and should be developed as a novel candidate for the treatment of liver fibrosis in the future. PMID:25853178

  16. Three-dimensional ordered macroporous (3DOM) composite for electrochemical study on acetylcholinesterase inhibition induced by endogenous neurotoxin.

    PubMed

    Teng, Yingqiao; Fu, Ying; Xu, Lili; Lin, Bin; Wang, Zhongchuan; Xu, Zhiai; Jin, Litong; Zhang, Wen

    2012-09-13

    In this paper, an electrochemical acetylcholinesterase (AChE) inhibition assay based on three-dimensional ordered macroporous (3DOM) composite was conducted. The 3DOM composite was first fabricated on the glassy carbon electrode by electropolymerization of aniline in the presence of ionic liquid (IL) on a sacrificial silica nanospheres template. After the silica nanospheres were etched, an IL-doped polyaniline (IL-PANI) film with 3DOM morphology was formed. Then, gold nanoparticles (AuNPs) were decorated on the IL-PANI film by electrodeposition. The immobilized AChE on the 3DOM composite displayed favorable affinity to substrate acetylthiocholine chloride (ATCh), and the 3DOM composite showed excellent electrocatalytic effect on thiocholine, the hydrolysis product of ATCh. The presence of IL and AuNPs could improve the sensitivity by accelerating the electron transfer. The designed AChE biosensor was successfully applied to evaluate the AChE inhibition induced by endogenous neurotoxin 1(R),2N-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline [(R)-NMSal]. The results demonstrate that (R)-NMSal exerts a considerable effect on AChE activity, and the inhibition is reversible. The developed method offers a new approach for AChE inhibition assay, which is of great benefit in understanding the mechanism behind neurotoxin-induced neurodegenerative disorders.

  17. Neurotoxin-induced DNA damage is persistent in SH-SY5Y cells and LC neurons.

    PubMed

    Wang, Yan; Musich, Phillip R; Cui, Kui; Zou, Yue; Zhu, Meng-Yang

    2015-05-01

    Degeneration of the noradrenergic neurons has been reported in the brain of patients suffering from neurodegenerative diseases. However, their pathological characteristics during the neurodegenerative course and underlying mechanisms remain to be elucidated. In the present study, we used the neurotoxin camptothecin (CPT) to induce the DNA damage response in neuroblastoma SH-SY5Y cells, normal fibroblast cells, and primarily cultured locus coeruleus (LC) and raphe neurons to examine cellular responses and repair capabilities after neurotoxin exposure. To our knowledge, the present study is the first to show that noradrenergic SH-SY5Y cells are more sensitive to CPT-induced DNA damage and deficient in DNA repair, as compared to fibroblast cells. Furthermore, similar to SH-SY5Y cells, primarily cultured LC neurons are more sensitive to CPT-induced DNA damage and show a deficiency in repairing this damage. Moreover, while N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) exposure also results in DNA damage in cultured LC neurons, neither CPT nor DSP4 induce DNA damage in neuronal cultures from the raphe nuclei. Taken together, noradrenergic SH-SY5Y cells and LC neurons are sensitive to CPT-induced DNA damage and exhibit a repair deficiency, providing a mechanistic explanation for the pathological characteristics of LC degeneration when facing endogenous and environmental DNA-damaging insults in vivo.

  18. Characterization and immunological activity of different forms of recombinant secreted Hc of botulinum neurotoxin serotype B products expressed in yeast.

    PubMed

    Liu, Bo; Shi, DanYang; Chang, ShaoHong; Gong, Xin; Yu, YunZhou; Sun, ZhiWei; Wu, Jun

    2015-01-01

    The recombinant Hc proteins of botulinum neurotoxins and tetanus toxin are exclusively produced by intracellular heterologous expression in Pichia pastoris for use in subunit vaccines; the same Hc proteins produced by secreted heterologous expression are hyper-glycosylated and immunologically inert. Here, several different recombinant secreted Hc proteins of botulinum neurotoxin serotype B (BHc) were expressed in yeast and we characterized and assessed their immunological activity in detail. Recombinant low-glycosylated secreted BHc products (BSK) were also immunologically inert, similar to hyper-glycosylated BHc products (BSG), although deglycosylation restored their immunological activities. Unexpectedly, deglycosylated proBHc contained an unexpected pro-peptide of an α-factor signal and fortuitous N-linked glycosylation sites in the non-cleaved pro-peptide sequences, but not in the BHc sequences. Notably, a non-glycosylated secreted homogeneous BHc isoform (mBHc), which we successfully prepared after deleting the pro-peptide and removing its single potential glycosylation site, was immunologically active and could confer effective protective immunity, similarly to non-glycosylated rBHc. In summary, we conclude that a non-glycosylated secreted BHc isoform can be prepared in yeast by deleting the pro-peptide of the α-factor signal and mutating its single potential glycosylation site. This approach provides a rational and feasible strategy for the secretory expression of botulism or other toxin antigens. PMID:25567004

  19. Cloning and characterization of an alpha-neurotoxin-type protein specific for the coral snake Micrurus corallinus.

    PubMed

    Silveira de Oliveira, J; Rossan de Brandão Prieto da Silva, A; Soares, M B; Stephano, M A; de Oliveira Dias, W; Raw, I; Ho, P L

    2000-01-27

    During the cloning of abundant cDNAs expressed in the Micrurus corallinus coral snake venom gland, we cloned an alpha-neurotoxin homologue cDNA (nxh1). Two others isoforms were also cloned (nxh3 and nxh7, respectively). The nxh1 cDNA codes for a potential coral snake toxin with a signal peptide of 21 amino acids plus a predicted mature peptide with 57 amino acids. The deduced protein is highly similar to known toxic three-finger alpha-neurotoxins, with four deduced S-S bridges at the same conserved positions. This is the first cDNA coding for a three-finger related protein described so far for coral snakes. However, the predicted protein does not possess some of the important amino acids for the nicotinic acetylcholine receptor interaction. This protein was expressed in Escherichia coli as a His-tagged protein that allowed the rapid purification of the recombinant protein. This protein was used to generate antibodies which recognized the recombinant protein in Western blot and also a single band present in the M. corallinus venom, but not in the venom of 10 other Micrurus species.

  20. The biological activity of botulinum neurotoxin type C is dependent upon novel types of ganglioside binding sites.

    PubMed

    Strotmeier, Jasmin; Gu, Shenyan; Jutzi, Stephan; Mahrhold, Stefan; Zhou, Jie; Pich, Andreas; Eichner, Timo; Bigalke, Hans; Rummel, Andreas; Jin, Rongsheng; Binz, Thomas

    2011-07-01

    The seven botulinum neurotoxins (BoNT) cause muscle paralysis by selectively cleaving core components of the vesicular fusion machinery. Their extraordinary activity primarily relies on highly specific entry into neurons. Data on BoNT/A, B, E, F and G suggest that entry follows a dual receptor interaction with complex gangliosides via an established ganglioside binding region and a synaptic vesicle protein. Here, we report high resolution crystal structures of the BoNT/C cell binding fragment alone and in complex with sialic acid. The WY-motif characteristic of the established ganglioside binding region was located on an exposed loop. Sialic acid was co-ordinated at a novel position neighbouring the binding pocket for synaptotagmin in BoNT/B and G and the sialic acid binding site in BoNT/D and TeNT respectively. Employing synaptosomes and immobilized gangliosides binding studies with BoNT/C mutants showed that the ganglioside binding WY-loop, the newly identified sialic acid-co-ordinating pocket and the area corresponding to the established ganglioside binding region of other BoNTs are involved in ganglioside interaction. Phrenic nerve hemidiaphragm activity tests employing ganglioside deficient mice furthermore evidenced that the biological activity of BoNT/C depends on ganglioside interaction with at least two binding sites. These data suggest a unique cell binding and entry mechanism for BoNT/C among clostridial neurotoxins. PMID:21542861

  1. Development of a Highly Sensitive Cell-Based Assay for Detecting Botulinum Neurotoxin Type A through Neural Culture Media Optimization.

    PubMed

    Hong, Won S; Pezzi, Hannah M; Schuster, Andrea R; Berry, Scott M; Sung, Kyung E; Beebe, David J

    2016-01-01

    Botulinum neurotoxin (BoNT) is the most lethal naturally produced neurotoxin. Due to the extreme toxicity, BoNTs are implicated in bioterrorism, while the specific mechanism of action and long-lasting effect was found to be medically applicable in treating various neurological disorders. Therefore, for both public and patient safety, a highly sensitive, physiologic, and specific assay is needed. In this paper, we show a method for achieving a highly sensitive cell-based assay for BoNT/A detection using the motor neuron-like continuous cell line NG108-15. To achieve high sensitivity, we performed a media optimization study evaluating three commercially available neural supplements in combination with retinoic acid, purmorphamine, transforming growth factor β1 (TGFβ1), and ganglioside GT1b. We found nonlinear combinatorial effects on BoNT/A detection sensitivity, achieving an EC50 of 7.4 U ± 1.5 SD (or ~7.9 pM). The achieved detection sensitivity is comparable to that of assays that used primary and stem cell-derived neurons as well as the mouse lethality assay.

  2. Toxic effects of potential environmental neurotoxins related to 1-methyl-4-phenylpyridinium on cultured rat dopaminergic neurons

    SciTech Connect

    Michel, P.P.; Dandapani, B.K.; Sanchez-Ramos, J.; Efange, S.; Pressman, B.C.; Hefti, F.

    1989-02-01

    Dopaminergic rat mesencephalic neurons in culture were exposed to a group of potential environmental neurotoxins. These cultures, which contained 0.5 to 1% dopaminergic neurons, were a suitable tool for determining nonselective and selective dopaminergic cytotoxicity. Selective toxicity was quantitated as the concentration which destroyed half of the population of dopaminergic neurons as visualized by tyrosine hydroxylase immunocytochemistry. Nonselective toxicity was defined as the concentration of test drug which destroyed half of the entire population of cultured cells as visualized by phase contrast microscopy. The compounds tested were selected to fulfill two molecular criteria underlying the toxic activity of 1-methyl-4-phenylpyridinium (MPP+), the active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toward dopaminergic cells: 1) to be a substrate for the selective uptake system of the dopaminergic neurons and 2) to possess a delocalized positive charge related to their ability to inhibit mitochondrial electron transport. Of a total number of 29 compounds tested, MPP+ and its close derivatives, 2'-methyl-MPP+ and p-amino-MPP+, exhibited highly selective dopaminergic toxicity, hence the requirements for a selective dopaminergic neurotoxin are rather strict.

  3. Selection of RNA Aptamers Against Botulinum Neurotoxin Type A Light Chain Through a Non-Radioactive Approach.

    PubMed

    Chang, Tzuu-Wang; Janardhanan, Pavithra; Mello, Charlene M; Singh, Bal Ram; Cai, Shuowei

    2016-09-01

    Botulinum neurotoxin (BoNT), a category A agent, is the most toxic molecule known to mankind. The endopeptidase activity of light chain domain of BoNT is the cause for the inhibition of the neurotransmitter release and the flaccid paralysis that leads to lethality in botulism. Currently, antidotes are not available to reverse the flaccid paralysis caused by BoNT. In the present study, a non-radioactive-based systematic evolution of ligands by exponential enrichment (SELEX) process is developed by utilizing surface plasmon resonance to monitor the binding enrichment. Two RNA aptamers have been identified as strong binders against light chain of botulinum neurotoxin type A. These two aptamers showed strong inhibition activity on LCA, with IC50 in nanomolar range. Inhibition kinetic studies reveal mid nanomolar KI and non-competitive nature of their inhibition, suggesting that they have strong potential as antidotes that can reverse the symptom caused by BoNT/A. More importantly, we observed that the 2'-fluorine-pyrimidine-modified RNA aptamers identified here do not change their binding and biological activities. This observation could lead to a cost-effective way for SELEX, by using regular nucleotide during SELEX, and 2'-fluorine-pyrimidine-modified nucleotide for final application to enhance their RNase-resistance. PMID:27085355

  4. Development of Germline-Humanized Antibodies Neutralizing Botulinum Neurotoxin A and B

    PubMed Central

    Liu, Yvonne; Tierney, Robert; Rasetti-Escargueil, Christine; Avril, Arnaud; Frenzel, André; Thullier, Philippe; Pelat, Thibaut; Urbain, Remi; Fontayne, Alexandre; Sesardic, Dorothea; Hust, Michael; Popoff, Michel Robert

    2016-01-01

    Botulinum neurotoxins (BoNTs) are counted among the most toxic substances known and are responsible for human botulism, a life-threatening disease characterized by flaccid muscle paralysis that occurs naturally by food poisoning or colonization of the gastrointestinal tract by BoNT-producing clostridia. To date, 7 serologically distinct serotypes of BoNT (serotype A-G) are known. Due to the high toxicity of BoNTs the Centers for Disease Control and Prevention (CDC) have classified BoNTs as category A agent, including the six biological agents with the highest potential risk of use as bioweapons. Well tolerated antibodies neutralizing BoNTs are required to deal with the potential risk. In a previous work, we described the development of scFv and scFv-Fc (Yumab) from macaque origin (Macaca fascicularis) neutralizing BoNT/A and B by targeting the heavy and light chain of each serotype. In the present study, we humanized the macaque antibodies SEM120-IIIC1 (anti-BoNT/A light chain), A1HC38 (anti-BoNT/A heavy chain), BLC3 (anti-BoNT/B light chain) and B2-7 (anti-BoNT/B heavy chain) by germline-humanization to obtain a better potential immunotolerance in humans. We increased the Germinality Index (GI) of SEM120-IIIC1 to 94.5%, for A1HC38, to 95% for BLC3 and to 94.4% for B2-7. Furthermore, the neutralization efficacies of the germline-humanized antibodies were analyzed in lethal and non-lethal in vivo mouse assays as full IgG. The germline-humanized IgGs hu8SEM120-IIIC1, hu8A1HC38, hu8BLC3 and hu8B2-7 were protective in vivo, when anti-heavy and anti-light chain antibodies were combined. The synergistic effect and high humanness of the selected IgGs makes them promising lead candidates for further clinical development. PMID:27560688

  5. Crystal Structure of the Botulinum Neurotoxin Type G Binding Domain: Insight into Cell Surface Binding

    SciTech Connect

    Stenmark, Pål; Dong, Min; Dupuy, Jérôme; Chapman, Edwin R.; Stevens, Raymond C.

    2011-11-02

    Botulinum neurotoxins (BoNTs) typically bind the neuronal cell surface via dual interactions with both protein receptors and gangliosides. We present here the 1.9-{angstrom} X-ray structure of the BoNT serotype G (BoNT/G) receptor binding domain (residues 868-1297) and a detailed view of protein receptor and ganglioside binding regions. The ganglioside binding motif (SxWY) has a conserved structure compared to the corresponding regions in BoNT serotype A and BoNT serotype B (BoNT/B), but several features of interactions with the hydrophilic face of the ganglioside are absent at the opposite side of the motif in the BoNT/G ganglioside binding cleft. This may significantly reduce the affinity between BoNT/G and gangliosides. BoNT/G and BoNT/B share the protein receptor synaptotagmin (Syt) I/II. The Syt binding site has a conserved hydrophobic plateau located centrally in the proposed protein receptor binding interface (Tyr1189, Phe1202, Ala1204, Pro1205, and Phe1212). Interestingly, only 5 of 14 residues that are important for binding between Syt-II and BoNT/B are conserved in BoNT/G, suggesting that the means by which BoNT/G and BoNT/B bind Syt diverges more than previously appreciated. Indeed, substitution of Syt-II Phe47 and Phe55 with alanine residues had little effect on the binding of BoNT/G, but strongly reduced the binding of BoNT/B. Furthermore, an extended solvent-exposed hydrophobic loop, located between the Syt binding site and the ganglioside binding cleft, may serve as a third membrane association and binding element to contribute to high-affinity binding to the neuronal membrane. While BoNT/G and BoNT/B are homologous to each other and both utilize Syt-I/Syt-II as their protein receptor, the precise means by which these two toxin serotypes bind to Syt appears surprisingly divergent.

  6. Glycosylated SV2 and Gangliosides as Dual Receptors for Botulinum Neurotoxin Serotype F

    SciTech Connect

    Fu, Zhuji; Chen, Chen; Barbieri, Joseph T.; Kim, Jung-Ja P.; Baldwin, Michael R.

    2010-02-22

    Botulinum neurotoxin causes rapid flaccid paralysis through the inhibition of acetylcholine release at the neuromuscular junction. The seven BoNT serotypes (A-G) have been proposed to bind motor neurons via ganglioside-protein dual receptors. To date, the structure-function properties of BoNT/F host receptor interactions have not been resolved. Here, we report the crystal structures of the receptor binding domains (HCR) of BoNT/A and BoNT/F and the characterization of the dual receptors for BoNT/F. The overall polypeptide fold of HCR/A is essentially identical to the receptor binding domain of the BoNT/A holotoxin, and the structure of HCR/F is very similar to that of HCR/A, except for two regions implicated in neuronal binding. Solid phase array analysis identified two HCR/F binding glycans: ganglioside GD1a and oligosaccharides containing an N-acetyllactosamine core. Using affinity chromatography, HCR/F bound native synaptic vesicle glycoproteins as part of a protein complex. Deglycosylation of glycoproteins using {alpha}(1-3,4)-fucosidase, endo-{beta}-galactosidase, and PNGase F disrupted the interaction with HCR/F, while the binding of HCR/B to its cognate receptor, synaptotagmin I, was unaffected. These data indicate that the HCR/F binds synaptic vesicle glycoproteins through the keratan sulfate moiety of SV2. The interaction of HCR/F with gangliosides was also investigated. HCR/F bound specifically to gangliosides that contain {alpha}2,3-linked sialic acid on the terminal galactose of a neutral saccharide core (binding order GT1b = GD1a GM3; no binding to GD1b and GM1a). Mutations within the putative ganglioside binding pocket of HCR/F decreased binding to gangliosides, synaptic vesicle protein complexes, and primary rat hippocampal neurons. Thus, BoNT/F neuronal discrimination involves the recognition of ganglioside and protein (glycosylated SV2) carbohydrate moieties, providing a structural basis for the high affinity and specificity of BoNT/F for neurons.

  7. Human monoclonal ScFv that inhibits cellular entry and metalloprotease activity of tetanus neurotoxin.

    PubMed

    Indrawattana, Nitaya; Sookrung, Nitat; Kulkeaw, Kasem; Seesuay, Watee; Kongngoen, Thida; Chongsa-nguan, Manas; Tungtrongchitr, Anchalee; Chaicumpa, Wanpen

    2010-03-01

    Tetanus is a deadly disease of warm blooded animals and humans caused by an exotoxin called tetanospasmin or tetanus neurotoxin (TeNT) produced by anaerobic bacterium named Clostridium tetani TeNT is an A-B toxin; each molecule consists of a heavy chain (HC) containing cellular receptor binding domain and a light chain (LC) with zinc metalloprotease activity. TeNT produced in the infected tissue by the bacteria grown under anaerobic condition binds to ganglioside receptors of peripheral nerve, and endocytosed. The A subunit exits from the endosome and undergoes a retrograde transport via the nerve axon to the spinal cord. This highly toxic enzyme specifically cleaves one of the nerve cell SNARE proteins, i.e., synaptobrevin, resulting in inhibition of the release of neurotransmitters (glycine and GABA) from inhibitory interneuron causing spastic paralysis, the characteristic of tetanus. Current treatment mainstay of human tetanus is by passively administering anti-tetanus toxin produced from animals immunized with adjuvanted tetanus toxoid (TT). There are several obstacles in production and use of the animal derived therapeutic antibody especially the allergic reaction and serum sickness induced by the host immune response to the foreign protein. The animal antibody, mainly IgG, blocks nerve cell entry of the TeNT but does not neutralize the TeNT protease activity per se and cannot reverse the tetanus symptoms. In this study, fully human single chain antibody fragments (HuScFv) were produced from a human antibody phage display library. TT was used as antigen in a single round phage bio-panning to select phage clones that display TT bound-HuScFv from the library. HuScFv from 4 selected huscfv-phagemid transformed E. coli clones inhibited binding of the native TeNT to retinoic acid pulsed human neuroblastoma cells when used at the molecular TeNT:HuScFv ratio of 1:100. HuScFv from one of the 4 clones also inhibited the TeNT mediated cleavage of recombinant

  8. No Decrease in Muscle Strength after Botulinum Neurotoxin-A Injection in Children with Cerebral Palsy

    PubMed Central

    Eek, Meta N.; Himmelmann, Kate

    2016-01-01

    Spasticity and muscle weakness is common in children with cerebral palsy (CP). Spasticity can be treated with botulinum neurotoxin-A (BoNT-A), but this drug has also been reported to induce muscle weakness. Our purpose was to describe the effect on muscle strength in the lower extremities after BoNT-A injections in children with CP. A secondary aim was to relate the effect of BoNT-A to gait pattern and range of motion. Twenty children with spastic CP were included in the study, 8 girls and 12 boys (mean age 7.7 years). All were able to walk without support, but with increased muscle tone interfering with motor function and gait pattern. Sixteen children had unilateral spastic CP and four bilateral spastic CP. Twenty-four legs received injections with BoNT-A in the plantar flexor muscles. The children were tested before treatment, around 6 weeks after at the peak effect of BoNT-A, and at 6 months after treatment, with measurement of muscle strength, gait analysis, and range of motion. There were no differences in muscle strength in plantar flexors of treated legs at peak effect compared to baseline. Six months after treatment, there was still no change in untreated plantar flexor muscles, but an increasing trend in plantar flexor strength in legs treated with BoNT-A. Parents reported positive effects in all children, graded as: small in three children, moderate in eight, and large in nine children. The gait analysis showed a small improvement in knee extension at initial contact, and there was a small increase in passive range of motion for ankle dorsiflexion. Two children had a period with transient weakness and pain. We found that voluntary force production in plantar flexor muscles did not decrease after BoNT-A, instead there was a trend to increased muscle strength at follow-up. The increase may be explained as an effect of the blocking of involuntary nerve impulses, leading to an opportunity to using and training the muscles with voluntary control. Adequate

  9. Snake neurotoxin α-bungarotoxin is an antagonist at native GABAA receptors

    PubMed Central

    Hannan, Saad; Mortensen, Martin; Smart, Trevor G.

    2015-01-01

    The snake neurotoxin α-bungarotoxin (α-Bgtx) is a competitive antagonist at nicotinic acetylcholine receptors (nAChRs) and is widely used to study their function and cell-surface expression. Increasingly, α-Bgtx is also used as an imaging tool for fluorophore-labelling studies, and given the structural conservation within the pentameric ligand-gated ion channel family, we assessed whether α-Bgtx could bind to recombinant and native γ-aminobutyric type-A receptors (GABAARs). Applying fluorophore-linked α-Bgtx to recombinant αxβ1/2γ2 GABAARs expressed in HEK-293 cells enabled clear cell-surface labelling of α2β1/2γ2 contrasting with the weaker staining of α1/4β1/2γ2, and no labelling for α3/5/6β1/2γ2. The labelling of α2β2γ2 was abolished by bicuculline, a competitive antagonist at GABAARs, and by d-tubocurarine (d-Tc), which acts in a similar manner at nAChRs and GABAARs. Labelling by α-Bgtx was also reduced by GABA, suggesting that the GABA binding site at the receptor β–α subunit interface forms part of the α-Bgtx binding site. Using whole-cell recording, high concentrations of α-Bgtx (20 μM) inhibited GABA-activated currents at all αxβ2γ2 receptors examined, but at lower concentrations (5 μM), α-Bgtx was selective for α2β2γ2. Using α-Bgtx, at low concentrations, permitted the selective inhibition of α2 subunit-containing GABAARs in hippocampal dentate gyrus granule cells, reducing synaptic current amplitudes without affecting the GABA-mediated tonic current. In conclusion, α-Bgtx can act as an inhibitor at recombinant and native GABAARs and may be used as a selective tool to inhibit phasic but not tonic currents in the hippocampus. PMID:25634239

  10. Evidence-based review and assessment of botulinum neurotoxin for the treatment of urologic conditions.

    PubMed

    Chancellor, Michael B; Elovic, Elie; Esquenazi, Alberto; Naumann, Markus; Segal, Karen R; Schiavo, Giampietro; Smith, Christopher P; Ward, Anthony B

    2013-06-01

    Botulinum neurotoxin (BoNT) can be injected to achieve therapeutic benefit across a large range of clinical conditions. To assess the efficacy and safety of BoNT injections for the treatment of certain urologic conditions, including detrusor sphincter dyssynergia (DSD), lower urinary tract symptoms due to benign prostatic hyperplasia (BPH), and detrusor overactivity (both neurogenic [NDO] and idiopathic [IDO]), an expert panel reviewed evidence from the published literature. Data sources included English-language studies identified via MEDLINE, EMBASE, CINAHL, Current Contents, and the Cochrane Central Register of Controlled Trials. Evidence tables generated in the 2008 Report of the Therapeutics and Technology Assessment Subcommittee of the American Academy of Neurology (AAN) review of the use of BoNT for autonomic disorders were also reviewed and updated. The panel evaluated evidence at several levels, supporting BoNT as a class, for the serotypes BoNT-A and BoNT-B, as well as for the four individual commercially available formulations: abobotulinumtoxinA (A/Abo), onabotulinumtoxinA (A/Ona), incobotulinumtoxinA (A/Inco), and rimabotulinumtoxinB (B/Rima). The panel ultimately made recommendations on the use of BoNT for the management of these urologic conditions based upon the strength of clinical evidence and following the AAN classification scale. For the treatment of DSD, the evidence supported a Level B recommendation for the use of A/Ona; A/Abo, A/Inco, and B/Rima received a Level U recommendation. For the treatment of NDO, there was sufficient clinical evidence to support a Level A recommendation for BoNT-A as well as for both A/Ona and A/Abo; no published data were identified for either A/Inco or B/Rima (Level U). For the treatment of IDO, the evidence supported a Level A recommendation for A/Ona; A/Inco, A/Abo, and B/Rima received a Level U recommendation. For the management of BPH, the evidence supported a Level B recommendation for BoNT and A/Ona; no

  11. Development of Germline-Humanized Antibodies Neutralizing Botulinum Neurotoxin A and B.

    PubMed

    Miethe, Sebastian; Mazuet, Christelle; Liu, Yvonne; Tierney, Robert; Rasetti-Escargueil, Christine; Avril, Arnaud; Frenzel, André; Thullier, Philippe; Pelat, Thibaut; Urbain, Remi; Fontayne, Alexandre; Sesardic, Dorothea; Hust, Michael; Popoff, Michel Robert

    2016-01-01

    Botulinum neurotoxins (BoNTs) are counted among the most toxic substances known and are responsible for human botulism, a life-threatening disease characterized by flaccid muscle paralysis that occurs naturally by food poisoning or colonization of the gastrointestinal tract by BoNT-producing clostridia. To date, 7 serologically distinct serotypes of BoNT (serotype A-G) are known. Due to the high toxicity of BoNTs the Centers for Disease Control and Prevention (CDC) have classified BoNTs as category A agent, including the six biological agents with the highest potential risk of use as bioweapons. Well tolerated antibodies neutralizing BoNTs are required to deal with the potential risk. In a previous work, we described the development of scFv and scFv-Fc (Yumab) from macaque origin (Macaca fascicularis) neutralizing BoNT/A and B by targeting the heavy and light chain of each serotype. In the present study, we humanized the macaque antibodies SEM120-IIIC1 (anti-BoNT/A light chain), A1HC38 (anti-BoNT/A heavy chain), BLC3 (anti-BoNT/B light chain) and B2-7 (anti-BoNT/B heavy chain) by germline-humanization to obtain a better potential immunotolerance in humans. We increased the Germinality Index (GI) of SEM120-IIIC1 to 94.5%, for A1HC38, to 95% for BLC3 and to 94.4% for B2-7. Furthermore, the neutralization efficacies of the germline-humanized antibodies were analyzed in lethal and non-lethal in vivo mouse assays as full IgG. The germline-humanized IgGs hu8SEM120-IIIC1, hu8A1HC38, hu8BLC3 and hu8B2-7 were protective in vivo, when anti-heavy and anti-light chain antibodies were combined. The synergistic effect and high humanness of the selected IgGs makes them promising lead candidates for further clinical development. PMID:27560688

  12. Structural basis for recognition of synaptic vesicle protein 2C by botulinum neurotoxin A

    NASA Astrophysics Data System (ADS)

    Benoit, Roger M.; Frey, Daniel; Hilbert, Manuel; Kevenaar, Josta T.; Wieser, Mara M.; Stirnimann, Christian U.; McMillan, David; Ceska, Tom; Lebon, Florence; Jaussi, Rolf; Steinmetz, Michel O.; Schertler, Gebhard F. X.; Hoogenraad, Casper C.; Capitani, Guido; Kammerer, Richard A.

    2014-01-01

    Botulinum neurotoxin A (BoNT/A) belongs to the most dangerous class of bioweapons. Despite this, BoNT/A is used to treat a wide range of common medical conditions such as migraines and a variety of ocular motility and movement disorders. BoNT/A is probably best known for its use as an antiwrinkle agent in cosmetic applications (including Botox and Dysport). BoNT/A application causes long-lasting flaccid paralysis of muscles through inhibiting the release of the neurotransmitter acetylcholine by cleaving synaptosomal-associated protein 25 (SNAP-25) within presynaptic nerve terminals. Two types of BoNT/A receptor have been identified, both of which are required for BoNT/A toxicity and are therefore likely to cooperate with each other: gangliosides and members of the synaptic vesicle glycoprotein 2 (SV2) family, which are putative transporter proteins that are predicted to have 12 transmembrane domains, associate with the receptor-binding domain of the toxin. Recently, fibroblast growth factor receptor 3 (FGFR3) has also been reported to be a potential BoNT/A receptor. In SV2 proteins, the BoNT/A-binding site has been mapped to the luminal domain, but the molecular details of the interaction between BoNT/A and SV2 are unknown. Here we determined the high-resolution crystal structure of the BoNT/A receptor-binding domain (BoNT/A-RBD) in complex with the SV2C luminal domain (SV2C-LD). SV2C-LD consists of a right-handed, quadrilateral β-helix that associates with BoNT/A-RBD mainly through backbone-to-backbone interactions at open β-strand edges, in a manner that resembles the inter-strand interactions in amyloid structures. Competition experiments identified a peptide that inhibits the formation of the complex. Our findings provide a strong platform for the development of novel antitoxin agents and for the rational design of BoNT/A variants with improved therapeutic properties.

  13. Llama-derived single-domain antibodies for the detection of botulinum A neurotoxin

    PubMed Central

    Swain, Marla D.; Anderson, George P.; Zabetakis, Dan; Bernstein, Rachael D.; Liu, Jinny L.; Sherwood, Laura J.; Hayhurst, Andrew

    2011-01-01

    Single-domain antibodies (sdAb) specific for botulinum neurotoxin serotype A (BoNT A) were selected from an immune llama phage display library derived from a llama that was immunized with BoNT A toxoid. The constructed phage library was panned using two methods: panning on plates coated with BoNT A toxoid (BoNT A Td) and BoNT A complex toxoid (BoNT Ac Td) and panning on microspheres coupled to BoNT A Td and BoNT A toxin (BoNT A Tx). Both panning methods selected for binders that had identical sequences, suggesting that panning on toxoided material may be as effective as panning on bead-immobilized toxin for isolating specific binders. All of the isolated binders tested were observed to recognize bead-immobilized BoNT A Tx in direct binding assays, and showed very little cross-reactivity towards other BoNT serotypes and unrelated protein. Sandwich assays that incorporated selected sdAb as capture and tracer elements demonstrated that all of the sdAb were able to recognize soluble (“live”) BoNT A Tx and BoNT Ac Tx with virtually no cross-reactivity with other BoNT serotypes. The isolated sdAb did not exhibit the high degree of thermal stability often associated with these reagents; after the first heating cycle most of the binding activity was lost, but the portion of the protein that did refold and recover antigen-binding activity showed only minimal loss on subsequent heating and cooling cycles. The binding kinetics of selected binders, assessed by both an equilibrium fluid array assay as well as surface plasmon resonance (SPR) using toxoided material, gave dissociation constants (KD) in the range 2.2×10−11 to 1.6×10−10M. These high-affinity binders may prove beneficial to the development of recombinant reagents for the rapid detection of BoNT A, particularly in field screening and monitoring applications. PMID:20582697

  14. Sifuvirtide, a potent HIV fusion inhibitor peptide

    SciTech Connect

    Wang, Rui-Rui; Yang, Liu-Meng; Wang, Yun-Hua; Pang, Wei; Tam, Siu-Cheung; Tien, Po; Zheng, Yong-Tang

    2009-05-08

    Enfuvirtide (ENF) is currently the only FDA approved HIV fusion inhibitor in clinical use. Searching for more drugs in this category with higher efficacy and lower toxicity seems to be a logical next step. In line with this objective, a synthetic peptide with 36 amino acid residues, called Sifuvirtide (SFT), was designed based on the crystal structure of gp41. In this study, we show that SFT is a potent anti-HIV agent with relatively low cytotoxicity. SFT was found to inhibit replication of all tested HIV strains. The effective concentrations that inhibited 50% viral replication (EC{sub 50}), as determined in all tested strains, were either comparable or lower than benchmark values derived from well-known anti-HIV drugs like ENF or AZT, while the cytotoxic concentrations causing 50% cell death (CC{sub 50}) were relatively high, rendering it an ideal anti-HIV agent. A GST-pull down assay was performed to confirm that SFT is a fusion inhibitor. Furthermore, the activity of SFT on other targets in the HIV life cycle was also investigated, and all assays showed negative results. To further understand the mechanism of action of HIV peptide inhibitors, resistant variants of HIV-1{sub IIIB} were derived by serial virus passage in the presence of increasing doses of SFT or ENF. The results showed that there was cross-resistance between SFT and ENF. In conclusion, SFT is an ideal anti-HIV agent with high potency and low cytotoxicity, but may exhibit a certain extent of cross-resistance with ENF.

  15. Potent antibacterial nanoparticles for pathogenic bacteria.

    PubMed

    Lai, Hong-Zheng; Chen, Wei-Yu; Wu, Ching-Yi; Chen, Yu-Chie

    2015-01-28

    Antibiotic-resistant bacteria have emerged because of the prevalent use of antibacterial agents. Thus, new antibacterial agents and therapeutics that can treat bacterial infections are necessary. Vancomycin is a potent antibiotic. Unfortunately, some bacterial strains have developed their resistance toward vancomycin. Nevertheless, it has been demonstrated that vancomycin-immobilized nanoparticles (NPs) are capable to be used in inhibition of the cell growth of vancomycin-resistant bacterial strains through multivalent interactions. However, multistep syntheses are usually necessary to generate vancomycin-immobilized NPs. Thus, maintaining the antibiotic activity of vancomycin when the drug is immobilized on the surface of NPs is challenging. In this study, a facile approach to generate vancomycin immobilized gold (Van-Au) NPs through one-pot stirring of vancomycin with aqueous tetrachloroauric acid at pH 12 and 25 °C for 24 h was demonstrated. Van-Au NPs (8.4 ± 1.3 nm in size) were readily generated. The generated Van-Au NPs maintained their antibiotic activities and inhibited the cell growth of pathogens, which included Gram-positive and Gram-negative bacteria as well as antibiotic-resistant bacterial strains. Furthermore, the minimum inhibitory concentration of the Van-Au NPs against bacteria was lower than that of free-form vancomycin. Staphylococcus aureus-infected macrophages were used as the model samples to examine the antibacterial activity of the Van-Au NPs. Macrophages have the tendency to engulf Van-Au NPs through endocytosis. The results showed that the cell growth of S. aureus in the macrophages was effectively inhibited, suggesting the potential of using the generated Van-Au NPs as antibacterial agents for bacterial infectious diseases.

  16. Sifuvirtide, a potent HIV fusion inhibitor peptide.

    PubMed

    Wang, Rui-Rui; Yang, Liu-Meng; Wang, Yun-Hua; Pang, Wei; Tam, Siu-Cheung; Tien, Po; Zheng, Yong-Tang

    2009-05-01

    Enfuvirtide (ENF) is currently the only FDA approved HIV fusion inhibitor in clinical use. Searching for more drugs in this category with higher efficacy and lower toxicity seems to be a logical next step. In line with this objective, a synthetic peptide with 36 amino acid residues, called Sifuvirtide (SFT), was designed based on the crystal structure of gp41. In this study, we show that SFT is a potent anti-HIV agent with relatively low cytotoxicity. SFT was found to inhibit replication of all tested HIV strains. The effective concentrations that inhibited 50% viral replication (EC(50)), as determined in all tested strains, were either comparable or lower than benchmark values derived from well-known anti-HIV drugs like ENF or AZT, while the cytotoxic concentrations causing 50% cell death (CC(50)) were relatively high, rendering it an ideal anti-HIV agent. A GST-pull down assay was performed to confirm that SFT is a fusion inhibitor. Furthermore, the activity of SFT on other targets in the HIV life cycle was also investigated, and all assays showed negative results. To further understand the mechanism of action of HIV peptide inhibitors, resistant variants of HIV-1(IIIB) were derived by serial virus passage in the presence of increasing doses of SFT or ENF. The results showed that there was cross-resistance between SFT and ENF. In conclusion, SFT is an ideal anti-HIV agent with high potency and low cytotoxicity, but may exhibit a certain extent of cross-resistance with ENF.

  17. Growth cone collapse and inhibition of neurite growth by Botulinum neurotoxin C1: a t-SNARE is involved in axonal growth

    PubMed Central

    1996-01-01

    The growth cone is responsible for axonal growth, where membrane expansion is most likely to occur. Several recent reports have suggested that presynaptic proteins are involved in this process; however, the molecular mechanism details are unclear. We suggest that by cleaving a presynaptic protein syntaxin, which is essential in targeting synaptic vesicles as a target SNAP receptor (t-SNARE), neurotoxin C1 of Clostridium botulinum causes growth cone collapse and inhibits axonal growth. Video-enhanced microscopic studies showed (a) that neurotoxin C1 selectively blocked the activity of the central domain (the vesicle-rich region) at the initial stage, but not the lamellipodia in the growth cone; and (b) that large vacuole formation occurred probably through the fusion of smaller vesicles from the central domain to the most distal segments of the neurite. The total surface area of the accumulated vacuoles could explain the membrane expansion of normal neurite growth. The gradual disappearance of the surface labeling by FITC-WGA on the normal growth cone, suggesting membrane addition, was inhibited by neurotoxin C1. The experiments using the peptides derived from syntaxin, essential for interaction with VAMP or alpha-SNAP, supported the results using neurotoxin C1. Our results demonstrate that syntaxin is involved in axonal growth and indicate that syntaxin may participate directly in the membrane expansion that occurs in the central domain of the growth cone, probably through association with VAMP and SNAPs, in a SNARE-like way. PMID:8698815

  18. Explaining human recreational use of 'pesticides': The neurotoxin regulation model of substance use vs. the hijack model and implications for age and sex differences in drug consumption.

    PubMed

    Hagen, Edward H; Roulette, Casey J; Sullivan, Roger J

    2013-01-01

    Most globally popular drugs are plant neurotoxins or their close chemical analogs. These compounds evolved to deter, not reward or reinforce, consumption. Moreover, they reliably activate virtually all toxin defense mechanisms, and are thus correctly identified by human neurophysiology as toxins. Acute drug toxicity must therefore play a more central role in drug use theory. We accordingly challenge the popular idea that the rewarding and reinforcing properties of drugs "hijack" the brain, and propose instead that the brain evolved to carefully regulate neurotoxin consumption to minimize fitness costs and maximize fitness benefits. This perspective provides a compelling explanation for the dramatic changes in substance use that occur during the transition from childhood to adulthood, and for pervasive sex differences in substance use: because nicotine and many other plant neurotoxins are teratogenic, children, and to a lesser extent women of childbearing age, evolved to avoid ingesting them. However, during the course of human evolution many adolescents and adults reaped net benefits from regulated intake of plant neurotoxins.

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

    PubMed

    Li, Jing; Zhang, Huayuan; Liu, Jing; Xu, Kangsen

    2006-09-01

    Three-finger toxins are a family of low-molecular-mass toxins (<10 kDa) having very similar three-dimensional structures. In the present study, 19 novel cDNAs coding three-finger toxins were cloned from the venom gland of Ophiophagus hannah (king cobra). Alignment analysis showed that the putative peptides could be divided into six kinds of three-finger toxins: LNTXs (long-chain neurotoxins), short-chain neurotoxins, cardiotoxins (CTXs), weak neurotoxins, muscarinic toxins and a toxin with a free SH group. Furthermore, a phylogenetic tree was established on the basis of the toxin cDNAs and the previously reported similar nucleotide sequences from the same source venom. It indicated that three-finger-toxin genes in O. hannah diverged early in the course of evolution by long- and short-type pathways. Two LNTXs, namely rLNTX1 (recombinant LNTX1) and rLNTX3, were expressed and showed cytolytic activity in addition to their neurotoxic function. By comparing the functional residues, we offer some possible explanations for the differences in their neurotoxic function. Moreover, a plausible elucidation of the additonal cytolytic activity was achieved by hydropathy-profile analysis. This, to our knowledge, is the first observation that recombinant long chain alpha-neurotoxins have a CTX-like cytolytic activity. PMID:16689684

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

    PubMed

    Li, Jing; Zhang, Huayuan; Liu, Jing; Xu, Kangsen

    2006-09-01

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

  1. Crystal Structure of Botulinum Neurotoxin Type a in Complex With the Cell Surface Co-Receptor GT1b-Insight Into the Toxin-Neuron Interaction

    SciTech Connect

    Stenmark, P.; Dupuy, J.; Inamura, A.; Kiso, M.; Stevens, R.C.

    2009-05-26

    Botulinum neurotoxins have a very high affinity and specificity for their target cells requiring two different co-receptors located on the neuronal cell surface. Different toxin serotypes have different protein receptors; yet, most share a common ganglioside co-receptor, GT1b. We determined the crystal structure of the botulinum neurotoxin serotype A binding domain (residues 873-1297) alone and in complex with a GT1b analog at 1.7 A and 1.6 A, respectively. The ganglioside GT1b forms several key hydrogen bonds to conserved residues and binds in a shallow groove lined by Tryptophan 1266. GT1b binding does not induce any large structural changes in the toxin; therefore, it is unlikely that allosteric effects play a major role in the dual receptor recognition. Together with the previously published structures of botulinum neurotoxin serotype B in complex with its protein co-receptor, we can now generate a detailed model of botulinum neurotoxin's interaction with the neuronal cell surface. The two branches of the GT1b polysaccharide, together with the protein receptor site, impose strict geometric constraints on the mode of interaction with the membrane surface and strongly support a model where one end of the 100 A long translocation domain helix bundle swing into contact with the membrane, initiating the membrane anchoring event.

  2. Structural analysis and the effect of cyclo(His-Pro) dipeptide on neurotoxins--a dynamics and density functional theory study.

    PubMed

    Abiram, Angamuthu; Kolandaivel, Ponmalai

    2010-02-01

    The switching propensity and maximum probability of occurrence of the side chain imidazole group in the dipeptide cyclo(His-Pro) (CHP) were studied by applying molecular dynamics simulations and density functional theory. The atomistic behaviour of CHP with the neurotoxins glutamate (E) and paraquat (Pq) were also explored; E and Pq engage in hydrogen bond formation with the diketopiperazine (DKP) ring of the dipeptide, with which E shows a profound interaction, as confirmed further by NH and CO stretching vibrational frequencies. The effect of CHP was found to be greater on E than on Pq neurotoxin. A ring puckering study indicated a twist boat conformation for the six-membered DKP ring. Molecular electrostatic potential (MESP) mapping was also used to explore the hydrogen bond interactions prevailing between the neurotoxins and the DKP ring. The results of this study reveal that the DKP ring of the dipeptide CHP can be expected to play a significant role in reducing effects such as oxidative stress and cell death caused by neurotoxins.

  3. Fluorescent transgenic zebrafish Tg(nkx2.2a:mEGFP) provides a highly sensitive monitoring tool for neurotoxins.

    PubMed

    Zhang, Xiaoyan; Gong, Zhiyuan

    2013-01-01

    Previously a standard toxicological test termed as DarT (Danio rerio Teratogenic assay) using wild type zebrafish embryos has been established and it is widely applied in toxicological and chemical screenings. As an increasing number of fluorescent transgenic zebrafish lines with specific fluorescent protein expression specifically expressed in different organs and tissues, we envision that the fluorescent markers may provide more sensitive endpoints for monitoring chemical induced phenotypical changes. Here we employed Tg(nkx2.2a:mEGFP) transgenic zebrafish which have GFP expression in the central nervous system to investigate its potential for screening neurotoxic chemicals. Five potential neurotoxins (acetaminophen, atenolol, atrazine, ethanol and lindane) and one neuroprotectant (mefenamic acid) were tested. We found that the GFP-labeled ventral axons from trunk motoneurons, which were easily observed in live fry and measured for quantification, were a highly sensitive to all of the five neurotoxins and the length of axons was significantly reduced in fry which looked normal based on DarT endpoints at low concentrations of neurotoxins. Compared to the most sensitive endpoints of DarT, ventral axon marker could improve the detection limit of these neurotoxins by about 10 fold. In contrast, there was no improvement for detection of the mefenamic acid compared to all DarT endpoints. Thus, ventral axon lengths provide a convenient and measureable marker specifically for neurotoxins. Our study may open a new avenue to use other fluorescent transgenic zebrafish embryos/fry to develop sensitive and specific toxicological tests for different categories of chemicals.

  4. Exchange of the H(CC) domain mediating double receptor recognition improves the pharmacodynamic properties of botulinum neurotoxin.

    PubMed

    Rummel, Andreas; Mahrhold, Stefan; Bigalke, Hans; Binz, Thomas

    2011-12-01

    The four-domain structure of botulinum neurotoxins (BoNTs) reflects their multistep intoxication process. The high toxicity of BoNTs primarily results from specific binding and uptake into neurons mediated by their 50-kDa cell-binding fragment (H(C) ). X-ray crystallography data have revealed that the H(C) fragment consists of two domains of equal size, named the 25-kDa N-terminal half of H(C) (H(CN) ) and the 25-kDa C-terminal half of H(C) (H(CC) ). In recent years, the ganglioside-binding sites of all seven BoNT serotypes have been allocated to the H(CC) domain. For BoNT/A, BoNT/B and BoNT/G, the protein receptor-binding site has been also been localized to the H(CC) domain. Here, we demonstrate that the H(CC) serotype can modulate the affinity of the H(C) fragment for neuronal membranes as well as the potency of full-length BoNT by replacing the BoNT/A H(CC) domain with the BoNT/B H(CC) , BoNT/C H(CC) and BoNT/E H(CC) domains, which exhibit higher affinity for synaptosomes. Indeed, the hybrids H(C) AB and H(C) AC display a higher affinity than wild-type H(C) A. Furthermore, the potency of a BoNT/A-based full-length hybrid containing the H(CC) B domain (AAAB; letters represent the serotype origin of the four domains) was quadrupled as compared with wild-type BoNT/A. Analogously, exchange of the H(C) fragment (AABB) yielded a neurotoxin with four-fold higher potency. As BoNT/A and BoNT/B are extensively used to treat neurological disorders, thereby facing the problem of BoNT neutralizing antibody formation, a BoNT with increased potency would lower the repeatedly administered protein dosage while maintaining the clinical benefit. Such a lowered protein load will delay the onset of neurotoxin antibody formation in patients.

  5. Cross-Neutralisation of In Vitro Neurotoxicity of Asian and Australian Snake Neurotoxins and Venoms by Different Antivenoms

    PubMed Central

    Silva, Anjana; Hodgson, Wayne C.; Isbister, Geoffrey K.

    2016-01-01

    There is limited information on the cross-neutralisation of neurotoxic venoms with antivenoms. Cross-neutralisation of the in vitro neurotoxicity of four Asian and four Australian snake venoms, four post-synaptic neurotoxins (α-bungarotoxin, α-elapitoxin-Nk2a, α-elapitoxin-Ppr1 and α-scutoxin; 100 nM) and one pre-synaptic neurotoxin (taipoxin; 100 nM) was studied with five antivenoms: Thai cobra antivenom (TCAV), death adder antivenom (DAAV), Thai neuro polyvalent antivenom (TNPAV), Indian Polyvalent antivenom (IPAV) and Australian polyvalent antivenom (APAV). The chick biventer cervicis nerve-muscle preparation was used for this study. Antivenom was added to the organ bath 20 min prior to venom. Pre- and post-synaptic neurotoxicity of Bungarus caeruleus and Bungarus fasciatus venoms was neutralised by all antivenoms except TCAV, which did not neutralise pre-synaptic activity. Post-synaptic neurotoxicity of Ophiophagus hannah was neutralised by all antivenoms, and Naja kaouthia by all antivenoms except IPAV. Pre- and post-synaptic neurotoxicity of Notechis scutatus was neutralised by all antivenoms, except TCAV, which only partially neutralised pre-synaptic activity. Pre- and post-synaptic neurotoxicity of Oxyuranus scutellatus was neutralised by TNPAV and APAV, but TCAV and IPAV only neutralised post-synaptic neurotoxicity. Post-synaptic neurotoxicity of Acanthophis antarcticus was neutralised by all antivenoms except IPAV. Pseudonaja textillis post-synaptic neurotoxicity was only neutralised by APAV. The α-neurotoxins were neutralised by TNPAV and APAV, and taipoxin by all antivenoms except IPAV. Antivenoms raised against venoms with post-synaptic neurotoxic activity (TCAV) cross-neutralised the post-synaptic activity of multiple snake venoms. Antivenoms raised against pre- and post-synaptic neurotoxic venoms (TNPAV, IPAV, APAV) cross-neutralised both activities of Asian and Australian venoms. While acknowledging the limitations of adding antivenom prior to

  6. Botulinum neurotoxins for the treatment of focal dystonias: Review of rating tools used in clinical trials.

    PubMed

    Del Sorbo, Francesca; Albanese, Alberto

    2015-12-01

    Botulinum neurotoxins (BoNTs) are used to achieve therapeutic benefit in focal dystonia. An expert panel recently reviewed published evidence on the efficacy of BoNTs for the treatment of focal dystonias and produced recommendations for clinical practice. Another panel reviewed the clinimetric properties of rating scales for dystonia and produced recommendations for current usage and future directions. Considering that the strength of evidence derives not only from the quality of the study design, but also from usage of validated outcome measures, we combined the information provided by these two recent reviews and assessed the appropriateness of the rating instruments used in clinical trials on BoNT treatment in focal dystonia. Data sources included all the publications on BoNT treatment for focal dystonias reviewed by the recent evidence-based analysis. We reviewed all rating instruments used to assess primary and secondary outcome following BoNT treatment. The publications were allocated into five topics according to the focal dystonia type reviewed in the meta-analysis: blepharospasm, oromandibular dystonia, cervical dystonia, upper limb dystonia, and laryngeal dystonia. For each topic, papers were divided, according to the terminology used in the meta-analysis, into placebo-controlled, active comparator and methodological or uncontrolled. For each topic we identified the rating tools used in each study class and annotated which were the mostly used in each focal dystonia type. Outcome measures included tools related to motor and non-motor features, such as pain and depression, and functional as well as health-related quality of life features. Patient- and investigator-reported outcomes were also included. Rating instruments were classified as recommended, suggested, listed or not included, based on recommendations produced by the rating scale task force. Both primary and secondary outcome measures were assessed. As a final step we compared current practice, as

  7. Interneuronal Transfer and Distal Action of Tetanus Toxin and Botulinum Neurotoxins A and D in Central Neurons.

    PubMed

    Bomba-Warczak, Ewa; Vevea, Jason D; Brittain, Joel M; Figueroa-Bernier, Annette; Tepp, William H; Johnson, Eric A; Yeh, Felix L; Chapman, Edwin R

    2016-08-16

    Recent reports suggest that botulinum neurotoxin (BoNT) A, which is widely used clinically to inhibit neurotransmission, can spread within networks of neurons to have distal effects, but this remains controversial. Moreover, it is not known whether other members of this toxin family are transferred between neurons. Here, we investigate the potential distal effects of BoNT/A, BoNT/D, and tetanus toxin (TeNT), using central neurons grown in microfluidic devices. Toxins acted upon the neurons that mediated initial entry, but all three toxins were also taken up, via an alternative pathway, into non-acidified organelles that mediated retrograde transport to the somato-dendritic compartment. Toxins were then released into the media, where they entered and exerted their effects upon upstream neurons. These findings directly demonstrate that these agents undergo transcytosis and interneuronal transfer in an active form, resulting in long-distance effects. PMID:27498860

  8. Tryptophan-47 rotational isomerization in variant-3 scorpion neurotoxin. A combination thermodynamic perturbation and umbrella sampling study.

    PubMed Central

    Haydock, C; Sharp, J C; Prendergast, F G

    1990-01-01

    A combination thermodynamic perturbation and umbrella sampling study predicts two free energy wells for the rotational isomerization of the variant-3 scorpion neurotoxin tryptophan-47 indole side chain. One well has the indole side chain in the crystallographic orientation; the other has the indole rotated approximately 220 degrees to form a new conformation with a relative free energy of 3 +/- 2 kcal/mol. The activation barrier is 8.5 kcal/mol from the crystallographic well, from which transition state theory predicts a rate of escape of 2 x 10(5) s-1. Correlations in the displacements of side chains neighboring tryptophan-47 and the isomerization reaction coordinate last up to 20 ps. Favorable conditions of experimental verification are discussed. PMID:2393708

  9. LC-Biosensor System for the Determination of the Neurotoxin β-N-Oxalyl-l-α,β-diaminopropionic Acid.

    PubMed

    Belay, A; Ruzgas, T; Csöregi, E; Moges, G; Tessema, M; Solomon, T; Gorton, L

    1997-09-01

    An analysis system is described for the determination of the neurotoxin β-N-oxalyl-l-α,β-diaminopropionic acid (β-ODAP). The system is based on liquid chromatographic separation of β-ODAP from interfering amino acids on an anion exchange column coupled with an amperometric enzyme electrode for the registration of β-ODAP. The electrode is based on a graphite rod modified with an Os(2+/3+) redox polymer cross-linked with l-glutamate oxidase and horseradish peroxidase. This LC-bienzyme electrode analytical system enabled monitoring of as low as 4 μM β-ODAP (injection volume 100 μL). The β-ODAP content in real grass pea samples was measured to range between 3.3 and 5.2 g kg(-)(1) in dry grass pea. PMID:21639270

  10. Structure-function relationships in scorpion neurotoxins. Identification of the supperreactive lysine residue in toxin I of Androctonus australis Hector.

    PubMed

    Sampieri, F; Habersetzer-Rochat, C

    1978-07-21

    In a previous article (Habersetzer-Rochat, C. and Sampieri, R. (1976) Biochemistry 15, 2254--2261) it was demonstrated that the toxin I of the North African Scorpion Androctonus australis Hector was inactivated after reaction with iodoacetate; the toxicity loss in mice was correlated with the carboxymethylation of one superreactive residue. In the present work, alkylation of toxin I was performed with iodo[14C]-acetate. Hence, it was possible, after reduction, S-methylation and chymotryptic hydrolysis of this toxin, to isolate the peptide containing the labelled lysine residue. By automatic Edman degradation, this residue was identified as being the penultimate lysine at position 56 in the primary sequence. Comparison of three primary structures of scorpion neurotoxins and comparison in different kinds of activity seem to indicate that this lysine residue is mainly important for toxicity in mice.

  11. Identification of the environmental neurotoxins annonaceous acetogenins in an Annona cherimolia Mill. Alcoholic Beverage Using HPLC-ESI-LTQ-Orbitrap.

    PubMed

    Le Ven, Jessica; Schmitz-Afonso, Isabelle; Lewin, Guy; Brunelle, Alain; Touboul, David; Champy, Pierre

    2014-08-27

    Epidemiological and toxicological studies have suggested Annonaceaeous acetogenins to be environmental neurotoxins responsible for sporadic atypical parkinsonism/dementia in tropical areas. These compounds are present in the tropical genus Annona (Annonaceae), known for its fruit-yielding cultivated species such as Annona cherimolia. This species is widely cultivated in South America, Spain, and Portugal and yields acetogenins in its seeds, stems, and roots. The presence of these compounds in the pulp of its fruit and in derived food products is unclear. An innovative and sensitive methodology by HPLC-ESI-LTQ-Orbitrap with postcolumn infusion of lithium iodide was used to identify the presence of low levels of acetogenins in an A. cherimolia Mill. fruit-based commercial alcoholic beverage. More than 80 representatives were detected, and the 31 most intense acetogenins were identified. All together these findings indicate that this species should be considered as a risk factor within the framework of a worldwide problem of food toxicity.

  12. A scorpion venom neurotoxin paralytic to insects that affects sodium current inactivation: Purification, primary structure, and mode of action

    SciTech Connect

    Eitan, M.; Fowler, E.; Herrmann, R.; Duval, A.; Pelhate, M.; Zlotkin, E. )

    1990-06-26

    A new toxin, Lqh alpha IT, which caused a unique mode of paralysis of blowfly larvae, was purified from the venom of the scorpion Leiurus quinquestriatus hebraeus, and its structural and pharmacological properties were compared to those of three other groups of neurotoxins found in Buthinae scorpion venoms. Like the excitatory and depressant insect-selective neurotoxins, Lqh alpha IT was highly toxic to insects, but it differed from these toxins in two important characteristics: (a) Lqh alpha IT lacked strict selectivity for insects; it was highly toxic to crustaceans and had a measurable but low toxicity to mice. (b) It did not displace an excitatory insect toxin, 125I-AaIT, from its binding sites in the insect neuronal membrane; this indicates that the binding sites for Lqh alpha IT are different from those shared by the excitatory and depressant toxins. However, in its primary structure and its effect on excitable tissues, Lqh alpha IT strongly resembled the well-characterized alpha scorpion toxins, which affect mammals. The amino acid sequence was identical with alpha toxin sequences in 55%-75% of positions. This degree of similarity is comparable to that seen among the alpha toxins themselves. Voltage- and current-clamp studies showed that Lqh alpha IT caused an extreme prolongation of the action potential in both cockroach giant axon and rat skeletal muscle preparations as a result of the slowing and incomplete inactivation of the sodium currents. These observations indicate that Lqh alpha IT is an alpha toxin which acts on insect sodium channels.

  13. Expression, purification and characterization of the receptor-binding domain of botulinum neurotoxin serotype B as a vaccine candidate.

    PubMed

    Ben David, Alon; Torgeman, Amram; Barnea, Ada; Zichel, Ran

    2015-06-01

    The receptor-binding domain of botulinum neurotoxins (the HC fragment) is a promising vaccine candidate. Among the HC fragments of the seven BoNT serotypes, the expression of HC/B in Escherichia coli is considered especially challenging due to its accumulation as a non-soluble protein aggregate. In this study, the effects of different parameters on the expression of soluble HC/B were evaluated using a screening assay that included growing the bacterium at a small scale, a chemical cell lysis step, and a specific ELISA. The highest soluble HC/B expression levels were obtained when the bacterium E. coli BL21(DE3)+pET-9a-HC/B was grown in terrific broth media at 18°C without induction. Under these conditions, the yield was an order of magnitude higher than previously reported. Standard purification of the protein using a nickel column resulted in a low purity of HC/B. However, the addition of an acidic wash step prior to protein elution released a major protein contaminant and significantly increased the purity level. Mass spectrometry analysis identified the contaminant as ArnA, an E. coli protein that often contaminates recombinant His-tagged protein preparations. The purified HC/B was highly immunogenic, protecting mice from a 10(6) LD50 challenge after a single vaccination and generating a neutralizing titer of 50IU/ml after three immunizations. Moreover, the functionality of the protein was preserved, as it inhibited BoNT/B intoxication in vivo, presumably due to blockade of the neurotoxin protein receptor synaptotagmin.

  14. Characterization of the binding of the Ptychodiscus brevis neurotoxin T17 to sodium channels in rat brain synaptosomes

    SciTech Connect

    Poli, M.A.

    1985-01-01

    The lipid-soluble polyether neurotoxins isolated from the marine dinoflagellate Ptychodiscus brevis (formerly Gymnodinium breve) have been determined to bind to a unique receptor site associated with the voltage-sensitive sodium channel in rat brain synaptosomes. Reduction of the C/sub 42/ aldehyde function of T34 to the alcohol function of T17 using NaB/sup 3/H/sub 4/ yielded /sup 3/H-T17 with a specific activity of 15 Ci;/mmol. Using this specific probe, binding to sodium channels was measured at 4/sup 0/CC, 22/sup 0/C, and 37/sup 0/C. Rosenthal analysis of the binding data yielded a K/sub d/ of 2.9 nM and B/sub max/ of 6.8 pmoles /sup 3/H-T17 per mg of synaptosomal protein at 4/sup 0/C. Both K/sub d/ and B/sub max/ were found to be temperature dependent. Depolarization of the synaptosomes by osmotic lysis resulted in the loss of 34% of the available receptor sites, with no decrease in binding affinity. Unlabeled T17, T34, and synthetic T17 (reduced T34) were equipotent in their ability to displace /sup 3/H-T17 from its specific receptor site. Competition experiments using natural toxin probes specific for sites I-IV on the voltage-sensitive sodium channel demonstrate that /sup 3/H-T17 does not bind to any of the previously-described neurotoxin receptor sites. A fifth site is proposed.

  15. Detection, differentiation, and identification of botulinum neurotoxin serotypes C, CD, D, and DC by highly specific immunoassays and mass spectrometry.

    PubMed

    Hansbauer, Eva-Maria; Skiba, Martin; Endermann, Tanja; Weisemann, Jasmin; Stern, Daniel; Dorner, Martin B; Finkenwirth, Friedrich; Wolf, Jessica; Luginbühl, Werner; Messelhäußer, Ute; Bellanger, Laurent; Woudstra, Cédric; Rummel, Andreas; Fach, Patrick; Dorner, Brigitte G

    2016-09-21

    Botulinum neurotoxin (BoNT) serotypes C and D and their mosaic variants CD and DC cause severe cases of botulism in animal husbandry and wildlife. Epidemiological data on the exact serotype or toxin variant causing outbreaks are rarely available, mainly because of their high sequence identity and the lack of fast and specific screening tools to detect and differentiate the four similar toxins. To fill this gap, we developed four highly specific sandwich enzyme-linked immunosorbent assays (ELISAs) able to detect and differentiate botulinum neurotoxins type BoNT/C, D, CD, and DC based on four distinct combinations of specific monoclonal antibodies targeting both conserved and divergent subdomains of the four toxins. Here, highly sensitive detection with detection limits between 2 and 24 pg mL(-1) was achieved. The ELISAs were extensively validated and results were compared with data obtained by quantitative real-time PCR using a panel of Clostridium botulinum strains, real sample materials from veterinary botulism outbreaks, and non-BoNT-producing Clostridia. Additionally, in order to verify the results obtained by ELISA screening, the new monoclonal antibodies were used for BoNT enrichment and subsequent detection (i) on a functional level by endopeptidase mass spectrometry (Endopep-MS) assays and (ii) on a protein sequence level by LC-MS/MS spectrometry. Based on all technical information gathered in the validation study, the four differentiating ELISAs turned out to be highly reliable screening tools for the rapid analysis of veterinary botulism cases and should aid future field investigations of botulism outbreaks and the acquisition of epidemiological data. PMID:27353114

  16. CRISPR/Cas9-Mediated Genomic Deletion of the Beta-1, 4 N-acetylgalactosaminyltransferase 1 Gene in Murine P19 Embryonal Carcinoma Cells Results in Low Sensitivity to Botulinum Neurotoxin Type C

    PubMed Central

    Tsukamoto, Kentaro; Ozeki, Chikako; Kohda, Tomoko; Tsuji, Takao

    2015-01-01

    Botulinum neurotoxins produced by Clostridium botulinum cause flaccid paralysis by inhibiting neurotransmitter release at peripheral nerve terminals. Previously, we found that neurons derived from the murine P19 embryonal carcinoma cell line exhibited high sensitivity to botulinum neurotoxin type C. In order to prove the utility of P19 cells for the study of the intracellular mechanism of botulinum neurotoxins, ganglioside-knockout neurons were generated by deletion of the gene encoding beta-1,4 N-acetylgalactosaminyltransferase 1 in P19 cells using the clustered regularly interspaced short palindromic repeats combined with Cas9 (CRISPR/Cas9) system. By using this system, knockout cells could be generated more easily than with previous methods. The sensitivity of the generated beta-1,4 N-acetylgalactosaminyltransferase 1-depleted P19 neurons to botulinum neurotoxin type C was decreased considerably, and the exogenous addition of the gangliosides GD1a, GD1b, and GT1b restored the susceptibility of P19 cells to botulinum neurotoxin type C. In particular, addition of a mixture of these three ganglioside more effectively recovered the sensitivity of knockout cells compared to independent addition of GD1a, GD1b, or GT1b. Consequently, the genome-edited P19 cells generated by the CRISPR/Cas9 system were useful for identifying and defining the intracellular molecules involved in the toxic action of botulinum neurotoxins. PMID:26177297

  17. Discovery of 3,3'-diindolylmethanes as potent antileishmanial agents.

    PubMed

    Bharate, Sandip B; Bharate, Jaideep B; Khan, Shabana I; Tekwani, Babu L; Jacob, Melissa R; Mudududdla, Ramesh; Yadav, Rammohan R; Singh, Baljinder; Sharma, P R; Maity, Sudip; Singh, Baldev; Khan, Ikhlas A; Vishwakarma, Ram A

    2013-05-01

    An efficient protocol for synthesis of 3,3'-diindolylmethanes using recyclable Fe-pillared interlayered clay (Fe-PILC) catalyst under aqueous medium has been developed. All synthesized 3,3'-diindolylmethanes showed promising antileishmanial activity against Leishmania donovani promastigotes as well as axenic amastigotes. Structure-activity relationship analysis revealed that nitroaryl substituted diindolylmethanes showed potent antileishmanial activity. The 4-nitrophenyl linked 3,3'-diindolylmethane 8g was found to be the most potent antileishmanial analog showing IC50 values of 7.88 and 8.37 μM against both L. donovani promastigotes and amastigotes, respectively. Further, a pharmacophore based QSAR model was established to understand the crucial molecular features of 3,3'-diindolylmethanes essential for potent antileishmanial activity. These compounds also exhibited promising antifungal activity against Cryptococcus neoformans, wherein fluorophenyl substituted 3,3'-diindolylmethanes were found to be most potent antifungal agents. Developed synthetic protocol will be useful for economical and eco-friendly synthesis of potent antileishmanial and antifungal 3,3'-diindolylmethane class of compounds. PMID:23517732

  18. Differential myotoxic and cytotoxic activities of pre-synaptic neurotoxins from Papuan taipan (Oxyuranus scutellatus) and Irian Jayan death adder (Acanthophis rugosus) venoms.

    PubMed

    Chaisakul, Janeyuth; Parkington, Helena C; Isbister, Geoffrey K; Konstantakopoulos, Nicki; Hodgson, Wayne C

    2013-05-01

    Pre-synaptic PLA(2) neurotoxins are important components of many Australasian elapid snake venoms. These toxins disrupt neurotransmitter release. Taipoxin, a pre-synaptic neurotoxin isolated from the venom of the coastal taipan (Oxyuranus scutellatus), causes necrosis and muscle degeneration. The present study examined the myotoxic and cytotoxic activities of venoms from the Papuan taipan (O. scutellatus) and Irian Jayan death adder (Acanthophis rugosus), and also tested their pre-synaptic neurotoxins: cannitoxin and P-EPTX-Ar1a. Based on size-exclusion chromatography analysis, cannitoxin represents 16% of O. scutellatus venom, while P-EPTX-Ar1a represents 6% of A. rugosus venom. In the chick biventer cervicis nerve-muscle preparation, A. rugosus venom displayed significantly higher myotoxic activity than O. scutellatus venom as indicated by inhibition of direct twitches, and an increase in baseline tension. Both cannitoxin and P-EPTX-Ar1a displayed marked myotoxic activity. A. rugosus venom (50-300 μg/ml) produced concentration-dependent inhibition of cell proliferation in a rat skeletal muscle cell line (L6), while 300 μg/ml of O. scutellatus venom was required to inhibit cell proliferation, following 24-hr incubation. P-EPTX-Ar1a had greater cytotoxicity than cannitoxin, inhibiting cell proliferation after 24-hr incubation in L6 cells. Lactate dehydrogenase levels were increased after 1-hr incubation with A. rugosus venom (100-250 μg/ml), O. scutellatus venom (200-250 μg/ml) and P-EPTX-Ar1a (1-2 μM), but not cannitoxin (1-2 μM), suggesting venoms/toxin generated cell necrosis. Thus, A. rugosus and O. scutellatus venoms possess different myotoxic and cytotoxic activities. The proportion of pre-synaptic neurotoxin in the venoms and PLA(2) activity of the whole venoms are unlikely to be responsible for these activities.

  19. Gangliosides in human, cow and goat milk, and their abilities as to neutralization of cholera toxin and botulinum type A neurotoxin.

    PubMed

    Iwamori, Masao; Takamizawa, Kotarou; Momoeda, Mikio; Iwamori, Yuriko; Taketani, Yuji

    2008-10-01

    To elucidate the potential of mammalian milk as to protection of infants from infections, we determined the ganglioside compositions of human, cow and goat milk in relation with cholera toxin and botulinum type A neurotoxin-receptors. Gangliosides accounted for 1 to 2 micromol of lipid-bound sialic acid (LSA) in 100 ml of milk, and GD3 comprised about 69% of LSA in all milk samples. Among the milk samples examined, goat milk was found to contain an amount of gangliosides belonging to the b-pathway representing 15.8% of the total LSA. Accordingly, botulinum neurotoxin bound to GT1b and GQ1b in goat milk, but not to any gangliosides in human or cow milk. On the other hand, GM1, the cholera toxin receptor, was found to be present in all milk samples at concentrations of 0.02% to 0.77% of the total LSA and to be maintained at a relatively constant level in human milk during the postpartum period. Gangliosides from 1 ml of pooled human milk exhibited the ability to attenuate the binding of cholera toxin (30 ng) to GM1 by 93%, and those from 500 microl of goat milk completely inhibited the binding of botulinum type A neurotoxin 1.5 microg to GT1b.

  20. 9-Benzoyl 9-deazaguanines as potent xanthine oxidase inhibitors.

    PubMed

    Rodrigues, Marili V N; Barbosa, Alexandre F; da Silva, Júlia F; dos Santos, Deborah A; Vanzolini, Kenia L; de Moraes, Marcela C; Corrêa, Arlene G; Cass, Quezia B

    2016-01-15

    A novel potent xanthine oxidase inhibitor, 3-nitrobenzoyl 9-deazaguanine (LSPN451), was selected from a series of 10 synthetic derivatives. The enzymatic assays were carried out using an on-flow bidimensional liquid chromatography (2D LC) system, which allowed the screening¸ the measurement of the kinetic inhibition constant and the characterization of the inhibition mode. This compound showed a non-competitive inhibition mechanism with more affinity for the enzyme-substrate complex than for the free enzyme, and inhibition constant of 55.1±9.80 nM, about thirty times more potent than allopurinol. Further details of synthesis and enzymatic studies are presented herein.

  1. Molecular cloning of the Clostridium botulinum structural gene encoding the type B neurotoxin and determination of its entire nucleotide sequence.

    PubMed Central

    Whelan, S M; Elmore, M J; Bodsworth, N J; Brehm, J K; Atkinson, T; Minton, N P

    1992-01-01

    DNA fragments derived from the Clostridium botulinum type A neurotoxin (BoNT/A) gene (botA) were used in DNA-DNA hybridization reactions to derive a restriction map of the region of the C. botulinum type B strain Danish chromosome encoding botB. As the one probe encoded part of the BoNT/A heavy (H) chain and the other encoded part of the light (L) chain, the position and orientation of botB relative to this map were established. The temperature at which hybridization occurred indicated that a higher degree of DNA homology occurred between the two genes in the H-chain-encoding region. By using the derived restriction map data, a 2.1-kb BglII-XbaI fragment encoding the entire BoNT/B L chain and 108 amino acids of the H chain was cloned and characterized by nucleotide sequencing. A contiguous 1.8-kb XbaI fragment encoding a further 623 amino acids of the H chain was also cloned. The 3' end of the gene was obtained by cloning a 1.6-kb fragment amplified from genomic DNA by inverse polymerase chain reaction. Translation of the nucleotide sequence derived from all three clones demonstrated that BoNT/B was composed of 1,291 amino acids. Comparative alignment of its sequence with all currently characterized BoNTs (A, C, D, and E) and tetanus toxin (TeTx) showed that a wide variation in percent homology occurred dependent on which component of the dichain was compared. Thus, the L chain of BoNT/B exhibits the greatest degree of homology (50% identity) with the TeTx L chain, whereas its H chain is most homologous (48% identity) with the BoNT/A H chain. Overall, the six neurotoxins were shown to be composed of highly conserved amino acid domains interceded with amino acid tracts exhibiting little overall similarity. In total, 68 amino acids of an average of 442 are absolutely conserved between L chains and 110 of 845 amino acids are conserved between H chains. Conservation of Trp residues (one in the L chain and nine in the H chain) was particularly striking. The most

  2. Endangered North Atlantic right whales (Eubalaena glacialis) experience repeated, concurrent exposure to multiple environmental neurotoxins produced by marine algae.

    PubMed

    Doucette, Gregory J; Mikulski, Christina M; King, Kristen L; Roth, Patricia B; Wang, Zhihong; Leandro, Luis F; DeGrasse, Stacey L; White, Kevin D; De Biase, Daniela; Gillett, Roxanne M; Rolland, Rosalind M

    2012-01-01

    The western North Atlantic population of right whales (Eubalaena glacialis) is one of the most critically endangered of any whale population in the world. Among the factors considered to have potentially adverse effects on the health and reproduction of E. glacialis are biotoxins produced by certain microalgae responsible for causing harmful algal blooms. The worldwide incidence of these events has continued to increase dramatically over the past several decades and is expected to remain problematic under predicted climate change scenarios. Previous investigations have demonstrated that N. Atlantic right whales are being exposed to at least two classes of algal-produced environmental neurotoxins-paralytic shellfish toxins (PSTs) and domoic acid (DA). Our primary aims during this six-year study (2001-2006) were to assess whether the whales' exposure to these algal biotoxins occurred annually over multiple years, and to what extent individual whales were exposed repeatedly and/or concurrently to one or both toxin classes. Approximately 140 right whale fecal samples obtained across multiple habitats in the western N. Atlantic were analyzed for PSTs and DA. About 40% of these samples were attributed to individual whales in the North Atlantic Right Whale Catalog, permitting analysis of biotoxin exposure according to sex, age class, and reproductive status/history. Our findings demonstrate clearly that right whales are being exposed to both of these algal biotoxins on virtually an annual basis in multiple habitats for periods of up to six months (April through September), with similar exposure rates for females and males (PSTs: ∼70-80%; DA: ∼25-30%). Notably, only one of 14 lactating females sampled did not contain either PSTs or DA, suggesting the potential for maternal toxin transfer and possible effects on neonatal animals. Moreover, 22% of the fecal samples tested for PSTs and DA showed concurrent exposure to both neurotoxins, leading to questions of interactive

  3. The Analgesic Effect on Neuropathic Pain of Retrogradely Transported botulinum Neurotoxin A Involves Schwann Cells and Astrocytes

    PubMed Central

    Ricordy, Ruggero; Uggenti, Carolina; Tata, Ada Maria; Luvisetto, Siro; Pavone, Flaminia

    2012-01-01

    In recent years a growing debate is about whether botulinum neurotoxins are retrogradely transported from the site of injection. Immunodetection of cleaved SNAP-25 (cl-SNAP-25), the protein of the SNARE complex targeted by botulinum neurotoxin serotype A (BoNT/A), could represent an excellent approach to investigate the mechanism of action on the nociceptive pathways at peripheral and/or central level. After peripheral administration of BoNT/A, we analyzed the expression of cl-SNAP-25, from the hindpaw’s nerve endings to the spinal cord, together with the behavioral effects on neuropathic pain. We used the chronic constriction injury of the sciatic nerve in CD1 mice as animal model of neuropathic pain. We evaluated immunostaining of cl-SNAP-25 in the peripheral nerve endings, along the sciatic nerve, in dorsal root ganglia and in spinal dorsal horns after intraplantar injection of saline or BoNT/A, alone or colocalized with either glial fibrillar acidic protein, GFAP, or complement receptor 3/cluster of differentiation 11b, CD11b, or neuronal nuclei, NeuN, depending on the area investigated. Immunofluorescence analysis shows the presence of the cl-SNAP-25 in all tissues examined, from the peripheral endings to the spinal cord, suggesting a retrograde transport of BoNT/A. Moreover, we performed in vitro experiments to ascertain if BoNT/A was able to interact with the proliferative state of Schwann cells (SC). We found that BoNT/A modulates the proliferation of SC and inhibits the acetylcholine release from SC, evidencing a new biological effect of the toxin and further supporting the retrograde transport of the toxin along the nerve and its ability to influence regenerative processes. The present results strongly sustain a combinatorial action at peripheral and central neural levels and encourage the use of BoNT/A for the pathological pain conditions difficult to treat in clinical practice and dramatically impairing patients’ quality of life. PMID:23110146

  4. Endangered North Atlantic right whales (Eubalaena glacialis) experience repeated, concurrent exposure to multiple environmental neurotoxins produced by marine algae.

    PubMed

    Doucette, Gregory J; Mikulski, Christina M; King, Kristen L; Roth, Patricia B; Wang, Zhihong; Leandro, Luis F; DeGrasse, Stacey L; White, Kevin D; De Biase, Daniela; Gillett, Roxanne M; Rolland, Rosalind M

    2012-01-01

    The western North Atlantic population of right whales (Eubalaena glacialis) is one of the most critically endangered of any whale population in the world. Among the factors considered to have potentially adverse effects on the health and reproduction of E. glacialis are biotoxins produced by certain microalgae responsible for causing harmful algal blooms. The worldwide incidence of these events has continued to increase dramatically over the past several decades and is expected to remain problematic under predicted climate change scenarios. Previous investigations have demonstrated that N. Atlantic right whales are being exposed to at least two classes of algal-produced environmental neurotoxins-paralytic shellfish toxins (PSTs) and domoic acid (DA). Our primary aims during this six-year study (2001-2006) were to assess whether the whales' exposure to these algal biotoxins occurred annually over multiple years, and to what extent individual whales were exposed repeatedly and/or concurrently to one or both toxin classes. Approximately 140 right whale fecal samples obtained across multiple habitats in the western N. Atlantic were analyzed for PSTs and DA. About 40% of these samples were attributed to individual whales in the North Atlantic Right Whale Catalog, permitting analysis of biotoxin exposure according to sex, age class, and reproductive status/history. Our findings demonstrate clearly that right whales are being exposed to both of these algal biotoxins on virtually an annual basis in multiple habitats for periods of up to six months (April through September), with similar exposure rates for females and males (PSTs: ∼70-80%; DA: ∼25-30%). Notably, only one of 14 lactating females sampled did not contain either PSTs or DA, suggesting the potential for maternal toxin transfer and possible effects on neonatal animals. Moreover, 22% of the fecal samples tested for PSTs and DA showed concurrent exposure to both neurotoxins, leading to questions of interactive

  5. Rapid assembly of diverse and potent allosteric Akt inhibitors.

    PubMed

    Wu, Zhicai; Robinson, Ronald G; Fu, Sheng; Barnett, Stanley F; Defeo-Jones, Deborah; Jones, Raymond E; Kral, Astrid M; Huber, Hans E; Kohl, Nancy E; Hartman, George D; Bilodeau, Mark T

    2008-03-15

    This paper describes the rapid assembly of four different classes of potent Akt inhibitors from a common intermediate. Among them, a pyridopyrimidine series displayed the best intrinsic and cell potency against Akt1 and Akt2. This series also showed a promising pharmacokinetic profile and excellent selectivity over other closely related kinases.

  6. DIMETHYLARSINE AND TRIMETHYLARSINE ARE POTENT GENOTOXINS IN VITRO.

    EPA Science Inventory

    Dimethylarsine and Trimethylarsine are potent genotoxins in vitro
    Andrewes, P; Kitchin, KT; and Wallace, KA

    Abstract
    The mechanism of arsenic carcinogenesis is unclear. A complicating factor receiving increasing attention is that arsenic is biomethylated to form vari...

  7. Total synthesis of the potent microtubule-stabilizing agent (+)-discodermolide.

    PubMed

    Harried, Scott S; Lee, Christopher P; Yang, Ge; Lee, Tony I H; Myles, David C

    2003-08-22

    The total synthesis of the potent microtubule-stabilizing, antimitotic agent (+)-discodermolide is described. The convergent synthetic strategy takes advantage of the diastereoselective alkylation of a ketone enolate to establish the key C15-C16 bond. The synthesis is amenable to preparation of gram-scale quantities of (+)-discodermolide and analogues.

  8. Potent Antioxidant Dendrimers Lacking Pro-oxidant Activity

    PubMed Central

    Lee, Choon Young; Sharma, Ajit; Uzarski, Rebecca L.; Cheong, Jae Eun; Xu, Hao; Held, Rich A.; Upadhaya, Samik K.; Nelson, Julie L.

    2010-01-01

    It is well known that antioxidants have protective effects against oxidative stress. Unfortunately, in the presence of transition metals, antioxidants including polyphenols with potent antioxidant activities may also exhibit pro-oxidant effects, which may irreversibly damage DNA. Therefore, antioxidants with strong free radical scavenging abilities and devoid of pro-oxidant effects would be of immense biological importance. We report two antioxidant dendrimers with a surface rich in multiple phenolic hydroxyl groups, benzylic hydrogens and electron donating ring substituents that contribute to their potent free radical quenching property. In order to minimize their pro-oxidant effects, the dendrimers were designed with a metal chelating tris(2-aminoethyl)amine (TREN) core. The dendritic antioxidants were prepared by attachment of six syringaldehyde or vanillin molecules to TREN by reductive amination. They exhibited potent radical scavenging properties: 5 times stronger than quercetin and 15 times more potent than Trolox according to the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The antioxidant dendrimers also protected low-density lipoprotein, lysozyme and DNA against 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced free radical damage. More importantly, unlike quercetin and Trolox, the two TREN antioxidant dendrimers did not damage DNA via their pro-oxidant effects when incubated with physiological amounts of copper ions. The dendrimers also showed no cytotoxicity towards Chinese hamster ovary cells. PMID:20977937

  9. Potent pyrrolidine- and piperidine-based BACE-1 inhibitors

    SciTech Connect

    Iserloh, U.; Wu, Y.; Cumming, J.N.; Pan, J.; Wang, L.Y.; Stamford, A.W.; Kennedy, M.E.; Kuvelkar, R.; Chen, X.; Parker, E.M.; Strickland, C.; Voigt, J.

    2008-08-18

    Based on lead compound 1 identified from the patent literature, we developed novel patentable BACE-1 inhibitors by introducing a cyclic amine scaffold. Extensive SAR studies on both pyrrolidines and piperidines ultimately led to inhibitor 2f, one of the most potent inhibitors synthesized to date. The discovery and development of novel BACE-1 inhibitors incorporating a cyclic amine scaffold is described.

  10. The cyanobacterial neurotoxin β-N-methylamino-l-alanine (BMAA) induces neuronal and behavioral changes in honeybees.

    PubMed

    Okle, Oliver; Rath, Lisa; Galizia, C Giovanni; Dietrich, Daniel R

    2013-07-01

    The cyanobacterially produced neurotoxin beta-N-methylamino-l-alanine (BMAA) is thought to induce amyotrophic lateral sclerosis/Parkinsonism dementia complex (ALS/PDC)-like symptoms. However, its mechanism of action and its pathway of intoxication are yet unknown. In vivo animal models suitable for investigating the neurotoxic effect of BMAA with applicability to the human are scarce. Hence, we used the honeybee (Apis mellifera) since its nervous system is relatively simple, yet having cognitive capabilities. Bees fed with BMAA-spiked sugar water had an increased mortality rate and a reduced ability to learn odors in a classical conditioning paradigm. Using (14)C-BMAA we demonstrated that BMAA is biologically available to the bee, and is found in the head, thorax and abdomen with little to no excretion. BMAA is also transferred from one bee to the next via trophallaxis resulting in an exposure of the whole beehive. BMAA bath application directly onto the brain leads to an altered Ca(2+) homeostasis and to generation of reactive oxygen species. These behavioral and physiological observations suggest that BMAA may have effects on bee brains similar to those assumed to occur in humans. Therefore the bee could serve as a surrogate model system for investigating the neurological effects of BMAA.

  11. The neurotoxin 1-methyl-4-phenylpyridinium (MPP+) alters hippocampal excitatory synaptic transmission by modulation of the GABAergic system

    PubMed Central

    Huang, YuYing; Chen, JunFang; Chen, Ying; Zhuang, YingHan; Sun, Mu; Behnisch, Thomas

    2015-01-01

    The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces Parkinson’s disease-like symptoms following administration to mice, monkeys, and humans. A common view is that MPTP is metabolized to 1-methyl-4-phenylpyridinium ion (MPP+) to induce its neurodegenerative effects on dopaminergic neurons in the substantia nigra (SN). Moreover, the hippocampus contains dopaminergic fibers, which are projecting from the ventral tegmental area, SN and pars compacta and contain the whole machinery required for dopamine synthesis making them sensitive to MPTP and MPP+. Here, we present data showing that acute bath-application of MPP+ elicited a dose-dependent facilitation followed by a depression of synaptic transmission of hippocampal Schaffer collaterals-CA1 synapses in mice. The effects of MPP+ were not mediated by D1/D5- and D2-like receptor activation. Inhibition of the dopamine transporters did not prevent but increased the depression of excitatory post-synaptic field potentials. In the search for a possible mechanism, we observed that MPP+ reduced the appearance of polyspikes in population spikes recorded in str. pyramidale and increased the frequency of miniature inhibitory post-synaptic currents. The acute effect of MPP+ on synaptic transmission was attenuated by co-application of a GABAA receptor antagonist. Taking these data together, we suggest that MPP+ affects hippocampal synaptic transmission by enhancing some aspects of the hippocampal GABAergic system. PMID:26300734

  12. The use of Endopep-MS to detect multiple subtypes of botulinum neurotoxins A, B, E, and F

    NASA Astrophysics Data System (ADS)

    Kalb, Suzanne R.; Smith, Theresa J.; Moura, Hercules; Hill, Karen; Lou, Jianlong; Geren, Isin N.; Garcia-Rodriguez, Consuelo; Marks, James D.; Smith, Leonard A.; Pirkle, James L.; Barr, John R.

    2008-12-01

    Botulinum neurotoxins (BoNTs) cause the disease botulism, which can be lethal if untreated. Rapid determination of exposure to BoNT is an important public health goal. Previous work in our laboratory focused on the development of Endopep-MS, a mass spectrometry-based endopeptidase method for detecting and differentiating BoNT A-G in buffer and BoNT A, B, E, and F in clinical samples. We introduce here the use of Endopep-MS to detect non-commercial subtypes of BoNT A, B, E, and F which have been associated with botulism outbreaks. We have now tested and successfully detected 15 of the 17 known subtypes of BoNT A, B, E, and F by Endopep-MS. Extraction of BoNT A and B from a complex mixture prior to analysis is accomplished by using monoclonal antibodies specific for the catalytically inactive heavy chain of the toxin. These antibodies have high-binding affinities and do not interfere with the catalytic activity of the light chain resulting in a lower limit of detection for BoNT A and B than previously reported. We also report for the first time limits of detection for BoNT A2, A3, B2, and bivalent B using Endopep-MS.

  13. A venom-derived neurotoxin, CsTx-1, from the spider Cupiennius salei exhibits cytolytic activities.

    PubMed

    Kuhn-Nentwig, Lucia; Fedorova, Irina M; Lüscher, Benjamin P; Kopp, Lukas S; Trachsel, Christian; Schaller, Johann; Vu, Xuan Lan; Seebeck, Thomas; Streitberger, Kathrin; Nentwig, Wolfgang; Sigel, Erwin; Magazanik, Lev G

    2012-07-20

    CsTx-1, the main neurotoxic acting peptide in the venom of the spider Cupiennius salei, is composed of 74 amino acid residues, exhibits an inhibitory cysteine knot motif, and is further characterized by its highly cationic charged C terminus. Venom gland cDNA library analysis predicted a prepropeptide structure for CsTx-1 precursor. In the presence of trifluoroethanol, CsTx-1 and the long C-terminal part alone (CT1-long; Gly-45-Lys-74) exhibit an α-helical structure, as determined by CD measurements. CsTx-1 and CT1-long are insecticidal toward Drosophila flies and destroys Escherichia coli SBS 363 cells. CsTx-1 causes a stable and irreversible depolarization of insect larvae muscle cells and frog neuromuscular preparations, which seem to be receptor-independent. Furthermore, this membranolytic activity could be measured for Xenopus oocytes, in which CsTx-1 and CT1-long increase ion permeability non-specifically. These results support our assumption that the membranolytic activities of CsTx-1 are caused by its C-terminal tail, CT1-long. Together, CsTx-1 exhibits two different functions; as a neurotoxin it inhibits L-type Ca(2+) channels, and as a membranolytic peptide it destroys a variety of prokaryotic and eukaryotic cell membranes. Such a dualism is discussed as an important new mechanism for the evolution of spider venomous peptides.

  14. Chemical modification of cationic groups of a novel alpha-neurotoxin (Oh-4) from king cobra (Ophiophagus hannah) venom.

    PubMed

    Lin, S R; Chi, S H; Chang, L S; Kuo, K W; Chang, C C

    1995-08-01

    The cationic groups of arginine and lysine residues in Oh-4, a novel alpha-neurotoxin from king cobra (Ophiophagus hannah) venom were subjected to modification with p-hydroxyphenylglyoxal (HPG) and trinitrobenzene sulfonate (TNBS), respectively. Monoderivatization of Arg-35, resulted in a drastic loss in neurotoxicity to 25% of the native toxin. The activity was decreased to a greater extent with the derivative extensively modified on Arg-35, -9, and -37. The Arg-35-modified derivative retained about a half of the antigenicity of the native toxin, and extensive modification on Arg-9 and Arg-37 caused a further decrease in the antigenicity of the toxin molecule. Selective trinitrophenylation (TNP-) of Lys-51 caused losses of neurotoxicity and antigenicity by 77 and 83%, respectively. These results indicate that Arg-35 and Lys-51 in Oh-4 have important roles in the neurotoxicity. In contrast to the Arg residues at 9, 35, and 37, Lys-51 plays a more critical role in the antigenicity.

  15. Effect of nitrogen on cellular production and release of the neurotoxin anatoxin-a in a nitrogen-fixing cyanobacterium.

    PubMed

    Gagnon, Alexis; Pick, Frances R

    2012-01-01

    Anatoxin-a (ANTX) is a neurotoxin produced by several freshwater cyanobacteria and implicated in lethal poisonings of domesticated animals and wildlife. The factors leading to its production in nature and in culture are not well understood. Resource availability may influence its cellular production as suggested by the carbon-nutrient hypothesis, which links the amount of secondary metabolites produced by plants or microbes to the relative abundance of nutrients. We tested the effects of nitrogen supply (as 1, 5, and 100% N of standard cyanobacterial medium corresponding to 15, 75, and 1500 mg L(-1) of NaNO(3) respectively) on ANTX production and release in a toxic strain of the planktonic cyanobacterium Aphanizomenon issatschenkoi (Nostocales). We hypothesized that nitrogen deficiency might constrain the production of ANTX. However, the total concentration and more significantly the cellular content of anatoxin-a peaked (max. 146 μg/L and 1683 μg g(-1) dry weight) at intermediate levels of nitrogen supply when N-deficiency was evident based on phycocyanin to chlorophyll a and carbon to nitrogen ratios. The results suggest that the cellular production of anatoxin-a may be stimulated by moderate nitrogen stress. Maximal cellular contents of other cyanotoxins have recently been reported under severe stress conditions in another Nostocales species.

  16. Label-free electrochemical detection of botulinum neurotoxin type E based on its enzymatic activity using interdigitated electrodes

    NASA Astrophysics Data System (ADS)

    Hyun, Sang Hwa; Park, Dae Keun; Kang, Aeyeon; Kim, Soohyun; Kim, Daehee; Shin, Yu Mi; Song, Ji-Joon; Yun, Wan Soo

    2016-02-01

    We report a simple label-free electrochemical method of detecting low concentrations of botulinum neurotoxin type E light chain (BoNT/E LC) based on its peptide cleavage activity. Dual-mode cyclic voltammetry was employed to observe changes in the redox signal of ferri-/ferro-cyanide on interdigitated microelectrodes, whose surfaces were covered by peptides designed from synaptosomal-associated protein 25 to be cleaved by BoNT/E LC. With the introduction of BoNT/E LC, the redox signal showed a time-dependent increase due to cleavage of the immobilized peptide molecules. In addition to the increased redox signal intensity, its time-dependence can be considered as a strong evidence of BoNT/E sensing, since the time-dependent increase can only result from the enzymatic activity of BoNT/E LC. Using this method, BoNT/E LC, at concentrations as low as 5 pg/ml, was readily measurable with only an hour of incubation.

  17. Effects of solution conditions and surface chemistry on the adsorption of three recombinant botulinum neurotoxin antigens to aluminum salt adjuvants.

    PubMed

    Vessely, Christina; Estey, Tia; Randolph, Theodore W; Henderson, Ian; Nayar, Rajiv; Carpenter, John F

    2007-09-01

    Botulinum neurotoxin (BoNT) is a biological warfare threat. Protein antigens have been developed against the seven major BoNT serotypes for the development of a recombinant protein vaccine. This study is an evaluation of adsorption profiles for three of the recombinant protein antigens to aluminum salt adjuvants in the development of a trivalent vaccine against BoNT. Adsorption profiles were obtained over a range of protein concentrations. The results document that charge-charge interactions dominate the adsorption of antigen to adjuvant. Optimal conditions for adsorption were determined. However, potency studies and solution stability studies indicated the necessity of using aluminum hydroxide adjuvant at low pH. To improve the adsorption profiles to AlOH adjuvant, phosphate ions were introduced into the adsorption buffers. The resulting change in the adjuvant chemistry led to an improvement of adsorption of the BoNT antigens to aluminum hydroxide adjuvant while maintaining potency. Competitive adsorption profiles were also determined, and showed changes in maximum adsorption from mixed solutions compared to adsorption from individual protein solutions. The adsorption profiles for each protein varied due to differences in adsorption mechanism and affinity for the adjuvant surface. These results emphasize the importance of evaluating competitive adsorption in the development of multivalent vaccine products. PMID:17518359

  18. Depolarization after resonance energy transfer (DARET): a sensitive fluorescence-based assay for botulinum neurotoxin protease activity.

    PubMed

    Gilmore, Marcella A; Williams, Dudley; Okawa, Yumiko; Holguin, Bret; James, Nicholas G; Ross, Justin A; Roger Aoki, K; Jameson, David M; Steward, Lance E

    2011-06-01

    The DARET (depolarization after resonance energy transfer) assay is a coupled Förster resonance energy transfer (FRET)-fluorescence polarization assay for botulinum neurotoxin type A or E (BoNT/A or BoNT/E) proteolytic activity that relies on a fully recombinant substrate. The substrate consists of blue fluorescent protein (BFP) and green fluorescent protein (GFP) flanking SNAP-25 (synaptosome-associated protein of 25 kDa) residues 134-206. In this assay, the substrate is excited with polarized light at 387 nm, which primarily excites the BFP, whereas emission from the GFP is monitored at 509 nm. Energy transfer from the BFP to the GFP in the intact substrate results in a substantial depolarization of the GFP emission. The energy transfer is eliminated when the fluorescent domains separate on cleavage by the endopeptidase, and emission from the directly excited GFP product fragment is then highly polarized, resulting in an overall increase in polarization. This increase in polarization can be monitored to assay the proteolytic activity of BoNT/A and BoNT/E in real time. It allows determination of the turnover rate of the substrate and the kinetic constants (V(max) and k(cat)) based on the concentration of cleaved substrate determined directly from the measurements using the additivity properties of polarization. The assay is amenable to high-throughput applications.

  19. Effect of Nitrogen on Cellular Production and Release of the Neurotoxin Anatoxin-A in a Nitrogen-Fixing Cyanobacterium

    PubMed Central

    Gagnon, Alexis; Pick, Frances R.

    2012-01-01

    Anatoxin-a (ANTX) is a neurotoxin produced by several freshwater cyanobacteria and implicated in lethal poisonings of domesticated animals and wildlife. The factors leading to its production in nature and in culture are not well understood. Resource availability may influence its cellular production as suggested by the carbon-nutrient hypothesis, which links the amount of secondary metabolites produced by plants or microbes to the relative abundance of nutrients. We tested the effects of nitrogen supply (as 1, 5, and 100% N of standard cyanobacterial medium corresponding to 15, 75, and 1500 mg L−1 of NaNO3 respectively) on ANTX production and release in a toxic strain of the planktonic cyanobacterium Aphanizomenon issatschenkoi (Nostocales). We hypothesized that nitrogen deficiency might constrain the production of ANTX. However, the total concentration and more significantly the cellular content of anatoxin-a peaked (max. 146 μg/L and 1683 μg g−1 dry weight) at intermediate levels of nitrogen supply when N-deficiency was evident based on phycocyanin to chlorophyll a and carbon to nitrogen ratios. The results suggest that the cellular production of anatoxin-a may be stimulated by moderate nitrogen stress. Maximal cellular contents of other cyanotoxins have recently been reported under severe stress conditions in another Nostocales species. PMID:22701451

  20. The neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) alters hippocampal excitatory synaptic transmission by modulation of the GABAergic system.

    PubMed

    Huang, YuYing; Chen, JunFang; Chen, Ying; Zhuang, YingHan; Sun, Mu; Behnisch, Thomas

    2015-01-01

    The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces Parkinson's disease-like symptoms following administration to mice, monkeys, and humans. A common view is that MPTP is metabolized to 1-methyl-4-phenylpyridinium ion (MPP(+)) to induce its neurodegenerative effects on dopaminergic neurons in the substantia nigra (SN). Moreover, the hippocampus contains dopaminergic fibers, which are projecting from the ventral tegmental area, SN and pars compacta and contain the whole machinery required for dopamine synthesis making them sensitive to MPTP and MPP(+). Here, we present data showing that acute bath-application of MPP(+) elicited a dose-dependent facilitation followed by a depression of synaptic transmission of hippocampal Schaffer collaterals-CA1 synapses in mice. The effects of MPP(+) were not mediated by D1/D5- and D2-like receptor activation. Inhibition of the dopamine transporters did not prevent but increased the depression of excitatory post-synaptic field potentials. In the search for a possible mechanism, we observed that MPP(+) reduced the appearance of polyspikes in population spikes recorded in str. pyramidale and increased the frequency of miniature inhibitory post-synaptic currents. The acute effect of MPP(+) on synaptic transmission was attenuated by co-application of a GABAA receptor antagonist. Taking these data together, we suggest that MPP(+) affects hippocampal synaptic transmission by enhancing some aspects of the hippocampal GABAergic system. PMID:26300734