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Sample records for neurotoxin crotoxin complex

  1. Structural studies of the oligomeric formation crotoxin and their complexes; Estudos estruturais da formacao oligomerica da crotoxina e seus complexos

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    Morelli, Humberto A.S.; Fernandes, Carlos A.H.; Magro, Angelo J.; Fontes, Marcos R.M.; Fernandez, Roberto M., E-mail: rmorato@ibb.unesp.br [Universidade Estadual Paulista Julio de Mesquita Filho (UNESP), Botucatu, SP (Brazil). Inst. de Biociencias. Dept. de Fisica e Biofisica; Bicev, Renata N.; Oliveira, Cristiano L.P. de [Universidade de Sao Paulo (USP), Sao Paulo, SP (Brazil). Inst. de Fisica. Dept. de Fisica Experimental; Barioni, Marina B.; Ito, Amando S. [Universidade de Sao Paulo (USP), Ribeirao Preto, SP (Brazil). Fac. de Filosofia, Ciencias e Letras. Dept. de Fisica

    2013-08-15

    The crotoxin is a phospholipase A{sub 2} that is the mainly responsible for the neurotoxic effect of the snakes' venom of the species Crotalus durissus, popularly known as rattlesnakes. In the crotoxin there are two subunits: an acidic, non-toxic and devoid of enzymatic action, (called crotoxin A or CA); and a basic, toxic with enzymatic activity (called crotoxin B or CB). The main objective of this work is to study the CA, CB and the complex CA + CB using the Small-Angle X-ray Scattering (SAXS), Optical absorption spectrometry and static fluorescence techniques. (author)

  2. Presynaptic Neurotoxins: Biochemistry, Molecular Biology, Immunology and Other Exploratory Studies

    Science.gov (United States)

    1994-04-01

    residues are conserved in PLA2s that exist as aggregates (e.g. crotoxin, Mojave toxin, vipoxin from Vipera ammodytes , the complex from Pseudocerastes...success of others in using the site-directed polyclonal antibody approach to localize the toxic site in the ammodytoxins from the venom of Vipera ... ammodytes (Curin-Serbec et al, 1991). Molecular Biology. Descriptions of the organization of genomic presynaptic neurotoxin genes is presently in its

  3. Crotoxin in humans: analysis of the effects on extraocular and facial muscles

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    Geraldo de Barros Ribeiro

    2012-12-01

    Full Text Available PURPOSE: Crotoxin is the main neurotoxin of South American rattlesnake Crotalus durissus terrificus. The neurotoxic action is characterized by a presynaptic blockade. The purpose of this research is to assess the ability of crotoxin to induce temporary paralysis of extraocular and facial muscles in humans. METHODS: Doses of crotoxin used ranged from 2 to 5 units (U, each unit corresponding to one LD50. We first applied 2U of crotoxin in one of the extraocular muscles of 3 amaurotic individuals to be submitted to ocular evisceration. In the second stage, we applied crotoxin in 12 extraocular muscles of 9 patients with strabismic amblyopia. In the last stage, crotoxin was used in the treatment of blepharospasm in another 3 patients. RESULTS: No patient showed any systemic side effect or change in vision or any eye structure problem after the procedure. The only local side effects observed were slight conjunctival hyperemia, which recovered spontaneously. In 2 patients there was no change in ocular deviation after 2U crotoxin application. Limitation of the muscle action was observed in 8 of the 12 applications. The change in ocular deviation after application of 2U of crotoxin (9 injections was in average 15.7 prism diopters (PD. When the dose was 4U (2 applications the change was in average 37.5 PD and a single application of 5U produced a change of 16 PD in ocular deviation. This effect lasted from 1 to 3 months. Two of the 3 patients with blepharospasm had the hemifacial spasm improved with crotoxin, which returned after 2 months. CONCLUSIONS: This study provides data suggesting that crotoxin may be a useful new therapeutic option for the treatment of strabismus and blepharospasm. We expect that with further studies crotoxin could be an option for many other medical areas.

  4. Structural Studies on Intact Clostridium Botulinum Neurotoxins Complexed with Inhibitors Leading to Drug Design

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    2006-02-01

    structure1. Introduction Tetanus neurotoxin (TeNT) produced by Clostridium tetani and the seven antigenically distinct botulinum neurotoxins (BoNT/A-G...2-0011 TITLE: Structural Studies on Intact Clostridium Botulinum Neurotoxins Complexed with Inhibitors Leading to Drug...DATES COVERED (From - To) 28 Jan 2005 – 27 Jan 2006 4. TITLE AND SUBTITLE Structural Studies on Intact Clostridium Botulinum Neurotoxins Complexed

  5. Preliminary X-ray crystallographic studies of a tetrameric phospholipase A{sub 2} formed by two isoforms of crotoxin B from Crotalus durissus terrificus venom

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    Marchi-Salvador, D. P.; Corrêa, L. C.; Salvador, G. H. M.; Magro, A. J. [Departamento de Física e Biofísica, Instituto de Biociências, UNESP, CP 510, 18618-000 Botucatu-SP (Brazil); Oliveira, C. Z. [Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, FCFRP, USP, Ribeirão Preto-SP (Brazil); Iulek, J. [Departamento de Química, UEPG, Ponta Grossa-PR (Brazil); Soares, A. M. [Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, FCFRP, USP, Ribeirão Preto-SP (Brazil); Fontes, M. R. M., E-mail: fontes@ibb.unesp.br [Departamento de Física e Biofísica, Instituto de Biociências, UNESP, CP 510, 18618-000 Botucatu-SP (Brazil)

    2007-12-01

    Crotoxin B is a basic phospholipase A{sub 2} found in the venom of C. durissus terrificus and is one of the subunits that constitute crotoxin. Here, the crystallization, X-ray diffraction data collection and molecular-replacement solution of a novel tetrameric complex formed by two dimers of crotoxin B isoforms are presented. Crotoxin B is a basic phospholipase A{sub 2} found in the venom of Crotalus durissus terrificus and is one of the subunits that constitute crotoxin. This heterodimeric toxin, which is the main component of C. d. terrificus venom, is completed by an acidic, nontoxic and non-enzymatic component (crotoxin A) and is involved in important envenomation effects, such as neurological disorders, myotoxicity and renal failure. Although crotoxin was first crystallized in 1938, no crystal structure is currently available for crotoxin, crotoxin A or crotoxin B. In this work, the crystallization, X-ray diffraction data collection to 2.28 Å resolution and molecular-replacement solution of a novel tetrameric complex formed by two dimers of crotoxin B isoforms (CB1 and CB2) is presented.

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

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    Gu, Shenyan; Rumpel, Sophie; Zhou, Jie; Strotmeier, Jasmin; Bigalke, Hans; Perry, Kay; Shoemaker, Charles B.; Rummel, Andreas; Jin, Rongsheng (Cornell); (Tufts); (Hannover-MED); (Sanford-Burnham)

    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.

  7. Intraspecies variation in the venom of the rattlesnake Crotalus simus from Mexico: different expression of crotoxin results in highly variable toxicity in the venoms of three subspecies.

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    Castro, Edgar Neri; Lomonte, Bruno; del Carmen Gutiérrez, María; Alagón, Alejandro; Gutiérrez, José María

    2013-07-11

    The composition and toxicological profile of the venom of the rattlesnake Crotalus simus in Mexico was analyzed at the subspecies and individual levels. Venoms of the subspecies C. s. simus, C. s. culminatus and C. s. tzabcan greatly differ in the expression of the heterodimeric neurotoxin complex 'crotoxin', with highest concentrations in C. s. simus, followed by C. s. tzabcan, whereas the venom of C. s. culminatus is almost devoid of this neurotoxic PLA2. This explains the large variation in lethality (highest in C. s. simus, which also exerts higher myotoxicity). Coagulant activity on plasma and fibrinogen occurs with the venoms of C. s. simus and C. s. tzabcan, being absent in C. s. culminatus which, in turn, presents higher crotamine-like activity. Proteomic analysis closely correlates with toxicological profiles, since the venom of C. s. simus has high amounts of crotoxin and of serine proteinases, whereas the venom of C. s. culminatus presents higher amounts of metalloproteinases and crotamine. This complex pattern of intraspecies venom variation provides valuable information for the diagnosis and clinical management of envenoming by this species in Mexico, as well as for the preparation of venom pools for the production and quality control of antivenoms. This study describes the variation in venom composition and activities of the three subspecies of Crotalus simus from Mexico. Results demonstrate that there is a notorious difference in these venoms, particularly regarding the content of the potent neurotoxic phospholipase A2 complex 'crotoxin'. In addition, other differences were observed regarding myotoxic and coagulant activities, and expression of the myotoxin 'crotamine'. These findings have implications in, at least, three levels: (a) the adaptive role of variations in venom composition; (b) the possible differences in the clinical manifestations of envenomings by these subspecies in Mexico; and (c) the design of venom mixtures for the preparation of

  8. Structural Studies on Intact Clostridium botulinum Neurotoxins Complexed with Inhibitors Leading to Drug Design

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    2008-02-01

    Clostridium botulinum Neurotoxins Complexed with Inhibitors Leading to Drug Design PRINCIPAL INVESTIGATOR: Subramanyam Swaminathan...Inhibitors Leading to Drug Design 5b. GRANT NUMBER DAMD17-02-2-0011 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) Subramanyam Swaminathan, Ph.D. 5d...on Intact Clostridium botulinum Neurotoxins Complexed with Inhibitors Leading to Drug Design Annual Report for the Period ending January 2008

  9. Crotoxin, the major toxin from the rattlesnake Crotalus durissus terrificus, inhibits ³H-choline uptake in guinea pig ileum

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    L.S. Kattah

    2000-09-01

    Full Text Available We examined the effect of crotoxin, the neurotoxic complex from the venom of the South American rattlesnake Crotalus durissus terrificus, on the uptake of ³H-choline in minces of smooth muscle myenteric plexus from guinea pig ileum. In the concentration range used (0.03-1 µM and up to 10 min of treatment, crotoxin decreased ³H-choline uptake by 50-75% compared to control. This inhibition was time dependent and did not seem to be associated with the disruption of the neuronal membrane, because at least for the first 20 min of tissue exposure to the toxin (up to 1 µM the levels of lactate dehydrogenase (LDH released into the supernatant were similar to those of controls. Higher concentrations of crotoxin or more extensive incubation times with this toxin resulted in elevation of LDH activity detected in the assay supernatant. The inhibitory effect of crotoxin on ³H-choline uptake seems to be associated with its phospholipase activity since the equimolar substitution of Sr2+ for Ca2+ in the incubation medium or the modification of the toxin with p-bromophenacyl bromide substantially decreased this effect. Our results show that crotoxin inhibits ³H-choline uptake with high affinity (EC25 = 10 ± 5 nM. We suggest that this inhibition could explain, at least in part, the blocking effect of crotoxin on neurotransmission.

  10. Effect of gamma irradiation on the behavioral properties of crotoxin

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    E.G. Moreira

    1997-02-01

    Full Text Available Crotoxin has been detoxified with gamma radiation in order to improve crotalic antiserum production. Nevertheless, present knowledge of the biological characteristics of irradiated crotoxin is insufficient to propose it as an immunizing agent. Crotoxin is known to increase the emotional state of rats and to decrease their exploratory behavior (Moreira EG, Nascimento N, Rosa GJM, Rogero JR and Vassilieff VS (1996 Brazilian Journal of Medical and Biological Research, 29: 629-632. Therefore, we decided 1 to evaluate the effects of crotoxin in the social interaction test, which has been widely used for the evaluation of anxiogenic drugs, and 2 to determine if irradiated crotoxin induces behavioral alterations similar to those of crotoxin in the social interaction, open-field and hole-board tests. Male Wistar rats (180-220 g were used. Crotoxin (100, 250, and 500 µg/kg was injected intraperitoneally 2 h before the social interaction test. Similarly, irradiated crotoxin (2000 Gy gamma radiation from a 60Co source was administered at the doses of 100, 250, and 500 µg/kg for the hole-board test, and at the doses of 1000 and 2500 µg/kg for the open-field and social interaction tests. ANOVA complemented with the Dunnett test was used for statistical analysis (P<0.05. Crotoxin decreased the social interaction time (s at the doses of 100, 250 and 500 µg/kg (means ± SEM from 51.6 ± 4.4 to 32.6 ± 3.7, 28.0 ± 3.6 and 31.6 ± 4.4, respectively. Irradiated crotoxin did not induce behavioral alterations. These results indicate that 1 crotoxin may be an anxiogenic compound, and 2 in contrast to crotoxin, irradiated crotoxin was unable to induce behavioral alterations, which makes it a promising compound for the production of crotalic antiserum

  11. Comparative nephrotoxicity of native or Co-60 gamma rays irradiated crotoxin in mice

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    Rocha, Andre Moreira; Alves, Glaucie J.; Aires, Raquel da Silva; Turibio, Thompson O.; Thomazi, Gabriela O. Coelho; Spencer, Patrick J.; Nascimento, Nanci do, E-mail: andrerocha@usp.br [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil); Nascimento-Rocha, Josefa M.; Magalhaes Filho, Asterio Souza, E-mail: 0304@prof.itpacporto.com.br [Instituto Tocantinense Presidente Antonio Carlos Porto (ITPAC), Porto Nacional, TO (Brazil)

    2015-07-01

    Snake venoms are complex mixtures of proteins and peptides with a wide spectrum of physiological targets such as the blood coagulation and cardiovascular systems and the motor end plate among others. Acute renal failure is a common complication in accidents with the South American rattlesnake. The toxin involved in this pathology is the crotoxin, a major component of the venom in terms of concentration and toxicity. Snake venoms, when irradiated with {sup 60}Co gamma rays present a significant decrease in toxicity while the immunogenic properties of its components are preserved. The use of irradiated venom is an attractive alternative for antisera production since it might reduce the appearance of renal lesions improving the welfare and lifespan of those animals employed on antivenom production. At the present work, we have compared the effects of native and irradiated crotoxin on the mice renal function. Tubular lesions were observed in all the samples from the animal group injected with native crotoxin. Animals injected with the irradiated toxin presented alteration only after 30 minutes and 1 hour after injection. These data suggest that the onset of the renal lesions is delayed and that the severity of the lesions might be lower when using irradiated crotoxin. (author)

  12. Purification and characterization of neurotoxin complex from a dual toxin gene containing Clostridium Botulinum Strain PS-5.

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    Singh, Ajay K; Sachdeva, Amita; Degrasse, Jeffrey A; Croley, Timothy R; Stanker, Larry H; Hodge, David; Sharma, Shashi K

    2013-04-01

    Botulinum neurotoxins are produced as a toxin complex (TC) which consists of neurotoxin (NT) and neurotoxin associated proteins. The characterization of NT in its native state is an essential step for developing diagnostics and therapeutic countermeasures against botulism. The presence of NT genes was validated by PCR amplification of toxin specific fragments from genomic DNA of Clostridium botulinum strain PS-5 which indicated the presence of both serotype A and B genes on PS-5 genome. Further, TC was purified and characterized by Western blotting, Digoxin-enzyme linked immunosorbent assay, endopeptidase activity assay, and Liquid chromatography-Mass spectrometry. The data showed the presence of serotype A specific neurotoxin. Based on the analysis of neurotoxin genes and characterization of TC, PS-5 strain appears as a serotype A (B) strain of C. botulinum which produces only serotype A specific TC in the cell culture medium.

  13. Study of crotoxin mechanism of action to mammary carcinomas and evaluation of its potential as a radiopharmaceutical; Estudo do mecanismo de acao da crotoxina em tumores mamarios e avaliacao do seu potenctial radiofarmaceutico

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    Silveira, Marina Bicalho

    2010-07-01

    Crotoxin, the main component of Crotalus durissus terrificus snake venom, has been studied since 1938. It is a natural polypeptidic complex with pharmacological potential because of its antitumoral properties which has attracted great interest for diagnosis and therapy of oncological diseases. However, Crotoxin mechanism of action and sites of specific interaction on tumor cells are still misunderstood. Breast cancer is the second most frequent type in the world and the most common cancer in women. About 30 to 60% of mammary tumors overexpress epidermal growth factor receptor (EGFR), a transmembrane protein related to cell proliferation. Since literature has reported that Crotoxin antitumoral effect is more potent on cells with EGFR overexpression the objectives of this work were to evaluate Crotoxin cytotoxic effects on mammary tumor cells human breast carcinoma (MCF-7) and Ehrlich tumor cells (murine ascitics carcinoma), and to investigate the specific molecular interaction of Crotoxin on Ehrlich tumor cells. Initially, Crotoxin was radiolabelled with iodine-125 ({sup 125}I-Crotoxin) and iodine-131 ({sup 131}I-Crotoxin). Saturation and competition assay were carried out to characterize Crotoxin in vitro interaction; Crotoxin biodistribution studies and singlephoton emission computed tomography (SPECT) of mice bearing Ehrlich tumor have been evaluated to describe in vivo interaction. Our results showed that Crotoxin presented cytotoxic effect against Ehrlich with DL{sub 50} in vitro (concentration of compound which is lethal for 50% of cells) of about one micromolar, but did not present significant effect against MCF-7. Morphological alterations characteristic of apoptosis suggests programmed cell death. {sup 125}I-Crotoxin interaction with Ehrlich tumor cells was saturable with approximately 70% specificity, and presented K{sub d}=24.98 nmol/L and B{sub max}=16,570 sites/cell for low affinity binding sites and K{sub d}=0.06 nmol/L and B{sub max}=210 sites

  14. Presence of presynaptic neurotoxin complexes in the venoms of Australo-Papuan death adders (Acanthophis spp.).

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    Blacklow, Benjamin; Konstantakopoulos, Nicki; Hodgson, Wayne C; Nicholson, Graham M

    2010-06-01

    Australo-papuan death adders (Acanthophis spp.) are a cause of serious envenomations in Papua New Guinea and northern Australia often resulting in neurotoxic paralysis. Furthermore, victims occasionally present with delayed-onset neurotoxicity that sometimes responds poorly to antivenom or anticholinesterase treatment. This clinical outcome could be explained by the presence of potent snake presynaptic phospholipase A(2) neurotoxin (SPAN) complexes and monomers, in addition to long- and short-chain postsynaptic alpha-neurotoxins, that bind irreversibly, block neurotransmitter release and result in degeneration of the nerve terminal. The present study therefore aimed to determine within-genus variations in expression of high molecular mass SPAN complexes in the venoms of six major species of Acanthophis, four geographic variants of Acanthophis antarcticus. Venoms were separated by size-exclusion liquid chromatography under non-denaturing conditions and fractions corresponding to proteins in the range of 22 to >60 kDa were subjected to pharmacological characterization using the isolated chick biventer cervicis nerve-muscle (CBCNM) preparation. All venoms, except Acanthophis wellsi and Acanthophis pyrrhus, contained high mass fractions with phospholipase A(2) activity that inhibited twitch contractions of the CBCNM preparation. This inhibition was of slow onset, and responses to exogenous nicotinic agonists were not blocked, consistent with the presence of SPAN complexes. The results of the present study indicate that clinicians may need to be aware of possible prejunctional neurotoxicity following envenomations from A. antarcticus (all geographic variants except perhaps South Australia), Acanthophis praelongus, Acanthophis rugosus and Acanthophis. laevis species, and that early antivenom intervention is important in preventing further development of toxicity.

  15. High-resolution crystal structure of HA33 of botulinum neurotoxin type B progenitor toxin complex

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    Lee, Kwangkook; Lam, Kwok-Ho; Kruel, Anna Magdalena; Perry, Kay; Rummel, Andreas; Jin, Rongsheng

    2014-01-01

    Botulinum neurotoxins (BoNTs) are produced as progenitor toxin complexes (PTCs) by Clostridium botulinum. The PTCs are composed of BoNT and non-toxic neurotoxin-associated proteins (NAPs), which serve to protect and deliver BoNT through the gastrointestinal tract in food borne botulism. HA33 is a key NAP component that specifically recognizes host carbohydrates and helps enrich PTC on the intestinal lumen preceding its transport across the epithelial barriers. Here, we report the crystal structure of HA33 of type B PTC (HA33/B) in complex with lactose at 1.46 Å resolution. The structural comparisons among HA33 of serotypes A–D reveal two different HA33–glycan interaction modes. The glycan-binding pockets on HA33/A and B are more suitable to recognize galactose-containing glycans in comparison to the equivalent sites on HA33/C and D. On the contrary, HA33/C and D could potentially recognize Neu5Ac as an independent receptor, whereas HA33/A and B do not. These findings indicate that the different oral toxicity and host susceptibility observed among different BoNT serotypes could be partly determined by the serotype-specific interaction between HA33 and host carbohydrate receptors. Furthermore, we have identified a key structural water molecule that mediates the HA33/B–lactose interactions. It provides the structural basis for development of new receptor-mimicking compounds, which have enhanced binding affinity with HA33 through their water-displacing moiety. PMID:24631690

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

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    Kwangkook Lee

    Full Text Available 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.

  17. Immunoprecipitation of native botulinum neurotoxin complexes from Clostridium botulinum subtype A strains.

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    Lin, Guangyun; Tepp, William H; Bradshaw, Marite; Fredrick, Chase M; Johnson, Eric A

    2015-01-01

    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.

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

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    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.

  19. Molecular composition and extinction coefficient of native botulinum neurotoxin complex produced by Clostridium botulinum hall A strain.

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    Bryant, Anne-Marie; Davis, Jenny; Cai, Shuowei; Singh, Bal Ram

    2013-02-01

    Seven distinct strains of Clostridium botulinum (type A to G) each produce a stable complex of botulinum neurotoxin (BoNT) along with neurotoxin-associated proteins (NAPs). Type A botulinum neurotoxin (BoNT/A) is produced with a group of NAPs and is commercially available for the treatment of numerous neuromuscular disorders and cosmetic purposes. Previous studies have indicated that BoNT/A complex composition is specific to the strain, the method of growth and the method of purification; consequently, any variation in composition of NAPs could have significant implications to the effectiveness of BoNT based therapeutics. In this study, a standard analytical technique using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and densitometry analysis was developed to accurately analyze BoNT/A complex from C. botulinum type A Hall strain. Using 3 batches of BoNT/A complex the molar ratio was determined as neurotoxin binding protein (NBP, 124 kDa), heavy chain (HC, 90 kDa), light chain (LC, 53 kDa), NAP-53 (50 kDa), NAP-33 (36 kDa), NAP-22 (24 kDa), NAP-17 (17 kDa) 1:1:1:2:3:2:2. With Bradford, Lowry, bicinchoninic acid (BCA) and spectroscopic protein estimation methods, the extinction coefficient of BoNT/A complex was determined as 1.54 ± 0.26 (mg/mL)(-1)cm(-1). These findings of a reproducible BoNT/A complex composition will aid in understanding the molecular structure and function of BoNT/A and NAPs.

  20. Structural analysis of irradiated crotoxin by spectroscopic techniques

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    Oliveira, Karina C. de; Fucase, Tamara M.; Silva, Ed Carlos S. e; Chagas, Bruno B.; Buchi, Alisson T.; Viala, Vincent L.; Spencer, Patrick J.; Nascimento, Nanci do, E-mail: kcorleto@usp.br [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil). Centro de Biotecnologia

    2013-07-01

    Snake bites are a serious public health problem, especially in subtropical countries. In Brazil, the serum, the only effective treatment in case of snake bites, is produced in horses which, despite of their large size, have a reduced lifespan due to the high toxicity of the antigen. Ionizing radiation has been successfully employed to attenuate the biological activity of animal toxins. Crotoxin, the main toxic compound from Crotalus durissus terrificus (Cdt), is a heterodimeric protein composed of two subunits: crotapotin and phospholipase A{sub 2}. Previous data indicated that this protein, following irradiation process, undergoes unfolding and/or aggregation, resulting in a much lower toxic antigen. The exact mechanisms and structural modifications involved in aggregation process are not clear yet. This work investigates the effects of ionizing radiation on crotoxin employing Infrared Spectroscopy, Circular Dichroism and Dynamic Light Scattering techniques. The infrared spectrum of lyophilized crotoxin showed peaks corresponding to the vibrational spectra of the secondary structure of crotoxin, including β-sheet, random coil, α-helix and β-turns. We calculated the area of these spectral regions after adjusting for baseline and normalization using the amide I band (1590-1700 cm{sup -1}), obtaining the variation of secondary structures of the toxin following irradiation. The Circular Dichroism spectra of native and irradiated crotoxin suggests a conformational change within the molecule after the irradiation process. This data indicates structural changes between the samples, apparently from ordered conformation towards a random coil. The analyses by light scattering indicated that the irradiated crotoxin formed multimers with an average molecular radius 100 folds higher than the native toxin. (author)

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

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    Inui, Ken [Department of Food and Cosmetic Science, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493 (Japan); Japan Society for the Promotion of Science, 1-8 Chiyoda-ku, Tokyo 102-8472 (Japan); Sagane, Yoshimasa [Department of Food and Cosmetic Science, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493 (Japan); Miyata, Keita [Department of Food and Cosmetic Science, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493 (Japan); Japan Society for the Promotion of Science, 1-8 Chiyoda-ku, Tokyo 102-8472 (Japan); Miyashita, Shin-Ichiro [Department of Food and Cosmetic Science, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493 (Japan); Suzuki, Tomonori [Department of Bacteriology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558 (Japan); Shikamori, Yasuyuki [Agilent Technologies International Japan, Ltd. Takaura-cho 9-1, Hachioji-shi, Tokyo 192-0033 (Japan); Ohyama, Tohru; Niwa, Koichi [Department of Food and Cosmetic Science, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493 (Japan); Watanabe, Toshihiro, E-mail: t-watana@bioindustry.nodai.ac.jp [Department of Food and Cosmetic Science, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri 099-2493 (Japan)

    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.

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

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    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.

  3. Biochemical and immunological characterization of the main products of crotoxin irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Nascimento, M. [Instituto de Pesquisas Energeticas e Nucleares (IPEN), Sao Paulo, SP (Brazil). Dept. de Bioengenharia

    1996-07-01

    Irradiation of crotoxin and its subunits with 2,000 Gy of gamma-rays from {sup 60} Co source leads to aggregation and generation of lower molecular weight breakdown products. Aggregates separated by gel filtration retain at least part of their higher-ordered structure, based on their reactivity with monoclonal antibodies, known to react with conformational epitopes in native crotoxin. Linear epitopes are also preserved, as demonstrated by peptide mapping of the aggregates. These same aggregates can function as antigens to raise antisera which cross-react and neutralize crotoxin. Compared with crotoxin, the aggregates appear to be less myotoxic, largely devoid of phospholipase activity and virtually non-toxic in mice. These results indicate that the irradiation of toxic proteins can promote significant detoxification, but still retain many of the original antigenic and immunological properties of native crotoxin. (author)

  4. Construction of "Toxin Complex" in a Mutant Serotype C Strain of Clostridium botulinum Harboring a Defective Neurotoxin Gene.

    Science.gov (United States)

    Suzuki, Tomonori; Nagano, Thomas; Niwa, Koichi; Uchino, Masataka; Tomizawa, Motohiro; Sagane, Yoshimasa; Watanabe, Toshihiro

    2017-01-01

    A non-toxigenic mutant of the toxigenic serotype C Clostridium botulinum strain Stockholm (C-St), C-N71, does not produce the botulinum neurotoxin (BoNT). However, the original strain C-St produces botulinum toxin complex, in which BoNT is associated with non-toxic non-hemagglutinin (NTNHA) and three hemagglutinin proteins (HA-70, HA-33, and HA-17). Therefore, in this study, we aimed to elucidate the effects of bont gene knockout on the formation of the "toxin complex." Nucleotide sequence analysis revealed that a premature stop codon was introduced in the bont gene, whereas other genes were not affected by this mutation. Moreover, we successfully purified the "toxin complex" produced by C-N71. The "toxin complex" was identified as a mixture of NTNHA/HA-70/HA-17/HA-33 complexes with intact NTNHA or C-terminally truncated NTNHA, without BoNT. These results indicated that knockout of the bont gene does not affect the formation of the "toxin complex." Since the botulinum toxin complex has been shown to play an important role in oral toxin transport in the human and animal body, a non-neurotoxic "toxin complex" of C-N71 may be valuable for the development of an oral drug delivery system.

  5. Comparative sequence analyses of the neurotoxin complex genes in Clostridium botulinum serotypes A, B, E, and F

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    Ajay K. Singh

    2012-09-01

    Full Text Available Neurotoxin complex (NTC genes are arranged in two known hemagglutinin (HA and open reading frame X (ORFX clusters. NTC genes have been analyzed in four serotypes A, B, E and F of Clostridium botulinum causing human botulism. Analysis of amino acid sequences of NT genes demonstrated significant differences among subtypes and four serotypes. Phylogram tree of NT genes reveals that serotypes A1 and B1 are much closer compared to serotype E1 and F1. However, non-toxic non-hemagglutinin (NTNH gene is highly conserved among four serotypes. Analysis of phylogram tree of NTNH gene reveals that serotypes A and F are more closely related compared to serotype B and E. Additionally, sequences of HAs and ORFX genes are very divergent but these genes are specific in subtypes and serotypes of Clostridium botulinum. Information derived from sequence analyses of NTC has direct implication in development of detection tools and therapeutic countermeasures for botulism.

  6. Obtainment of crotoxin/phema-g-LDPE and crotoxin/PCL systems;Obtencao dos sistemas bioconjugados crotoxina/PEBD-g-phema e crotoxina/PCL

    Energy Technology Data Exchange (ETDEWEB)

    Lorenzetti, Solange Gubbelini

    2006-07-01

    The aim of the work was the obtainment of polymeric matrices immobilized with crotoxin purified from crude venom of rattle snake. A matrix was processed by gamma irradiation by the grafting of a hydrogel onto a polymeric film which resulted in a copolymer for the chemical immobilization of crotoxin. The second matrix was attained by the entrapment of crotoxin in microspheres of epsilon-polycaprolactone. After the purification, the crotoxin proceeding from the snake Crotalus durissus terrificus was evaluated biochemical and biologically. The lethal dose (LD50%) of the toxin was 0.09/kg animal. The test of cytotoxicity not revealed any significant difference between the tumoral cells and the respective normal control cells in culture. Grafting copolymers were used as scaffold for the chemical immobilization of the purified crotoxin. For this purpose the low density polyethylene (LDPE) and the hydrophilic monomer 2-hydroxy-ethyl-methacrylate (HEMA) were copolymerized in a {sup 60}Co source. The copolymers (LDPE-g-PHEMA) showed grafting levels in the range of 2 and 50 %. In the infrared spectroscopy analysis (FTIR-ATR) it was observed in the copolymer, carbonyl groups (C=O) and hydroxyl groups -OH due to the grafting of PHEMA. The MEV micrographs showed a smooth surface for the virgin LDPE and a rough surface for the LDPE-g-PHEMA, owing to the presence of grafted PHEMA. The hydrophilicity was observed by the determination of water content in the copolymer after immersion in water. By the diffusion coefficient it was noted that from 30 % grafting degree, the copolymers become less hydrophilic due to the crosslinking increase among the chains in PHEMA. The biocompatibility of the LDPE-g-PHEMA was proved by the cytotoxicity test. At the end, the immobilized copolymer, the entrapped crotoxin and the free crotoxin was tested 'in vivo'. During 20 days, C3H strain mice were observed in their weight, behavior and motor changes. The results demonstrated that the group

  7. Crystal Structure of Botulinum Neurotoxin Type a in Complex With the Cell Surface Co-Receptor GT1b-Insight Into the Toxin-Neuron Interaction

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    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.

  8. Study of crotoxin on the induction of paralysis in extraocular muscle in animal model

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    Geraldo de Barros Ribeiro

    2012-10-01

    Full Text Available PURPOSE: Crotoxin is the major toxin of the venom of the South American rattlesnake Crotalus durissus terrificus, capable of causing a blockade of the neurotransmitters at the neuromuscular junction. The objective of this study was to appraise the action and effectiveness of the crotoxin induced paralysis of the extraocular muscle and to compare its effects with the botulinum toxin type A (BT-A. METHODS: The crotoxin, with LD50 of 1.5 µg, was injected into the superior rectus muscle in ten New Zealand rabbits. The concentration variance was 0.015 up to 150 µg. Two rabbits received 2 units of botulinum toxin type A for comparative analysis. The evaluation of the paralysis was performed using serial electromyography. After the functional recovery of the muscles, which occurred after two months, six rabbits were sacrificed for anatomopathology study. RESULTS: The animals did not show any evidence of systemic toxicity. Transitory ptosis was observed in almost every animal and remained up to fourteen days. These toxins caused immediate blockade of the electrical potentials. The recovery was gradual in the average of one month with regeneration signs evident on the electromyography. The paralysis effect of the crotoxin on the muscle was proportional to its concentration. The changes with 1.5 µg crotoxin were similar to those produced by the botulinum toxin type A. The histopathology findings were localized to the site of the injection. No signs of muscle fiber's necrosis were seen in any sample. The alterations induced by crotoxin were also proportional to the concentration and similar to botulinum toxin type A in concentration of 1.5 µg. CONCLUSION: Crotoxin was able to induce transitory paralysis of the superior rectus muscle. This effect was characterized by reduction of action potentials and non-specific signs of fibrillation. Crotoxin, in concentration of 1.5 µg was able to induce similar effects as botulinum toxin type A.

  9. Structural aspects of crotoxin modified by ionizing radiation; Aspectos estruturais da crotoxina modificada pela radiacao ionizante

    Energy Technology Data Exchange (ETDEWEB)

    Oliveira, Karina Corleto de; Albero, Felipe Guimaraes; Zezell, Denise Maria; Spencer, Patrick Jack; Nascimento, Nanci do, E-mail: kcorleto@usp.b [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)

    2010-03-15

    Ionizing radiation has proven to be an excellent tool for reducing the toxicity of venoms and isolated toxins, resulting in better immunogens for serum production, the only effective treatment in case of snake bites, and contributing to the welfare of serum-producing animals. Since the action of gamma radiation of venoms and toxins has not been yet fully clarified from the structural point of view, we proposed in this paper, to characterize the crotoxin, a venom protein of the species Crotalus durissus terrificus by Circular Dichroism (CD) and Infrared Spectroscopy (FTIR) techniques. After chromatographic techniques, crotoxin was irradiated with 2.0 kGy ({sup 60}Co source). The CD spectra obtained of native and irradiated crotoxin solutions showed changes between the samples in characteristic regions of -sheet and-helix . The Infrared analyse showed expressive changes in the spectra of the native and irradiated crotoxin (amide I band region). These tests showed that crotoxin when subjected to gamma radiation, showed changes in their structural conformation compared with the samples in the native state. Such changes probably occur in the secondary structure and may explain its neurotoxic activity loss. (author)

  10. Crotoxin in humans: analysis of the effects on extraocular and facial muscles Crotoxina em humanos: estudo da ação em músculos extraoculares e faciais

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    Geraldo de Barros Ribeiro

    2012-12-01

    Full Text Available PURPOSE: Crotoxin is the main neurotoxin of South American rattlesnake Crotalus durissus terrificus. The neurotoxic action is characterized by a presynaptic blockade. The purpose of this research is to assess the ability of crotoxin to induce temporary paralysis of extraocular and facial muscles in humans. METHODS: Doses of crotoxin used ranged from 2 to 5 units (U, each unit corresponding to one LD50. We first applied 2U of crotoxin in one of the extraocular muscles of 3 amaurotic individuals to be submitted to ocular evisceration. In the second stage, we applied crotoxin in 12 extraocular muscles of 9 patients with strabismic amblyopia. In the last stage, crotoxin was used in the treatment of blepharospasm in another 3 patients. RESULTS: No patient showed any systemic side effect or change in vision or any eye structure problem after the procedure. The only local side effects observed were slight conjunctival hyperemia, which recovered spontaneously. In 2 patients there was no change in ocular deviation after 2U crotoxin application. Limitation of the muscle action was observed in 8 of the 12 applications. The change in ocular deviation after application of 2U of crotoxin (9 injections was in average 15.7 prism diopters (PD. When the dose was 4U (2 applications the change was in average 37.5 PD and a single application of 5U produced a change of 16 PD in ocular deviation. This effect lasted from 1 to 3 months. Two of the 3 patients with blepharospasm had the hemifacial spasm improved with crotoxin, which returned after 2 months. CONCLUSIONS: This study provides data suggesting that crotoxin may be a useful new therapeutic option for the treatment of strabismus and blepharospasm. We expect that with further studies crotoxin could be an option for many other medical areas.OBJETIVO: A crotoxina é a principal neurotoxina da cascavel sul-americana Crotalus durissus terrificus e sua ação neurotóxica caracteriza-se por um bloqueio pr

  11. Catalytic Features of the Botulinum Neurotoxin A Light Chain Revealed by High Resolution Structure of an Inhibitory Peptide Complex

    Energy Technology Data Exchange (ETDEWEB)

    Silvaggi,N.; Wilson, D.; Tzipori, S.; Allen, K.

    2008-01-01

    The Clostridium botulinum neurotoxin serotype A light chain (BoNT/A-LC) is a Zn(II)-dependent metalloprotease that blocks the release of acetylcholine at the neuromuscular junction by cleaving SNAP-25, one of the SNARE proteins required for exocytosis. Because of the potential for use of the toxin in bioterrorism and the increasingly widespread application of the toxin in the medical field, there is significant interest in the development of small-molecule inhibitors of the metalloprotease. Efforts to design such inhibitors have not benefited from knowledge of how peptides bind to the active site since the enzyme-peptide structures available previously either were not occupied in the vicinity of the catalytic Zn(II) ion or did not represent the product of SNAP-25 substrate cleavage. Herein we report the 1.4 Angstroms-resolution X-ray crystal structure of a complex between the BoNT/A-LC and the inhibitory peptide N-Ac-CRATKML, the first structure of the light chain with an inhibitory peptide bound at the catalytic Zn(II) ion. The peptide is bound with the Cys S? atom coordinating the metal ion. Surprisingly, the cysteine sulfur is oxidized to the sulfenic acid form. Given the unstable nature of this species in solution, is it likely that oxidation occurs on the enzyme. In addition to the peptide-bound structure, we report two structures of the unliganded light chain with and without the Zn(II) cofactor bound at 1.25 and 1.20 Angstroms resolution, respectively. The two structures are nearly identical, confirming that the Zn(II) ion plays a purely catalytic role. Additionally, the structure of the Zn(II)-bound uncomplexed enzyme allows identification of the catalytic water molecule and a second water molecule that occupies the same position as the peptidic oxygen in the tetrahedral intermediate. This observation suggests that the enzyme active site is prearranged to stabilize the tetrahedral intermediate of the protease reaction.

  12. Clinical equivalence of conventional OnabotulinumtoxinA (900 KDa) and IncobotulinumtoxinA (neurotoxin free from complexing proteins - 150 KDa): 2012 multidisciplinary French consensus in aesthetics.

    Science.gov (United States)

    Poulain, Bernard; Trevidic, Patrick; Clave, Micheline; Aharoni, Claude; Baspeyras, Martine; Bui, Patrick; Cartier, Hugues; Charavel, Marie-Helene; Coulon, Pierre; Dahan, Serge; Dallara, Jean-Marie; Delonca, Denis; Dumas, Laurent; Essayagh, Eric; Galatoire, Olivier; Georgieu, Nicolas; Grangier, Yann; Humbert, Philippe; Le Pillouer-Prost, Anne; Mojallal, Alain

    2013-12-01

    Botulinum neurotoxins are the most popular non-surgical treatments for aesthetic indications, but there is uncertainty about whether certain formulations are comparable in efficacy and safety and can be substituted for one another by a simple one to one dose conversion ratio. An expert panel of French practitioners was convened to establish a consensus on the clinical equivalence in efficacy and safety of OnabotulinumtoxinA (900 KDa) and IncobotulinumtoxinA (neurotoxin free from complexing proteins - 150 KDa). The consensus was divided into three sections incorporating a biological, bibliographic and clinical analysis of the two toxins. This included a review of the published data that have directly compared the two toxins for aesthetic indications and a survey of the panel's extensive clinical experience with the two toxins in terms of efficacy and safety. All panel members reviewed and endorsed the content of each section. Among this expert panel of French aesthetic physicians and biologists there was consensus that OnabotulinumtoxinA and IncobotulinumtoxinA are clinically equivalent in terms of efficacy and safety, and that a switch from one drug to the other can be made using a simple 1:1 conversion ratio.

  13. The Structure of the Neurotoxin- Associated Protein HA33/A from Clostridium botulinum Suggests a Reoccurring Beta-Trefoil Fold in the Progenitor Toxin Complex

    Science.gov (United States)

    2007-11-02

    sequence similarity to the b-trefoil- containing binding domains of BoNT/A and tetanus neurotoxin (TeNT) from Clostridium tetani with sequence...doi:10.1016/j.jmb.2004.12.039 J. Mol. Biol. (2005) 346, 1083–1093The Structure of the Neurotoxin-associated Protein HA33/A from Clostridium botulinum...correspond stevens@scripps.eduThe hemagglutinating protein HA33 from Clostridium botulinum is associated with the large botulinum neurotoxin secreted

  14. Crotoxin: Structural Studies, Mechanism of Action and Cloning of Its Gene

    Science.gov (United States)

    1987-03-01

    in our snake facility, which we obtained from Mr. Hugh Quinn, curator of reptiles at the Houston zoo. These will be available for future work. We...successful in obtaining crystals suitable for x-ray diffraction studies. Detailed x-ray structural analysis should provide valuable insight into...center well is also eviden , in Figs. 1A and 18. An earlier report noted that reconstituted crotoxin antiserum formed a line of identity against intact

  15. Qualitative and Quantitative Detection of Botulinum Neurotoxins from Complex Matrices: Results of the First International Proficiency Test

    Directory of Open Access Journals (Sweden)

    Sylvia Worbs

    2015-11-01

    Full Text Available In the framework of the EU project EQuATox, a first international proficiency test (PT on the detection and quantification of botulinum neurotoxins (BoNT was conducted. Sample materials included BoNT serotypes A, B and E spiked into buffer, milk, meat extract and serum. Different methods were applied by the participants combining different principles of detection, identification and quantification. Based on qualitative assays, 95% of all results reported were correct. Successful strategies for BoNT detection were based on a combination of complementary immunological, MS-based and functional methods or on suitable functional in vivo/in vitro approaches (mouse bioassay, hemidiaphragm assay and Endopep-MS assay. Quantification of BoNT/A, BoNT/B and BoNT/E was performed by 48% of participating laboratories. It turned out that precise quantification of BoNT was difficult, resulting in a substantial scatter of quantitative data. This was especially true for results obtained by the mouse bioassay which is currently considered as “gold standard” for BoNT detection. The results clearly demonstrate the urgent need for certified BoNT reference materials and the development of methods replacing animal testing. In this context, the BoNT PT provided the valuable information that both the Endopep-MS assay and the hemidiaphragm assay delivered quantitative results superior to the mouse bioassay.

  16. Qualitative and Quantitative Detection of Botulinum Neurotoxins from Complex Matrices: Results of the First International Proficiency Test

    Science.gov (United States)

    Worbs, Sylvia; Fiebig, Uwe; Zeleny, Reinhard; Schimmel, Heinz; Rummel, Andreas; Luginbühl, Werner; Dorner, Brigitte G.

    2015-01-01

    In the framework of the EU project EQuATox, a first international proficiency test (PT) on the detection and quantification of botulinum neurotoxins (BoNT) was conducted. Sample materials included BoNT serotypes A, B and E spiked into buffer, milk, meat extract and serum. Different methods were applied by the participants combining different principles of detection, identification and quantification. Based on qualitative assays, 95% of all results reported were correct. Successful strategies for BoNT detection were based on a combination of complementary immunological, MS-based and functional methods or on suitable functional in vivo/in vitro approaches (mouse bioassay, hemidiaphragm assay and Endopep-MS assay). Quantification of BoNT/A, BoNT/B and BoNT/E was performed by 48% of participating laboratories. It turned out that precise quantification of BoNT was difficult, resulting in a substantial scatter of quantitative data. This was especially true for results obtained by the mouse bioassay which is currently considered as “gold standard” for BoNT detection. The results clearly demonstrate the urgent need for certified BoNT reference materials and the development of methods replacing animal testing. In this context, the BoNT PT provided the valuable information that both the Endopep-MS assay and the hemidiaphragm assay delivered quantitative results superior to the mouse bioassay. PMID:26703724

  17. Qualitative and Quantitative Detection of Botulinum Neurotoxins from Complex Matrices: Results of the First International Proficiency Test.

    Science.gov (United States)

    Worbs, Sylvia; Fiebig, Uwe; Zeleny, Reinhard; Schimmel, Heinz; Rummel, Andreas; Luginbühl, Werner; Dorner, Brigitte G

    2015-11-26

    In the framework of the EU project EQuATox, a first international proficiency test (PT) on the detection and quantification of botulinum neurotoxins (BoNT) was conducted. Sample materials included BoNT serotypes A, B and E spiked into buffer, milk, meat extract and serum. Different methods were applied by the participants combining different principles of detection, identification and quantification. Based on qualitative assays, 95% of all results reported were correct. Successful strategies for BoNT detection were based on a combination of complementary immunological, MS-based and functional methods or on suitable functional in vivo/in vitro approaches (mouse bioassay, hemidiaphragm assay and Endopep-MS assay). Quantification of BoNT/A, BoNT/B and BoNT/E was performed by 48% of participating laboratories. It turned out that precise quantification of BoNT was difficult, resulting in a substantial scatter of quantitative data. This was especially true for results obtained by the mouse bioassay which is currently considered as "gold standard" for BoNT detection. The results clearly demonstrate the urgent need for certified BoNT reference materials and the development of methods replacing animal testing. In this context, the BoNT PT provided the valuable information that both the Endopep-MS assay and the hemidiaphragm assay delivered quantitative results superior to the mouse bioassay.

  18. Biochemical and immunological characterization of the main products of crotoxin irradiation; Caracterizacao bioquimica e imunologica dos principais produtos gerados pela irradiacao de crotoxina

    Energy Technology Data Exchange (ETDEWEB)

    Nascimento, Nanci do

    1995-07-01

    Irradiation of crotoxin and its subunits with 2,000 Gy of {gamma}-rays from {sup 60} Co source leads to aggregation and generation of lower molecular wight breakdown products. Aggregates separated by gel filtration retain at least part of their higher-ordered structure, based on their reactivity with monoclonal antibodies known to react with conformation epitopes in native crotoxin. These same aggregates can serve as antigens to raise antisera that cross-reacts and neutralizes crotoxin. Compared with native crotoxin, aggregates appears less myotoxic, are largely devoid of phospholipase activity, and are virtually non-toxic in mice. These results indicate that irradiation of toxic proteins can promote significant detoxification, but still retain many of the original antigenic and immunological properties of native crotoxin. (author)

  19. Comparison of sea snake (Hydrophiidae) neurotoxin to cobra (Naja) neurotoxin.

    Science.gov (United States)

    Komori, Yumiko; Nagamizu, Masaya; Uchiya, Kei-Ichi; Nikai, Toshiaki; Tu, Anthony T

    2009-12-01

    Both sea snakes and cobras have venoms containing postsynaptic neurotoxins. Comparison of the primary structures indicates many similarities, especially the positions of the four disulfide bonds. However, detailed examination reveals differences in several amino acid residues. Amino acid sequences of sea snake neurotoxins were determined, and then compared to cobra neurotoxins by computer modeling. This allowed for easy comparison of the similarities and differences between the two types of postsynaptic neurotoxins. Comparison of computer models for the toxins of sea snakes and cobra will reveal the three dimensional difference of the toxins much clearer than the amino acid sequence alone.

  20. Binding studies of the antitumoral radiopharmaceutical 125I-Crotoxin to Ehrlich ascites tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Silveira, Marina B.; Santos, Raquel G. dos [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil); Dias, Consuelo L. Fortes [Fundacao Ezequiel Dias (FUNED), Belo Horizonte, MG (Brazil)], e-mail: consuelo@pq.cnpq.br; Cassali, Geovanni D. [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Lab. de Patologia Comparada], e-mail: cassalig@icb.ufmg.br

    2009-07-01

    The development of tools for functional diagnostic imaging is mainly based on radiopharmaceuticals that specifically target membrane receptors. Crotoxin (Crtx), a polypeptide isolated from Crotalus durissus terrificus venom, has been shown to have an antitumoral activity and is a promising bioactive tracer for tumor detection. More specific radiopharmaceuticals are being studied to complement the techniques applied in the conventional medicine against breast cancer, the most frequent cause of death from malignant disease in women. Crtx's effect has been shown to be related with the overexpression of epidermal growth factor receptor (EGFR), present in high levels in 30 to 60% of breast tumor cells. Our objective was to evaluate Crtx as a tracer for cancer diagnosis, investigating its properties as an EGFR-targeting agent. Ehrlich ascites tumor cells (EAT cells) were used due to its origin and similar characteristics to breast tumor cells, specially the presence of EGFR. Crtx was labeled with 125I and binding experiments were performed. To evaluate the specific binding in vitro of Crtx, competition binding assay was carried out in the presence of increasing concentrations of non-labelled crotoxin and epidermal growth factor (EGF). Specific binding of 125I-Crtx to EAT cells was determined and the binding was considered saturable, with approximately 70% of specificity, high affinity (Kd = 19.7 nM) and IC50 = 1.6 x 10-11 M. Our results indicate that Crtx's interaction with EAT cells is partially related with EGFR and increases the biotechnological potential of Crtx as a template for radiopharmaceutical design for cancer diagnosis. (author)

  1. Crotoxin from Crotalus durissus terrificus is able to down-modulate the acute intestinal inflammation in mice.

    Directory of Open Access Journals (Sweden)

    Caroline de Souza Almeida

    Full Text Available Inflammatory bowel diseases (IBD is the result of dysregulation of mucosal innate and adaptive immune responses. Factors such as genetic, microbial and environmental are involved in the development of these disorders. Accordingly, animal models that mimic human diseases are tools for the understanding the immunological processes of the IBD as well as to evaluate new therapeutic strategies. Crotoxin (CTX is the main component of Crotalus durissus terrificus snake venom and has an immunomodulatory effect. Thus, we aimed to evaluate the modulatory effect of CTX in a murine model of colitis induced by 2,4,6- trinitrobenzene sulfonic acid (TNBS. The CTX was administered intraperitoneally 18 hours after the TNBS intrarectal instillation in BALB/c mice. The CTX administration resulted in decreased weight loss, disease activity index (DAI, macroscopic tissue damage, histopathological score and myeloperoxidase (MPO activity analyzed after 4 days of acute TNBS colitis. Furthermore, the levels of TNF-α, IL-1β and IL-6 were lower in colon tissue homogenates of TNBS-mice that received the CTX when compared with untreated TNBS mice. The analysis of distinct cell populations obtained from the intestinal lamina propria showed that CTX reduced the number of group 3 innate lymphoid cells (ILC3 and Th17 population; CTX decreased IL-17 secretion but did not alter the frequency of CD4+Tbet+ T cells induced by TNBS instillation in mice. In contrast, increased CD4+FoxP3+ cell population as well as secretion of TGF-β, prostaglandin E2 (PGE2 and lipoxin A4 (LXA4 was observed in TNBS-colitis mice treated with CTX compared with untreated TNBS-colitis mice. In conclusion, the CTX is able to modulate the intestinal acute inflammatory response induced by TNBS, resulting in the improvement of clinical status of the mice. This effect of CTX is complex and involves the suppression of the pro-inflammatory environment elicited by intrarectal instillation of TNBS due to the

  2. Unique Ganglioside Recognition Strategies for Clostridial Neurotoxins

    Energy Technology Data Exchange (ETDEWEB)

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

    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.

  3. Crotamine and crotoxin interact with tumor cells and trigger cell death

    Energy Technology Data Exchange (ETDEWEB)

    Soares, Marcella Araugio; Pujatti, Priscilla Brunelli; Santos, Raquel Gouvea dos [Centro de Desenvolvimento da Tecnologia Nuclear CDTN/CNEN-MG, Belo Horizonte, MG (Brazil)]. E-mails: maso@cdtn.br; santosr@cdtn.br; Dias, Consuelo Latorre Fortes [Fundacao Ezequiel Dias FUNED, Belo Horizonte, MG (Brazil); Chavez Olortegui, Carlos Delfin [Universidade Federal de Minas Gerais UFMG, Belo Horizonte, MG (Brazil). Inst. de Ciencias Biologicas; Santos, Wagner Gouvea dos [Medical College of Virginia, Richmond, VA (United States). Neurosurgery Dept.

    2007-07-01

    Crotoxin (Crtx) and Crotamine (Crota) are polypeptides isolated from Crotalus durissus terrificus snake venom (CV). Previous reports have been shown therapeutic effects of Crotalus durissus terrificus venom and Crtx on skin, breast and lung tumours, although, the mechanisms of this antitumoral effect are still unknown. The aim of this work was to investigate the antitumoral effect of Crtx and Crota on brain tumours cells (GH3 and RT2) in vitro and their capacity of interaction with these tumour cells membranes. Cell survival after Crtx and Crota treatment was evaluated by MTT assay in different times post-treatment and apoptosis was evaluated by DAPI staining. In order to evaluate the specific interaction of Crtx and Crota, these polypeptides were radiolabelled, using {sup 125}I as radiotracer and binding assays were performed. The results were compared with the binding in nontumoral brain tissue. Crtx and Crota induced apoptosis on both tumour cells lineages but, Crota was more powerful than Crtx 90% and 20% cell death for RT2 cells; 80% and 20% cell death for GH3 cells, respectively). Both {sup 125}I-Crtx and {sup 125}I-Crota bound specifically in glioblastoma membranes. Nonetheless, CV polypeptides recognised glioblastoma cells with higher specificity than normal brain tissue. These results suggest that the Crtx and Crota interactions with the plasmatic membrane of tumour cells may be the first step of the cascade of signalling that trigger their antitumoral effect. (author)

  4. Walker 256 Tumor Growth Suppression by Crotoxin Involves Formyl Peptide Receptors and Lipoxin A4

    Science.gov (United States)

    Brigatte, Patrícia; Faiad, Odair Jorge; Ferreira Nocelli, Roberta Cornélio; Landgraf, Richardt G.; Palma, Mario Sergio; Cury, Yara; Curi, Rui; Sampaio, Sandra Coccuzzo

    2016-01-01

    We investigated the effects of Crotoxin (CTX), the main toxin of South American rattlesnake (Crotalus durissus terrificus) venom, on Walker 256 tumor growth, the pain symptoms associated (hyperalgesia and allodynia), and participation of endogenous lipoxin A4. Treatment with CTX (s.c.), daily, for 5 days reduced tumor growth at the 5th day after injection of Walker 256 carcinoma cells into the plantar surface of adult rat hind paw. This observation was associated with inhibition of new blood vessel formation and decrease in blood vessel diameter. The treatment with CTX raised plasma concentrations of lipoxin A4 and its natural analogue 15-epi-LXA4, an effect mediated by formyl peptide receptors (FPRs). In fact, the treatment with Boc-2, an inhibitor of FPRs, abolished the increase in plasma levels of these mediators triggered by CTX. The blockage of these receptors also abolished the inhibitory action of CTX on tumor growth and blood vessel formation and the decrease in blood vessel diameter. Together, the results herein presented demonstrate that CTX increases plasma concentrations of lipoxin A4 and 15-epi-LXA4, which might inhibit both tumor growth and formation of new vessels via FPRs. PMID:27190493

  5. Analysis of the neurotoxin complex genes in Clostridium botulinum A1-A4 and B1 strains: BoNT/A3, /Ba4 and /B1 clusters are located within plasmids.

    Directory of Open Access Journals (Sweden)

    Theresa J Smith

    Full Text Available BACKGROUND: Clostridium botulinum and related clostridial species express extremely potent neurotoxins known as botulinum neurotoxins (BoNTs that cause long-lasting, potentially fatal intoxications in humans and other mammals. The amino acid variation within the BoNT is used to categorize the species into seven immunologically distinct BoNT serotypes (A-G which are further divided into subtypes. The BoNTs are located within two generally conserved gene arrangements known as botulinum progenitor complexes which encode toxin-associated proteins involved in toxin stability and expression. METHODOLOGY/PRINCIPAL FINDINGS: Because serotype A and B strains are responsible for the vast majority of human botulism cases worldwide, the location, arrangement and sequences of genes from eight different toxin complexes representing four different BoNT/A subtypes (BoNT/A1-Ba4 and one BoNT/B1 strain were examined. The bivalent Ba4 strain contained both the BoNT/A4 and BoNT/bvB toxin clusters. The arrangements of the BoNT/A3 and BoNT/A4 subtypes differed from the BoNT/A1 strains and were similar to those of BoNT/A2. However, unlike the BoNT/A2 subtype, the toxin complex genes of BoNT/A3 and BoNT/A4 were found within large plasmids and not within the chromosome. In the Ba4 strain, both BoNT toxin clusters (A4 and bivalent B were located within the same 270 kb plasmid, separated by 97 kb. Complete genomic sequencing of the BoNT/B1 strain also revealed that its toxin complex genes were located within a 149 kb plasmid and the BoNT/A3 complex is within a 267 kb plasmid. CONCLUSIONS/SIGNIFICANCE: Despite their size differences and the BoNT genes they contain, the three plasmids containing these toxin cluster genes share significant sequence identity. The presence of partial insertion sequence (IS elements, evidence of recombination/gene duplication events, and the discovery of the BoNT/A3, BoNT/Ba4 and BoNT/B1 toxin complex genes within plasmids illustrate the

  6. Prediction of antigenic epitopes and MHC binders of neurotoxin ...

    African Journals Online (AJOL)

    PRECIOUS

    2009-12-01

    Dec 1, 2009 ... Hydrophobicity plot of neurotoxin alpha-KTx 3.8 by Hopp TP, Woods KR (1981). tion methods are ... protein is hydrophobic in nature and contains segments of low complexity ..... Hydrophobicity indices at ph 3.4 determined by ...

  7. Renal and vascular effects of Crotalus durissus cumanensis venom and its crotoxin fraction

    Directory of Open Access Journals (Sweden)

    TP Pereira

    2011-01-01

    Full Text Available In this study, we evaluated the actions of Crotalus durissus cumanensis venom (CDCmV, and its crotoxin (Crtx fraction, on renal and vascular functions in Wistar rats. In isolated perfused kidneys, CDCmV (10 µg/mL significantly increased the perfusion pressure (PP from 110.7 ± 2.4 to 125.3 ± 2.8 mmHg after 30 minutes. This effect was accompanied by an increased renal vascular resistance (RVR from 5.4 ± 0.1 to 6.2 ± 0.2 mmHg/mL.g-1.min-1. We observed decreases in urinary flow (UF from 0.13 ± 0.01 to 0.05 ± 001 mL.g-1.min-1 and glomerular filtration rate (GFR from 0.66 ± 0.06 to 0.18 ± 0.02 mL.g-1.min-1. Crtx did not change PP or RVR, but diminished GFR (from 0.65 ± 0.05 to 0.26 ± 003 mL.g-1.min-1 and UF (from 0.11 ± 0.008 to 0.09 ± 0.008 mL.g-1.min-1. Both CDCmV and Crtx reduced the percentage of tubular transport of sodium, chloride and potassium. The cytotoxicity of these substances against MDCK cells was tested by the MTT method: only CDCmV caused a decrease in the cell viability with an IC50 of 5.4 µg/mL. In endothelium-intact isolated aortic rings, CDCmV (0.1 to 30 µg/mL increased the sustained phenylephrine-induced contraction to a value of 130.0 ± 6.6% of its corresponding control, but showed a relaxant effect in endothelium-denuded preparations. Similar results were observed in aortic rings contracted with potassium (40 mM. Crtx was ineffective in aortic ring assays. Thus, it is reasonable to suggest that the renal effects induced by the CDCmV may be due to its influence on the endothelium's ability to release factors that can alter the contractile behavior of vascular smooth muscle. In conclusion, CDCmV is toxic to kidney cells. It changes parameters of the renal function including the glomerular filtration rate, renal vascular resistance and tubular transport. The actions induced by CDCmV also involve endothelium-dependent vasoactive properties. Their effects may be only partially attributed to Crtx.

  8. BOTDB: A Database for the Clostridial Neurotoxins

    Science.gov (United States)

    2003-07-01

    alpha-test phase of development and will become a publicly available web site. INTRODUCTION The neurotoxins from Clostridium botulinum and related...tetanus neurotoxin (TeNT) from C. tetani , in contrast, causes spastic paralysis due to its net disinhibitory effect within the central nervous system... Clostridium botulinum and its neurotoxins: a metabolic and cellular perspective. Toxicon 2001;39:1703-1722. 6. Rossetto O, Seveso M, Caccin P, Schiavo G

  9. Zebrafish Sensitivity to Botulinum Neurotoxins

    Directory of Open Access Journals (Sweden)

    Kamalakar Chatla

    2016-05-01

    Full Text Available 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.

  10. PEG precipitation coupled with chromatography is a new and sufficient method for the purification of botulinum neurotoxin type B [corrected].

    Directory of Open Access Journals (Sweden)

    Yao Zhao

    Full Text Available Clostridium botulinum neurotoxins are used to treat a variety of neuro-muscular disorders, as well as in cosmetology. The increased demand requires efficient methods for the production and purification of these toxins. In this study, a new purification process was developed for purifying type B neurotoxin. The kinetics of C.botulinum strain growth and neurotoxin production were determined for maximum yield of toxin. The neurotoxin was purified by polyethylene glycol (PEG precipitation and chromatography. Based on design of full factorial experiment, 20% (w/v PEG-6000, 4 °C, pH 5.0 and 0.3 M NaCl were optimal conditions to obtain a high recovery rate of 87% for the type B neurotoxin complex, as indicated by a purification factor of 61.5 fold. Furthermore, residual bacterial cells, impurity proteins and some nucleic acids were removed by PEG precipitation. The following purification of neurotoxin was accomplished by two chromatography techniques using Sephacryl™ S-100 and phenyl HP columns. The neurotoxin was recovered with an overall yield of 21.5% and the purification factor increased to 216.7 fold. In addition, a mouse bioassay determined the purified neurotoxin complex possessed a specific toxicity (LD(50 of 4.095 ng/kg.

  11. Botulinum neurotoxin homologs in non-Clostridium species.

    Science.gov (United States)

    Mansfield, Michael J; Adams, Jeremy B; Doxey, Andrew C

    2015-01-30

    Clostridial neurotoxins (CNTs) are the deadliest toxins known and the causative agents of botulism and tetanus. Despite their structural and functional complexity, no CNT homologs are currently known outside Clostridium. Here, we report the first homologs of Clostridium CNTs within the genome of the rice fermentation organism Weissella oryzae SG25. One gene in W. oryzae S25 encodes a protein with a four-domain architecture and HExxH protease motif common to botulinum neurotoxins (BoNTs). An adjacent gene with partial similarity to CNTs is also present, and both genes seem to have been laterally transferred into the W. oryzae genome from an unknown source. Identification of mobile, CNT-related genes outside of Clostridium has implications for our understanding of the evolution of this important toxin family. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  12. Biodistribution and SPECT imaging of {sup 125/131}I-crotoxin on mice bearing Ehrlich solid tumour

    Energy Technology Data Exchange (ETDEWEB)

    Soares, Marcella Araugio; Santos, Raquel Gouvea dos [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil)], e-mail: marcellaaraugio@yahoo.com.br, e-mail: santosr@cdtn.br; Silveira, Marina B.; Simal, Carlos [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Faculdade de Medicina; Dias, Consuelo L. Fortes [Fundacao Ezequiel Dias (FUNED), Belo Horizonte, MG (Brazil)

    2009-07-01

    The search of specific radiopharmaceuticals to be used in breast tumour diagnosis is relevant to complement the techniques applied in conventional medicine. Crotalus durissus terrificus venom (CV) and its main polypeptide, Crotoxin (Crtx), are natural source of several bioactive substances with therapeutical potential. The aim of this work was to evaluate the binding of Crtx with tumour targets in vivo, as well as, evaluate its applicability for breast tumours diagnosis. Crtx was labelled with {sup 125/131}I using lactoperoxidase method and radiochemical analysis was performed by chromatography. {sup 125}I-Crtx was used for biodistribution and pharmacokinetics studies on swiss mice bearing Ehrlich solid tumour, while {sup 131}I-Crtx was used for single photon emission computed tomography (SPECT) imaging. Crtx presented specific binding sites on Ehrlich tumour cells and had a rapid blood clearance (T{sub 1/2}= 201.1 min.). Intratumoral administration increased significantly the activity delivered into the tumour site (128-fold higher) and reduced the kidney burden (7.2-fold lower). {sup 131}I-Crxt demonstrated to interact with tumour cells for until 72 hours allowing good quality images of tumour. Our results indicate the biotechnological potential of Crtx as template for radiopharmaceutical design for cancer diagnosis. (author)

  13. Botulinum neurotoxin: a marvel of protein design.

    Science.gov (United States)

    Montal, Mauricio

    2010-01-01

    Botulinum neurotoxin (BoNT), the causative agent of botulism, is acknowledged to be the most poisonous protein known. BoNT proteases disable synaptic vesicle exocytosis by cleaving their cytosolic SNARE (soluble NSF attachment protein receptor) substrates. BoNT is a modular nanomachine: an N-terminal Zn(2+)-metalloprotease, which cleaves the SNAREs; a central helical protein-conducting channel, which chaperones the protease across endosomes; and a C-terminal receptor-binding module, consisting of two subdomains that determine target specificity by binding to a ganglioside and a protein receptor on the cell surface and triggering endocytosis. For BoNT, functional complexity emerges from its modular design and the tight interplay between its component modules--a partnership with consequences that surpass the simple sum of the individual component's action. BoNTs exploit this design at each step of the intoxication process, thereby achieving an exquisite toxicity. This review summarizes current knowledge on the structure of individual modules and presents mechanistic insights into how this protein machine evolved to this level of sophistication. Understanding the design principles underpinning the function of such a dynamic modular protein remains a challenging task.

  14. Sensing the deadliest toxin: technologies for botulinum neurotoxin detection.

    Science.gov (United States)

    Capek, 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.

  15. Autophagy is involved in cytotoxic effects of crotoxin in human breast cancer cell line MCF-7 cells

    Institute of Scientific and Technical Information of China (English)

    Ci-hui YAN; Ya-ping YANG; Zheng-hong QIN; Zhen-lun GU; Paul REID; Zhong-qin LIANG

    2007-01-01

    Aim: To investigate the role of crotoxin (CrTX)-induced autophagy in the death of MCF-7 cells, a caspase-3-deficient, human breast cancer cell line. Methods: Cul-tured MCF-7 cells were treated with various doses of CrTX, a phospholipase A2(PLA2) isolated from the venom of the South American rattlesnake, Crotalus durissus terrificus. The cytotoxicity of CrTX in the presence and absence of caspase inhibitors was measured with methyl thiazolyl tetrazolium (MTT) and lactate dehydrogenase (LDH) leakage assays. The activation of autophagy was determined with transmission electron microscope and monodansylcadaverin(MDC) labeling. The upregulation of lysosomal enzymes, the release of cyto-chrome c (cyto-c), and the nuclear translocation of the apoptosis inducing factor(AIF) were examined by immunoblotting and immunofluorescence. Results: CrTX inhibited the viability of MCF-7 cells in a dose- and time-dependent manner. CrTX-activated autophagy was revealed by punctuate MDC labeling, and an increase in the formation of autophagosomes as well as apoptosis, as evidenced by nuclear condensation and fragmentation. The activation of cathepsin B, D, and L, in addition to the release of cytochrome c and the relocation of AIF into nuclei, were observed after CrTX treatment. Autophagy inhibitors 3-methyladenine (3-MA),NH4Cl, and the pan-caspase inhibitor, Z-Val-Ala-Asp-fluoromethylketone (Z-Vad-fmk), attenuated CrTX-induced cell death. Conclusion: An autophagic mecha-nism contributes to the apoptosis of MCF-7 cells induced by CrTX.

  16. Substrate Binding Mode and Its Implication on Drug Design for Botulinum Neurotoxin A

    Science.gov (United States)

    2008-09-26

    1000165 Editor: C. Erec Stebbins , The Rockefeller University, United States of America Received May 30, 2008; Accepted August 28, 2008; Published...dash lines while zinc co-ordination is shown in solid line . doi:10.1371/journal.ppat.1000165.g003 Neurotoxin-Substrate Complex PLoS Pathogens

  17. Ciguatera neurotoxin poisoning mimicking burning mouth syndrome.

    Science.gov (United States)

    Heir, Gary M

    2005-01-01

    Burning mouth syndrome is a condition in which the patient perceives a sensation of intraoral burning, typically of the anterior tongue. This article presents a case report of a patient presenting for orofacial pain evaluation in whom ciguatera neurotoxin poisoning is diagnosed. The clinician should be aware of neurotoxin poisoning as a possible cause of symptoms of burning mouth, especially among patients who have recently traveled to a tropical area. Recognition of this condition in this case highlights the need for a detailed and accurate patient history.

  18. Physical Characterization of Clostridium Botulinum Neurotoxin Genes

    Science.gov (United States)

    1993-10-01

    Clostridium tetani (Welloner, 1982). They differ in that whereas BoNT acts at the nerve periphery, TeTx blocks the release of inhibitory amino acids...AD-A27 2 939 GRANT NO: DAMDl7-90-Z-0033 TITLE: PHYSICAL CHARACTERIZATION OF CLOSTRIDIUM BOTULINUM NEUROTOXIN GENES PRINCIPAL INVESTIGATOR: Nigel P...Characterization of Clostridium Grant No. Botulinum. Neurotoxin Genes DAMD 17-90- Z-0033 6. AUTHOR(S) Nigel P. Minton, Ph.D. 7. PERFORMING ORGANIZATION NAME(S

  19. Molecular Structures and Functional Relationships in Clostridial Neurotoxins

    Energy Technology Data Exchange (ETDEWEB)

    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.

  20. Involvement of formyl peptide receptors in the stimulatory effect of crotoxin on macrophages co-cultivated with tumour cells.

    Science.gov (United States)

    Costa, E S; Faiad, O J; Landgraf, R G; Ferreira, A K; Brigatte, P; Curi, R; Cury, Y; Sampaio, S C

    2013-11-01

    Crotoxin (CTX) is the main neurotoxic component of Crotalus durissus terrificus snake venom. It inhibits tumour growth and modulates the function of macrophages, which are essential cells in the tumour microenvironment. The present study investigated the effect of CTX on the secretory activity of monocultured macrophages and macrophages co-cultivated with LLC-WRC 256 cells. The effect of the macrophage secretory activities on tumour cell proliferation was also evaluated. Macrophages pre-treated with CTX (0.3 μg/mL) for 2 h were co-cultivated with LLC-WRC 256 cells, and the secretory activity of the macrophages was determined after 12, 24 and 48 h. The co-cultivation of CTX-treated macrophages with the tumour cells caused a 20% reduction in tumour cell proliferation. The production of both H2O2 and NO was increased by 41% and 29% after 24 or 48 h of co-cultivation, respectively, compared to the values for the co-cultures of macrophages of control. The level of secreted IL-1β increased by 3.7- and 3.2-fold after 12 h and 24 h of co-cultivation, respectively. Moreover, an increased level of LXA4 (25%) was observed after 24 h of co-cultivation, and a 2.3- and 2.1-fold increased level of 15-epi-LXA4 was observed after 24 h and 48 h, respectively. Boc-2, a selective antagonist of formyl peptide receptors, blocked both the stimulatory effect of CTX on the macrophage secretory activity and the inhibitory effect of these cells on tumour cell proliferation. Taken together, these results indicate that CTX enhanced the secretory activity of macrophages, which may contribute to the antitumour activity of these cells, and that activation of formyl peptide receptors appears to play a major role in this effect.

  1. Fetal exposure to environmental neurotoxins in Taiwan.

    Directory of Open Access Journals (Sweden)

    Chuen-Bin Jiang

    Full Text Available 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.

  2. Neurotoxin Binding Site on the Acetylcholine Receptor

    Science.gov (United States)

    1987-03-01

    Neurotoxins fron snakes belonging to the families Elapidae (cobras, kraits, mambas , and others) and Hydrophidae (sea snakes) are polypep- tides of 60-74... black mice were used as immunizing anrmals and as a soirce of feeder spleen cells. Mice were injected subcutaneously with j2-mer (50 nmol) emulsified in

  3. The neurotoxin BMAA in aquatic systems

    NARCIS (Netherlands)

    Faassen, E.J.

    2016-01-01

    Eutrophication is a major water quality issue and in many aquatic systems, it leads to the proliferation of toxic phytoplankton species. The neurotoxin β-N-methylamino-L-alanine (BMAA) is one of the compounds that can be present in phytoplankton. BMAA has been suggested to play a role in the ne

  4. Comparative morphocytologic study of native and irradiated crotoxin on CBA/J mice tissues and cells; Estudo morfocitologico comparativo de crotoxina nativa e irradiada em tecidos e celulas de camundongos CBA/J

    Energy Technology Data Exchange (ETDEWEB)

    Cardi, Bruno A.

    1995-12-31

    Snake venoms submitted to gamma radiation are better immunogens, have less toxic activity and maintain immunogenic and antigenic properties, favoring its use in anti-sera production which is the main therapy in human accidents. Severe human accidents attributed to neurotoxicity, are caused by Crotalus durissus terrificus, which main toxin - crotoxin - acts on neuro muscular junctions, as detected by electrophysiological studies and could be detoxified by gamma radiation. In order to evaluate the biological effects of this irradiated protein, we analyzed, at morphological and immunohistochemical levels, organs and cells from CBA/J mice challenged with crude venom or purified crotoxin, in an acute intoxication model. Both toxins were submitted to 2 kGy irradiation with a Co-60 source, in water solution. (author). 140 refs., 10 figs., 1 tab.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  6. Organization and regulation of the neurotoxin genes in Clostridium botulinum and Clostridium tetani.

    Science.gov (United States)

    Raffestin, Stéphanie; Marvaud, Jean Christophe; Cerrato, Rosario; Dupuy, Bruno; Popoff, Michel R

    2004-04-01

    Botulinum and tetanus neurotoxins are structurally and functionally related 150 kDa proteins that are potent inhibitors of neuroexocytosis. Botulinum neurotoxin associates with non-toxic proteins to form complexes of various sizes. The botulinum neurotoxin and non-toxic protein genes are clustered in a DNA segment called the botulinum locus. This locus is probably located on a mobile or degenerate mobile element, which accounts for the various genomic localizations (chromosome, plasmid, phage) in different Clostridium botulinum types. The botulinum neurotoxin and non-toxic protein genes are organized in two polycistronic operons (ntnh-bont and ha operons) transcribed in opposite orientations. The gene that separates the two operons of the botulinum locus in C. botulinum A encodes a 21 kDa protein BotR/A, which is a positive regulator of the expression of the botulinum locus genes. Similarly, in Clostridium tetani, the gene located immediately upstream of the tetanus toxin gene, encodes a positive regulatory protein, TetR. BotR and TetR are possibly alternative sigma factors related to TxeR and UviA, which regulate C. difficile toxin and C. perfringens bacteriocin production, respectively. TxeR and UviA define a new sub-group of the sigma(70) family of RNA polymerase initiation factors. In addition, the C. botulinum genome contains predicted two-component system genes, some of which are possibly involved in regulation of toxinogenesis.

  7. Binding of long-chain α-neurotoxin would stabilize the resting state of nAChR: A comparative study with α-conotoxin

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    Naderi-Manesh Hossein

    2009-02-01

    Full Text Available Abstract Background The details of interaction in a complex between potent antagonists such as long chain α-neurotoxins and α-conotoxins with nicotinic acetylcholine receptor (nAChR, and conformational changes induced by these antagonists, are not yet clear. Modeling In order to uncover some of these critical structural features, we conducted a docking simulation and a molecular dynamics simulation (MD of a model of the ligand binding domain of nAChR in complex with a long-chain α-neurotoxin and an α-conotoxin. Results Our docking results confirm the claim that T.nAChR is in the basal or resting state, which favors binding to the alpha-neurotoxins. Moreover, more correct "hits" for the α/γ interface upon docking for conotoxin-nAChR confirm the preference of conotoxin GI for the α/γ interface. More importantly, upon binding of α-neurotoxin, ligand-bonded nAChR is less dynamic in certain domains than the apo form of the conotoxin-AChR complex. Some critical interactions in the binding site such as the salt bridge formed between K145/D200 in the neurotoxin-nAChR complex is further stabilized during the MD simulation, while it is obviously more labile in the apo form. Conclusion These observations could support the claim that alpha neurotoxins stabilize the nAChR resting state.

  8. Historical Perspectives and Guidelines for Botulinum Neurotoxin Subtype Nomenclature

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    Michael W. Peck

    2017-01-01

    Full Text Available Botulinum neurotoxins are diverse proteins. They are currently represented by at least seven serotypes and more than 40 subtypes. New clostridial strains that produce novel neurotoxin variants are being identified with increasing frequency, which presents challenges when organizing the nomenclature surrounding these neurotoxins. Worldwide, researchers are faced with the possibility that toxins having identical sequences may be given different designations or novel toxins having unique sequences may be given the same designations on publication. In order to minimize these problems, an ad hoc committee consisting of over 20 researchers in the field of botulinum neurotoxin research was convened to discuss the clarification of the issues involved in botulinum neurotoxin nomenclature. This publication presents a historical overview of the issues and provides guidelines for botulinum neurotoxin subtype nomenclature in the future.

  9. Historical Perspectives and Guidelines for Botulinum Neurotoxin Subtype Nomenclature

    Science.gov (United States)

    Peck, Michael W.; Smith, Theresa J.; Anniballi, Fabrizio; Austin, John W.; Bano, Luca; Bradshaw, Marite; Cuervo, Paula; Cheng, Luisa W.; Derman, Yagmur; Dorner, Brigitte G.; Fisher, Audrey; Hill, Karen K.; Kalb, Suzanne R.; Korkeala, Hannu; Lindström, Miia; Lista, Florigio; Lúquez, Carolina; Mazuet, Christelle; Pirazzini, Marco; Popoff, Michel R.; Rossetto, Ornella; Rummel, Andreas; Sesardic, Dorothea; Singh, Bal Ram; Stringer, Sandra C.

    2017-01-01

    Botulinum neurotoxins are diverse proteins. They are currently represented by at least seven serotypes and more than 40 subtypes. New clostridial strains that produce novel neurotoxin variants are being identified with increasing frequency, which presents challenges when organizing the nomenclature surrounding these neurotoxins. Worldwide, researchers are faced with the possibility that toxins having identical sequences may be given different designations or novel toxins having unique sequences may be given the same designations on publication. In order to minimize these problems, an ad hoc committee consisting of over 20 researchers in the field of botulinum neurotoxin research was convened to discuss the clarification of the issues involved in botulinum neurotoxin nomenclature. This publication presents a historical overview of the issues and provides guidelines for botulinum neurotoxin subtype nomenclature in the future. PMID:28106761

  10. Mechanism of Action of Presynaptic Neurotoxins

    Science.gov (United States)

    1985-09-01

    cell system in order to directly assess the physiological relevance of the tetanus toxin receptor in the intoxication process and to study the events...the interactions of the Clostridlal neurotoxins with their physiological target tissues. The NI1-RE-105 cell line has receptors that are analogous to...L. A., and Tischler, A. S. (1982) PC12 Pheochromocytoma Cultures in Neurobiological Research. Adv. Cell. Neurobiol. •, 373-415. 18 Gundersen, C. B

  11. Cycad neurotoxins, consumption of flying foxes, and ALS-PDC disease in Guam.

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    Cox, Paul Alan; Sacks, Oliver W

    2002-03-26

    The Chamorro people of Guam have been afflicted with a complex of neurodegenerative diseases (now known as ALS-PDC) with similarities to ALS, AD, and PD at a far higher rate than other populations throughout the world. Chamorro consumption of flying foxes may have generated sufficiently high cumulative doses of plant neurotoxins to result in ALS-PDC neuropathologies, since the flying foxes forage on neurotoxic cycad seeds.

  12. Universal and specific quantitative detection of botulinum neurotoxin genes

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    Arnon Stephen S

    2010-10-01

    Full Text Available Abstract Background Clostridium botulinum, an obligate anaerobic spore-forming bacterium, produces seven antigenic variants of botulinum toxin that are distinguished serologically and termed "serotypes". Botulinum toxin blocks the release of acetylcholine at neuromuscular junctions resulting in flaccid paralysis. The potential lethality of the disease warrants a fast and accurate means of diagnosing suspected instances of food contamination or human intoxication. Currently, the Food and Drug Administration (FDA-accepted assay to detect and type botulinum neurotoxins (BoNTs is the mouse protection bioassay. While specific and sensitive, this assay requires the use of laboratory animals, may take up to four days to achieve a diagnosis, and is unsuitable for high-throughput analysis. We report here a two-step PCR assay that identifies all toxin types, that achieves the specificity of the mouse bioassay while surpassing it in equivalent sensitivity, that has capability for high-throughput analysis, and that provides quantitative results within hours. The first step of our assay consists of a conventional PCR that detects the presence of C. botulinum regardless of the neurotoxin type. The second step uses quantitative PCR (qPCR technology to determine the specific serotype of the neurotoxin. Results We assayed purified C. botulinum DNA and crude toxin preparations, as well as food and stool from healthy individuals spiked with purified BoNT DNA, and one stool sample from a case of infant botulism for the presence of the NTNH gene, which is part of the BoNT gene cluster, and for the presence of serotype-specific BoNT genes. The PCR surpassed the mouse bioassay both in specificity and sensitivity, detecting positive signals in BoNT preparations containing well below the 1 LD50 required for detection via the mouse bioassay. These results were type-specific and we were reliably able to quantify as few as 10 genomic copies. Conclusions While other studies

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

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    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

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

    Science.gov (United States)

    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.

  15. Insights into the evolutionary origins of clostridial neurotoxins from analysis of the Clostridium botulinum strain A neurotoxin gene cluster

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    Meiering Elizabeth M

    2008-11-01

    Full Text Available Abstract Background Clostridial neurotoxins (CNTs are the most deadly toxins known and causal agents of botulism and tetanus neuroparalytic diseases. Despite considerable progress in understanding CNT structure and function, the evolutionary origins of CNTs remain a mystery as they are unique to Clostridium and possess a sequence and structural architecture distinct from other protein families. Uncovering the origins of CNTs would be a significant contribution to our understanding of how pathogens evolve and generate novel toxin families. Results The C. botulinum strain A genome was examined for potential homologues of CNTs. A key link was identified between the neurotoxin and the flagellin gene (CBO0798 located immediately upstream of the BoNT/A neurotoxin gene cluster. This flagellin sequence displayed the strongest sequence similarity to the neurotoxin and NTNH homologue out of all proteins encoded within C. botulinum strain A. The CBO0798 gene contains a unique hypervariable region, which in closely related flagellins encodes a collagenase-like domain. Remarkably, these collagenase-containing flagellins were found to possess the characteristic HEXXH zinc-protease motif responsible for the neurotoxin's endopeptidase activity. Additional links to collagenase-related sequences and functions were detected by further analysis of CNTs and surrounding genes, including sequence similarities to collagen-adhesion domains and collagenases. Furthermore, the neurotoxin's HCRn domain was found to exhibit both structural and sequence similarity to eukaryotic collagen jelly-roll domains. Conclusion Multiple lines of evidence suggest that the neurotoxin and adjacent genes evolved from an ancestral collagenase-like gene cluster, linking CNTs to another major family of clostridial proteolytic toxins. Duplication, reshuffling and assembly of neighboring genes within the BoNT/A neurotoxin gene cluster may have lead to the neurotoxin's unique architecture. This

  16. Neurotoxins from Marine Dinoflagellates: A Brief Review

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    Da-Zhi Wang

    2008-06-01

    Full Text Available Dinoflagellates are not only important marine primary producers and grazers, but also the major causative agents of harmful algal blooms. It has been reported that many dinoflagellate species can produce various natural toxins. These toxins can be extremely toxic and many of them are effective at far lower dosages than conventional chemical agents. Consumption of seafood contaminated by algal toxins results in various seafood poisoning syndromes: paralytic shellfish poisoning (PSP, neurotoxic shellfish poisoning (NSP, amnesic shellfish poisoning (ASP, diarrheic shellfish poisoning (DSP, ciguatera fish poisoning (CFP and azaspiracid shellfish poisoning (ASP. Most of these poisonings are caused by neurotoxins which present themselves with highly specific effects on the nervous system of animals, including humans, by interfering with nerve impulse transmission. Neurotoxins are a varied group of compounds, both chemically and pharmacologically. They vary in both chemical structure and mechanism of action, and produce very distinct biological effects, which provides a potential application of these toxins in pharmacology and toxicology. This review summarizes the origin, structure and clinical symptoms of PSP, NSP, CFP, AZP, yessotoxin and palytoxin produced by marine dinoflagellates, as well as their molecular mechanisms of action on voltage-gated ion channels.

  17. Neurotoxins from marine dinoflagellates: a brief review.

    Science.gov (United States)

    Wang, Da-Zhi

    2008-06-11

    Dinoflagellates are not only important marine primary producers and grazers, but also the major causative agents of harmful algal blooms. It has been reported that many dinoflagellate species can produce various natural toxins. These toxins can be extremely toxic and many of them are effective at far lower dosages than conventional chemical agents. Consumption of seafood contaminated by algal toxins results in various seafood poisoning syndromes: paralytic shellfish poisoning (PSP), neurotoxic shellfish poisoning (NSP), amnesic shellfish poisoning (ASP), diarrheic shellfish poisoning (DSP), ciguatera fish poisoning (CFP) and azaspiracid shellfish poisoning (ASP). Most of these poisonings are caused by neurotoxins which present themselves with highly specific effects on the nervous system of animals, including humans, by interfering with nerve impulse transmission. Neurotoxins are a varied group of compounds, both chemically and pharmacologically. They vary in both chemical structure and mechanism of action, and produce very distinct biological effects, which provides a potential application of these toxins in pharmacology and toxicology. This review summarizes the origin, structure and clinical symptoms of PSP, NSP, CFP, AZP, yessotoxin and palytoxin produced by marine dinoflagellates, as well as their molecular mechanisms of action on voltage-gated ion channels.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  19. Substrate binding mode and its implication on drug design for botulinum neurotoxin A.

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    Desigan Kumaran

    Full Text Available 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 (197QRATKM(202 and its variant (197RRATKM(202 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.

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

    Science.gov (United States)

    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.

  1. CRYSTAL STRUCTURE OF CLOSTRIDIUM BOTULINUM NEUROTOXIN SEROTYPE B.

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    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].

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

    Science.gov (United States)

    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.

  3. Study of crotoxin on the induction of paralysis in extraocular muscle in animal model Estudo da crotoxina na indução de paralisia da musculatura extraocular em modelo animal

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    Geraldo de Barros Ribeiro

    2012-10-01

    Full Text Available PURPOSE: Crotoxin is the major toxin of the venom of the South American rattlesnake Crotalus durissus terrificus, capable of causing a blockade of the neurotransmitters at the neuromuscular junction. The objective of this study was to appraise the action and effectiveness of the crotoxin induced paralysis of the extraocular muscle and to compare its effects with the botulinum toxin type A (BT-A. METHODS: The crotoxin, with LD50 of 1.5 µg, was injected into the superior rectus muscle in ten New Zealand rabbits. The concentration variance was 0.015 up to 150 µg. Two rabbits received 2 units of botulinum toxin type A for comparative analysis. The evaluation of the paralysis was performed using serial electromyography. After the functional recovery of the muscles, which occurred after two months, six rabbits were sacrificed for anatomopathology study. RESULTS: The animals did not show any evidence of systemic toxicity. Transitory ptosis was observed in almost every animal and remained up to fourteen days. These toxins caused immediate blockade of the electrical potentials. The recovery was gradual in the average of one month with regeneration signs evident on the electromyography. The paralysis effect of the crotoxin on the muscle was proportional to its concentration. The changes with 1.5 µg crotoxin were similar to those produced by the botulinum toxin type A. The histopathology findings were localized to the site of the injection. No signs of muscle fiber's necrosis were seen in any sample. The alterations induced by crotoxin were also proportional to the concentration and similar to botulinum toxin type A in concentration of 1.5 µg. CONCLUSION: Crotoxin was able to induce transitory paralysis of the superior rectus muscle. This effect was characterized by reduction of action potentials and non-specific signs of fibrillation. Crotoxin, in concentration of 1.5 µg was able to induce similar effects as botulinum toxin type A.OBJETIVO: A

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

    Science.gov (United States)

    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

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

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    Skadi Kull

    Full Text Available 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

  6. Tratamiento de epicondilitis refractaria con neurotoxina botulínica tipo A libre de complejo proteínico Treament of refractory epicondilitis with botulin neurotoxin type A free of protein complex

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    B. Santos

    2013-02-01

    conservative measures are failing. Material and methods: We present 3 patients diagnosed of epicondylitis for more than 6 months, and which had been subjected to the usual treatment without success, which will manage with free protein complex botulinum toxin (50 IU at a single point of application on an outpatient basis. The intensity of the pain in the 3 patients were evaluated using an analog scale visual (EVA in which 0 represents no pain and 10 means more intense pain experienced with resisted wrist extension maneuver both palpation. Results: We obtained a significant reduction of pain in 3 patients treated to the first month of his administration with total resolution at 3 months. This benefit was maintained at least during 6 months. Conclusions: refractory to conservative treatment can be effectively treated with free protein complex botulinum toxin.

  7. Use of Botulinum Neurotoxin in Ophthalmology

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    Emel Başar

    2016-12-01

    Full Text Available Botulinum neurotoxin (BoNT is the first biological toxin used in the treatment of ophthalmic diseases and to decrease skin wrinkles as an aesthetic agent. When used appropriately, it weakens the force of muscular contraction and/or inhibits glandular secretion. The most common areas for botulinum toxin treatment are the upper face, including the glabella, forehead, brows, and lateral canthal lines or crow’s feet. By relaxing the muscles causing wrinkles, non-permanent results may be achieved with its use. BoNT has gained widespread use in a variety of ophthalmic diseases. The effect of BoNT is temporary, but the therapeutic benefit is usually maintained even after repeated injections. Treatment is usually well tolerated. Complications and side effects associated with the treatment are rare and temporary. Complications occur due to weakness (chemodenervation of adjacent muscle groups, immunological mechanisms and injection technique. Current therapeutic indications, doses, complications and contraindications of BoNT use in the following disorders related to ophthalmology were investigated: aesthetic use, strabismus, blepharospasm, hemifacial spasm, eyelid retraction, entropion, lacrimal hypersecretion syndrome, and facial paralysis.

  8. Use of Botulinum Neurotoxin in Ophthalmology

    Science.gov (United States)

    Başar, Emel; Arıcı, Ceyhun

    2016-01-01

    Botulinum neurotoxin (BoNT) is the first biological toxin used in the treatment of ophthalmic diseases and to decrease skin wrinkles as an aesthetic agent. When used appropriately, it weakens the force of muscular contraction and/or inhibits glandular secretion. The most common areas for botulinum toxin treatment are the upper face, including the glabella, forehead, brows, and lateral canthal lines, or crow’s feet. By relaxing the muscles causing wrinkles, non-permanent results may be achieved with its use. BoNT has gained widespread use in a variety of ophthalmic diseases. The effect of BoNT is temporary, but the therapeutic benefit is usually maintained even after repeated injections. Treatment is usually well tolerated. Complications and side effects associated with the treatment are rare and temporary. Complications occur due to weakness (chemodenervation) of adjacent muscle groups, immunological mechanisms and injection technique. Current therapeutic indications, doses, complications and contraindications of BoNT use in the following disorders related to ophthalmology were investigated: aesthetic use, strabismus, blepharospasm, hemifacial spasm, eyelid retraction, entropion, lacrimal hypersecretion syndrome, and facial paralysis. PMID:28050326

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

    Science.gov (United States)

    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.

  10. A historical and proteomic analysis of botulinum neurotoxin type/G

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    Rees Jon

    2011-10-01

    Full Text Available Abstract Background Clostridium botulinum is the taxonomic designation for at least six diverse species that produce botulinum neurotoxins (BoNTs. There are seven known serotypes of BoNTs (/A through/G, all of which are potent toxins classified as category A bioterrorism agents. BoNT/G is the least studied of the seven serotypes. In an effort to further characterize the holotoxin and neurotoxin-associated proteins (NAPs, we conducted an in silico and proteomic analysis of commercial BoNT/G complex. We describe the relative quantification of the proteins present in the/G complex and confirm our ability to detect the toxin activity in vitro. In addition, we review previous literature to provide a complete description of the BoNT/G complex. Results An in-depth comparison of protein sequences indicated that BoNT/G shares the most sequence similarity with the/B serotype. A temperature-modified Endopep-MS activity assay was successful in the detection of BoNT/G activity. Gel electrophoresis and in gel digestions, followed by MS/MS analysis of/G complex, revealed the presence of four proteins in the complexes: neurotoxin (BoNT and three NAPs--nontoxic-nonhemagglutinin (NTNH and two hemagglutinins (HA70 and HA17. Rapid high-temperature in-solution tryptic digestions, coupled with MS/MS analysis, generated higher than previously reported sequence coverages for all proteins associated with the complex: BoNT 66%, NTNH 57%, HA70 91%, and HA17 99%. Label-free relative quantification determined that the complex contains 30% BoNT, 38% NTNH, 28% HA70, and 4% HA17 by weight comparison and 17% BoNT, 23% NTNH, 42% HA70, and 17% HA17 by molecular comparison. Conclusions The in silico protein sequence comparisons established that the/G complex is phenetically related to the other six serotypes of C. botulinum. Proteomic analyses and Endopep-MS confirmed the presence of BoNT and NAPs, along with the activity of the commercial/G complex. The use of data

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Yunhua Zhong

    Full Text Available 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.

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

    Science.gov (United States)

    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

  15. Antibody protection against botulinum neurotoxin intoxication in mice.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    Carter, Andrew T; Peck, Michael W

    2015-05-01

    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.

  17. Historical Perspectives and Guidelines for Botulinum Neurotoxin Subtype Nomenclature

    Science.gov (United States)

    2016-08-26

    monoclonal antibody-based immunoassay for detecting type B Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system...Characterization of botulinum neurotoxin A subtypes 1 through 5 by investigation of activities in mice, in neuronal cell cultures , and in vitro, Infect

  18. Use of Botulinum Neurotoxin for the Treatment of Movement Disorders

    Science.gov (United States)

    ... carefully reviewed all available scientific studies on the effect of BoNT for treating movement disorders. What is botulinum neurotoxin and how does it work? BoNT is a drug made by bacteria that causes some forms of food poisoning. Neurologists inject small and safe doses of ...

  19. Rattlesnake Neurotoxin Structure, Mechanism of Action, Immunology and Molecular Biology

    Science.gov (United States)

    1992-09-10

    phospholipase A2 presynaptic neurotoxin from ViDera ammodvtes ammodytes venom. Hydropathy profiles of B. asoer phospholipase and the ammodytoxins also show great...from 8, a•pe and V8 phospholipase A2 from Vipera russellii (provided by John Middlebrook, USAMRIID). In neither case was an enhancement in toxin

  20. Botulinum Neurotoxin Serotypes Detected by Electrochemical Impedance Spectroscopy

    Science.gov (United States)

    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

  1. Botulinum Neurotoxin Serotypes Detected by Electrochemical Impedance Spectroscopy

    Directory of Open Access Journals (Sweden)

    Alison C. Savage

    2015-05-01

    Full Text Available 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.

  2. Study of uptake and endocytosis of gamma rays-irradiated crotoxin by mice peritoneal macrophages; Avaliacao do mecanismo de captacao e endocitose de crotoxina submetida a acao da radiacao, por macrofagos peritoneais de camundongos

    Energy Technology Data Exchange (ETDEWEB)

    Cardi, Bruno Andrade

    1999-07-01

    The purpose was to investigate the uptake and endocytosis of 2000 Gy {sup 60}Co irradiated crotoxin through mouse peritoneal macrophages, correlating with native one and another non related protein, the ovalbumin. Native (CTXN) or 2000 Gy {sup 60} Co {gamma}-rays (dose rate 540 Gy/hour) irradiated crotoxin (CTXI) or ovalbumin processed of same manner (OVAN - OVAI) were offered to mouse peritoneal macrophages and their uptake was evaluated by immunohistochemistry and quantitative in situ ELISA. The involvement of scavenger receptors (ScvR) was evaluated by using blockers drugs (Probuco-PBC or Dextran Sulfate - SD) or with nonspecific blocking using fetal calf serum (FBS). The morphology and viability of macrophages were preserved during the experiments. CTXI showed irradiation-induced aggregates and formation of oxidative changing were observed on this protein after gamma rays treatment. By immunohistochemistry we could observe heavy stained phagocytic vacuole on macrophages incubated with CTXI, as compared with CTXN. Quantitatively by in situ ELISA, the sema pattern was observed, displaying a 2-fold CTXI incorporation. In presence of PBC or SD we could find a significant decrease of CTXI uptake but not of CTXN. However the CTXN uptake was depressed by FBS, not observed with CTXI. OVA, after gamma rays treatment, underwent a high degradation suffering a potent incorporation and metabolism by macrophages, with a major uptake of OVAI in longer incubation (120 minutes). Gamma rays ({sup 60} Co) produced oxidative changes on CTX molecule, leading to a uptake by ScvR-mice peritoneal macrophages, suggesting that the relation antigen-presenting cells and gamma rays-modified proteins are responsible for the better immune response presented by irradiated antigens. (author)

  3. Conjugative botulinum neurotoxin-encoding plasmids in Clostridium botulinum.

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    Kristin M Marshall

    Full Text Available BACKGROUND: Clostridium botulinum produces seven distinct serotypes of botulinum neurotoxins (BoNTs. The genes encoding different subtype neurotoxins of serotypes A, B, F and several dual neurotoxin-producing strains have been shown to reside on plasmids, suggesting that intra- and interspecies transfer of BoNT-encoding plasmids may occur. The objective of the present study was to determine whether these C. botulinum BoNT-encoding plasmids are conjugative. METHODOLOGY/PRINCIPAL FINDINGS: C. botulinum BoNT-encoding plasmids pBotCDC-A3 (strain CDC-A3, pCLJ (strain 657Ba and pCLL (strain Eklund 17B were tagged with the erythromycin resistance marker (Erm using the ClosTron mutagenesis system by inserting a group II intron into the neurotoxin genes carried on these plasmids. Transfer of the tagged plasmids from the donor strains CDC-A3, 657Ba and Eklund 17B to tetracycline-resistant recipient C. botulinum strains was evaluated in mating experiments. Erythromycin and tetracycline resistant transconjugants were isolated from donor:recipient mating pairs tested. Transfer of the plasmids to the transconjugants was confirmed by pulsed-field gel electrophoresis (PFGE and Southern hybridizations. Transfer required cell-to-cell contact and was DNase resistant. This indicates that transfer of these plasmids occurs via a conjugation mechanism. CONCLUSIONS/SIGNIFICANCE: This is the first evidence supporting conjugal transfer of native botulinum neurotoxin-encoding plasmids in C. botulinum, and provides a probable mechanism for the lateral distribution of BoNT-encoding plasmids to other C. botulinum strains. The potential transfer of C. botulinum BoNT-encoding plasmids to other bacterial hosts in the environment or within the human intestine is of great concern for human pathogenicity and necessitates further characterization of these plasmids.

  4. Structural And Biochemical Studies of Botulinum Neurotoxin Serotype C1 Light Chain Protease: Implications for Dual Substrate Specificity

    Energy Technology Data Exchange (ETDEWEB)

    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.

  5. Sequencing and phylogenetic analysis of neurotoxin gene from an environmental isolate of Clostridium sp.: comparison with other clostridial neurotoxins.

    Science.gov (United States)

    Dixit, Aparna; Alam, Syed Imteyaz; Singh, Lokendra

    2006-07-01

    A Clostridium sp. isolated from intestine of decaying fish exhibited 99% sequence identity with C. tetani at 16S rRNA level. It produced a neurotoxin that was neutralized by botulinum antitoxin (A+B+E) as well as tetanus antitoxin. The gene fragments for light chain, C-terminal and N-terminal regions of the heavy chain of the toxin were amplified using three reported primer sets for tetanus neurotoxin (TeNT). The neurotoxin gene fragments were cloned in Escherichia coli and sequenced. The sequences obtained exhibited approximately 98, 99 and 98% sequence identity with reported gene sequences of TeNT/LC, TeNT/HC and TeNT/HN, respectively. The phylogenetic interrelationship between the neurotoxin gene of Clostridium sp. with previously reported gene sequences of Clostridium botulinum A to G and C. tetani was examined by analysis of differences in the nucleotide sequences. Six amino acids were substituted at four different positions in the light chain of neurotoxin from the isolate when compared with the reported closest sequence of TeNT. Of these, four were located in the beta15 motif at a solvent inaccessible, buried region of the protein molecule. One of these substitutions were on the solvent accessible surface residue of alpha1 motif, previously shown to have strong sequence conservation. A substitution of two amino acids observed in N-terminal region of heavy chain were buried residues, located in the beta21 and beta37 motifs showing variability in other related sequences. The C-terminal region responsible for binding to receptor was conserved, showing no changes in the amino acid sequence.

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

    Science.gov (United States)

    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?

  7. Characterization of Clostridium sp. RKD producing botulinum-like neurotoxin.

    Science.gov (United States)

    Dixit, Aparna; Dhaked, Ram Kumar; Alam, Syed Imteyaz; Singh, Lokendra

    2005-07-01

    A Gram positive, motile, rod-shaped, strictly anaerobic bacterium isolated from intestine of decaying fish was identified as Clostridium sp. RKD and produced a botulinum type B-like neurotoxin as suggested by mouse bioassay and protection with anti botulinum antibodies. The neurotoxicity was functionally characterized by the phrenic nerve hemi-diaphragm assay. Phylogenetic analysis based on 16S rDNA sequence, placed it at a different position from the reported strains of Clostridium botulinum. The strain exhibited differences from both Clostridium botulinum and Clostridium tetani with respect to morphological, biochemical and chemotaxonomic characteristics. Botulinum group specific and serotype specific primers amplified the DNA fragments of 260 and 727 bp, respectively, indicating presence of botulinum type 'B' toxin gene. Sequence of nearly 700 bp amplified using primers specific for botulinum neurotoxin type B gene, did not show any significant match in the database when subjected to BLAST search.

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

    OpenAIRE

    Sayler, Gary S.; Cusick, Kathleen D.

    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 neurotoxin...

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  11. Adaptive Simulated Annealing Based Protein Loop Modeling of Neurotoxins

    Institute of Scientific and Technical Information of China (English)

    陈杰; 黄丽娜; 彭志红

    2003-01-01

    A loop modeling method, adaptive simulated annealing, for ab initio prediction of protein loop structures, as an optimization problem of searching the global minimum of a given energy function, is proposed. An interface-friendly toolbox-LoopModeller in Windows and Linux systems, VC++ and OpenGL environments is developed for analysis and visualization. Simulation results of three short-chain neurotoxins modeled by LoopModeller show that the method proposed is fast and efficient.

  12. Central Nervous System Toxicity After Botulinum Neurotoxin Injection

    Science.gov (United States)

    Ilkhchoui, Yashar; Ghaly, Ramsis F.; Knezevic, N. Nick; Candido, Kenneth D

    2013-01-01

    Since Its first description of botulism toxicity in 1820s, specific formulations of botulinum neurotoxin (BoNT) have been introduced with different clinical benefits. However, there is increasing number of adverse events reported to Food and Drug Administration. This report presents the case of 62-year-old woman with Parkinson’s disease who received BoNT injections to treat painful spasticity in her hands. She developed severe generalized dystonia shortly after BoNT injections. PMID:24223367

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

    OpenAIRE

    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 develop...

  14. Rational Design of Therapeutic and Diagnostic Against Botulinum Neurotoxin

    Science.gov (United States)

    2006-12-01

    Gram-positive bacilli bacterium, Clostridium botulinum, which can be found worldwide in soil. Clostridium tetani is a similar bacterium to C...B: Biological Sciences, 354, 259-268. 6. Nantel, A.J. (1999). Clostridium Botulinum. WHO international programme on chemical safety poisons...of Clostridium botulinum C1 neurotoxin. Nucleic Acids Research, 18, 4924. 20. Binz, T., Kurazono, H., Popoff, M.R., Eklund, M.W., Sakaguchi, G

  15. Electrochemical impedance spectroscopy biosensor for detection of active botulinum neurotoxin

    Directory of Open Access Journals (Sweden)

    Jennifer Halliwell

    2014-12-01

    Full Text Available The standard method for the detection of botulinum neurotoxin is currently the mouse bioassay which is considered to be the most reliable method for the detection of the active form of this toxin. Despite this it is a time-consuming and expensive assay to run and as such many alternative assays have recently been proposed. Herein we report the development of two electrochemical assays for the detection of active botulinum neurotoxin in a pharmaceutical sample. Gold electrodes were modified with self-assembled monolayers of the SNARE protein SNAP-25 which is selectively cleaved by active botulinum neurotoxin A. Cyclic voltammetry and electrochemical impedance spectroscopy were performed on the modified working electrodes to observe changes to the layer on addition of the toxin. Both methods were able to distinguish the difference between the presence of the active toxin and a placebo containing the excipients of the pharmaceutical product. The electrochemical impedance spectroscopy assay also allowed for detection of the active toxin at concentrations as low as 25 fg/ml, with results being obtained in under an hour outperforming the mouse bioassay.

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

    Science.gov (United States)

    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.

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

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    Jasmin Weisemann

    2016-05-01

    Full Text Available 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.

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

    Science.gov (United States)

    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. Excitatory amino acid b-N-methylamino-L-alanine is a putative environmental neurotoxin

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    VLADIMIR NEDELJKOV

    2011-04-01

    Full Text Available The amino acid b-N-methylamino-L-alanine (L-BMAA has been associated with the amyotrophic lateral sclerosis/parkinsonism-dementia complex in three distinct western Pacific populations. The putative neurotoxin is produced by cyanobacteria, which live symbiotically in the roots of cycad trees. L-BMAA was thought to be a threat only to those few populations whose diet and medicines rely heavily on cycad seeds. However, the recent discovery that cyanobacteria from diverse terrestrial, freshwater, and saltwater ecosystems around the world produce the toxin requires a reassessment of whether it poses a larger health threat. Therefore, it is proposed that monitoring L-BMAA levels in cyanobacteria-contaminated water supplies might be prudent.

  20. Snake venomics of the Central American rattlesnake Crotalus simus and the South American Crotalus durissus complex points to neurotoxicity as an adaptive paedomorphic trend along Crotalus dispersal in South America.

    Science.gov (United States)

    Calvete, Juan J; Sanz, Libia; Cid, Pedro; de la Torre, Pilar; Flores-Díaz, Marietta; Dos Santos, M Cristina; Borges, Adolfo; Bremo, Adolfo; Angulo, Yamileth; Lomonte, Bruno; Alape-Girón, Alberto; Gutiérrez, José María

    2010-01-01

    We report a comparative venomic and antivenomic characterization of the venoms of newborn and adult specimens of the Central American rattlesnake, Crotalus simus, and of the subspecies cumanensis, durissus, ruruima, and terrificus of South American Crotalus durissus. Neonate and adult C. simus share about 50% of their venom proteome. The venom proteome of 6-week-old C. simus is predominantly made of the neurotoxic heterodimeric phospholipase A(2) (PLA(2) crotoxin) (55.9%) and serine proteinases (36%), whereas snake venom Zn(2+)-metalloproteinases (SVMPs), exclusively of class PIII, represent only 2% of the total venom proteins. In marked contrast, venom from adult C. simus comprises toxins from 7 protein families. A large proportion (71.7%) of these toxins are SVMPs, two-thirds of which belong to the PIII class. These toxin profiles correlate well with the overall biochemical and pharmacological features of venoms from adult (hemorrhagic) and newborn (neurotoxic) C. simus specimens. The venoms of the South American Crotalus subspecies belong to one of two distinct phenotypes. C. d. cumanensis exhibits high levels of SVMPs and low lethal potency (LD(50)), whereas C. d. subspecies terrificus, ruruima, and durissus have low SVMP activity and high neurotoxicity to mice. Their overall toxin compositions explain the outcome of envenomation by these species. Further, in all C. simus and C. durissus venoms, the concentration of neurotoxins (crotoxin and crotamine) is directly related with lethal activity, whereas lethality and metalloproteinase activity show an inverse relationship. The similar venom toxin profiles of newborn C. simus and adult C. durissus terrificus, ruruima, and durissus subspecies strongly suggests that the South American taxa have retained juvenile venom characteristics in the adult form (paedomorphism) along their North-South stepping-stone dispersal. The driving force behind paedomorphism is often competition or predation pressure. The increased

  1. Botulinum neurotoxins A and E undergo retrograde axonal transport in primary motor neurons.

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    Laura Restani

    2012-12-01

    Full Text Available The striking differences between the clinical symptoms of tetanus and botulism have been ascribed to the different fate of the parental neurotoxins once internalised in motor neurons. Tetanus toxin (TeNT is known to undergo transcytosis into inhibitory interneurons and block the release of inhibitory neurotransmitters in the spinal cord, causing a spastic paralysis. In contrast, botulinum neurotoxins (BoNTs block acetylcholine release at the neuromuscular junction, therefore inducing a flaccid paralysis. Whilst overt experimental evidence supports the sorting of TeNT to the axonal retrograde transport pathway, recent findings challenge the established view that BoNT trafficking is restricted to the neuromuscular junction by highlighting central effects caused by these neurotoxins. These results suggest a more complex scenario whereby BoNTs also engage long-range trafficking mechanisms. However, the intracellular pathways underlying this process remain unclear. We sought to fill this gap by using primary motor neurons either in mass culture or differentiated in microfluidic devices to directly monitor the endocytosis and axonal transport of full length BoNT/A and BoNT/E and their recombinant binding fragments. We show that BoNT/A and BoNT/E are internalised by spinal cord motor neurons and undergo fast axonal retrograde transport. BoNT/A and BoNT/E are internalised in non-acidic axonal carriers that partially overlap with those containing TeNT, following a process that is largely independent of stimulated synaptic vesicle endo-exocytosis. Following intramuscular injection in vivo, BoNT/A and TeNT displayed central effects with a similar time course. Central actions paralleled the peripheral spastic paralysis for TeNT, but lagged behind the onset of flaccid paralysis for BoNT/A. These results suggest that the fast axonal retrograde transport compartment is composed of multifunctional trafficking organelles orchestrating the simultaneous transfer

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

    Science.gov (United States)

    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...

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

    Science.gov (United States)

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

    2012-12-04

    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.

  4. Dolichospermum and Aphanizomenon as neurotoxins producers in some Russian freshwaters.

    Science.gov (United States)

    Chernova, Ekaterina; Sidelev, Sergey; Russkikh, Iana; Voyakina, Ekaterina; Babanazarova, Olga; Romanov, Roman; Kotovshchikov, Anton; Mazur-Marzec, Hanna

    2017-05-01

    Last decades, cyanobacterial blooms have been commonly reported in Russia. Among the boom-forming species, potential toxin producers have been identified. The aim of this paper was to study the presence of neurotoxic compounds - saxitoxins and anatoxin-a - in water bodies from different regions of Russia. We also made attempts to identify the neurotoxin-producing genera. The good convergence of the results obtained by light microscopy, PCR and LC-MS/MS analyses indicated the presence of active neurotoxin producing species in all investigated water bodies. Saxitoxin was detected in phytoplankton from 4 water bodies in Central European Russia and West Siberia, including lake and reservoirs used as a source for potable water. The water bodies differed with the respect of saxitoxin producers which belonged to Aphanizomenon and/or Dolichospermum genera. For the first time, we obtained quantitative data on the intracellular saxitoxin concentration in Russian freshwaters using LC-MS/MS. Anatoxin-a was detected only in lakes of Northwestern Russia. In the eutrophic shallow Lower Suzdal Lake, Aphanizomenon was the stated anatoxin-a-producing genus. In the large shallow artificial hypertrophic Sestroretskij Razliv Lake, it was very likely that both dominant species - Aphanizomenon flos-aquae and Dolichospermum planctonicum - were anatoxin-a producers. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

    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

  6. Botulinum neurotoxin type A in the masseter muscle: Effects on incisor eruption in rabbits

    Science.gov (United States)

    Navarrete, Alfonso L.; Rafferty, Katherine L.; Liu, Zi Jun; Ye, Wenmin; Greenlee, Geoffrey M.; Herring, Susan W.

    2015-01-01

    Introduction Botulinum neurotoxins are responsible for the paralytic food poisoning, botulism. Commercial formulations such as botulinum neurotoxin type A are increasingly used for various conditions, including cosmetic recontouring of the lower face by injection of the large masseter muscles. The paralysis of a major muscle of mastication lowers occlusal force and thus might affect tooth eruption. The purpose of this study was to investigate the effects of unilateral masseter muscle injection of botulinum neurotoxin type A on the rate of eruption of incisors in a rabbit model. We hypothesized that the teeth would overerupt in an underloaded environment. Methods Forty rabbits were injected with either botulinum neurotoxin type A or saline solution in 1 masseter muscle. Mastication and muscle force production were monitored, and incisor eruption rate was assessed by caliper measurement of grooved teeth. Results The injection of saline solution had no effect. The masseter muscle injected with botulinum neurotoxin type A showed a dramatic loss of force 3 weeks after injection despite apparently normal mastication. Incisor eruption rate was significantly decreased for the botulinum neurotoxin type A group, an effect attributed to decreased attrition. Conclusions This study has implications for orthodontics. Although findings from ever-growing rabbit incisors cannot be extrapolated to human teeth, it is clear that botulinum neurotoxin type A caused a decrease in bite force that could influence dental eruption. PMID:23561411

  7. Structure-function relationships in the neurotoxin Vipoxin from the venom of Vipera ammodytes meridionalis

    Science.gov (United States)

    Georgieva, Dessislava Nikolova; Genov, Nicolay; Nikolov, Peter; Aleksiev, Boris; Rajashankar, Kanagalaghatta R.; Voelter, Wolfgang; Betzel, Christian

    2003-02-01

    The neurotoxic complex Vipoxin is the lethal component of the venom of Vipera ammodytes meridionalis, the most toxic snake in Europe. It is a complex between a toxic phospholipase A 2 (PLA 2) and a non-toxic and catalytically inactive protein, stabilizing the enzyme and reducing the activity and toxicity. Structure-function relationships in this complex were studied by spectroscopic methods. A good correlation between the ionization behaviour and accessible surface area (ASA) of the tyrosyl residues was observed. In the toxic PLA 2 subunit phenolic groups participate in H-bonding network that stabilizes the catalytically and pharmacologically active conformation. The tryptophan fluorescence decay of Vipoxin is well fitted by two exponentials with lifetimes of 0.1 (54%) and 2.5 (46%) ns. W20P, W31P and W31I are located in the interface between the two subunits and participate in hydrophobic interactions stabilizing the complex. Dissociation of the complex leads to a transition of the tryptophans from hydrophobic to hydrophilic environment, which influences mainly τ2. The longer lifetime is more sensitive to the polarity of the environment. Circular dichroism measurements demonstrate that the two components of the neurotoxin preserve their secondary structure after dissociation of the complex. The results of the spectroscopic studies are in accordance with a mechanism of blockade of transmission across the neuromuscular junctions of the breathing muscles by interaction of a dissociated toxic PLA 2 with a membrane. The loss of toxicity is connected with slight changes in the secondary structure of PLA 2. CD studies also show a substantial contribution of disulfide bonds to the stability of the neurotoxic complex and its components.

  8. Computational Insights of the Interaction among Sea Anemones Neurotoxins and Kv1.3 Channel

    OpenAIRE

    2014-01-01

    Sea anemone neurotoxins are peptides that interact with Na+ and K+ channels, resulting in specific alterations on their functions. Some of these neurotoxins (1ROO, 1BGK, 2K9E, 1BEI) are important for the treatment of about 80 autoimmune disorders because of their specificity for Kv1.3 channel. The aim of this study was to identify the common residues among these neurotoxins by computational methods, and establish whether there is a pattern useful for the future generation of a treatment for a...

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  10. Recommended Mass Spectrometry-Based Strategies to Identify Botulinum Neurotoxin-Containing Samples

    Directory of Open Access Journals (Sweden)

    Suzanne R. Kalb

    2015-05-01

    Full Text Available 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.

  11. Molecular Methods for Identification of Clostridium tetani by Targeting Neurotoxin.

    Science.gov (United States)

    Nagoba, Basavraj; Dharne, Mahesh; Gohil, Kushal N

    2017-01-01

    Tetanus is a potentially fatal muscle spasm disease. It is an important public health problem, especially in rural/tribal areas of developing countries. Tetanus toxin, a neurotoxin (tetanospasmin ), is the most important virulence factor that plays a key role in the pathogenicity of tetanus . Confirmation of virulence by confirming the production of tetanospasmin by infecting species forms the most important part in the diagnosis of tetanus . Various molecular methods have been devised for confirmation of diagnosis by targeting different genes. The most common molecular methods are tetanospasmin producing (TetX) gene-targeted methods using TetX-specific primers. Here, we describe various molecular methods targeting TetX gene such as polymerase chain reaction, pulsed-field gel electrophoresis, Southern blotting, loop-mediated isothermal amplification assay, etc. to confirm the virulence of Cl. tetani.

  12. Structural Analysis of Botulinum Neurotoxin Type G Receptor Binding

    Energy Technology Data Exchange (ETDEWEB)

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

    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.

  13. Botulinum Neurotoxin for Pain Management: Insights from Animal Models

    Directory of Open Access Journals (Sweden)

    Siro Luvisetto

    2010-12-01

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  15. Salsolinol, a catechol neurotoxin, induces oxidative modification of cytochrome c

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    Jung Hoon Kang

    2013-02-01

    Full Text Available Methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (salsolinol,an endogenous neurotoxin, is known to perform a role in thepathogenesis of Parkinson’s disease (PD. In this study, weevaluated oxidative modification of cytochrome c occurring afterincubation with salsolinol. When cytochrome c was incubatedwith salsolinol, protein aggregation increased in a dosedependentmanner. The formation of carbonyl compounds andthe release of iron were obtained in salsolinol- treated cytochromec. Salsolinol also led to the release of iron fromcytochrome c. Reactive oxygen species (ROS scavengers andiron specific chelator inhibited the salsolinol-mediated cytochromec modification and carbonyl compound formation. It issuggested that oxidative damage of cytochrome c by salsolinolmight induce the increase of iron content in cells, subsequentlyleading to the deleterious condition which was observed. Thismechanism may, in part, provide an explanation for thedeterioration of organs under neurodegenerative disorders suchas PD. [BMB Reports 2013; 46(2: 119-123

  16. Genetic Diversity Among Botulinum Neurotoxin Producing Clostridial Strains

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Yağmur Derman

    2016-09-01

    Full Text Available 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.

  19. The primary structure of a short neurotoxin homologue from Dendroaspis polylepis polylepis (Black mamba venom

    Directory of Open Access Journals (Sweden)

    F. J. Joubert

    1984-03-01

    Full Text Available Toxin CM-7 was purified from black mamba venom by gel filtration on Sephadex G-50 followed by ion exchange chromotography on CM-cellulose. It contains 60 amino acids, including eight half-cystines. The complete amino acid sequence of toxin CM-7 has been elucidated. In toxin CM-7 the eleven structurally invariant amino acids of the neurotoxins and cardiotoxins are conserved, but it has only one of the five functionally invariant amino acids of the neurotoxins. The eight cysteine residues of toxin CM-7 are in the same locations as those in short neurotoxins of known structures and are presumed to be similarly cross-linked. The sequence of CM-7 is structurally homologous with the short neurotoxins, but it is less toxic.

  20. Characterization of the Ptychodiscus brevis Polyether Neurotoxin Binding Component in Excitable Membranes

    Science.gov (United States)

    1986-07-31

    Centruroides suffusus suffusus toxin II to neurotoxin receptoi Site 4 by the brevetoxins. Catterall and Gainer (16) suggested that the brevetoxins bind at a...binding of 1251 Centruroides suffusus suffusus toxin 11 on neurotoxin receptor site 4. Purification of both alpha- and beta-scorpion toxins is...guinguestraitus venom (site 3), or Centruroides sculpturatus venom (site 4) (Fig 13 ). Tritiated brevetoxin binding is slightly enhanced (5-10%) in the presence

  1. Spectroscopic investigation of calcium binding sites in the neurotoxin Vipoxin and its components-relation with the X-ray structure

    Science.gov (United States)

    Georgieva, Dessislava N.; Betzel, Christian; Aleksiev, Boris; Genov, Nicolay

    2000-12-01

    Vipoxin is a neurotoxin from the venom of Vipera ammodytes meridionalis, the most toxic snake in Europe. It is a unique complex of a toxic phospholipase A 2 (PLA 2) and a non-toxic PLA 2-like protein inhibitor (Inh) which probably evolved from the enzyme and reduces its activity and toxicity. The enzymatic activity of Vipoxin is Ca 2+-dependent and the interaction of this metal ion with the neurotoxic complex and its separated components was investigated using the fluorescent probe ANS. Vipoxin binds two calcium ions, one per each subunit. The X-ray model of the Ca 2+-free neurotoxin shows that the potential metal-binding sites require minor structural changes to bind calcium. The dissociation constants K Ca2+ of the calcium complexes of Vipoxin and its components, PLA 2 and Inh, were determined to be 16, 10 and 9 mM, respectively. The affinity for calcium of Vipoxin is reduced in comparison to those of PLA 2 and Inh. The X-ray model shows that the potential Ca 2+-binding sites in the two components are partially 'shielded' in the complex. The affinity of the neurotoxin to Sr 2+ and Ba 2+ is lower and the respective K Ca2+ are 20 and 30 mM. The saturation of Ca 2+-binding sites increased the melting point Tm of Vipoxin by 11°C and the activation energy for the thermal deactivation of the excited tryptophans Ea by 11 kJ mol -1. Ca 2+ is important not only for the enzymatic activity of Vipoxin but also for its thermostability.

  2. Enhanced neutralization potency of botulinum neurotoxin antibodies using a red blood cell-targeting fusion protein.

    Directory of Open Access Journals (Sweden)

    Sharad P Adekar

    Full Text Available Botulinum neurotoxin (BoNT potently inhibits cholinergic signaling at the neuromuscular junction. The ideal countermeasures for BoNT exposure are monoclonal antibodies or BoNT antisera, which form BoNT-containing immune complexes that are rapidly cleared from the general circulation. Clearance of opsonized toxins may involve complement receptor-mediated immunoadherence to red blood cells (RBC in primates or to platelets in rodents. Methods of enhancing immunoadherence of BoNT-specific antibodies may increase their potency in vivo. We designed a novel fusion protein (FP to link biotinylated molecules to glycophorin A (GPA on the RBC surface. The FP consists of an scFv specific for murine GPA fused to streptavidin. FP:mAb:BoNT complexes bound specifically to the RBC surface in vitro. In a mouse model of BoNT neutralization, the FP increased the potency of single and double antibody combinations in BoNT neutralization. A combination of two antibodies with the FP gave complete neutralization of 5,000 LD50 BoNT in mice. Neutralization in vivo was dependent on biotinylation of both antibodies and correlated with a reduction of plasma BoNT levels. In a post-exposure model of intoxication, FP:mAb complexes gave complete protection from a lethal BoNT/A1 dose when administered within 2 hours of toxin exposure. In a pre-exposure prophylaxis model, mice were fully protected for 72 hours following administration of the FP:mAb complex. These results demonstrate that RBC-targeted immunoadherence through the FP is a potent enhancer of BoNT neutralization by antibodies in vivo.

  3. Pentavalent replicon vaccines against botulinum neurotoxins and tetanus toxin using DNA-based Semliki Forest virus replicon vectors

    OpenAIRE

    2014-01-01

    The clostridial neurotoxin (CNT) family includes botulinum neurotoxin (BoNT), serotypes A, B, E, and F of which can cause human botulism, and tetanus neurotoxin (TeNT), which is the causative agent of tetanus. This suggests that the greatest need is for a multivalent or multiagent vaccine that provides protection against all 5 agents. In this study, we investigated the feasibility of generating several pentavalent replicon vaccines that protected mice against BoNTs and TeNT. First, we evaluat...

  4. A Monoclonal Antibody Based Capture ELISA for Botulinum Neurotoxin Serotype B: Toxin Detection in Food

    Directory of Open Access Journals (Sweden)

    Larry H. Stanker

    2013-11-01

    Full Text Available 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.

  5. Mode of VAMP Substrate Recognition and Inhibition of Clostridium botulinum Neurotoxin F

    Energy Technology Data Exchange (ETDEWEB)

    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.

  6. Mode of VAMP substrate recognition and inhibition of Clostridium botulinum neurotoxin F.

    Science.gov (United States)

    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.

  7. Probiotic Microorganisms Inhibit Epithelial Cell Internalization of Botulinum Neurotoxin Serotype A

    Directory of Open Access Journals (Sweden)

    Tina I. Lam

    2016-12-01

    Full Text Available Botulinum neurotoxins (BoNTs are some of the most poisonous natural toxins known to man and are threats to public health and safety. Previous work from our laboratory showed that both BoNT serotype A complex and holotoxin can bind and transit through the intestinal epithelia to disseminate in the blood. The timing of BoNT/A toxin internalization was shown to be comparable in both the Caco-2 in vitro cell culture and in the oral mouse intoxication models. Probiotic microorganisms have been extensively studied for their beneficial effects in not only maintaining the normal gut mucosa but also protection from allergens, pathogens, and toxins. In this study, we evaluate whether probiotic microorganisms will block BoNT/A uptake in the in vitro cell culture system using Caco-2 cells. Several probiotics tested (Saccharomyces boulardii, Lactobacillus acidophilus, Lactobacillus rhamnosus LGG, and Lactobacillus reuteri blocked BoNT/A uptake in a dose-dependent manner whereas a non-probiotic strain of Escherichia coli did not. We also showed that inhibition of BoNT/A uptake was not due to the degradation of BoNT/A nor by sequestration of toxin via binding to probiotics. These results show for the first time that probiotic treatment can inhibit BoNT/A binding and internalization in vitro and may lead to the development of new therapies.

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

    Directory of Open Access Journals (Sweden)

    Luisa W. Cheng

    2015-11-01

    Full Text Available 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.

  9. Molecular Evolution of Antibody Cross-Reactivity for Two Subtypes of Type a Botulinum Neurotoxin

    Energy Technology Data Exchange (ETDEWEB)

    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.

  10. Plastic enzyme-linked immunosorbent assays (ELISA)-on-a-chip biosensor for botulinum neurotoxin A.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  12. Tetanus neurotoxin-induced epilepsy in mouse visual cortex.

    Science.gov (United States)

    Mainardi, Marco; Pietrasanta, Marta; Vannini, Eleonora; Rossetto, Ornella; Caleo, Matteo

    2012-07-01

    Tetanus neurotoxin (TeNT) is a metalloprotease that cleaves the synaptic protein VAMP/synaptobrevin, leading to focal epilepsy. Although this model is widely used in rats, the time course and spatial specificity of TeNT proteolytic action have not been precisely defined. Here we have studied the biochemical, electrographic, and anatomic characteristics of TeNT-induced epilepsy in mouse visual cortex (V1). We found that VAMP cleavage peaked at 10 days, was reduced at 21 days, and completely extinguished 45 days following TeNT delivery. VAMP proteolysis was restricted to the injected V1 and ipsilateral thalamus, whereas it was undetectable in other cortical areas. Electrographic epileptiform activity was evident both during and after the time window of TeNT effects, indicating development of chronic epilepsy. Anatomic analyses found no evidence for long-term tissue damage, such as neuronal loss or microglia activation. These data show that TeNT reliably induces nonlesional epilepsy in mouse cortex. Due to the excellent physiologic knowledge of the visual cortex and the availability of mouse transgenic strains, this model will be useful for examining the network and cellular alterations underlying hyperexcitability within an epileptic focus.

  13. Complexity

    CERN Document Server

    Gershenson, Carlos

    2011-01-01

    The term complexity derives etymologically from the Latin plexus, which means interwoven. Intuitively, this implies that something complex is composed by elements that are difficult to separate. This difficulty arises from the relevant interactions that take place between components. This lack of separability is at odds with the classical scientific method - which has been used since the times of Galileo, Newton, Descartes, and Laplace - and has also influenced philosophy and engineering. In recent decades, the scientific study of complexity and complex systems has proposed a paradigm shift in science and philosophy, proposing novel methods that take into account relevant interactions.

  14. Structural and mutational analyses of the receptor binding domain of botulinum D/C mosaic neurotoxin: Insight into the ganglioside binding mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Nuemket, Nipawan [Graduate School of Life Sciences, Hokkaido University, Sapporo 060-0810 (Japan); Tanaka, Yoshikazu [Creative Research Institution ' Sousei,' Hokkaido University, Sapporo 001-0021 (Japan); Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810 (Japan); Tsukamoto, Kentaro; Tsuji, Takao [Department of Microbiology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192 (Japan); Nakamura, Keiji; Kozaki, Shunji [Department of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka 598-8531 (Japan); Yao, Min [Graduate School of Life Sciences, Hokkaido University, Sapporo 060-0810 (Japan); Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810 (Japan); Tanaka, Isao, E-mail: tanaka@castor.sci.hokudai.ac.jp [Graduate School of Life Sciences, Hokkaido University, Sapporo 060-0810 (Japan); Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810 (Japan)

    2011-07-29

    Highlights: {yields} We determined the crystal structure of the receptor binding domain of BoNT in complex with 3'-sialyllactose. {yields} An electron density derived from the 3'-sialyllactose was confirmed at the cleft in the C-terminal subdomain. {yields} Alanine site-directed mutagenesis showed that GBS and GBL are important for ganglioside binding. {yields} A cell binding mechanism, which involves cooperative contribution of two sites, was proposed. -- Abstract: Clostridium botulinum type D strain OFD05, which produces the D/C mosaic neurotoxin, was isolated from cattle killed by the recent botulism outbreak in Japan. The D/C mosaic neurotoxin is the most toxic of the botulinum neurotoxins (BoNT) characterized to date. Here, we determined the crystal structure of the receptor binding domain of BoNT from strain OFD05 in complex with 3'-sialyllactose at a resolution of 3.0 A. In the structure, an electron density derived from the 3'-sialyllactose was confirmed at the cleft in the C-terminal subdomain. Alanine site-directed mutagenesis showed the significant contribution of the residues surrounding the cleft to ganglioside recognition. In addition, a loop adjoining the cleft also plays an important role in ganglioside recognition. In contrast, little effect was observed when the residues located around the surface previously identified as the protein receptor binding site in other BoNTs were substituted. The results of cell binding analysis of the mutants were significantly correlated with the ganglioside binding properties. Based on these observations, a cell binding mechanism of BoNT from strain OFD05 is proposed, which involves cooperative contribution of two ganglioside binding sites.

  15. Synergistic capture of Clostridium botulinum Type A neurotoxin by scFv antibodies to novel epitopes

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Science.gov (United States)

    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.

  17. Complex

    African Journals Online (AJOL)

    CLEMENT O BEWAJI

    Schiff bases and their complex compounds have been studied for their .... establishing coordination of the N–(2 – hydroxybenzyl) - L - α - valine Schiff base ..... (1967); “Spectrophotometric Identification of Organic Compounds”, Willey, New.

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

    Science.gov (United States)

    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.

  19. A camelid single-domain antibody neutralizes botulinum neurotoxin A by blocking host receptor binding

    Energy Technology Data Exchange (ETDEWEB)

    Yao, Guorui; Lam, Kwok-ho; Weisemann, Jasmin; Peng, Lisheng; Krez, Nadja; Perry, Kay; Shoemaker, Charles B.; Dong, Min; Rummel, Andreas; Jin, Rongsheng (BCH); (Cornell); (Tufts CTSI); (UCI); (MHH)

    2017-08-07

    Antibody treatment is currently the only available countermeasure for botulism, a fatal illness caused by flaccid paralysis of muscles due to botulinum neurotoxin (BoNT) intoxication. Among the seven major serotypes of BoNT/A-G, BoNT/A poses the most serious threat to humans because of its high potency and long duration of action. Prior to entering neurons and blocking neurotransmitter release, BoNT/A recognizes motoneurons via a dual-receptor binding process in which it engages both the neuron surface polysialoganglioside (PSG) and synaptic vesicle glycoprotein 2 (SV2). Previously, we identified a potent neutralizing antitoxin against BoNT/A1 termed ciA-C2, derived from a camelid heavy-chain-only antibody (VHH). In this study, we demonstrate that ciA-C2 prevents BoNT/A1 intoxication by inhibiting its binding to neuronal receptor SV2. Furthermore, we determined the crystal structure of ciA-C2 in complex with the receptor-binding domain of BoNT/A1 (HCA1) at 1.68 Å resolution. The structure revealed that ciA-C2 partially occupies the SV2-binding site on HCA1, causing direct interference of HCA1 interaction with both the N-glycan and peptide-moiety of SV2. Interestingly, this neutralization mechanism is similar to that of a monoclonal antibody in clinical trials, despite that ciA-C2 is more than 10-times smaller. Taken together, these results enlighten our understanding of BoNT/A1 interactions with its neuronal receptor, and further demonstrate that inhibiting toxin binding to the host receptor is an efficient countermeasure strategy.

  20. Therapeutic attenuation of mitochondrial dysfunction and oxidative stress in neurotoxin models of Parkinson's disease

    OpenAIRE

    Stack, Edward C.; Ferro, Joellyn L.; Kim, Jinho; Del Signore, Steven J.; Goodrich, Sarah; Matson, Samantha; Hunt, Bonnie B.; Cormier, Kerry; Smith, Karen; Matson, Wayne R.; Ryu, Hoon; Ferrante, Robert J.

    2008-01-01

    Therapeutic attenuation of mitochondrial dysfunction and oxidative stress in neurotoxin models of Parkinson?s disease correspondence: Corresponding author. GRECC Unit 182B, Bedford VA Medical Center, 200 Springs Road, Bedford, MA 01730, USA. Tel.: +1 781 687 2908; fax: +1 781 687 3515. (Ferrante, Robert J.) (Ferrante, Robert J.) Department of Neurology, Boston University School of Medicine - Boston--> , MA 02118--> ...

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

    Science.gov (United States)

    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 ...

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

    Science.gov (United States)

    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 ...

  3. [A crustacean-specific neurotoxin from the venom of the black widow spider Latrodectus mactans tredecimguttatus].

    Science.gov (United States)

    Krasnoperov, V G; Shamotienko, O G; Grishin, E V

    1990-11-01

    A method of the isolation of a crustacea-specific neurotoxin from the venom of the Latrodectus mactans tredecimguttatus spider by means of ion exchange chromatography on Mono Q and Mono S columns and hydrophobic chromatography on Phenyl-Superose column has been developed. LD50 of the toxin has been elucidated.

  4. [Isolation and properties of insect-specific neurotoxins from venoms of the spider Lactodectus mactans tredecimguttatus].

    Science.gov (United States)

    Krasnoperov, V G; Shamotienko, O G; Grishin, E V

    1990-08-01

    A method has been developed for isolating five insect-specific neurotoxins and alpha-latrotoxin from venom of the Latrodectus mactans tredecimguttatus spider by means of ion exchange chromatography on Mono Q and Mono S columns and chromatography on hydroxylapatite column. LD 50 of all the toxins are determined.

  5. Structural basis for recognition of synaptic vesicle protein 2C by botulinum neurotoxin A

    NARCIS (Netherlands)

    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 ag

  6. A comparative study on three analytical methods for the determination of the neurotoxin BMAA in cyanobacteria

    NARCIS (Netherlands)

    Faassen, E.J.; Gillissen, F.; Lürling, M.

    2012-01-01

    The cyanobacterial neurotoxin β-N-methylamino-L-alanine (BMAA) has been considered a serious health threat because of its putative role in multiple neurodegenerative diseases. First reports on BMAA concentrations in cyanobacteria were alarming: nearly all cyanobacteria were assumed to contain high B

  7. A comparative study on three analytical methods for the determination of the neurotoxin BMAA in cyanobacteria

    NARCIS (Netherlands)

    Faassen, E.J.; Gillissen, F.; Lurling, M.

    2012-01-01

    The cyanobacterial neurotoxin ß-N-methylamino-L-alanine (BMAA) has been considered a serious health threat because of its putative role in multiple neurodegenerative diseases. First reports on BMAA concentrations in cyanobacteria were alarming: nearly all cyanobacteria were assumed to contain high B

  8. Actions of Ethanol on Voltage-Sensitive Sodium Channels: Effects on Neurotoxin Binding

    Science.gov (United States)

    1987-01-01

    sodium inhibitory effect of ethanol on channel - mediated sodium influx channels ...Exprnmantal Trherpeutics Ped in I.SA. Actions of Ethanol on Voltage-Sensitive Sodium Channels : Effects on Neurotoxin Binding1 MICHAEL J. MULLIN 2 and... sodium channels . This indirect allosteric mechanism for inhibition of [H]BTX-B binding. effect orethanol was concentration-dependent and was

  9. Innovative mode of action based in vitro assays for detection of marine neurotoxins

    NARCIS (Netherlands)

    Nicolas, J.A.Y.

    2015-01-01

    Innovative mode of action based in vitro assays for detection of marine neurotoxins J. Nicolas, P.J.M. Hendriksen, T.F.H. Bovee, I.M.C.M. Rietjens Marine biotoxins are naturally occurring compounds produced by particular phytoplankton species. These toxins often accumulate in seafood and thereby

  10. Trans generational effects of the neurotoxin BMAA on the aquatic grazer Daphnia magna

    NARCIS (Netherlands)

    Faassen, Elisabeth J.; García-Altares, María; Mendes e Mello, Mariana; Lürling, Miquel

    2015-01-01

    Abstract 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

  11. Trans generational effects of the neurotoxin BMAA on the aquatic grazer Daphnia magna

    NARCIS (Netherlands)

    Faassen, E.J.; Garcia-Altares, M.; Mendes e Mello, M.; Lurling, M.F.L.L.W.

    2015-01-01

    ß-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

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

    Science.gov (United States)

    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 (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.

  13. Independent evolution of neurotoxin and flagellar genetic loci in proteolytic Clostridium botulinum

    Directory of Open Access Journals (Sweden)

    Twine Susan M

    2009-03-01

    Full Text Available Abstract Background Proteolytic Clostridium botulinum is the causative agent of botulism, a severe neuroparalytic illness. Given the severity of botulism, surprisingly little is known of the population structure, biology, phylogeny or evolution of C. botulinum. The recent determination of the genome sequence of C. botulinum has allowed comparative genomic indexing using a DNA microarray. Results Whole genome microarray analysis revealed that 63% of the coding sequences (CDSs present in reference strain ATCC 3502 were common to all 61 widely-representative strains of proteolytic C. botulinum and the closely related C. sporogenes tested. This indicates a relatively stable genome. There was, however, evidence for recombination and genetic exchange, in particular within the neurotoxin gene and cluster (including transfer of neurotoxin genes to C. sporogenes, and the flagellar glycosylation island (FGI. These two loci appear to have evolved independently from each other, and from the remainder of the genetic complement. A number of strains were atypical; for example, while 10 out of 14 strains that formed type A1 toxin gave almost identical profiles in whole genome, neurotoxin cluster and FGI analyses, the other four strains showed divergent properties. Furthermore, a new neurotoxin sub-type (A5 has been discovered in strains from heroin-associated wound botulism cases. For the first time, differences in glycosylation profiles of the flagella could be linked to differences in the gene content of the FGI. Conclusion Proteolytic C. botulinum has a stable genome backbone containing specific regions of genetic heterogeneity. These include the neurotoxin gene cluster and the FGI, each having evolved independently of each other and the remainder of the genetic complement. Analysis of these genetic components provides a high degree of discrimination of strains of proteolytic C. botulinum, and is suitable for clinical and forensic investigations of botulism

  14. Allosteric modulation of neurotoxin binding to voltage-sensitive sodium channels by Ptychodiscus brevis toxin 2.

    Science.gov (United States)

    Sharkey, R G; Jover, E; Couraud, F; Baden, D G; Catterall, W A

    1987-03-01

    The effects of Ptychodiscus brevis toxin 2 (PbTx-2) on the binding of neurotoxins at four different neurotoxin receptor sites on voltage-sensitive sodium channels in rat brain synaptosomes were examined. Binding of saxitoxin at neurotoxin receptor site 1 and Leiurus quinquestriatus alpha-scorpion toxin (LqTx) at neurotoxin receptor site 3 was unaffected. PbTx-2 enhanced binding of batrachotoxinin A 20-alpha-benzoate (BTX-B) to neurotoxin receptor site 2 and Centruroides suffusus suffusus beta-scorpion toxin (CsTx II) to site 4 on sodium channels. These results support the proposal that PbTx-2 and related toxins act at a new receptor site (site 5) that has not been previously analyzed in binding experiments. Half-maximal effects of PbTx-2 were observed in the range of 20-50 nM PbTx-2. The enhancement of BTX-B binding was reduced by depolarization. Saturating concentrations of PbTx-2 reduced KD values for binding of BTX-B and CsTx-II 2.9-fold and 2.6-fold, respectively. The effects of PbTx-2 and LqTx in enhancing BTX-B binding were synergistic. A model involving both preferential binding of BTX-B, PbTx-2, LqTx, and CsTx II to active states of sodium channels and allosteric interactions among the four receptor sites at which these toxins act accommodates these and previous results.

  15. Glycosylated SV2 and Gangliosides as Dual Receptors for Botulinum Neurotoxin Serotype F

    Energy Technology Data Exchange (ETDEWEB)

    Fu, Zhuji; Chen, Chen; Barbieri, Joseph T.; Kim, Jung-Ja P.; Baldwin, Michael R.; (MCW)

    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.

  16. Two-component systems are involved in the regulation of botulinum neurotoxin synthesis in Clostridium botulinum type A strain Hall.

    Science.gov (United States)

    Connan, Chloé; Brüggemann, Holger; Brueggemann, Holger; Mazuet, Christelle; Raffestin, Stéphanie; Cayet, Nadège; Popoff, Michel R

    2012-01-01

    Clostridium botulinum synthesizes a potent neurotoxin (BoNT) which associates with non-toxic proteins (ANTPs) to form complexes of various sizes. The bont and antp genes are clustered in two operons. In C. botulinum type A, bont/A and antp genes are expressed during the end of the exponential growth phase and the beginning of the stationary phase under the control of an alternative sigma factor encoded by botR/A, which is located between the two operons. In the genome of C. botulinum type A strain Hall, 30 gene pairs predicted to encode two-component systems (TCSs) and 9 orphan regulatory genes have been identified. Therefore, 34 Hall isogenic antisense strains on predicted regulatory genes (29 TCSs and 5 orphan regulatory genes) have been obtained by a mRNA antisense procedure. Two TCS isogenic antisense strains showed more rapid growth kinetics and reduced BoNT/A production than the control strain, as well as increased bacterial lysis and impairment of the bacterial cell wall structure. Three other TCS isogenic antisense strains induced a low level of BoNT/A and ANTP production. Interestingly, reduced expression of bont/A and antp genes was shown to be independent of botR/A. These results indicate that BoNT/A synthesis is under the control of a complex network of regulation including directly at least three TCSs.

  17. Two-component systems are involved in the regulation of botulinum neurotoxin synthesis in Clostridium botulinum type A strain Hall.

    Directory of Open Access Journals (Sweden)

    Chloé Connan

    Full Text Available Clostridium botulinum synthesizes a potent neurotoxin (BoNT which associates with non-toxic proteins (ANTPs to form complexes of various sizes. The bont and antp genes are clustered in two operons. In C. botulinum type A, bont/A and antp genes are expressed during the end of the exponential growth phase and the beginning of the stationary phase under the control of an alternative sigma factor encoded by botR/A, which is located between the two operons. In the genome of C. botulinum type A strain Hall, 30 gene pairs predicted to encode two-component systems (TCSs and 9 orphan regulatory genes have been identified. Therefore, 34 Hall isogenic antisense strains on predicted regulatory genes (29 TCSs and 5 orphan regulatory genes have been obtained by a mRNA antisense procedure. Two TCS isogenic antisense strains showed more rapid growth kinetics and reduced BoNT/A production than the control strain, as well as increased bacterial lysis and impairment of the bacterial cell wall structure. Three other TCS isogenic antisense strains induced a low level of BoNT/A and ANTP production. Interestingly, reduced expression of bont/A and antp genes was shown to be independent of botR/A. These results indicate that BoNT/A synthesis is under the control of a complex network of regulation including directly at least three TCSs.

  18. Computer-aided lead optimization: improved small-molecule inhibitor of the zinc endopeptidase of botulinum neurotoxin serotype A.

    Directory of Open Access Journals (Sweden)

    Jing Tang

    Full Text Available Optimization of a serotype-selective, small-molecule inhibitor of botulinum neurotoxin serotype A (BoNTA endopeptidase is a formidable challenge because the enzyme-substrate interface is unusually large and the endopeptidase itself is a large, zinc-binding protein with a complex fold that is difficult to simulate computationally. We conducted multiple molecular dynamics simulations of the endopeptidase in complex with a previously described inhibitor (K(i (app of 7+/-2.4 microM using the cationic dummy atom approach. Based on our computational results, we hypothesized that introducing a hydroxyl group to the inhibitor could improve its potency. Synthesis and testing of the hydroxyl-containing analog as a BoNTA endopeptidase inhibitor showed a twofold improvement in inhibitory potency (K(i (app of 3.8+/-0.8 microM with a relatively small increase in molecular weight (16 Da. The results offer an improved template for further optimization of BoNTA endopeptidase inhibitors and demonstrate the effectiveness of the cationic dummy atom approach in the design and optimization of zinc protease inhibitors.

  19. Two-component signal transduction system CBO0787/CBO0786 represses transcription from botulinum neurotoxin promoters in Clostridium botulinum ATCC 3502.

    Science.gov (United States)

    Zhang, Zhen; Korkeala, Hannu; Dahlsten, Elias; Sahala, Elina; Heap, John T; Minton, Nigel P; Lindström, Miia

    2013-03-01

    Blocking neurotransmission, botulinum neurotoxin is the most poisonous biological substance known to mankind. Despite its infamy as the scourge of the food industry, the neurotoxin is increasingly used as a pharmaceutical to treat an expanding range of muscle disorders. Whilst neurotoxin expression by the spore-forming bacterium Clostridium botulinum appears tightly regulated, to date only positive regulatory elements, such as the alternative sigma factor BotR, have been implicated in this control. The identification of negative regulators has proven to be elusive. Here, we show that the two-component signal transduction system CBO0787/CBO0786 negatively regulates botulinum neurotoxin expression. Single insertional inactivation of cbo0787 encoding a sensor histidine kinase, or of cbo0786 encoding a response regulator, resulted in significantly elevated neurotoxin gene expression levels and increased neurotoxin production. Recombinant CBO0786 regulator was shown to bind to the conserved -10 site of the core promoters of the ha and ntnh-botA operons, which encode the toxin structural and accessory proteins. Increasing concentration of CBO0786 inhibited BotR-directed transcription from the ha and ntnh-botA promoters, demonstrating direct transcriptional repression of the ha and ntnh-botA operons by CBO0786. Thus, we propose that CBO0786 represses neurotoxin gene expression by blocking BotR-directed transcription from the neurotoxin promoters. This is the first evidence of a negative regulator controlling botulinum neurotoxin production. Understanding the neurotoxin regulatory mechanisms is a major target of the food and pharmaceutical industries alike.

  20. Differentiating Botulinum Neurotoxin-Producing Clostridia with a Simple, Multiplex PCR Assay.

    Science.gov (United States)

    Williamson, Charles H D; Vazquez, Adam J; Hill, Karen; Smith, Theresa J; Nottingham, Roxanne; Stone, Nathan E; Sobek, Colin J; Cocking, Jill H; Fernández, Rafael A; Caballero, Patricia A; Leiser, Owen P; Keim, Paul; Sahl, Jason W

    2017-09-15

    Diverse members of the genus Clostridium produce botulinum neurotoxins (BoNTs), which cause a flaccid paralysis known as botulism. While multiple species of clostridia produce BoNTs, the majority of human botulism cases have been attributed to Clostridium botulinum groups I and II. Recent comparative genomic studies have demonstrated the genomic diversity within these BoNT-producing species. This report introduces a multiplex PCR assay for differentiating members of C. botulinum group I, C. sporogenes, and two major subgroups within C. botulinum group II. Coding region sequences unique to each of the four species/subgroups were identified by in silico analyses of thousands of genome assemblies, and PCR primers were designed to amplify each marker. The resulting multiplex PCR assay correctly assigned 41 tested isolates to the appropriate species or subgroup. A separate PCR assay to determine the presence of the ntnh gene (a gene associated with the botulinum neurotoxin gene cluster) was developed and validated. The ntnh gene PCR assay provides information about the presence or absence of the botulinum neurotoxin gene cluster and the type of gene cluster present (ha positive [ha(+)] or orfX(+)). The increased availability of whole-genome sequence data and comparative genomic tools enabled the design of these assays, which provide valuable information for characterizing BoNT-producing clostridia. The PCR assays are rapid, inexpensive tests that can be applied to a variety of sample types to assign isolates to species/subgroups and to detect clostridia with botulinum neurotoxin gene (bont) clusters.IMPORTANCE Diverse clostridia produce the botulinum neurotoxin, one of the most potent known neurotoxins. In this study, a multiplex PCR assay was developed to differentiate clostridia that are most commonly isolated in connection with human botulism cases: C. botulinum group I, C. sporogenes, and two major subgroups within C. botulinum group II. Since BoNT-producing and

  1. Cobra ( Naja spp. ) nicotinic acetylcholine receptor exhibits resistance to Erabu sea snake ( Laticauda semifasciata) short-chain alpha-neurotoxin.

    Science.gov (United States)

    Takacs, Zoltan; Wilhelmsen, Kirk C; Sorota, Steve

    2004-05-01

    Snake alpha-neutotoxins of Elapidae venoms are grouped into two structural classes, short-chain and long-chain alpha-neutotoxins. While these two classes share many chemical and biological characteristics, there are also distinct dissimilarities between them, including their binding site on the nicotinic acetylcholine receptor (nAChR), specificity among species of Chordata, and the associated pharmacological effects. In the present study we test the hypothesis that structural motifs that evolved to confer natural resistance against conspecific long-chain alpha-neurotoxins in Elapidae snakes also interfere with the biological action of short-chain alpha-neurotoxins. We expressed functional nAChRs that contains segments or single residues of the Elapidae nAChR ligand binding domain and tested the effect of short-chain alpha-neurotoxin erabutoxin-a (ETX-a) from the Erabu sea snake Laticauda semifasciata on the acetylcholine-induced currents as measured by two-microelectrode voltage clamp. Our results show that the Elapidae nAChR alpha subunit segment T(154)-L(208) ligand binding domain has an inhibitory effect on the pharmacological action of ETX-a. This effect is primarily attributed to the presence of glycosylation at position N(189). If the glycosylation is removed from the T(154)-L(208) segment, the nAChR will be inhibited, however, to a lesser extent than seen in the mouse. This effect correlates with the variations in alpha-neurotoxin sensitivity of different species and, importantly, reflects the evolutionary conservation of the binding site on the nAChR polypeptide backbone per se. Phylogenetic analysis of alpha-neurotoxin resistance suggests that alpha-neurotoxin-resistant nAChR evolved first, which permitted the evolution of snake venom alpha-neurotoxins. A model describing alpha-neurotoxin resistance in Elapidae snakes is presented.

  2. Biochemistry of snake venom neurotoxins and their application to the study of synapse. [Neurotoxins isolated from venom of the Formosan banded krait

    Energy Technology Data Exchange (ETDEWEB)

    Hanley, M.R.

    1978-11-01

    The crude venom of the Formosan banded krait, Bungarus multicinctus, was separated into eleven lethal protein fractions. Nine fractions were purified to final homogeneous toxins, designated ..cap alpha..-bungarotoxin, ..beta..-bungarotoxin, and toxins 7, 8, 9A, 11, 12, 13, and 14. Three of the toxins, ..cap alpha..-bungarotoxin, 7, and 8, were identified as post-synaptic curarimimetic neurotoxins. The remaining toxins were identified as pre-synaptic neurotoxins. ..cap alpha..-Bungarotoxin, toxin 7, and toxin 8 are all highly stable basic polypeptides of approx. 8000 daltons molecular weight. The pre-synaptic toxins fell into two structural groups: toxin 9A and 14 which were single basic chains of approx. 14,000 daltons, and ..beta..-bungarotoxin, and toxins 11 thru 13 which were composed of two chains of approx. 8000 and approx. 13,000 daltons covalently linked by disulfides. All the pre-synaptic neurotoxins were shown to have intrinsic calcium-dependent phospholipase A activities. Under certain conditions, intact synaptic membranes were hydrolyzed more rapidly than protein-free extracted synaptic-lipid liposomes which, in turn, were hydrolyzed more rapidly than any other tested liposomes. It was speculated that cell-surface arrays of phosphatidyl serine/glycolipids created high affinity target sites for ..beta..-bungarotoxin. Single-chain toxins were found to be qualitatively different from the two-chain toxins in their ability to block the functioning of acetylcholine receptors, and were quantitatively different in their enzymatic and membrane disruptive activities. ..beta..-Bungarotoxin was shown to be an extremely potent neuronal lesioning agent. There was no apparent selectivity for cholinergic over non-cholinergic neurons, nor for nerve terminals over cell bodies. It was suggested that ..beta..-bungarotoxin can be considered a useful new histological tool, which may exhibit some regional selectivity.

  3. Holotoxin Activity of Botulinum Neurotoxin Subtype A4 Originating from a Nontoxigenic Clostridium botulinum Expression System.

    Science.gov (United States)

    Bradshaw, Marite; Tepp, William H; Whitemarsh, Regina C M; Pellett, Sabine; Johnson, Eric A

    2014-12-01

    Clostridium botulinum subtype A4 neurotoxin (BoNT/A4) is naturally expressed in the dual-toxin-producing C. botulinum strain 657Ba at 100× lower titers than BoNT/B. In this study, we describe purification of recombinant BoNT/A4 (rBoNT/A4) expressed in a nonsporulating and nontoxigenic C. botulinum expression host strain. The rBoNT/A4 copurified with nontoxic toxin complex components provided in trans by the expression host and was proteolytically cleaved to the active dichain form. Activity of the recombinant BoNT/A4 in mice and in human neuronal cells was about 1,000-fold lower than that of BoNT/A1, and the recombinant BoNT/A4 was effectively neutralized by botulism heptavalent antitoxin. A previous report using recombinant truncated BoNT/A4 light chain (LC) expressed in Escherichia coli has indicated reduced stability and activity of BoNT/A4 LC compared to BoNT/A1 LC, which was surmounted by introduction of a single-amino-acid substitution, I264R. In order to determine whether this mutation would also affect the holotoxin activity of BoNT/A4, a recombinant full-length BoNT/A4 carrying this mutation as well as a second mutation predicted to increase solubility (L260F) was produced in the clostridial expression system. Comparative analyses of the in vitro, cellular, and in vivo activities of rBoNT/A4 and rBoNT/A4-L260F I264R showed 1,000-fold-lower activity than BoNT/A1 in both the mutated and nonmutated BoNT/A4. This indicates that these mutations do not alter the activity of BoNT/A4 holotoxin. In summary, a recombinant BoNT from a dual-toxin-producing strain was expressed and purified in an endogenous clostridial expression system, allowing analysis of this toxin.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  6. [The chromosomal genes for black widow spider neurotoxins do not contain introns].

    Science.gov (United States)

    Danilevich, V N; Grishin, E V

    2000-12-01

    The overlapping fragments of the chromosomal DNA from black widow spider Latrodectus mactans carrying genes for high-molecular-mass protein neurotoxins, alpha- and delta-latroinsectotoxins (alpha-LIT and delta-LIT) and alpha-latrotoxin (alpha-LTX), were PCR-amplified and cloned. Restriction analysis of the PCR products showed that the distribution and sizes of the restriction fragments coincided with those deduced from the earlier sequencing of cDNAs of the corresponding genes. It thus followed that the alpha-LIT and delta-LIT genes are intronless. Along with our data on the structure of the alpha-latrocrustotoxin (alpha-LCT), this implies that the lack of introns is a common feature of the black widow spider genes encoding high molecular mass neurotoxins.

  7. The analgesic effect on neuropathic pain of retrogradely transported botulinum neurotoxin A involves Schwann cells and astrocytes.

    Directory of Open Access Journals (Sweden)

    Sara Marinelli

    Full Text Available 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.

  8. The Analgesic Effect on Neuropathic Pain of Retrogradely Transported botulinum Neurotoxin A Involves Schwann Cells and Astrocytes

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    OpenAIRE

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

    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 ...

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

    Science.gov (United States)

    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.

  12. Biomagnification of cycad neurotoxins in flying foxes: implications for ALS-PDC in Guam.

    Science.gov (United States)

    Banack, Sandra Anne; Cox, Paul Alan

    2003-08-12

    Beta-methylamino-L-alanine (BMAA) occurs in higher levels in museum specimens of the Guamanian flying fox than in the cycad seeds the flying foxes feed on, confirming the hypothesis that cycad neurotoxins are biomagnified within the Guam ecosystem. Consumption of a single flying fox may have resulted in an equivalent BMAA dose obtained from eating 174 to 1,014 kg of processed cycad flour. Traditional feasting on flying foxes may be related to the prevalence of neuropathologic disease in Guam.

  13. A Review of the Disruptive Potential of Botulinum Neurotoxins as Chemical Warfare Agents

    Science.gov (United States)

    2011-10-01

    dangerous chemical weapon (persistence in vivo, exceedingly high potency, ease of distribution and ease of production) also make it highly...caused by ingestion of botulinum neurotoxin (BoNT) was first described as “ sausage poisoning” in 1820 and attributed to a bacterium in 1897 [1]. We...intoxication of susceptible hosts invariably results in neuromuscular paralysis. The same characteristics that make the BoNTs the most lethal substances

  14. Evaluation of Lateral-Flow Clostridium botulinum Neurotoxin Detection Kits for Food Analysis

    Science.gov (United States)

    2005-02-08

    samples for each food type (n 3). No mixed results were observed. 3938 SHARMA ET AL. APPL. ENVIRON. MICROBIOL. histolyticum, Clostridium tetani ...of Lateral-Flow Clostridium botulinum Neurotoxin Detection Kits for Food Analysis Shashi K. Sharma,1* Brian S. Eblen,1 Robert L. Bull,2 Donald H. Burr...detecting Clostridium botulinum neuro- toxins (BoNTs) in an assortment of foods were evaluated. Toxin extraction and preparation methods for various liquid

  15. Neurotoxins and their binding areas on voltage-gated sodium channels

    Directory of Open Access Journals (Sweden)

    Marijke eStevens

    2011-11-01

    Full Text Available Voltage-gated Sodium Channels (VGSCs are large transmembrane proteins that conduct sodium ions across the membrane and by doing so they generate signals of communication between many kinds of tissues. They are responsible for the generation and propagation of action potentials in excitable cells, in close collaboration with other channels like potassium channels. Genetic defects in sodium channel genes therefore can cause a wide variety of diseases, generally called ‘channelopathies’.The first insights into the mechanism of action potentials and the involvement of sodium channels originated from Hodgkin and Huxley for which they were awarded the Nobel Prize in 1963. Until now, these concepts still form the basis for understanding the functioning of VGSCs. When VGSCs sense a sufficient change in membrane potential, they are activated and will generate a massive influx of sodium ions. Immediately after, channels will start inactivating and currents decrease. In the inactivated state channels stay refractory for any new stimulus and they must return to the closed state before being susceptible to any new depolarization. On the other hand, studies with neurotoxins like tetrodotoxin (TTX and saxitoxin (STX also contributed largely to our today’s understanding of the structure and function of ion channels and specifically of VGSCs. Moreover, neurotoxins acting on ion channels turned out to be valuable tools in the development of new drugs for the enormous range of diseases in which ion channels are involved. A recent example of a synthetic neurotoxin that made it to the market is ziconotide (Prialt®, Elan. The original peptide, -MVIIA, is derived from the cone snail Conus magus and now FDA/EMEA-approved for the management of severe chronic pain by blocking the N-type voltage-gated calcium channels in neurons.This review focuses on the current status of research on neurotoxins acting on VGSC, their contribution to further unravel the

  16. Structure of botulinum neurotoxin type D light chain at 1.65 A resolution: repercussions for VAMP-2 substrate specificity.

    Science.gov (United States)

    Arndt, Joseph W; Chai, Qing; Christian, Todd; Stevens, Raymond C

    2006-03-14

    The seven serotypes (A-G) of botulinum neurotoxins (BoNTs) function through their proteolytic cleavage of one of three proteins (SNAP-25, Syntaxin, and VAMP) that form the SNARE complex required for synaptic vesicle fusion. The different BoNTs have very specific protease recognition requirements, between 15 and 50 amino acids in length depending on the serotype. However, the structural details involved in substrate recognition remain largely unknown. Here is reported the 1.65 A resolution crystal structure of the catalytic domain of BoNT serotype D (BoNT/D-LC), providing insight into the protein-protein binding interaction and final proteolysis of VAMP-2. Structural analysis has identified a hydrophobic pocket potentially involved in substrate recognition of the P1' VAMP residue (Leu 60) and a second remote site for recognition of the V1 SNARE motif that is critical for activity. A structural comparison of BoNT/D-LC with BoNT/F-LC that also recognizes VAMP-2 one residue away from the BoNT/D-LC site provides additional molecular details about the unique serotype specific activities. In particular, BoNT/D prefers a hydrophobic interaction for the V1 motif of VAMP-2, while BoNT/F adopts a more hydrophilic strategy for recognition of the same V1 motif.

  17. Detection of a novel neurotoxic metabolite of Parkinson's disease-related neurotoxin, 1-benzyl-1,2,3,4- tetrahydroisoquinoline.

    Science.gov (United States)

    Kotake, Yaichiro; Sekiya, Yoko; Okuda, Katsuhiro; Ohta, Shigeru

    2014-01-01

    Naturally occurring low-molecular weight compounds with a chemical structure like that of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, such as 1-benzyl-1,2,3,4-tetrahydroisoquinoline(1BnTIQ), are candidates for the endogenous neurotoxins that cause Parkinson's disease (PD). 1BnTIQ is an endogenous amine in human CSF and increases in the CSF of patients with PD. It inhibits complex Iand elicits PD-like behavioral abnormalities in monkey and mouse. In this study, we searched metabolites of 1BnTIQ by rat liver S9 using liquid chromatography-tandem mass spectrometry, and identified a dehydrated metabolite, 1-benzyl-3,4-dihydroisoquinoline (1BnDIQ). 1BnDIQ was identified by corresponding mass spectra and precursor ion scans in authentic and complete enzyme samples. Multiple reaction monitoring analysis showed microsome-dependent 1BnDIQ production. We previously reported that 1BnDIQ is more toxic than 1BnTIQ in cytotoxicity study in SH-SY5Y neuroblastoma cells. In addition, 1BnTIQ is reported to pass through the blood-brain barrier of the rat brain, and 1BnDIQ is supposed to be more lipophilic than 1BnTIQ. 1BnDIQ may easily reach the brain, and it might contribute to PD-related neurotoxicity.

  18. The cyanobacterial neurotoxin β-N-methylamino-l-alanine (BMAA) induces neuronal and behavioral changes in honeybees.

    Science.gov (United States)

    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.

  19. Occurrence of Clostridium botulinum neurotoxin in chronic disease of dairy cows.

    Science.gov (United States)

    Seyboldt, Christian; Discher, Sabrina; Jordan, Eva; Neubauer, Heinrich; Jensen, Katharina Charlotte; Campe, Amely; Kreienbrock, Lothar; Scheu, Theresa; Wichern, Anika; Gundling, Frieder; DoDuc, Phuong; Fohler, Svenja; Abdulmawjood, Amir; Klein, Günter; Hoedemaker, Martina

    2015-06-12

    Botulism caused by neurotoxins of Clostridium (C.) botulinum is a rare, but serious life-threatening disease in humans and animals. Botulism in livestock is usually caused by the oral uptake of C. botulinum neurotoxins (BoNT) via contaminated feed and is characterized by flaccid paralysis. In the recent past a new syndrome caused by BoNT in dairy cattle was postulated. It was supposed that C. botulinum is able to colonize the lower intestine and may subsequently produce neurotoxin. The continuous resorption of small amounts of these BoNT may then provoke the so called syndrome of "chronic" or "visceral" botulism involving unspecific clinical symptoms, reduced performance of dairy cows and massive animal losses in the affected herd. To test this hypothesis a case-control study was conducted involving 92 affected farms and 47 control farms located in Northern Germany. Fecal samples of 1388 animals were investigated for the presence of BoNT to verify the key requirement of the hypothesis of chronic botulism. BoNT was not detected in any of the fecal samples using the most sensitive standard method for BoNT detection, the mouse bioassay. Therefore, the existence of "chronic" or "visceral" botulism could not be proven.

  20. Weak neurotoxin from Naja kaouthia cobra venom affects haemodynamic regulation by acting on acetylcholine receptors.

    Science.gov (United States)

    Ogay, Alexey Ya; Rzhevsky, Dmitry I; Murashev, Arkady N; Tsetlin, Victor I; Utkin, Yuri N

    2005-01-01

    Recent in vitro studies of weak neurotoxins from snake venoms have demonstrated their ability to interact with both muscle-type and neuronal alpha7 nicotinic acetylcholine receptors (nAChR). However, the biological activity in vivo of weak neurotoxins remains largely unknown. We have studied the influence of weak neurotoxin (WTX) from the venom of cobra Naja kaouthia on arterial blood pressure (BP) and heart rate (HR) in rats and mice. It was found that intravenous injection of WTX induced a dose-dependent decrease in BP and an increase in HR in both species, the rats being more sensitive to WTX. Application of WTX following blockade of nAChRs or muscarinic acetylcholine receptors (mAChR) by hexamethonium or atropine, respectively, showed that both nAChRs and mAChRs are involved in the haemodynamic effects of WTX. Blockade of either nAChRs or mAChRs affected WTX action differently in rats and mice, thus reflecting interspecies differences in haemodynamic regulation.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Gary S. Sayler

    2013-03-01

    Full Text Available 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.

  3. Droplet microfluidics for characterizing the neurotoxin-induced responses in individual Caenorhabditis elegans.

    Science.gov (United States)

    Shi, Weiwei; Wen, Hui; Lu, Yao; Shi, Yang; Lin, Bingcheng; Qin, Jianhua

    2010-11-07

    A droplet-based microfluidic device integrated with a novel floatage-based trap array and a tapered immobilization channel array was presented for characterizing the neurotoxin-induced multiple responses in individual Caenorhabditis elegans (C. elegans) continuously. The established device enabled the evaluations of movement and fluorescence imaging analysis of individual C. elegans simultaneously. The utility of this device was demonstrated by the pharmacological evaluation of neurotoxin (6-hydroxydopamine, 6-OHDA) triggered mobility defects, neuron degeneration and oxidative stress in individual worms. Exposure of living worms to 6-OHDA could cause obvious mobility defects, selective degeneration of dopaminergic (DAergic) neurons, and increased oxidative stress in a dose dependent manner. These results are important towards the understanding of mechanisms leading to DAergic toxicity by neurotoxin and will be of benefit for the screening of new therapeutics for neurodegenerative diseases. This device was simple, stable and easy to operate, with the potential to facilitate whole-animal assays and drug screening in a high throughput manner at single animal resolution.

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

    Directory of Open Access Journals (Sweden)

    Jorge Lago

    2015-10-01

    Full Text Available 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.

  5. Enzyme-amplified protein micorarray and a fluidic renewable surface fluorescence immunoassay for botulinum neurotoxin detection using high-affinity recombinant antibodies

    Energy Technology Data Exchange (ETDEWEB)

    Varnum, Susan M.; Warner, Marvin G.; Dockendorff, Brian P.; Anheier, Norman C.; Lou, Jianlong; Marks, James D.; Smith, Leonard A.; Feldhaus, Michael J.; Grate, Jay W.; Bruckner-Lea, Cindy J.

    2006-06-16

    With the use of high-affinity recombinant monoclonal antibodies against the receptor binding domain of botulinum neurotoxin A (BoNT/A), two separate immunoassay platforms were developed for either the sensitive or the rapid detection of BoNT/A. An enzyme-linked immunosorbent assay (ELISA) microarray was developed for the specific and sensitive detection of BoNT in buffer and clinical fluids. This assay has the sensitivity to detect BoNT in diverse samples down to 14 fM (1.4 pg/mL). Using the recombinant monoclonal antibodies, a renewable surface microcolumn sensor was developed for the rapid detection of BoNT/A in an automated fluidic system. While the ELISA microarray assay, because of its sensitivity, offers an alternative to the mouse bioassay, the renewable surface assay has potential as a rapid screening assay for the analysis of complex environmental samples.

  6. Understanding peptide competitive inhibition of botulinum neurotoxin A binding to SV2 protein via molecular dynamics simulations.

    Science.gov (United States)

    Chang, Shan; He, Hong-Qiu; Shen, Lin; Wan, Hua

    2015-10-01

    Botulinum neurotoxins (BoNTs) are known as the most toxic natural substances. Synaptic vesicle protein 2 (SV2) has been proposed to be a protein receptor for BoNT/A. Recently, two short peptides (BoNT/A-A2 and SV2C-A3) were designed to inhibit complex formation between the BoNT/A receptor-binding domain (BoNT/A-RBD) and the synaptic vesicle protein 2C luminal domain (SV2C-LD). In this article, the two peptide complex systems are studied by molecular dynamics (MD) simulations. The structural stability analysis indicates that BoNT/A-A2 system is more stable than SV2C-A3 system. The conformational analysis implies that the β-sheet in BoNT/A-A2 system maintains its secondary structure but the two β-strands in SV2C-A3 system have remarkable conformational changes. Based on the calculation of hydrogen bonds, hydrophobic interactions and cation-π interactions, it is found that the internal hydrogen bonds play crucial roles in the structural stability of the peptides. Because of the stable secondary structure, the β-sheet in BoNT/A-A2 system establishes effective interactions at the interface and inhibits BoNT/A-RBD binding to SV2C-LD. In contrast, without other β-strands forming internal hydrogen bonds, the two isolated β-strands in SV2C-A3 system become the random coil. This conformational change breaks important hydrogen bonds and weakens cation-π interaction in the interface, so the complex formation is only partially inhibited by the two β-strands. These results are consistent with experimental studies and may be helpful in understanding the inhibition mechanisms of peptide inhibitors.

  7. Three classes of plasmid (47-63 kb) carry the type B neurotoxin gene cluster of group II Clostridium botulinum.

    Science.gov (United States)

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

    2014-08-01

    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.

  8. Nuclear magnetic resonance solution structure of the alpha-neurotoxin from the black mamba (Dendroaspis polylepis polylepis).

    Science.gov (United States)

    Brown, L R; Wüthrich, K

    1992-10-20

    The three-dimensional structure in solution of the alpha-neurotoxin from the black mamba (Dendroaspis polylepis polylepis) has been determined by nuclear magnetic resonance spectroscopy. A high quality structure for this 60-residue protein was obtained from 656 NOE distance constraints and 143 dihedral angle constraints, using the distance geometry program DIANA for the structure calculation and AMBER for restrained energy minimization. For a group of 20 conformers used to represent the solution structure, the average root-mean-square deviation value calculated for the polypeptide backbone heavy atoms relative to the mean structure was 0.45 A. The protein consists of a core region from which three finger-like loops extend outwards. It includes a short, two-stranded antiparallel beta-sheet of residues 1-5 and 13-17, a three-stranded antiparallel beta-sheet involving residues 23-31, 34-42 and 51-55, and four disulfide bridges in the core region. There is also extensive non-regular hydrogen bonding between the carboxy-terminal tail of the polypeptide chain and the rest of the core region. Comparison with the crystal structure of erabutoxin-b indicates that the structure of alpha-neurotoxin is quite similar to other neurotoxin structures, but that local structural differences are seen in regions thought to be important for binding of neurotoxins to the acetylcholine receptor. For two regions of the alpha-neurotoxin structure there is evidence for an equilibrium between multiple conformations, which might be related to conformational rearrangements upon binding to the receptor. Overall, the alpha-neurotoxin presents itself as a protein with a stable core and flexible surface areas that interact with the acetylcholine receptor in such a way that high affinity binding is achieved by conformational rearrangements of the deformable regions of the neurotoxin structure.

  9. Mechanism of Action of the Presynaptic Neurotoxins Tetanus Toxin

    Science.gov (United States)

    1992-01-25

    characterized and more work is needed to assess the physiological importance of gangliosides as binding molecules. It is now clear that the initial binding...Rogers, T.B. and Schnaar, R.L. Complex ganglioside expression and tetanus toxin binding by PC12 pheochromocytoma cells. J.Bil Chem. 263:2055-2063, 1983

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

    Directory of Open Access Journals (Sweden)

    Gabriel M. Filippelli

    2015-07-01

    Full Text Available Abstract 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 environmental 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 poorly studied, yet may pose a significant threat to 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 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.

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

    Science.gov (United States)

    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

  12. Bacterial Toxins and the Nervous System: Neurotoxins and Multipotential Toxins Interacting with Neuronal Cells

    Science.gov (United States)

    Popoff, Michel R.; Poulain, Bernard

    2010-01-01

    Toxins are potent molecules used by various bacteria to interact with a host organism. Some of them specifically act on neuronal cells (clostridial neurotoxins) leading to characteristics neurological affections. But many other toxins are multifunctional and recognize a wider range of cell types including neuronal cells. Various enterotoxins interact with the enteric nervous system, for example by stimulating afferent neurons or inducing neurotransmitter release from enterochromaffin cells which result either in vomiting, in amplification of the diarrhea, or in intestinal inflammation process. Other toxins can pass the blood brain barrier and directly act on specific neurons. PMID:22069606

  13. Bacterial toxins and the nervous system: neurotoxins and multipotential toxins interacting with neuronal cells.

    Science.gov (United States)

    Popoff, Michel R; Poulain, Bernard

    2010-04-01

    Toxins are potent molecules used by various bacteria to interact with a host organism. Some of them specifically act on neuronal cells (clostridial neurotoxins) leading to characteristics neurological affections. But many other toxins are multifunctional and recognize a wider range of cell types including neuronal cells. Various enterotoxins interact with the enteric nervous system, for example by stimulating afferent neurons or inducing neurotransmitter release from enterochromaffin cells which result either in vomiting, in amplification of the diarrhea, or in intestinal inflammation process. Other toxins can pass the blood brain barrier and directly act on specific neurons.

  14. Molecular size of different neurotoxin receptors on the voltage-sensitive Na+ channel.

    Science.gov (United States)

    Barhanin, J; Schmid, A; Lombet, A; Wheeler, K P; Lazdunski, M; Ellory, J C

    1983-01-25

    Measurements were made of the molecular sizes of two distinct receptors on the Na+ channel in rat brain synaptosomes that are specific for different neurotoxins. Radiation inactivation of the binding of radiolabeled derivatives of the toxins was consistent with Mr = 260,000 for the tetrodotoxin receptor and Mr = 266,000 for the receptor specific for two scorpion toxins, toxin II from Centruroides suffusus suffusus and toxin gamma from Tityus serrulatus serrulatus. Covalent cross-linking of the latter to its receptor similarly indicated Mr = 270,000. It seems most likely that these two distinct receptors reside on the same molecule.

  15. Identification and Biochemical Characterization of Small-Molecule Inhibitors of Clostridium Botulinum Neurotoxin Serotype A

    Science.gov (United States)

    2009-08-01

    post- exposure therapeutics. Methodology/Principal Findings: In this study, we report on five novel small-molecule BoNT/A inhibitors that have been...cellular and tissue-based assays intended to mimic BoNT exposure . These five compounds (at 15 mM) protected N2a cells from BoNT/A-mediated cleavage of SNAP...neurotoxin (rELC; residues 1 to 423) and truncated type A LC ( tALC ; residues 1 to 425) will be described elsewhere. Briefly, rELC with a C-terminal His6

  16. Two-component signal transduction system CBO0787/CBO0786 represses transcription from botulinum neurotoxin promoters in Clostridium botulinum ATCC 3502.

    Directory of Open Access Journals (Sweden)

    Zhen Zhang

    2013-03-01

    Full Text Available Blocking neurotransmission, botulinum neurotoxin is the most poisonous biological substance known to mankind. Despite its infamy as the scourge of the food industry, the neurotoxin is increasingly used as a pharmaceutical to treat an expanding range of muscle disorders. Whilst neurotoxin expression by the spore-forming bacterium Clostridium botulinum appears tightly regulated, to date only positive regulatory elements, such as the alternative sigma factor BotR, have been implicated in this control. The identification of negative regulators has proven to be elusive. Here, we show that the two-component signal transduction system CBO0787/CBO0786 negatively regulates botulinum neurotoxin expression. Single insertional inactivation of cbo0787 encoding a sensor histidine kinase, or of cbo0786 encoding a response regulator, resulted in significantly elevated neurotoxin gene expression levels and increased neurotoxin production. Recombinant CBO0786 regulator was shown to bind to the conserved -10 site of the core promoters of the ha and ntnh-botA operons, which encode the toxin structural and accessory proteins. Increasing concentration of CBO0786 inhibited BotR-directed transcription from the ha and ntnh-botA promoters, demonstrating direct transcriptional repression of the ha and ntnh-botA operons by CBO0786. Thus, we propose that CBO0786 represses neurotoxin gene expression by blocking BotR-directed transcription from the neurotoxin promoters. This is the first evidence of a negative regulator controlling botulinum neurotoxin production. Understanding the neurotoxin regulatory mechanisms is a major target of the food and pharmaceutical industries alike.

  17. Detection of Clostridium tetani in human clinical samples using tetX specific primers targeting the neurotoxin.

    Science.gov (United States)

    Ganesh, Madhu; Sheikh, Nasira K; Shah, Pooja; Mehetre, Gajanan; Dharne, Mahesh S; Nagoba, Basavraj S

    2016-01-01

    Tetanus resulting from ear injury remains an important health problem, particularly in the developing world. We report the successful detection of Clostridium tetani using tetX specific primers targeting the Cl. tetani neurotoxin. The sample was obtained from an ear discharge of a case of otogenic tetanus in a 2-year-old male child. Based on the culture results of the ear discharge, Gram staining and virulence testing by genotyping, a diagnosis of tetanus was confirmed. This is the first report from India on the successful detection of Cl. tetani in a human clinical sample using tetX specific primers targeting the Cl. tetani neurotoxin.

  18. Production of the Neurotoxin BMAA by a Marine Cyanobacterium

    Directory of Open Access Journals (Sweden)

    Paul Alan Cox

    2007-12-01

    Full Text Available Diverse species of cyanobacteria have recently been discovered to produce theneurotoxic non-protein amino acid β-methylamino-L-alanine (BMAA. In Guam, BMAAhas been studied as a possible environmental toxin in the diets of indigenous Chamorropeople known to have high levels of Amyotrophic Lateral Sclerosis/ ParkinsonismDementia Complex (ALS/PDC. BMAA has been found to accumulate in brain tissues ofpatients with progressive neurodegenerative illness in North America. In Guam, BMAAwas found to be produced by endosymbiotic cyanobacteria of the genus Nostoc which livein specialized cycad roots. We here report detection of BMAA in laboratory cultures of afree-living marine species of Nostoc. We successfully detected BMAA in this marinespecies of Nostoc with five different methods: HPLC-FD, UPLC-UV, Amino AcidAnalyzer, LC/MS, and Triple Quadrupole LC/MS/MS. This consensus of five differentanalytical methods unequivocally demonstrates the presence of BMAA in this marinecyanobacterium. Since protein-associated BMAA can accumulate in increasing levelswithin food chains, it is possible that biomagnification of BMAA could occur in marineecosystems similar to the biomagnification of BMAA in terrestrial ecosystems. Productionof BMAA by marine cyanobacteria may represent another route of human exposure toBMAA. Since BMAA at low concentrations causes the death of motor neurons, low levelsof BMAA exposure may trigger motor neuron disease in genetically vulnerableindividuals.

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

    Directory of Open Access Journals (Sweden)

    Phillip H Beske

    2015-04-01

    Full Text Available 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+ presynaptic 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.

  20. The cyanobacterial neurotoxin beta-N-methylamino-L-alanine (BMAA) induces neuronal and behavioral changes in honeybees

    Energy Technology Data Exchange (ETDEWEB)

    Okle, Oliver, E-mail: oliver.okle@uni-konstanz.de [Human and Environmental Toxicology, University of Konstanz, Jacob-Burckhardt-Strasse 25, 78457 Konstanz (Germany); Rath, Lisa; Galizia, C. Giovanni [Zoology and Neurobiology, University of Konstanz, Universitätsstraße 10, 78457 Konstanz (Germany); Dietrich, Daniel R., E-mail: daniel.dietrich@uni-konstanz.de [Human and Environmental Toxicology, University of Konstanz, Jacob-Burckhardt-Strasse 25, 78457 Konstanz (Germany)

    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 {sup 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{sup 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. - Highlights: • Investigating of neurotoxic effects of BMAA in honeybees • BMAA impairs ALS markers (ROS, Ca{sup 2+}, learning, memory, odor) in bees. • A method for the observation of ROS development in living bees brain was established. • Honeybees are a suitable model to explore neurodegenerative processes. • Neurotoxic BMAA can be spread in bee populations by trophallaxis.

  1. Suppression of oocyte release in rats by local administration of the noradrenergic neurotoxin DSP4.

    Science.gov (United States)

    Goldman, J M; Stoker, T E; Cooper, R L; McElroy, W K; Parrish, M B

    1996-03-01

    The presence of noradrenergic neuronal innervation in the ovaries and cyclic alterations in ovarian noradrenaline suggest a role for such innervation in oocyte release. The current experiments evaluated the relationship between ovulation and alterations in ovarian concentrations of noradrenaline induced by unilateral, intrabursal administration of the specific noradrenergic neurotoxin DSP4. Intrabursal injections of DSP4 (0-10 mumoles per ovary) given at 19:00 h at pro-oestrus induced a prompt, dose-related reduction in ovarian noradrenaline on the injected and non-injected sides. Although this result suggests that injected material was reaching the contralateral ovary, ovulation was suppressed only on the injected side. This suppression was persistent, and lasted through at least the next two cycles following either unilateral or bilateral treatment. The reductions in noradrenaline could be mostly, if not entirely, attenuated by prior administration of desipramine which blocks re-uptake of noradrenaline, while the ipsilateral ovulatory effects remained unchanged. Although it has been reported that DSP4 binds the opiate receptor, intrabursal co-administration of the antagonist naloxone was ineffective in altering ovulatory suppression. These results suggest that while decreases in ovarian noradrenaline in response to local exposure to a noradrenergic neurotoxin may accompany a reduction in oocyte release or a block in ovulation, the anti-ovulatory effect of DSP4 is independent of the changes in noradrenaline concentrations and may be due to some other ovarian response.

  2. Effect of neurotoxin DSP4 on EEG power spectra in the rat acute model of epilepsy.

    Science.gov (United States)

    Culić, M; Saponjić, J; Janković, B; Udović, S; Popović, S; Rakić, L

    1995-08-18

    The effect of the adrenergic neurotoxin N-(chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) on electroencephalographic (EEG) activity was studied in the model of epilepsy induced by systemic application of penicillin (1,000,000 IU/kg, i.p). DSP4 (50 mg/kg, i.p.) was administrated to male Wistar rats, while the control animals were rats from the same litters. EEG activity was recorded in acute and chronic experiments 3 or 4 weeks after DSP4 treatment, before and after penicillin administration. Occasional locus coeruleus (LC) stimulation served as an electrophysiological test of DSP4 toxic effect. EEG power spectra in DSP4 treated animals showed a tendency to be greater in lower frequency bands than in controls before penicillin administration; there was almost no effect of electrical LC stimulation, regardless on penicillin treatment. In the model of epilepsy, the mean total EEG power spectra were greater in the period of 135-330 min after penicillin administration, as well as during 345-540 min, in DSP4 treated animals as compared to the controls. It seems that neurotoxin DSP4 is an optimal tool for studying the removal of LC influence in the acute model of epilepsy. It is also suggested that norepinephrine (NE) may have a modulatory role in the systemic penicillin epilepsy.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  4. Neurotoxin-induced selective ubiquitination and regulation of MEF2A isoform in neuronal stress response.

    Science.gov (United States)

    She, Hua; Yang, Qian; Mao, Zixu

    2012-09-01

    The myocyte enhancer factor 2A-D (MEF2) proteins are members of the MCM1-agamous-deficiens-serum response factor family of transcription factors. Various MEF2 isoform proteins are enriched in neurons and exhibit distinct patterns of expression in different regions of the brain. In neurons, MEF2 functions as a converging factor to regulate many neuronal functions including survival. MEF2 activities are tightly controlled in neurons in response to stress. Whether stress signal may differentially regulate MEF2s remains largely unknown. In this work, we showed that MEF2A, but not MEF2C or MEF2D, was modified by ubiquitination in dopaminergic neuronal cell line SN4741 cells. MEF2A was ubiquitinated at its N'-terminus, and the ubiquitination of MEF2A was first detectable in the nuclear compartment and later in the cytoplasm. Ubiquitination of MEF2A correlated with reduced DNA-binding activity and transcriptional activity. More importantly, disturbing the degradation of ubiquitinated MEF2A through proteasome pathway by neurotoxins known to induce Parkinson's disease features in model animals caused accumulation of ubiquitinated MEF2A, reduced MEF2 activity, and impaired cellular viability. Our work thus provides the first evidence to demonstrate an isoforms-specific regulation of MEF2s by ubiquitination-proteasome pathway in dopaminergic neuronal cell by neurotoxins, suggesting that stress signal and cellular context-dependent dysregulation of MEF2s may underlie the loss of neuronal viability.

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

    Science.gov (United States)

    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.

  6. Neutralizing antibodies to botulinum neurotoxin type A in aesthetic medicine: five case reports

    Directory of Open Access Journals (Sweden)

    Torres S

    2013-12-01

    Full Text Available Sebastian Torres,1 Mark Hamilton,2 Elena Sanches,4 Polina Starovatova,3 Elena Gubanova,3 Tatiana Reshetnikova51Di Stefano Velona Clinic, Catania, Italy; 2Hamilton Face Clinic, Dublin, Ireland; 3Preventive Medicine Clinic "Vallex M", Moscow, Russia; 4EKLAN Co Ltd Medical Center for Aesthetic Correction, Moscow, Russia; 5Department of Dermatovenereology and Cosmetology, State Medical University, Novosibirsk, RussiaAbstract: Botulinum neurotoxin injections are a valuable treatment modality for many therapeutic indications as well as in the aesthetic field for facial rejuvenation. As successful treatment requires repeated injections over a long period of time, secondary resistance to botulinum toxin preparations after repeated injections is an ongoing concern. We report five case studies in which neutralizing antibodies to botulinum toxin type A developed after injection for aesthetic use and resulted in secondary treatment failure. These results add to the growing number of reports in the literature for secondary treatment failure associated with high titers of neutralizing antibodies in the aesthetic field. Clinicians should be aware of this risk and implement injection protocols that minimize resistance development.Keywords: aesthetic medicine, botulinum neurotoxin type A, neutralizing antibody, secondary treatment failure

  7. Expression of functional recombinant scorpion beta-neurotoxin Css II in E. coli.

    Science.gov (United States)

    Johnson, T M; Quick, M W; Sakai, T T; Krishna, N R

    2000-06-01

    The gene for a beta-neurotoxin [Centruroides suffusus suffusus toxin II (Css II)] from the scorpion C. suffusus suffusus was synthesized by recursive PCR and cloned into the expression vector, pET15b. This recombinant vector was transformed into a thioredoxin mutant host bacterial cell, AD 494(DE3)pLysS, and expression was induced with isopropyl thiogalactoside (IPTG). Although the level of expression was low, the recombinant toxin was found only in the soluble fraction with no evidence for the formation of inclusion bodies as had been observed previously with other scorpion toxins. The recombinant Css II was purified by successive ion-exchange and hydrophobic interaction chromatography. Nuclear magnetic resonance (NMR) and circular dichroism (CD) spectral measurements indicate that the protein has a native structure with no indication of denatured species. The recombinant neurotoxin inhibits the uptake of [(3)H]GABA [gamma-aminobutyric acid (GABA)] in neuronal cells as effectively as natural beta-toxins.

  8. Functional unit size of the neurotoxin receptors on the voltage-dependent sodium channel.

    Science.gov (United States)

    Angelides, K J; Nutter, T J; Elmer, L W; Kempner, E S

    1985-03-25

    Radiation inactivation was used in situ to determine the functional unit sizes of the neurotoxin receptors of the voltage-dependent sodium channel from rat brain. Frozen or lyophilized synaptosomes were irradiated with high energy electrons generated by a linear accelerator and assayed for [3H]saxitoxin, 125I-Leiurus quinquestriatus quinquestriatus (alpha-scorpion toxin), 125I-Centruroides suffusus suffusus (beta-scorpion toxin), and batrachotoxinin-A 20 alpha-[3H]benzoate binding activity. The functional unit size of the neurotoxin receptors determined in situ by target analysis are 220,000 for saxitoxin, 263,000 for alpha-scorpion toxin, and 45,000 for beta-scorpion toxin. Analysis of the inactivation curve for batrachotoxinin-A 20 alpha-benzoate binding to the channel yields two target sizes of Mr approximately 287,000 (50%) and approximately 51,000 (50%). The results are independent of the purity of the membrane preparation. Comparison of the radiation inactivation data with the protein composition of the rat brain sodium channel indicates that there are at least two functional components.

  9. [Botulism: the agent, mode of action of the botulinum neurotoxins, forms of acquisition, treatment and prevention].

    Science.gov (United States)

    Marvaud, Jean-Christophe; Raffestin, Stéphanie; Popoff, Michel R

    2002-08-01

    The botulinum neurotoxins are produced by anaerobic, spore-forming bacteria belonging to the Clostridium genus. They are synthesised as a single chain protein (150 kDa), which is not or weakly active. The active form results from a proteolysis cleaving the precursor in a light chain (about 50 kDa) and a heavy chain (about 100 kDa), which are linked by a disulfide bridge. The heavy chain is involved in the recognition of a specific neuronal surface receptor and mediates the internalization of the light chain into the cytosol. The light chain is responsible for the intracellular activity. It catalyses the proteolysis of SNARE proteins, which are involved in the exocytosis of synaptic vesicles containing acetylcholine. Hence, the release of acetylcholine at the neuromuscular junction is blocked, leading to a flaccid paralysis. Human botulism, usually type A, B or E, is associated with intoxination, ingestion of preformed toxin in food, with digestive toxi-infection, mainly in newborns (infant botulism), or with wound contamination (wound botulism). The treatment of botulism is usually symptomatic. The specific treatment is based on the serotherapy or on the use of purified specific antibodies. The vaccination against botulism is efficient. However, since the botulinum neurotoxins are widely used for the treatment of numerous dystonias, a generalised vaccination is not conceivable.

  10. Noradrenergic-Dopaminergic Interactions Due to DSP-4-MPTP Neurotoxin Treatments: Iron Connection.

    Science.gov (United States)

    Archer, Trevor

    The investigations of noradrenergic lesions and dopaminergic lesions have established particular profiles of functional deficits and accompanying alterations of biomarkers in brain regions and circuits. In the present account, the focus of these lesions is directed toward the effects upon dopaminergic neurotransmission and expression that are associated with the movement disorders and psychosis-like behavior. In this context, it was established that noradrenergic denervation, through administration of the selective noradrenaline (NA) neurotoxin, DSP-4, should be performed prior to the depletion of dopamine (DA) with the selective neurotoxin, MPTP. Employing this regime, it was shown that (i) following DSP-4 (50 mg/kg) pretreatment of C57/Bl6 mice, both the functional and neurochemical (DA loss) effects of MPTP (2 × 20 and 2 × 40 mg/kg) were markedly exacerbated, and (ii) following postnatal iron (Fe(2+), 7.5 mg/kg, on postnatal days 19-12), pretreatment with DSP-4 followed by the lower 2 × 20 mg/kg MPTP dose induced even greater losses of motor behavior and striatal DA. As yet, the combination of NA-DA depletions, and even more so Fe(2+)-NA-DA depletion, has been considered to present a movement disorder aspect although studies exploring cognitive domains are lacking. With intrusion of iron overload into this formula, the likelihood of neuropsychiatric disorder, as well, unfolds.

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

    Science.gov (United States)

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

    2015-09-08

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  14. Botulinum neurotoxin serotype A specific cell-based potency assay to replace the mouse bioassay.

    Science.gov (United States)

    Fernández-Salas, Ester; Wang, Joanne; Molina, Yanira; Nelson, Jeremy B; Jacky, Birgitte P S; Aoki, K Roger

    2012-01-01

    Botulinum neurotoxin serotype A (BoNT/A), a potent therapeutic used to treat various disorders, inhibits vesicular neurotransmitter exocytosis by cleaving SNAP25. Development of cell-based potency assays (CBPAs) to assess the biological function of BoNT/A have been challenging because of its potency. CBPAs can evaluate the key steps of BoNT action: receptor binding, internalization-translocation, and catalytic activity; and therefore could replace the current mouse bioassay. Primary neurons possess appropriate sensitivity to develop potential replacement assays but those potency assays are difficult to perform and validate. This report describes a CBPA utilizing differentiated human neuroblastoma SiMa cells and a sandwich ELISA that measures BoNT/A-dependent intracellular increase of cleaved SNAP25. Assay sensitivity is similar to the mouse bioassay and measures neurotoxin biological activity in bulk drug substance and BOTOX® product (onabotulinumtoxinA). Validation of a version of this CBPA in a Quality Control laboratory has led to FDA, Health Canada, and European Union approval for potency testing of BOTOX®, BOTOX® Cosmetic, and Vistabel®. Moreover, we also developed and optimized a BoNT/A CBPA screening assay that can be used for the discovery of novel BoNT/A inhibitors to treat human disease.

  15. Cyanobacterial neurotoxin β-N-methylamino-L-alanine (BMAA) in shark fins.

    Science.gov (United States)

    Mondo, Kiyo; Hammerschlag, Neil; Basile, Margaret; Pablo, John; Banack, Sandra A; Mash, Deborah C

    2012-02-01

    Sharks are among the most threatened groups of marine species. Populations are declining globally to support the growing demand for shark fin soup. Sharks are known to bioaccumulate toxins that may pose health risks to consumers of shark products. The feeding habits of sharks are varied, including fish, mammals, crustaceans and plankton. The cyanobacterial neurotoxin β-N-methylamino-L-alanine (BMAA) has been detected in species of free-living marine cyanobacteria and may bioaccumulate in the marine food web. In this study, we sampled fin clips from seven different species of sharks in South Florida to survey the occurrence of BMAA using HPLC-FD and Triple Quadrupole LC/MS/MS methods. BMAA was detected in the fins of all species examined with concentrations ranging from 144 to 1836 ng/mg wet weight. Since BMAA has been linked to neurodegenerative diseases, these results may have important relevance to human health. We suggest that consumption of shark fins may increase the risk for human exposure to the cyanobacterial neurotoxin BMAA.

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

    Science.gov (United States)

    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

  17. Noradrenergic neurotoxin, N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP-4), treatment eliminates estrogenic effects on song responsiveness in female zebra finches (Taeniopygia guttata).

    Science.gov (United States)

    Vyas, Akshat; Harding, Cheryl; McGowan, Joseph; Snare, Randall; Bogdan, Diane

    2008-10-01

    Female songbirds use male songs as an important criterion for mate selection. Several studies have reported that female songbirds prefer complex songs to other song types. In a recent study, the authors found that song responsiveness in female zebra finches (Taeniopygia guttata) is strongly modulated by circulating estrogen levels. The behavioral effects of estrogen are often mediated via norepinephrine (NE). The current study administered the noradrenergic neurotoxin, N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP-4) to estradiol-treated female zebra finches to investigate if estrogenic effects on song responsiveness are mediated via NE. The authors tested song responsiveness of adult female zebra finches for three acoustically different song types--simple, long-bout, and complex--under three treatment conditions, untreated, estradiol-treated, and estradiol + DSP-4-treated. Females only showed differential song responsiveness when treated with estradiol alone, responding more to complex songs. DSP-4 treatment eliminated this differential responsiveness. The results are discussed in the light of evidence from functional, neurochemical, and neuroanatomical studies that suggest that estrogenic effects on song processing might be mediated by NE.

  18. Effects of carbon dioxide on growth of proteolytic Clostridium botulinum, its ability to produce neurotoxin, and its transcriptome.

    Science.gov (United States)

    Artin, Ingrid; Mason, David R; Pin, Carmen; Schelin, Jenny; Peck, Michael W; Holst, Elisabet; Rådström, Peter; Carter, Andrew T

    2010-02-01

    The antimicrobial gas carbon dioxide is frequently used in modified atmosphere packaging. In the present study, the effects of CO2 (10 to 70%, vol/vol) on gene expression (measured using quantitative reverse transcription-PCR and a whole-genome DNA microarray) and neurotoxin formation (measured using an enzyme-linked immunosorbent assay [ELISA]) by proteolytic Clostridium botulinum type A1 strain ATCC 3502 were studied during the growth cycle. Interestingly, in marked contrast to the situation with nonproteolytic C. botulinum types B and E, CO2 had little effect on any of these parameters. At all CO2 concentrations, relative expression of neurotoxin cluster genes peaked in the transition between exponential and stationary phases, with evidence of a second rise in expression in late stationary phase. Microarray analysis enabled identification of coding sequences whose expression profiles matched those of the neurotoxin cluster. Further research is needed to determine whether these are connected to neurotoxin formation or are merely growth phase associated.

  19. MOLECULAR-BIOLOGY OF CLOSTRIDIAL TOXINS - EXPRESSION OF MESSENGER-RNAS ENCODING TETANUS AND BOTULINUM NEUROTOXINS IN APLYSIA NEURONS

    NARCIS (Netherlands)

    MOCHIDA, S; POULAIN, B; EISEL, U; BINZ, T; KURAZONO, H; NIEMANN, H; TAUC, L

    1990-01-01

    mRNAs encoding the light chain of tetanus and botulinum neurotoxins were transcribed, in vitro, from the cloned and specifically truncated genes of Clostridium tetani and Clostridium botulinum, respectively, and injected into presynaptic identified cholinergic neurons of the buccal ganglia of

  20. MOLECULAR-BIOLOGY OF CLOSTRIDIAL TOXINS - EXPRESSION OF MESSENGER-RNAS ENCODING TETANUS AND BOTULINUM NEUROTOXINS IN APLYSIA NEURONS

    NARCIS (Netherlands)

    MOCHIDA, S; POULAIN, B; EISEL, U; BINZ, T; KURAZONO, H; NIEMANN, H; TAUC, L

    1990-01-01

    mRNAs encoding the light chain of tetanus and botulinum neurotoxins were transcribed, in vitro, from the cloned and specifically truncated genes of Clostridium tetani and Clostridium botulinum, respectively, and injected into presynaptic identified cholinergic neurons of the buccal ganglia of Aplysi

  1. Destabilization of survival factor MEF2D mRNA by neurotoxin in models of Parkinson's disease.

    Science.gov (United States)

    Wang, Bao; Cai, Zhibiao; Lu, Fangfang; Li, Chen; Zhu, Xiaofei; Su, Linna; Gao, Guodong; Yang, Qian

    2014-09-01

    Progressive loss of dopaminergic (DA) neurons in the substantial nigra pars compacta (SNc) is an important pathological feature in Parkinson's disease (PD). Loss of transcription factor myocyte enhancer factor 2D (MEF2D), a key neuronal survival factor, has been shown to underlie the loss of DA neurons in SNc and the pathogenic process of PD. It is known that PD-associated neurotoxins reduce the level of MEF2D protein to trigger neuronal death. Although neurotoxins clearly destabilize MEF2D by post-translational mechanisms, it is not known whether regulation of MEF2D mRNA contributes to neurotoxin-induced decrease in MEF2D protein. In this work, we showed that MPP(+), the toxic metabolite of MPTP, caused a significant decrease in the half-life and total level of MEF2D mRNA in a DA neuronal cell line, SN4741 cells. Quantitative PCR analysis of the SNc DA neurons captured by immune-laser capture microdissection showed that exposure to MPTP led to a marked reduction in the level of MEF2D mRNA in SNc DA neurons compared to controls. Down-regulation of MEF2D mRNA alone reduced the viability of SN4741 cells and sensitized the cells to MPP(+)-induced toxicity. These results suggest that destabilization and reduction in MEF2D mRNA is in part responsible for neurotoxin-induced decrease in MEF2D protein and neuronal viability. Myocyte enhancer factor 2D (MEF2D) plays an important role in neuronal survival. How MEF2D mRNA is deregulated under toxic stress is unclear. We found that PD-associated neurotoxins destabilize MEF2D mRNA and reduce its level in vitro and in vivo. Reduction in MEF2D mRNA is sufficient to sensitize model cells to neurotoxin-induced toxicity, suggesting that destabilization of MEF2D mRNA is part of the mechanism by which neurotoxins trigger deregulation of neuronal survival.

  2. A long-form α-neurotoxin from cobra venom produces potent opioidindependent analgesia

    Institute of Scientific and Technical Information of China (English)

    Zhi-xin CHEN; Hui-ling ZHANG; Zhen-lun GU; Bo-wen CHEN; Rong HAN; Paul F REID; Laurence N RAYMOND; Zheng-hong QIN

    2006-01-01

    Aim:In light of the antinociceptive activity of the short-chain neurotoxin,cobrotoxin,and other acetylcholine antagonists,the antinociceptive activity and mechanisms of cobratoxin (CTX) ,a long-chain postsynaptic α-neurotoxin,was investigated in rodent pain models.Methods:CTX was administered intraperitoneally (30,45,68μg/kg) ,intra-cerebral ventricularly (4.5 μg/kg) or microinjected into periaqueductal gray (PAG;4.5 μg/kg).The antinociceptive action was tested using the hot.plate and acetic acid writhing tests in mice and rats.The involvement of the cholinergic system and opioid system in CTX-induced analgesia was examined by pretreatment of animals with atropine (0.5 mg/kg,im;or 10 mg/kg,ip) or naloxone (1 and 5 mg/kg,ip).The effect of CTX on motor activity was tested using the Animex test.Results:CTX exhibited a dose-dependent analgesic action in mice as determined by both the hot-plate and acetic acid writhing tests.The Deak effect of analgesia was seen 3 h after administration.In the mouse acetic acid writhing test,the intra-cerebral ventricular administration of CTX at 4.5μg/kg (1/12th of a systemic dose) produced marked analgesic effects.Microinjection of CTX (4.5μg/kg) into the PAG region did not elicit an analgesic action in rats in the hot-plate test.Atropine at 0.5 mg/kg (im) and naloxone at l and 5 mg/kg (ip) both failed to block the analgesic effects of CTX,but atropine at 1 0 mg/kg (ip) did antagonize the analgesia mediated bv CTX in the mouse acetic acid writhing test.Acetylsalicylic acid (300 mg/kg) did not enhance the analgesic effects of CTX.At the highest effective dose of 68μg/kg the neurotoxin did not change the spontaneous mobility of mice.Conclusion:CTX has analgesic effects.which are mediated in the central nervous system though not through the PAG.The central cholinergic system but not opioid system appears to be involved in the antinociceptive action of CTX.

  3. Botulinum Neurotoxins

    Science.gov (United States)

    2012-03-28

    and VI (Sobel, 2005) and include ocular disorders such as blurred vision, diplopia , ptosis and photophobia (Terranova et al., 1979). This is generally...Dysphagia 96 97 82 Dry mouth 83 100 93 Diplopia 90 92 39 Dysarthia 100 69 50 Upper extremity weakness 86 64 NA Lower

  4. Neurotoxin Mitigation

    Science.gov (United States)

    2007-11-01

    methylglyoxal involved in ligand binding and esterase activity. J Biol Chem 280:5724-5732. Benschop HP and Keijer JH (1966) On the mechanism of ageing of...identiWes hotspot site of modiWcation in human serum albumin by methylglyoxal involved in ligand binding and esterase activity, J. Biol. Chem. 280 (2005

  5. Synaptobrevin/vesicle-associated membrane protein (VAMP) of Aplysia californica: structure and proteolysis by tetanus toxin and botulinal neurotoxins type D and F.

    Science.gov (United States)

    Yamasaki, S; Hu, Y; Binz, T; Kalkuhl, A; Kurazono, H; Tamura, T; Jahn, R; Kandel, E; Niemann, H

    1994-01-01

    Synaptobrevin/vesicle-associated membrane protein (VAMP) and syntaxin are potential vesicle donor and target membrane receptors of a docking complex that requires N-ethylmaleimide-sensitive factor (NSF) and soluble NSF-attachment proteins as soluble factors for vesicle fusion with target membranes. Members of this docking complex are the target of clostridial neurotoxins that act as zinc-dependent proteases. Molecular cloning of the Aplysia californica synaptobrevin cDNA revealed a 180-residue polypeptide (M(r), 19,745) with a central transmembrane region and an atypically large C-terminal intravesicular domain. This polypeptide integrates into membranes at both the co- and posttranslational level, as shown by modification of an artificially introduced N-glycosylation site. The soluble and membrane-anchored forms of synaptobrevin are cleaved by the light chains of the botulinal toxins type D and F and by tetanus toxin involving the peptide bonds Lys49-Ile50, Gln48-Lys49, and Gln66-Phe67, respectively. The active center of teh tetanus toxin light chain was identified by site-specific mutagenesis. His233, His237, Glu234, and Glu270/271 are essential to this proteolytic activity. Modification of histidine residues resulted in loss of zinc binding, whereas a replacement of Glu234 only slightly reduced the zinc content. Images PMID:8197120

  6. Effects of the noradrenergic neurotoxin DSP4 on spatial memory in the rat.

    Science.gov (United States)

    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.

  7. First evidence of antimicrobial activity of neurotoxin 2 from Anemonia sulcata (Cnidaria

    Directory of Open Access Journals (Sweden)

    MR Trapani

    2014-06-01

    Full Text Available We investigated the antibacterial activity of Anemonia sulcata (Cnidaria, Anthozoa tentacle and body acidic extracts. Biochemical purification consisted of first step on solid phase Sep-Pak C8 column followed by several HPLC runs on C18 column using different conditions. Anti-Micrococcus lysodeikticus activity has been detected in 40 % acetonitrile fractions. The resulting purified molecule from tentacles had a molecular mass determined by MALDI-TOF mass spectrum of 4946,299 Da and has been completely sequenced. Its aa sequence revealed identity with the Neurotoxin 2 (ATX-II, a Na+ channel blocking toxins. Consequently, ATX-II appeared to display a dual role as toxin and as antibacterial.

  8. Botulinum neurotoxin type-A when utilized in animals with trigeminal sensitization induced a antinociceptive effect

    Directory of Open Access Journals (Sweden)

    Elcio J Piovesan

    2016-06-01

    Full Text Available ABSTRACT Purpose of the study was evaluate the possible antinociceptive effect of botulinum neurotoxin type-A (BoNT/A in an experimental model of trigeminal neuralgia. Method Neuropathic pain was induced by surgical constriction of the infraorbital nerve in rats. A control group underwent a sham procedure consisting of surgical exposure of the nerve. Subgroups of each group received either BoNT/A or isotonic saline solution. The clinical response was assessed with the -20°C test. Animals that underwent nerve constriction developed sensitization; the sham group did not. Results The sensitization was reversed by BoNT/A treatment evident 24 hours following application. Pronociceptive effect was observed in the sham group following BoNT/A. Conclusion BoNT/A has an antinociceptive effect in sensitized animals and a pronociceptive effect in non-sensitized animals.

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

    Science.gov (United States)

    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.

  10. Interference of the noradrenergic neurotoxin DSP4 with neuronal and nonneuronal monoamine transporters.

    Science.gov (United States)

    Wenge, Birger; Bönisch, Heinz

    2009-12-01

    The haloalkylamine DSP4 (N[-2-chloroethyl]-N-ethyl-2-bromobenzylamine) is a noradrenergic neurotoxin, which is used for the chemical denervation of noradrenergic neurons, and it has been proposed to be a selective substrate for the neuronal, Na(+)- and Cl(-)-dependent noradrenaline transporter (NAT). In the present study, we investigated whether DSP4 not only interacts with the human NAT (hNAT) but also with other neuronal monoamine transporters such as the transporters for dopamine (hDAT) and serotonin (hSERT) or with nonneuronal (Na(+)-independent) monoamine transporters also known as organic cation transporters (OCTs), such as hOCT(1), hOCT(2), and hOCT(3). Using human embryonic kidney HEK293 cells heterologously expressing the corresponding transporter, we show that DSP4 irreversibly inhibits the hNAT, hDAT, hSERT, and hOCT(3). However, this inhibition includes a reversible component at the hDAT, hSERT, and hOCT(3) but not at the hNAT. The inhibitory potency of DSP4 at the neuronal transporters was highest at the hNAT (IC(50) about 5 microM), and it was about five and 40 times lower at the hSERT and hDAT, respectively. DSP4 inhibited all three hOCTs with high potency (IC(50) about 1 microM) but in a completely reversible manner at hOCT(1) and hOCT(2). Cytotoxicity by 24-h exposure of hNAT- or hOCT-expressing cells to low DSP4 concentrations (DSP4's high-affinity uptake through the NAT together with its completely irreversible mode of interaction with the NAT may contribute to its selectivity as noradrenergic neurotoxin.

  11. Genetic diversity within Clostridium botulinum serotypes, botulinum neurotoxin gene clusters and toxin subtypes.

    Science.gov (United States)

    Hill, Karen K; Smith, Theresa J

    2013-01-01

    Clostridium botulinum is a species of spore-forming anaerobic bacteria defined by the expression of any one or two of seven serologically distinct botulinum neurotoxins (BoNTs) designated BoNT/A-G. This Gram-positive bacterium was first identified in 1897 and since then the paralyzing and lethal effects of its toxin have resulted in the recognition of different forms of the intoxication known as food-borne, infant, or wound botulism. Early microbiological and biochemical characterization of C. botulinum isolates revealed that the bacteria within the species had different characteristics and expressed different toxin types. To organize the variable bacterial traits within the species, Group I-IV designations were created. Interestingly, it was observed that isolates within different Groups could express the same toxin type and conversely a single Group could express different toxin types. This discordant phylogeny between the toxin and the host bacteria indicated that horizontal gene transfer of the toxin was responsible for the variation observed within the species. The recent availability of multiple C. botulinum genomic sequences has offered the ability to bioinformatically analyze the locations of the bont genes, the composition of their toxin gene clusters, and the genes flanking these regions to understand their variation. Comparison of the genomic sequences representing multiple serotypes indicates that the bont genes are not in random locations. Instead the analyses revealed specific regions where the toxin genes occur within the genomes representing serotype A, B, C, E, and F C. botulinum strains and C. butyricum type E strains. The genomic analyses have provided evidence of horizontal gene transfer, site-specific insertion, and recombination events. These events have contributed to the variation observed among the neurotoxins, the toxin gene clusters and the bacteria that contain them, and has supported the historical microbiological, and biochemical

  12. Environmental neurotoxins β-N-methylamino-l-alanine (BMAA) and mercury in shark cartilage dietary supplements.

    Science.gov (United States)

    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.

  13. Production and characterisation of a neutralising chimeric antibody against botulinum neurotoxin A.

    Directory of Open Access Journals (Sweden)

    Julie Prigent

    Full Text Available Botulinum neurotoxins, produced by Clostridium botulinum bacteria, are the causative agent of botulism. This disease only affects a few hundred people each year, thus ranking it among the orphan diseases. However, botulinum toxin type A (BoNT/A is the most potent toxin known to man. Due to their potency and ease of production, these toxins were classified by the Centers for Disease Control and Prevention (CDC as Category A biothreat agents. For several biothreat agents, like BoNT/A, passive immunotherapy remains the only possible effective treatment allowing in vivo neutralization, despite possible major side effects. Recently, several mouse monoclonal antibodies directed against a recombinant fragment of BoNT/A were produced in our laboratory and most efficiently neutralised the neurotoxin. In the present work, the most powerful one, TA12, was selected for chimerisation. The variable regions of this antibody were thus cloned and fused with the constant counterparts of human IgG1 (kappa light and gamma 1 heavy chains. Chimeric antibody production was evaluated in mammalian myeloma cells (SP2/0-Ag14 and insect cells (Sf9. After purifying the recombinant antibody by affinity chromatography, the biochemical properties of chimeric and mouse antibody were compared. Both have the same very low affinity constant (close to 10 pM and the chimeric antibody exhibited a similar capacity to its parent counterpart in neutralising the toxin in vivo. Its strong affinity and high neutralising potency make this chimeric antibody interesting for immunotherapy treatment in humans in cases of poisoning, particularly as there is a probable limitation of the immunological side effects observed with classical polyclonal antisera from heterologous species.

  14. Novel Ganglioside-mediated Entry of Botulinum Neurotoxin Serotype D into Neurons

    Energy Technology Data Exchange (ETDEWEB)

    Kroken, Abby R.; Karalewitz, Andrew P.-A.; Fu, Zhuji; Kim, Jung-Ja P.; Barbieri, Joseph T. (MCW)

    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.

  15. Snake venom toxins. The amino-acid sequence of a short-neurotoxin homologue from Dendroaspis polylepis polylepis (black mamba) venom.

    Science.gov (United States)

    Strydom, D J

    1977-06-01

    The third most abundant component of black mamba venom, named FS2, was sequenced with the aid of sequenator studies and peptides derived by tryptic and chymotryptic digestion. Cyanogen bromide digests provided extra information to support the proposed structure. This protein is a homologue of the short neurotoxins of snake venom, but is much less toxic. Its structure is quite different from both neurotoxins and the other mamba proteins, called angusticeps types (neurotoxin homologues). Comparison of the known angusticeps-type toxins from mamba venom with mamba neurotoxins and each other leads to proposals that these proteins of low toxicity are inventions of the group of mambas and that three different, as yet unknown, functions will be associated with the three subgroups that are discernable.

  16. Reinterpretation of luminiscence properties of neurotoxins from the venom of Middle-Asian corba Naja oxiana eichw.

    Science.gov (United States)

    Bukolova-Orlova, T G; Permyakov, E A; Burstein, E A; Yukelson, L Y

    1976-08-09

    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).

  17. The type F6 neurotoxin gene cluster locus of group II clostridium botulinum has evolved by successive disruption of two different ancestral precursors.

    Science.gov (United States)

    Carter, Andrew T; Stringer, Sandra C; Webb, Martin D; Peck, Michael W

    2013-01-01

    Genome sequences of five different Group II (nonproteolytic) Clostridium botulinum type F6 strains were compared at a 50-kb locus containing the neurotoxin gene cluster. A clonal origin for these strains is indicated by the fact that sequences were identical except for strain Eklund 202F, with 10 single-nucleotide polymorphisms and a 15-bp deletion. The essential topB gene encoding topoisomerase III was found to have been split by the apparent insertion of 34.4 kb of foreign DNA (in a similar manner to that in Group II C. botulinum type E where the rarA gene has been disrupted by a neurotoxin gene cluster). The foreign DNA, which includes the intact 13.6-kb type F6 neurotoxin gene cluster, bears not only a newly introduced topB gene but also two nonfunctional botulinum neurotoxin gene remnants, a type B and a type E. This observation combined with the discovery of bacteriophage integrase genes and IS4 elements suggest that several rounds of recombination/horizontal gene transfer have occurred at this locus. The simplest explanation for the current genotype is that the ancestral bacterium, a Group II C. botulinum type B strain, received DNA firstly from a strain containing a type E neurotoxin gene cluster, then from a strain containing a type F6 neurotoxin gene cluster. Each event disrupted the previously functional neurotoxin gene. This degree of successive recombination at one hot spot is without precedent in C. botulinum, and it is also the first description of a Group II C. botulinum genome containing more than one neurotoxin gene sequence.

  18. Neurotoxins from Clostridium botulinum (serotype A) isolated from the soil of Mendoza (Argentina) differ from the A-Hall archetype and from that causing infant botulism.

    Science.gov (United States)

    Caballero, P; Troncoso, M; Patterson, S I; López Gómez, C; Fernandez, R; Sosa, M A

    2016-10-01

    The type A of neurotoxin produced by Clostridium botulinum is the prevalent serotype in strains of Mendoza. The soil is the main reservoir for C.botulinum and is possibly one of the infection sources in infant botulism. In this study, we characterized and compared autochthonous C. botulinum strains and their neurotoxins. Bacterial samples were obtained from the soil and from fecal samples collected from children with infant botulism. We first observed differences in the appearance of the colonies between strains from each source and with the A Hall control strain. In addition, purified neurotoxins of both strains were found to be enriched in a band of 300 kDa, whereas the A-Hall strain was mainly made up of a band of ∼600 kDa. This finding is in line with the lack of hemagglutinating activity of the neurotoxins under study. Moreover, the proteolytic activity of C. botulinum neurotoxins was evaluated against SNARE (soluble N-ethylmaleimide-sensitive factor-attachment protein receptor) proteins from rat brain. It was observed that both, SNAP 25 (synaptosomal-associated protein 25) and VAMP 2 (vesicle-associated membrane protein) were cleaved by the neurotoxins isolated from the soil strains, whereas the neurotoxins from infant botulism strains only induced a partial cleavage of VAMP 2. On the other hand, the neurotoxin from the A-Hall strain was able to cleave both proteins, though at a lesser extent. Our data indicate that the C.botulinum strain isolated from the soil, and its BoNT, exhibit different properties compared to the strain obtained from infant botulism patients, and from the A-Hall archetype. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Proton nuclear magnetic resonance studies on the variant-3 neurotoxin from Centruroides sculpturatus Ewing: Sequential assignment of resonances

    Energy Technology Data Exchange (ETDEWEB)

    Nettesheim, D.G.; Klevit, R.E.; Drobny, G.; Watt, D.D.; Krishna, N.R. (Univ. of Alabama, Birmingham (USA))

    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.

  20. Development and Testing of an In Vitro Assay for Screening of Potential Therapeutic Agents against Na Channel Neurotoxins

    Science.gov (United States)

    1987-02-03

    organizations and trade names in this report do not constitute an official Department of the Army endorsement or approval of the products or services of... BTX -B binding in frozen synaptoneurosomes. 26 Figure 5. Stability of sodium channel neurotoxins to storage at -70C. 27 Figure 6. Inhibition of...voltage-sensitive sodium channel producing a block of ion flux. The lipid-soluble toxins, of which BTX is the most potent, bind at site 2 causing a shift

  1. Effects of algal-produced neurotoxins on metabolic activity in telencephalon, optic tectum and cerebellum of Atlantic salmon (Salmo salar)

    Energy Technology Data Exchange (ETDEWEB)

    Bakke, Marit Jorgensen [Department of Pharmacology and Toxicology, Norwegian School of Veterinary Science, PO Box 8146 Dep., N-0033 Oslo (Norway); Horsberg, Tor Einar [Department of Pharmacology and Toxicology, Norwegian School of Veterinary Science, PO Box 8146 Dep., N-0033 Oslo (Norway)], E-mail: tor.e.horsberg@veths.no

    2007-11-30

    Neurotoxins from algal blooms have been reported to cause mortality in a variety of species, including sea birds, sea mammals and fish. Farmed fish cannot escape harmful algal blooms and their potential toxins, thus they are more vulnerable for exposure than wild stocks. Sublethal doses of the toxins are likely to affect fish behaviour and may impair cognitive abilities. In the present study, changes in the metabolic activity in different parts of the Atlantic salmon (Salmo salar) brain involved in central integration and cognition were investigated after exposure to sublethal doses of three algal-produced neurotoxins; saxitoxin (STX), brevetoxin (BTX) and domoic acid (DA). Fish were randomly selected to four groups for i.p. injection of saline (control) or one of the neurotoxins STX (10 {mu}g STX/kg bw), BTX (68 {mu}g BTX/kg bw) or DA (6 mg DA/kg bw). In addition, {sup 14}C-2-deoxyglucose was i.m. injected to measure brain metabolic activity by autoradiography. The three regions investigated were telencephalon (Tel), optic tectum (OT) and cerebellum (Ce). There were no differences in the metabolic activity after STX and BTX exposure compared to the control in these regions. However, a clear increase was observed after DA exposure. When the subregions with the highest metabolic rate were pseudocoloured in the three brain regions, the three toxins caused distinct differences in the respective patterns of metabolic activation. Fish exposed to STX displayed similar patterns as the control fish, whereas fish exposed to BTX and DA showed highest metabolic activity in subregions different from the control group. All three neurotoxins affected subregions that are believed to be involved in cognitive abilities in fish.

  2. Preparation and properties of a neurotoxin purified from the venom of black widow spider (Latrodectus mactans tredecimguttatus).

    Science.gov (United States)

    Grasso, A

    1976-08-09

    A neurotoxin of the venom of the spider Latrodectus mactans tredecimguttatus, has been prepared in a homogeneous form and examined by a variety of techniques. The protein has a molecular weight of 130 000 and its toxicity in mice is about 49 000 LD50 mg pure protein/g body weight. The toxin releases norepinephrine from synaptosomes prepared from rat brain and shows most of the toxic effects of the crude venom preparation.

  3. Botulinum and Tetanus Neurotoxin Induced Blockage of Synaptic Transmission in Networked Cultures of Human and Rodent Neurons

    Science.gov (United States)

    2015-11-28

    spontaneous postsynaptic currents The clostridial neurotoxins (CNTs) are a family of closely related bacterial protein toxins that include tetanus...and cleavage of cognate SNARE proteins (Fig. 3D–F). Concentration-response curves were used to estimate toxin concentrations that caused proteolysis...Each BOTOX vial contains stabilizing excipients with 100 or 200 U of “complex” toxin , consisting of BoNT/A associated with naturally associated proteins

  4. Actions of Ethanol on Voltage-Sensitive Sodium Channels. Effects on Neurotoxin-Stimulated Sodium Uptake in Synaptosomes

    Science.gov (United States)

    1985-01-01

    concentration in the nonaqueuus (membrane) phase (Lyon et aL, 1981). Concentration- effect summarized in table 1 . When sodium channels were activated curves were...Voltage-Sensitive Sodium Channels : Effects on Neurotoxin-Stimulated Sodium Uptake in DT (7 Synaptosomes E L C MICHAEL J. MULLIN’ and WALTER A. HUNT...1984). At the present time, the 8 1 structural and functional properties of the voltage-sensitive sodium channels are understood most completely

  5. Botulinum Neurotoxin Serotype F: Identification of Substrate Recognition Requirements and Development of Inhibitors with Low Nanomolar Affinity

    Science.gov (United States)

    2007-11-02

    toxin, secreted by Clostridium tetani , and the seven serologically distinct botulinum toxins, designated types A-G, produced by various strains of... Clostridium botulinum, Clostridium baratii, and Clostridium butyricum (1-3). They are the most toxic substances known (4). Each neurotoxin is synthesized as...The latter characteristic distinguishes BoNT F from other zinc metalloendoproteases, including BoNTs A and B. 15. SUBJECT TERMS Clostridium botulinuim

  6. Effects of Megaplasmid Loss on Growth of Neurotoxigenic Clostridium butyricum Strains and Botulinum Neurotoxin Type E Expression

    OpenAIRE

    Concetta eScalfaro; Angelo eIacobino; Laura eGrande; Stefano eMorabito; Giovanna eFranciosa

    2016-01-01

    Clostridium butyricum strains that atypically produce the botulinum neurotoxin type E (BoNT/E) possess a megaplasmid of unknown functions in their genome. In this study, we cured two botulinum neurotoxigenic C. butyricum type E strains of their megaplasmids, and compared the obtained megaplasmid-cured strains to their respective wild-type parental strains. Our results showed that the megaplasmids do not confer beta-lactam resistance on the neurotoxigenic C. butyricum type E strains, although ...

  7. Comparison of the interactions of a specific neurotoxin (alpha-bungarotoxin) with the acetylcholine receptor in Torpedo californica and Electrophorus electricus membrane preparations.

    Science.gov (United States)

    Leprince, P; Noble, R L; Hess, G P

    1981-09-15

    alpha-Bungarotoxin, a snake neurotoxin, binds irreversibly and specifically to the acetylcholine receptor isolated from the electroplax of Electrophorus electricus and Torpedo species and has been an important tool in the study of the receptor-ligand binding mechanism. Two distinct kinetic processes have been observed in studies with membranes from E. electricus. A minimum mechanism for the toxin reaction involves (i) the reversible binding of two toxin molecules to the receptor prior to the irreversible formation of toxin receptor complexes and (ii) a toxin-induced conformational change of the receptor which leads to an increase in the affinity of the receptor binding sites for toxin [Hess, G. P., Bulger, J. E., Fu, J.-j. L., Hindy, E. F., & Silberstein, R. J. (1975) Biochem. Biophys. Res. Commun. 64, 1018-1027]. Only one process has been detected in Torpedo membranes. Here, we determine whether the receptors in Torpedo californica and E. electricus membranes have different properties or whether the measurements and their interpretation were responsible for the different results. Two methods which are frequently used in binding studies to separate free and bound toxin, a CM-52 cellulose minicolumn assay and DE-81 filter disk assay, have been compared. The results obtained indicate that the interaction of toxin with receptor from T. californica is similar to that observed with receptor from E. electricus. The apparent differences which have been reported in the literature are shown to have arisen from the design of the experiments in which T. californica membranes were used.

  8. Simultaneous and sensitive detection of six serotypes of botulinum neurotoxin using enzyme-linked immunosorbent assay-based protein antibody microarrays

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yanfeng; Lou, Jianlong; Jenko, Kathryn L.; Marks, James D.; Varnum, Susan M.

    2012-11-15

    Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, are a group of seven (A-G) immunologically distinct proteins and cause the paralytic disease botulism. These toxins are the most poisonous substances known to humans and are potential bioweapon agents. Therefore, it is necessary to develop highly sensitive assays for the detection of BoNTs in both clinical and environmental samples. In the present study, we have developed an ELISA-based protein antibody microarray for the sensitive and simultaneous detection of BoNT serotype A, B, C, D, E and F. With engineered high-affinity antibodies, the assays have sensitivities in buffer of 8 fM (1.2 pg/mL) for serotypes A and B, and 32 fM (4.9 pg/mL) for serotypes C, D, E, and F. Using clinical and environmental samples (serum and milk), the microarray is capable of detecting BoNT/A-F to the same levels as in standard buffer. Cross reactivity between assays for individual serotype was also analyzed. These simultaneous, rapid, and sensitive assays have the potential to measure botulinum toxins in a high-throughput manner in complex clinical or environmental samples.

  9. Extraction of BoNT/A, /B, /E, and /F with a single, high affinity monoclonal antibody for detection of botulinum neurotoxin by Endopep-MS.

    Directory of Open Access Journals (Sweden)

    Suzanne R Kalb

    Full Text Available Botulinum neurotoxins (BoNTs are extremely potent toxins that are capable of causing respiratory failure leading to long-term intensive care or death. The best treatment for botulism includes serotype-specific antitoxins, which are most effective when administered early in the course of the intoxication. Early confirmation of human exposure to any serotype of BoNT is an important public health goal. In previous work, we focused on developing Endopep-MS, a mass spectrometry-based endopeptidase method for detecting and differentiating the seven serotypes (BoNT/A-G in buffer and BoNT/A, /B, /E, and /F (the four serotypes that commonly affect humans in clinical samples. We have previously reported the success of antibody-capture to purify and concentrate BoNTs from complex matrices, such as clinical samples. However, to check for any one of the four serotypes of BoNT/A, /B, /E, or /F, each sample is split into 4 aliquots, and tested for the specific serotypes separately. The discovery of a unique monoclonal antibody that recognizes all four serotypes of BoNT/A, /B, /E and /F allows us to perform simultaneous detection of all of them. When applied in conjunction with the Endopep-MS assay, the detection limit for each serotype of BoNT with this multi-specific monoclonal antibody is similar to that obtained when using other serotype-specific antibodies.

  10. Identification of fibroblast growth factor receptor 3 (FGFR3 as a protein receptor for botulinum neurotoxin serotype A (BoNT/A.

    Directory of Open Access Journals (Sweden)

    Birgitte P S Jacky

    Full Text Available Botulinum neurotoxin serotype A (BoNT/A causes transient muscle paralysis by entering motor nerve terminals (MNTs where it cleaves the SNARE protein Synaptosomal-associated protein 25 (SNAP25206 to yield SNAP25197. Cleavage of SNAP25 results in blockage of synaptic vesicle fusion and inhibition of the release of acetylcholine. The specific uptake of BoNT/A into pre-synaptic nerve terminals is a tightly controlled multistep process, involving a combination of high and low affinity receptors. Interestingly, the C-terminal binding domain region of BoNT/A, HC/A, is homologous to fibroblast growth factors (FGFs, making it a possible ligand for Fibroblast Growth Factor Receptors (FGFRs. Here we present data supporting the identification of Fibroblast Growth Factor Receptor 3 (FGFR3 as a high affinity receptor for BoNT/A in neuronal cells. HC/A binds with high affinity to the two extra-cellular loops of FGFR3 and acts similar to an agonist ligand for FGFR3, resulting in phosphorylation of the receptor. Native ligands for FGFR3; FGF1, FGF2, and FGF9 compete for binding to FGFR3 and block BoNT/A cellular uptake. These findings show that FGFR3 plays a pivotal role in the specific uptake of BoNT/A across the cell membrane being part of a larger receptor complex involving ganglioside- and protein-protein interactions.

  11. Identification of fibroblast growth factor receptor 3 (FGFR3 as a protein receptor for botulinum neurotoxin serotype A (BoNT/A.

    Directory of Open Access Journals (Sweden)

    Birgitte P S Jacky

    Full Text Available Botulinum neurotoxin serotype A (BoNT/A causes transient muscle paralysis by entering motor nerve terminals (MNTs where it cleaves the SNARE protein Synaptosomal-associated protein 25 (SNAP25206 to yield SNAP25197. Cleavage of SNAP25 results in blockage of synaptic vesicle fusion and inhibition of the release of acetylcholine. The specific uptake of BoNT/A into pre-synaptic nerve terminals is a tightly controlled multistep process, involving a combination of high and low affinity receptors. Interestingly, the C-terminal binding domain region of BoNT/A, HC/A, is homologous to fibroblast growth factors (FGFs, making it a possible ligand for Fibroblast Growth Factor Receptors (FGFRs. Here we present data supporting the identification of Fibroblast Growth Factor Receptor 3 (FGFR3 as a high affinity receptor for BoNT/A in neuronal cells. HC/A binds with high affinity to the two extra-cellular loops of FGFR3 and acts similar to an agonist ligand for FGFR3, resulting in phosphorylation of the receptor. Native ligands for FGFR3; FGF1, FGF2, and FGF9 compete for binding to FGFR3 and block BoNT/A cellular uptake. These findings show that FGFR3 plays a pivotal role in the specific uptake of BoNT/A across the cell membrane being part of a larger receptor complex involving ganglioside- and protein-protein interactions.

  12. In vitro detection of cardiotoxins or neurotoxins affecting ion channels or pumps using beating cardiomyocytes as alternative for animal testing.

    Science.gov (United States)

    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. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. New Typical Vector of Neurotoxin β-N-Methylamino-l-Alanine (BMAA in the Marine Benthic Ecosystem

    Directory of Open Access Journals (Sweden)

    Aifeng Li

    2016-11-01

    Full Text Available The neurotoxin β-N-methylamino-l-alanine (BMAA has been identified as an environmental factor triggering neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS and Alzheimer’s disease (AD. We investigated the possible vectors of BMAA and its isomers 2,4-diaminobutyric acid (DAB and N-2(aminoethylglycine (AEG in marine mollusks collected from the Chinese coast. Sixty-eight samples of marine mollusks were collected along the Chinese coast in 2016, and were analyzed by an HILIC-MS/MS (hydrophilic interaction liquid chromatography with tandem quadrupole mass spectrometer method without derivatization. BMAA was detected in a total of five samples from three species: Neverita didyma, Solen strictus, and Mytilus coruscus. The top three concentrations of free-form BMAA (0.99~3.97 μg·g−1 wet weight were detected in N. didyma. DAB was universally detected in most of the mollusk samples (53/68 with no species-specific or regional differences (0.051~2.65 μg·g−1 wet weight. No AEG was detected in any mollusk samples tested here. The results indicate that the gastropod N. didyma might be an important vector of the neurotoxin BMAA in the Chinese marine ecosystem. The neurotoxin DAB was universally present in marine bivalve and gastropod mollusks. Since N. didyma is consumed by humans, we suggest that the origin and risk of BMAA and DAB toxins in the marine ecosystem should be further investigated in the future.

  14. Iptakalim hydrochloride protects cells against neurotoxin-induced glutamate transporter dysfunction in in vitro and in vivo models.

    Science.gov (United States)

    Yang, Yan-Ling; Meng, Chang-Hong; Ding, Jian-Hua; He, Hai-Rong; Ellsworth, Kevin; Wu, Jie; Hu, Gang

    2005-07-01

    Iptakalim hydrochloride (Ipt), a novel antihypertensive drug, exhibits K(ATP) channel activation. Here, we report that Ipt remarkably protects cells against neurotoxin-induced glutamate transporter dysfunction in in vitro and in vivo models. Chronic exposure of cultured PC12 cells to neurotoxins, such as 6-OHDA, MPP+, or rotenone, decreased overall [3H]-glutamate uptake in a concentration-dependent manner. Pre-treatment using 10 microM Ipt significantly protected cells against neurotoxin-induced glutamate uptake diminishment, and this protection was abolished by the K(ATP) channel blocker glibenclamide (20 microM), suggesting that the protective mechanisms may involve the opening of K(ATP) channels. In 6-OHDA-treated rats (as an in vivo Parkinson's disease model), [3H]-glutamate uptake was significantly lower in synaptosomes isolated from the striatum and cerebral cortex, but not the hippocampus. Pre-conditioning using 10, 50, and 100 microM Ipt significantly restored glutamate uptake impairment and these protections were abolished by blockade of K(ATP) channels. It is concluded that Ipt exhibits substantial protection of cells against neurotoxicity in in vitro and in vivo models. The cellular mechanisms of this protective effect may involve the opening of K(ATP) channels. Collectively, Ipt may serve as a novel and effective drug for PD therapy.

  15. New Typical Vector of Neurotoxin β-N-Methylamino-l-Alanine (BMAA) in the Marine Benthic Ecosystem.

    Science.gov (United States)

    Li, Aifeng; Song, Jialiang; Hu, Yang; Deng, Longji; Ding, Ling; Li, Meihui

    2016-11-04

    The neurotoxin β-N-methylamino-l-alanine (BMAA) has been identified as an environmental factor triggering neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS) and Alzheimer's disease (AD). We investigated the possible vectors of BMAA and its isomers 2,4-diaminobutyric acid (DAB) and N-2(aminoethyl)glycine (AEG) in marine mollusks collected from the Chinese coast. Sixty-eight samples of marine mollusks were collected along the Chinese coast in 2016, and were analyzed by an HILIC-MS/MS (hydrophilic interaction liquid chromatography with tandem quadrupole mass spectrometer) method without derivatization. BMAA was detected in a total of five samples from three species: Neverita didyma, Solen strictus, and Mytilus coruscus. The top three concentrations of free-form BMAA (0.99~3.97 μg·g(-1) wet weight) were detected in N. didyma. DAB was universally detected in most of the mollusk samples (53/68) with no species-specific or regional differences (0.051~2.65 μg·g(-1) wet weight). No AEG was detected in any mollusk samples tested here. The results indicate that the gastropod N. didyma might be an important vector of the neurotoxin BMAA in the Chinese marine ecosystem. The neurotoxin DAB was universally present in marine bivalve and gastropod mollusks. Since N. didyma is consumed by humans, we suggest that the origin and risk of BMAA and DAB toxins in the marine ecosystem should be further investigated in the future.

  16. Neurotoxin-induced DNA damage is persistentin SH-SY5Y cells and LC neurons

    Science.gov (United States)

    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

  17. Implementing the Bruker MALDI Biotyper in the Public Health Laboratory for C. botulinum Neurotoxin Detection

    Directory of Open Access Journals (Sweden)

    Michael J. Perry

    2017-03-01

    Full Text Available Currently, the gold standard method for active botulinum neurotoxin (BoNT detection is the mouse bioassay (MBA. A Centers for Disease Control and Prevention-developed mass spectrometry (MS-based assay that detects active BoNT was successfully validated and implemented in a public health laboratory in clinical matrices using the Bruker MALDI-TOF MS (Matrix-assisted laser desorption ionization–time of flight mass spectrometry Biotyper. For the first time, a direct comparison with the MBA was performed to determine MS-based assay sensitivity using the Bruker MALDI Biotyper. Mice were injected with BoNT/A, /B, /E, and /F at concentrations surrounding the established MS assay limit of detection (LOD and analyzed simultaneously. For BoNT/B, /E, and /F, MS assay sensitivity was equivalent or better than the MBA at 25, 0.3, and 8.8 mLD50, respectively. BoNT/A was detected by the MBA between 1.8 and 18 mLD50, somewhat more sensitive than the MS method of 18 mLD50. Studies were performed to compare assay performance in clinical specimens. For all tested specimens, the MS method rapidly detected BoNT activity and serotype in agreement with, or in the absence of, results from the MBA. We demonstrate that the MS assay can generate reliable, rapid results while eliminating the need for animal testing.

  18. Disulfide bond reduction corresponds to dimerization and hydrophobicity changes of Clostridium botulinum type A neurotoxin

    Institute of Scientific and Technical Information of China (English)

    Jiunn-jye WEY; Shiao-shek TANG; Tzong-yuan WU

    2006-01-01

    Aim: To determine the structure factors that mediate the intoxication process of botulinum neurotoxin type A (BoNT/A). Methods: Triton X-114 phase separation experiments and 1-anilino-8-naphthalene sulfonate binding assay were used to study the structural factor that corresponds to the hydrophobicity change of BoNT/A. In addition, sucrose density gradient centrifugation and a chemical crosslinking study were employed to determine the quaternary structure of BoNT/A. Results: Our results demonstrated that in other than acidic conditions, the disulfide reduction is the structural factor that corresponds to the hydrophobicity change of BoNT/A. The quaternary structure of BoNT/A exists as a dimmer in acidic solution (pH 4.5), although the monomeric structure of BoNT/A was reported based on X-ray crystallography. Conclusion: Disulfide bond reduction is critical for BoNT/A's channel formation and ability to cross endosome membranes. This result implies that compounds that block this disulfide bond reduction may serve as potential therapeutic agents for botulism.

  19. Camelid-derived heavy-chain nanobody against Clostridium botulinum neurotoxin E in Pichia pastoris.

    Science.gov (United States)

    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.

  20. Neurotoxin-Induced Catecholaminergic Loss in the Colonic Myenteric Plexus of Rhesus Monkeys

    Science.gov (United States)

    Shultz, Jeanette M; Resnikoff, Henry; Bondarenko, Viktorya; Joers, Valerie; Mejia, Andres; Simmons, Heather; Emborg, Marina E

    2017-01-01

    Objective Constipation is a common non-motor symptom of Parkinson’s disease (PD). Although pathology of the enteric nervous system (ENS) has been associated with constipation in PD, the contribution of catecholaminergic neurodegeneration to this symptom is currently debated. The goal of this study was to assess the effects of the neurotoxin 6-hydroxydopamine (6-OHDA) on the colonic myenteric plexus and shed light on the role of catecholaminergic innervation in gastrointestinal (GI) function. Methods Proximal colon tissue from 6-OHDA-treated (n=5) and age-matched control (n=5) rhesus monkeys was immunostained and quantified using ImageJ software. All animals underwent routine daily feces monitoring to assess for constipation or other GI dysfunction. Results Quantification of tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC)-immunoreactivity (-ir) revealed significant reduction in myenteric ganglia of 6-OHDA-treated animals compared to controls (TH-ir: 87.8%, P30% days) soft stool or diarrhea in 2 of the 5 6-OHDA-treated animals and 0 of the 5 control animals during the 2 months prior to necropsy, with no animals exhibiting signs of constipation. Conclusion Systemic administration of 6-OHDA to rhesus monkeys significantly reduced catecholaminergic expression in the colonic myenteric plexus without inducing constipation. These findings support the concept that ENS catecholaminergic loss is not responsible for constipation in PD. PMID:28090391

  1. In vitro detection and quantification of botulinum neurotoxin type E activity in avian blood

    Science.gov (United States)

    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.

  2. The Receptor Binding Domain of Botulinum Neurotoxin Stereotype C Binds Phosphoinositides

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    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.

  3. Clinical Uses of Botulinum Neurotoxins: Current Indications, Limitations and Future Developments

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    Sheng Chen

    2012-10-01

    Full Text Available 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.

  4. Eosinophil-Derived Neurotoxin Is Elevated in Patients with Amyotrophic Lateral Sclerosis

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    Guan-Ting Liu

    2013-01-01

    Full Text Available 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.

  5. Biodistribution and Lymphatic Tracking of the Main Neurotoxin of Micrurus fulvius Venom by Molecular Imaging

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    Irene Vergara

    2016-03-01

    Full Text Available 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.

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

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    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.

  7. Iterative structure-based peptide-like inhibitor design against the botulinum neurotoxin serotype A.

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    Jorge E Zuniga

    Full Text Available The botulinum neurotoxin serotype A light chain (BoNT/A LC protease is the catalytic component responsible for the neuroparalysis that is characteristic of the disease state botulism. Three related peptide-like molecules (PLMs were designed using previous information from co-crystal structures, synthesized, and assayed for in vitro inhibition against BoNT/A LC. Our results indicate these PLMS are competitive inhibitors of the BoNT/A LC protease and their K(i values are in the nM-range. A co-crystal structure for one of these inhibitors was determined and reveals that the PLM, in accord with the goals of our design strategy, simultaneously involves both ionic interactions via its P1 residue and hydrophobic contacts by means of an aromatic group in the P2' position. The PLM adopts a helical conformation similar to previously determined co-crystal structures of PLMs, although there are also major differences to these other structures such as contacts with specific BoNT/A LC residues. Our structure further demonstrates the remarkable plasticity of the substrate binding cleft of the BoNT/A LC protease and provides a paradigm for iterative structure-based design and development of BoNT/A LC inhibitors.

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

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

    2013-05-07

    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.

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

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    Lmar Babrak

    2016-01-01

    Full Text Available 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.

  10. Intraprostatic Botulinum Neurotoxin Type A Injection for Benign Prostatic Hyperplasia—A Spotlight in Reality

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    Yu-Chao Hsu

    2016-04-01

    Full Text Available 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.

  11. Botulinum neurotoxin and chronic migraine: muscle fiber chemodenervation or nociceptic system modulation?

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    A. R. Artemenko

    2013-01-01

    Full Text Available The results of controlled investigations suggest that botulinumtoxin type A (BTA leads to decrease headache intensity and prevent migraine attacks. The antinociceptive mechanisms of BTA action remain unclear. Modern and previous hypothesis of antinociceptive action BTA in chronic migraine (CM are discussed in details. Recent experimental and clinical evidence strongly suggest that BTA has aspecific antinociceptive effect realized through inhibition of proinflammatory neurotransmitters release not only from the sensory terminals but from muscle nociceptors. The mechanism of the action of BTA in CM has more than one target and is considered to involve different pathophysiological levels CM: neurogenic inflammation, peripheral and central sensitization. The administration of BTA on the PREEMPT principle (paradigm ensures optimal neurotoxin distribution in the anatomic areas in accordance with their sensory innervation by cervical segments and sensory fibers in the trigeminal system, the terminal branches which are the major target of BTA in the treatment of CM.

  12. Neutralization of botulinum neurotoxin by a human monoclonal antibody specific for the catalytic light chain.

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    Sharad P Adekar

    Full Text Available BACKGROUND: Botulinum neurotoxins (BoNT are a family of category A select bioterror agents and the most potent biological toxins known. Cloned antibody therapeutics hold considerable promise as BoNT therapeutics, but the therapeutic utility of antibodies that bind the BoNT light chain domain (LC, a metalloprotease that functions in the cytosol of cholinergic neurons, has not been thoroughly explored. METHODS AND FINDINGS: We used an optimized hybridoma method to clone a fully human antibody specific for the LC of serotype A BoNT (BoNT/A. The 4LCA antibody demonstrated potent in vivo neutralization when administered alone and collaborated with an antibody specific for the HC. In Neuro-2a neuroblastoma cells, the 4LCA antibody prevented the cleavage of the BoNT/A proteolytic target, SNAP-25. Unlike an antibody specific for the HC, the 4LCA antibody did not block entry of BoNT/A into cultured cells. Instead, it was taken up into synaptic vesicles along with BoNT/A. The 4LCA antibody also directly inhibited BoNT/A catalytic activity in vitro. CONCLUSIONS: An antibody specific for the BoNT/A LC can potently inhibit BoNT/A in vivo and in vitro, using mechanisms not previously associated with BoNT-neutralizing antibodies. Antibodies specific for BoNT LC may be valuable components of an antibody antidote for BoNT exposure.

  13. Construction of an insecticidal baculovirus expressing insect-specific neurotoxin AaIT

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    姚斌; 庞义; 范云六; 赵荣敏; 杨应昌; 王天原

    1996-01-01

    Considering the factors which affect gene transcription, translation and the stability of mRNA, without changing the amino acid composition of the encoded polypeptide, AaIT gene encoding insect-specific neurotoxin was designed and synthesized according to bias in codon choice, overall G+C content and G + C content of bases at the third position in codons of polyhedrin genes of baculovirus and of plant genes as well. AaIT gene was fused behind a synthetic gp67 signal sequence and then recombined into the genome of Trichoplusia ni nuclear polyhedrosis virus (TnNPV) by transfer vector pSXIV VI+X3. The recombinant virus TnNPV-AalT (occ+-gal-) was screened. The results of Southern blotting and SDS-PAGE demonstrated that AaIT gene had integrated into the genome of virus and expressed. Bioassays on the 3rd-instar Trichoplusia ni larvae showed that recombinant viruses TnNPV-AalT could shorten the time of killing insect and improve the efficiency of killing agronomically important insects.

  14. Cannabidiol Exposure During Neuronal Differentiation Sensitizes Cells Against Redox-Active Neurotoxins.

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    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.

  15. Botulinum neurotoxin D uses synaptic vesicle protein SV2 and gangliosides as receptors.

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    Lisheng Peng

    2011-03-01

    Full Text Available Botulinum neurotoxins (BoNTs include seven bacterial toxins (BoNT/A-G that target presynaptic terminals and act as proteases cleaving proteins required for synaptic vesicle exocytosis. Here we identified synaptic vesicle protein SV2 as the protein receptor for BoNT/D. BoNT/D enters cultured hippocampal neurons via synaptic vesicle recycling and can bind SV2 in brain detergent extracts. BoNT/D failed to bind and enter neurons lacking SV2, which can be rescued by expressing one of the three SV2 isoforms (SV2A/B/C. Localization of SV2 on plasma membranes mediated BoNT/D binding in both neurons and HEK293 cells. Furthermore, chimeric receptors containing the binding sites for BoNT/A and E, two other BoNTs that use SV2 as receptors, failed to mediate the entry of BoNT/D suggesting that BoNT/D binds SV2 via a mechanism distinct from BoNT/A and E. Finally, we demonstrated that gangliosides are essential for the binding and entry of BoNT/D into neurons and for its toxicity in vivo, supporting a double-receptor model for this toxin.

  16. Enhancing the Pharmacokinetic Properties of Botulinum Neurotoxin Serotype A Protease Inhibitors Through Rational Design.

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    Capek, Petr; Zhang, Yan; Barlow, Deborah J; Houseknecht, Karen L; Smith, Garry R; Dickerson, Tobin J

    2011-06-15

    Botulinum neurotoxin (BoNT), the etiological agent that causes the neuroparalytic disease botulism, has become a highly studied drug target in light of the potential abuse of this toxin as a weapon of bioterrorism. In particular, small molecule inhibitors of the light chain metalloprotease of BoNT serotype A have received significant attention and a number of small molecule and biologic inhibitors have been reported. However, all small molecules reported have been identified from either primary screens or medicinal chemistry follow-up studies, and the pharmacokinetic profiles of these compounds have not been addressed. In this study, we have removed the pharmacologic liabilities of one of the best compounds reported to date, 2,4-dichlorocinnamate hydroxamic acid, and in the process, uncovered a related class of benzothiophene hydroxamic acids that are significantly more potent inhibitors of the BoNT/A light chain, while also possessing greatly improved ADME properties, with the best compound showing the most potent inhibition of BoNT/A light chain reported (K(i) = 77 nM). Using a strategy of incorporating traditional drug development filters early into the discovery process, potential liabilities in BoNT/A lead compounds have been illuminated and removed, clearing the path for advancement into further pharmacologic optimization and in vivo efficacy testing.

  17. Botulinum neurotoxin: unique folding of enzyme domain of the most-poisonous poison.

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    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.

  18. Solution structure of robustoxin, the lethal neurotoxin from the funnel-web spider Atrax robustus.

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    Pallaghy, P K; Alewood, D; Alewood, P F; Norton, R S

    1997-12-15

    The solution structure of robustoxin, the lethal neurotoxin from the Sydney funnel-web spider Atrax robustus, has been determined from 2D 1H NMR data. Robustoxin is a polypeptide of 42 residues cross-linked by four disulphide bonds, the connectivities of which were determined from NMR data and trial structure calculations to be 1-15, 8-20, 14-31 and 16-42 (a 1-4/2-6/3-7/5-8 pattern). The structure consists of a small three-stranded, anti-parallel beta-sheet and a series of interlocking gamma-turns at the C-terminus. It also contains a cystine knot, thus placing it in the inhibitor cystine knot motif family of structures, which includes the omega-conotoxins and a number of plant and animal toxins and protease inhibitors. Robustoxin contains three distinct charged patches on its surface, and an extended loop that includes several aromatic and non-polar residues. Both of these structural features may play a role in its binding to the voltage-gated sodium channel.

  19. 1H NMR study of robustoxin, the lethal neurotoxin from the funnel web spider Atrax robustus.

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    Temple, M D; Hinds, M G; Sheumack, D D; Howden, M E; Norton, R S

    1999-03-01

    Robustoxin, the lethal neurotoxin from the Sydney funnel web spider Atrax robustus, is a polypeptide of 42 residues cross-linked by four disulfide bonds. This paper describes the sequence-specific assignment of resonances in the 1H nuclear magnetic resonance spectrum of robustoxin in aqueous solution. Several broad backbone amide resonances were encountered in spectra recorded at 27 degrees C, making the assignments at that temperature incomplete. In spectra recorded at lower temperatures these amide resonances became sharper, but others that were sharp at 27 degrees C became broad, indicative of conformational averaging on the millisecond timescale for certain regions of the structure. Nevertheless, it was possible to establish that robustoxin contains a small, triple-stranded, antiparallel beta-sheet and several reverse turns, but no alpha-helix. These observations indicate that this toxin may adopt the inhibitor cystine knot structure found in polypeptides from a diverse range of species, including a number of spiders. Analysis of the pH dependence of the spectrum yielded pKa values for Tyr22 and Tyr25, one of the three carboxyl groups, and the Lys residues.

  20. Botulinum neurotoxin type A for the treatment of pain: not just in migraine and trigeminal neuralgia.

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    Sandrini, Giorgio; De Icco, Roberto; Tassorelli, Cristina; Smania, Nicola; Tamburin, Stefano

    2017-12-01

    Despite their huge epidemiological impact, primary headaches, trigeminal neuralgia and other chronic pain conditions still receive suboptimal medical approach, even in developed countries. The limited efficacy of current pain-killers and prophylactic treatments stands among the main reasons for this phenomenon. Botulinum neurotoxin (BoNT) represents a well-established and licensed treatment for chronic migraine, but also an emerging treatment for other types of primary headache, trigeminal neuralgia, neuropathic pain, and an increasing number of pain conditions. We searched and critically reviewed evidence for the efficacy of BoNT for the treatment of chronic pain. Meta-analyses and randomized controlled trials (RCTs) suggest that BoNT potentially represents a multi-purpose drug for the treatment of pain in several disorders due to a favorable safety profile and a long-lasting relief after a single injection. BoNT is an emerging treatment in different pain conditions. Future RCTs should explore the use of BoNT injection therapy combined with systemic drugs and/or physical therapies as new pain treatment strategies.

  1. Effects on craniofacial growth and development of unilateral botulinum neurotoxin injection into the masseter muscle.

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    Tsai, Chi-Yang; Chiu, Wan Chi; Liao, Yi-Hsuan; Tsai, Chih-Mong

    2009-02-01

    The effects of botulinum neurotoxin type A (BoNT/A) on masseter muscles, when injected for cosmetic purposes (volumetric reduction) or treatment of excessive muscle activity (bruxism), have been investigated. However, the full anatomic effects of treatment are not known, particularly with respect to the mandible and relevant anthropometric measurements. The intent of this study was to use unilaterial BoNT/A injections to induce localized masseter atrophy and paresis and then to measure the effects of muscle influence on craniofacial growth and development. Growing male Wistar rats, 30 days old, were studied. The experimental group consisted of 8 rats. One side of the masseter muscle was injected with BoNT/A and the other side of the masseter muscle was injected with saline. The side with BoNT/A belonged to 1 group and the side with saline was the sham group. Three rats without injections was the control. After 45 days, the masseter muscles were dissected and weighed. Dry skulls were prepared, and anthropometric measurements determined. One-way ANOVA showed that the animals maintained their weight in both groups; however, the muscles injected with BoNT/A were smaller than the sham or control muscles. Anthropometric measurements of the bony structures attached to the masseter muscle showed a significant treatment effect. After localized masseter muscle atrophy induced by BoNT/A injection, alterations of craniofacial bone growth and development were seen. The results agree with the functional matrix theory that soft tissues regulate bone growth.

  2. Ouabain exacerbates botulinum neurotoxin-induced muscle paralysis via progression of muscle atrophy in mice.

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    Fujikawa, Ryu; Muroi, Yoshikage; Unno, Toshihiro; Ishii, Toshiaki

    2010-12-01

    Botulinum neurotoxin serotype A (BoNT/A) inhibits acetylcholine release at the neuromuscular junction in isolated muscles, and ouabain can partially block its effect. However, it is not clear whether ouabain attenuates BoNT/A-induced neuromuscular paralysis in vivo. In this work, we investigated the effects of ouabain on BoNT/A-induced neuromuscular paralysis in mice. Ouabain was administered to mice intraperitoneally immediately after a single injection of BoNT/A into skeletal muscle. The effects of ouabain on BoNT/A-induced muscle paralysis were assessed by quantitative monitoring of muscle tension and digit abduction via the digit abduction scoring (DAS) assay. A single administration of ouabain significantly prolonged BoNT/A-induced neuromuscular paralysis. Moreover, consecutive daily injection of ouabain exacerbated BoNT/A-induced neuromuscular paralysis, and led to a significant decrease in both twitch and tetanic forces as assayed in isolated BoNT/A-injected muscles. We next looked at the effects of ouabain on BoNT/A-induced muscle atrophy. Administration of ouabain led to a decrease in the myofibrillar cross-sectional area (CSAs) by 14 post-BoNT/A injection. In addition, repeated administration of ouabain increased mRNA expression levels of ubiquitin ligases, which are markers of muscle atrophy, in BoNT/A-injected muscle. These results suggest that ouabain exacerbates BoNT/A-induced neuromuscular paralysis via a marked progression of BoNT/A-induced muscle atrophy.

  3. Molecular Assembly of Clostridium botulinum progenitor M complex of type E.

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    Eswaramoorthy, Subramaniam; Sun, Jingchuan; Li, Huilin; Singh, Bal Ram; Swaminathan, Subramanyam

    2015-12-07

    Clostridium botulinum neurotoxin (BoNT) is released as a progenitor complex, in association with a non-toxic-non-hemagglutinin protein (NTNH) and other associated proteins. We have determined the crystal structure of M type Progenitor complex of botulinum neurotoxin E [PTC-E(M)], a heterodimer of BoNT and NTNH. The crystal structure reveals that the complex exists as a tight, interlocked heterodimer of BoNT and NTNH. The crystal structure explains the mechanism of molecular assembly of the complex and reveals several acidic clusters at the interface responsible for association at low acidic pH and disassociation at basic/neutral pH. The similarity of the general architecture between the PTC-E(M) and the previously determined PTC-A(M) strongly suggests that the progenitor M complexes of all botulinum serotypes may have similar molecular arrangement, although the neurotoxins apparently can take very different conformation when they are released from the M complex.

  4. Extraction and inhibition of enzymatic activity of botulinum neurotoxins/A1, /A2, and /A3 by a panel of monoclonal anti-BoNT/A antibodies.

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    Suzanne R Kalb

    Full Text Available Botulinum neurotoxins (BoNTs are extremely potent toxins that are capable of causing death or respiratory failure leading to long-term intensive care. Treatment includes serotype-specific antitoxins, which must be administered early in the course of the intoxication. Rapidly determining human exposure to BoNT is an important public health goal. In previous work, our laboratory focused on developing Endopep-MS, a mass spectrometry-based endopeptidase method for detecting and differentiating BoNT/A-G serotypes in buffer and BoNT/A, /B, /E, and /F in clinical samples. We have previously reported the effectiveness of antibody-capture to purify and concentrate BoNTs from complex matrices, such as clinical samples. Because some antibodies inhibit or neutralize the activity of BoNT, the choice of antibody with which to extract the toxin is critical. In this work, we evaluated a panel of 16 anti-BoNT/A monoclonal antibodies (mAbs for their ability to inhibit the in vitro activity of BoNT/A1, /A2, and /A3 complex as well as the recombinant LC of A1. We also evaluated the same antibody panel for the ability to extract BoNT/A1, /A2, and /A3. Among the mAbs, there were significant differences in extraction efficiency, ability to extract BoNT/A subtypes, and inhibitory effect on BoNT catalytic activity. The mAbs binding the C-terminal portion of the BoNT/A heavy chain had optimal properties for use in the Endopep-MS assay.

  5. Production and evaluation of a recombinant subunit vaccine against botulinum neurotoxin serotype B using a 293E expression system.

    Science.gov (United States)

    Yu, YunZhou; Shi, DanYang; Liu, Si; Gong, Zheng-Wei; Wang, Shuang; Sun, ZhiWei

    2015-01-01

    Although Escherichia coli and yeast were commonly used to express recombinant Hc of botulinum neurotoxins, as an alternative, in current study, a 293E expression system was used to express the Hc of botulinum neurotoxin serotype B (BHc) as soluble recombinant protein for experimental vaccine evaluation. Our results demonstrated that the 293E expression system could produce high level of recombinant secreted BHc protein, which was immunorecognized specifically by anti-botulinum neurotoxin serotype B (BoNT/B) sera and showed ganglioside binding activities. The serological response and efficacy of recombinant BHc formulated with aluminum hydroxide adjuvant were evaluated in mice. Immunization with Alhydrogel-formulated BHc subunit vaccine afforded the effective protection against BoNT/B challenge. A frequency- and dose-dependent effect to immunization with BHc subunit vaccine was observed and the ELISA antibody titers correlated well with neutralizing antibody titers and protection. And a solid-phase assay showed that the neutralizing antibodies from the BHc-immunized mice inhibited the binding of BHc to the ganglioside GT1b. Our results also show that the plasmid pABE293SBHc derived of the 293E expression system as DNA vaccine is capable of inducing stronger humoral response and protective efficacy against BoNT/B than the pVAX1SBHc. In summary, immunization with the 293E-expressed BHc protein generates effective immune protection against BoNT/B as E. coli or yeast-expressed BHc, so the efficient expression of botulinum Hc protein for experimental vaccine can be prepared using the 293E expression system.

  6. Recovery of central noradrenergic neurons one year after the administration of the neurotoxin DSP4.

    Science.gov (United States)

    Wolfman, C; Abó, V; Calvo, D; Medina, J; Dajas, F; Silveira, R

    1994-10-01

    The long-term effects of the systemic administration of DSP4 (N-(2-chloroethyl)N-ethyl-2-bromobenzylamine hydrochloride), a selective noradrenergic neurotoxin, on the endogenous levels of monoamines and their metabolites and on alpha- and beta-adrenoceptors in selected brain regions of the rat were examined. After 7 days, DSP4 caused a marked reduction (about 80%) of endogenous noradrenaline levels in locus coeruleus-innervated regions. At 90, 240 and 300 days after DSP4 injection, a partial and gradual recovery (50%, 41% and 25% of control values, respectively) of the noradrenaline cortical levels was evident. One year after DSP4 administration, brain regional noradrenaline stores were almost completely recovered. No changes in 5-hydroxytryptamine levels were observed in the three time intervals, but a mild decrease in cortical and hippocampal 5-hydroxyindolacetic acid levels was found 7 days after DSP4 injection. Following the profound noradrenaline depletion seen at 7 days, the cerebral cortical density of alpha 1-, alpha 2- and beta-adrenoceptors was significantly increased. Assessment of adrenergic receptors in cerebral cortex at 365 days after DSP4 injection, indicated that alpha 1- and alpha 2-adrenoceptor densities did not differ from control values; however, the density of beta-adrenoceptors remained increased. No changes were observed in the affinities of the three types of adrenoceptors studied. These results indicate that after a selective noradrenergic denervation induced by DSP4, there is a slow and gradual recovery of noradrenaline stores and of alpha 1- and alpha 2-adrenoceptor populations, suggesting a possible regrowth and/or collateral sprouting of noradrenergic terminals.

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

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    Yongfeng Fan

    2015-08-01

    Full Text Available 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.

  8. Botulinum Neurotoxin Type-A for the Treatment of Atypical Odontalgia.

    Science.gov (United States)

    Cuadrado, María-Luz; García-Moreno, Héctor; Arias, José-Antonio; Pareja, Juan A

    2016-09-01

    Atypical odontalgia (AO), a subform of persistent idiopathic facial pain, is defined as a continuous toothache in which a thorough examination reveals no dental pathology. AO is believed to be a neuropathic condition, given that some cases are preceded by dental procedures. Different topical and systemic medications have been used for the treatment of AO, but their effect is often unsatisfactory. The authors aimed to assess the effect and safety of botulinum neurotoxin type-A (BoNTA) in a series of patients with AO. Four patients with refractory AO (2 males and 2 females, aged 31-72) were treated with local injections of BoNTA to the painful area. BoNTA was injected at various sites into the gums, and two patients had additional injections in the hard palate or the upper lip. The total dose of BoNTA for each procedure was 15-30 U, and the total number of injection points was 6-12. The follow-up ranged from 6 to 20 months. Two patients received two cycles of BoNTA, while the remaining patients received three and five cycles each, respectively. All patients obtained significant relief with complete or almost complete reduction of pain. The analgesic effect was apparent after a latency period of 3-14 days, and the effect persisted for 2-6 months. There were no adverse events reported from any of the interventions. The responses to BoNTA injections in this series agree with those previously observed in neuropathic pain. BoNTA injections may be a safe and effective option for the treatment of AO. © 2016 American Academy of Pain Medicine. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  9. Inhibitory effects of recombinant neurotoxin BmK IM on seizures induced by pentylenetetrazol in Rats

    Institute of Scientific and Technical Information of China (English)

    何小华; 彭方; 章军建; 李文鑫; 曾宪春; 刘辉

    2003-01-01

    Objective To elucidate the inhibitory effects of recombinant Chinese scorpion neurotoxin BmK IM on seizures induced by pentylenetetrazol (PTZ) and the possible mechanism.Methods After purifying recombinant BmK IM from an E. coli cell line, its toxicity (both LD50 and minimum lethal dose) on rats was determined. BmK IM was then microinjected into the CA3 region of the right hippocampus and its ability to inhibit the effects of an intraperitoneal injection of PTZ was assessed. The effects of BmK IM on the electrophysiological properties of isolated CA3 pyramidal neurons were then studied using whole-cell patch clamp techniques.Results BmK IM can significantly prolong the latent period of epileptic seizures, decrease the degree of seizures, and decrease the frequency of epileptiform discharges induced by PTZ. At the same time, 24h after injection of BmK IM into the hippocampal tissue, BmK IM significantly reduces the concentration of the neurotransmitter glutamate and alleviates PTZ-induced lesions in the hippocampus. Whole-cell patch clamp recordings indicate that BmK IM inhibits INa of rat hippocampal neurons in a dose-dependent manner. BmK IM significantly shifts the activation curve of INa in a positive direction, indicating that BmK IM enhances the threshold potential of INa.Conclusions BmK IM has significant anti-epileptic properties, and may prove useful as a drug in the therapy of epilepsy. The inhibitory effects of BmK IM on seizures caused by pentylenetetrazol might depend on reductions in the release of presynaptic glutamate via the blocking of Na+ channels.

  10. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library.

    Science.gov (United States)

    Wang, Han; Yu, Rui; Fang, Ting; Yu, Ting; Chi, Xiangyang; Zhang, Xiaopeng; Liu, Shuling; Fu, Ling; Yu, Changming; Chen, Wei

    2016-09-11

    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.

  11. Production and evaluation of a recombinant chimeric vaccine against clostridium botulinum neurotoxin types C and D.

    Science.gov (United States)

    Gil, Luciana A F; da Cunha, Carlos Eduardo P; Moreira, Gustavo M S G; Salvarani, Felipe M; Assis, Ronnie A; Lobato, Francisco Carlos F; Mendonça, Marcelo; Dellagostin, Odir A; Conceição, Fabricio R

    2013-01-01

    Bovine botulism is a fatal disease that is caused by botulinum neurotoxins (BoNTs) produced by Clostridium botulinum serotypes C and D and that causes great economic losses, with nearly 100% lethality during outbreaks. It has also been considered a potential source of human food-borne illness in many countries. Vaccination has been reported to be the most effective way to control bovine botulism. However, the commercially available toxoid-based vaccines are difficult and hazardous to produce. Neutralizing antibodies targeted against the C-terminal fragment of the BoNT heavy chain (HC) are known to confer efficient protection against lethal doses of BoNTs. In this study, a novel recombinant chimera, consisting of Escherichia coli heat-labile enterotoxin B subunit (LTB), a strong adjuvant of the humoral immune response, fused to the HC of BoNT serotypes C and D, was produced in E. coli. Mice vaccinated with the chimera containing LTB and an equivalent molar ratio of the chimera without LTB plus aluminum hydroxide (Al(OH)3) developed 2 IU/mL of antitoxins for both serotypes. Guinea pigs immunized with the recombinant chimera with LTB plus Al(OH)3 developed a protective immune response against both BoNT/C (5 IU/mL) and BoNT/D (10 IU/mL), as determined by a mouse neutralization bioassay with pooled sera. The results achieved with guinea pig sera fulfilled the requirements of commercial vaccines for prevention of botulism, as determined by the Brazilian Ministry of Agriculture, Livestock and Food, Supply. The presence of LTB was essential for the development of a strong humoral immune response, as it acted in synergism with Al(OH)3. Thus, the vaccine described in this study is a strong candidate for the control of botulism in cattle.

  12. Production and evaluation of a recombinant chimeric vaccine against clostridium botulinum neurotoxin types C and D.

    Directory of Open Access Journals (Sweden)

    Luciana A F Gil

    Full Text Available Bovine botulism is a fatal disease that is caused by botulinum neurotoxins (BoNTs produced by Clostridium botulinum serotypes C and D and that causes great economic losses, with nearly 100% lethality during outbreaks. It has also been considered a potential source of human food-borne illness in many countries. Vaccination has been reported to be the most effective way to control bovine botulism. However, the commercially available toxoid-based vaccines are difficult and hazardous to produce. Neutralizing antibodies targeted against the C-terminal fragment of the BoNT heavy chain (HC are known to confer efficient protection against lethal doses of BoNTs. In this study, a novel recombinant chimera, consisting of Escherichia coli heat-labile enterotoxin B subunit (LTB, a strong adjuvant of the humoral immune response, fused to the HC of BoNT serotypes C and D, was produced in E. coli. Mice vaccinated with the chimera containing LTB and an equivalent molar ratio of the chimera without LTB plus aluminum hydroxide (Al(OH3 developed 2 IU/mL of antitoxins for both serotypes. Guinea pigs immunized with the recombinant chimera with LTB plus Al(OH3 developed a protective immune response against both BoNT/C (5 IU/mL and BoNT/D (10 IU/mL, as determined by a mouse neutralization bioassay with pooled sera. The results achieved with guinea pig sera fulfilled the requirements of commercial vaccines for prevention of botulism, as determined by the Brazilian Ministry of Agriculture, Livestock and Food, Supply. The presence of LTB was essential for the development of a strong humoral immune response, as it acted in synergism with Al(OH3. Thus, the vaccine described in this study is a strong candidate for the control of botulism in cattle.

  13. Neuroprotection effects of retained acupuncture in neurotoxin-induced Parkinson's disease mice.

    Science.gov (United States)

    Yang, Jen-Lin; Chen, Jay S C; Yang, Yi-Fei; Chen, Jyh-Cheng; Lin, Ching-Huang; Chang, Rong-Seng; Tsao, Po-Jui; Chen, Fang-Pey; Chern, Chang-Ming; Tsai, Tung-Hu; Chiu, Jen-Hwey

    2011-10-01

    The aim of this study was to investigate the role of retained acupuncture (RA) in neurotoxin-induced Parkinson's disease (PD) mice. Male C57BL/6 mice were injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to induce the PD model. The mice were divided into four groups, namely, (1) normal; (2) MPTP+retained acupuncture (RA); (3) MPTP+electroacupuncture (EA); (4) MPTP+sham acupuncture (SA). After mice being manipulated with/without acupuncture at acupoints (Daling, PC 7), groups 2-4 were injected with MPTP (15 mg/kg/d). The mice were evaluated for behavioral changes, in terms of time of landing, after acupuncture treatment. The animals were sacrificed and their brains assayed for dopamine and its metabolites and tyrosine hydroxylase (TH) expression by using HPLC and immunohistochemistry/Western blotting, respectively. [(123)I] IBZM-SPECT imaging between SA and RA groups were compared. The results showed that the time of landing of the three groups with treatment was significant longer than group 1 (normal) (4.33±0.15 s). Nonetheless, group 2 (RA) (7.13±0.20 s) had a shorter time of landing than group 4 (SA) (7.89±0.46 s). The number of TH (+) neurons and the expression of TH proteins were significantly higher in the RA group than in the SA/EA groups. RA also increased the uptake of [(123)I] IBZM into the triatum compared to the SA group. We conclude that RA possibly attenuates neuronal damage in MPTP-induced PD mice, which suggests RA may be useful as a complementary strategy when treating human PD.

  14. Pre-Clinical Study of a Novel Recombinant Botulinum Neurotoxin Derivative Engineered for Improved Safety.

    Science.gov (United States)

    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.

  15. A three monoclonal antibody combination potently neutralizes multiple botulinum neurotoxin serotype F subtypes

    Science.gov (United States)

    Fan, Yongfeng; Garcia-Rodriguez, Consuelo; Lou, Jianlong; Wen, Weihua; Conrad, Fraser; Zhai, Wenwu; Smith, Theresa J.; Smith, Leonard A.

    2017-01-01

    Human botulism is primarily caused by botulinum neurotoxin (BoNT) serotypes A, B and E, with around 1% caused by serotype F (BoNT/F). BoNT/F comprises at least seven different subtypes with the amino acid sequence difference between subtypes as high as 36%. The sequence differences present a significant challenge for generating monoclonal antibodies (mAbs) that can bind, detect and neutralize all BoNT/F subtypes. We used repertoire cloning of immune mouse antibody variable (V) regions and yeast display to generate a panel of 33 lead single chain Fv (scFv) mAbs that bound one or more BoNT/F subtypes with a median equilibrium dissociation constant (KD) of 4.06 × 10−9 M. By diversifying the V-regions of the lead mAbs and selecting for cross reactivity we generated five mAbs that bound each of the seven subtypes. Three scFv binding non-overlapping epitopes were converted to IgG that had KD for the different BoNT/F subtypes ranging from 2.2×10−8 M to 1.47×10−12 pM. An equimolar combination of the mAbs was able to potently neutralize BoNT/F1, F2, F4 and F7 in the mouse neutralization assay. The mAbs have potential utility as diagnostics capable of recognizing the known BoNT/F subtypes and could be developed as antitoxins to prevent and treat type F botulism. PMID:28323873

  16. Expression and biochemical characterization of light chains of Botulinum neurotoxin subtypes F5 and F7.

    Science.gov (United States)

    Guo, Jiubiao; Chen, Sheng

    2015-07-01

    Botulinum neurotoxins are the most potent protein toxins known to human. To date, seven subtypes of the BoNT/F serotype (BoNT/F1 to BoNT/F7) have been identified, among which BoNT/F5 and BoNT/F7 are the most divergent. However, little structural and functional information is available for these two subtypes due to a lack of suitable recombinant proteins for biochemical characterization, except that they appear to possess unique substrate recognition mechanisms, thereby impeding development of vaccine or inhibitors against these proteins. In the present study, we utilized a combinatorial approach which involved examining the effects of different affinity tags, mapping C-terminal truncation mutants and optimization of expression and purification conditions, that allowed us to successfully express and purify soluble and highly active recombinant LC/F5 and LC/F7 proteins. GST-LC/F5(1-450) and 6× His-LC/F5(1-405) were the formats which exhibit the highest level of solubility and activity, whereas GST-LC/F7(1-405) was the most active form of LC/F7. In comparison, GST-LC/F5(1-450) was more active than GST-LC/F7(1-405), which was in turn more active than the LC/F1 control. Our data suggest that solubility of these proteins strongly correlated with their catalytic activity. Successful expression and purification of LC/F5 and LC/F7 in this work will, for the first time, provide materials for further characterization of these two subtypes of BoNT/F, which is essential for future development of protective vaccine or other therapeutic strategies, as well as BoNT/F protein engineering. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. A novel paradigm for assessing efficacies of potential antidotes against neurotoxins in mice.

    Science.gov (United States)

    Crankshaw, Daune L; Goon, David J W; Briggs, Jacquie E; DeLong, David; Kuskowski, Michael; Patterson, Steven E; Nagasawa, Herbert T

    2007-12-10

    Historically, antidotal potencies of cyanide antagonists were measured as increases in the experimental LD(50) for cyanide elicited by the antidotes. This required the use of high doses of cyanide following pre-treatment with the putative antidote. Since IACUC guidelines at our institutions strongly discourage LD(50) determinations: we developed a new test paradigm that allowed for maximal survival of cyanide-treated animals with greatly reduced numbers of animals. Symptoms of cyanide toxicity include disruption of neuromuscular coordination, i.e., the righting reflex. Therefore, to establish a dose-response curve, the times required for recovery of this righting reflex with increasing doses of cyanide were measured. A cyanide dose that disrupted this righting reflex for approximately 1h with minimal deaths was then selected. Using this paradigm, the current cyanide antidotes, viz., nitrite plus thiosulfate and hydroxocobalamin, as well as some potential cyanide antidotes that we developed, were evaluated pre- and post-cyanide. This allowed, for the first time, the assessment of the post-cyanide effectiveness of the current antidotes against cyanide poisoning in a live animal. In addition, some prototype compounds were found to exhibit antidotal efficacy not only when injected i.p. following cyanide, but also when administered orally 30 min before cyanide. Pre-cyanide oral efficacy suggests that such compounds have the potential of being administered prophylactically before exposure to cyanide. This new test paradigm was found to be a powerful tool for assessing the efficacies of some novel antidotes against cyanide and should be equally applicable for evaluating putative antidotes for other neurotoxins.

  18. Accelerated neuronal cell recovery from Botulinum neurotoxin intoxication by targeted ubiquitination.

    Directory of Open Access Journals (Sweden)

    Chueh-Ling Kuo

    Full Text Available Botulinum neurotoxin (BoNT, a Category A biodefense agent, delivers a protease to motor neuron cytosol that cleaves one or more soluble NSF attachment protein receptors (SNARE proteins involved in neurotransmission to cause a flaccid paralysis. No antidotes exist to reverse symptoms of BoNT intoxication so severely affected patients require artificial respiration with prolonged intensive care. Time to recovery depends on toxin serotype because the intraneuronal persistence of the seven known BoNT serotypes varies widely from days to many months. Our therapeutic antidote strategy is to develop 'targeted F-box' (TFB agents that target the different intraneuronal BoNT proteases for accelerated degradation by the ubiquitin proteasome system (UPS, thus promoting rapid recovery from all serotypes. These agents consist of a camelid heavy chain-only V(H (VHH domain specific for a BoNT protease fused to an F-box domain recognized by an intraneuronal E3-ligase. A fusion protein containing the 14 kDa anti-BoNT/A protease VHH, ALcB8, joined to a 15 kDa F-box domain region of TrCP (D5 was sufficient to cause increased ubiquitination and accelerate turnover of the targeted BoNT/A protease within neurons. Neuronal cells expressing this TFB, called D5-B8, were also substantially resistant to BoNT/A intoxication and recovered from intoxication at least 2.5 fold quicker than control neurons. Fusion of D5 to a VHH specific for BoNT/B protease (BLcB10 led to accelerated turnover of the targeted protease within neurons, thus demonstrating the modular nature of these therapeutic agents and suggesting that development of similar therapeutic agents specific to all botulinum serotypes should be readily achievable.

  19. Widespread sequence variations in VAMP1 across vertebrates suggest a potential selective pressure from botulinum neurotoxins.

    Science.gov (United States)

    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

    2014-07-01

    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.

  20. Eosinophil-derived neurotoxin, elastase, and cytokine profile in effusion from eosinophilic otitis media.

    Science.gov (United States)

    Uchimizu, Hirotaka; Matsuwaki, Yoshinori; Kato, Masahiko; Otori, Nobuyosi; Kojima, Hiromi

    2015-09-01

    Eosinophilic otitis media (EOM) is an intractable disease characterized by a remarkably viscous effusion and accumulation of numerous eosinophils in both the middle ear effusion and the mucosa. The key factors in EOM pathogenesis remain unclear. The purpose of this study is to identify the important factors involved in EOM pathogenesis. Middle ear effusion samples were collected from 12 patients with EOM and 9 patients with secretory otitis media (SOM), as controls. Multiple cytokines in the effusion were measured using a Bio-Plex™ Human Cytokine 27-Plex panel. Eosinophil-derived neurotoxin (EDN) and elastase were measured by ELISA. The concentrations of EDN, elastase, and each cytokine were compared between the EOM and SOM groups. Furthermore, in the EOM group, each cytokine was examined for correlation with EDN and elastase. EDN and elastase concentrations were significantly higher in the EOM group than in the SOM group (p < 0.05). IL-5, IL-1β, MIP-1α, G-CSF, IL-1ra, IL-4, IFN-γ, MIP-1β, IL-10, TNF-α, VEGF, and IL-2 concentration was significantly higher in the EOM group than in the SOM group (p < 0.05). Significant positive correlations were found between EDN and IL-1ra, IL-2, IL-5, IL-9, IL-13, eotaxin, MIP-1α, PDGF-BB, and RANTES in the EOM group (p < 0.05). Our study showed that IL-5, IL-2, MIP-1α, and IL-1ra are the important factors involved in EOM pathogenesis. Furthermore, not only eosinophil, but also neutrophil are involved in middle ear inflammation of EOM. Copyright © 2015 Japanese Society of Allergology. Production and hosting by Elsevier B.V. All rights reserved.

  1. Monoclonal Antibodies Targeting the Alpha-Exosite of Botulinum Neurotoxin Serotype/A Inhibit Catalytic Activity.

    Directory of Open Access Journals (Sweden)

    Yongfeng Fan

    Full Text Available 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.

  2. Botulinum neurotoxin type A versus punctal plug insertion in the management of dry eye disease

    Directory of Open Access Journals (Sweden)

    Amal A Bukhari

    2014-01-01

    Full Text Available Purpose: To compare the efficacies of punctal plug insertion and Botulinum toxin injection in dry eye disease not responding to topical medications. Materials and Methods: A non-controlled randomized clinical trial of two parallel groups of 60 dry eye patients seen in the clinic not responding to topical medications were divided into two groups. One group received punctal plugs and the other group received Botulinum toxin injections to prevent lacrimal tear drainage. Results: Of a total of 36 patients with a mean age of 44.5 years who received punctal plugs, 50% of them experienced improvements in the clinical manifestations of their disease. 12/36 (33.3% developed plug extrusion, and 6/36 (16.7% patients developed conjunctival erosions with irritation that necessitated plug removal within one week of insertion. A total of 24 patients with a mean age of 47.5 years received injections of Botulinum toxin. Of these, 83.3% had improvement in all of the clinical manifestations of dry eye. 4/24 (16.7% had no improvement in the degrees to which they experienced foreign body sensations, 33.3% reported shampoo entering the eye while showering. All of the patients who received Botulinum toxin injections were satisfied with the results of their treatment, whereas only 72.3% of the patients who received punctal plugs were satisfied with their results. Conclusion: Botulinum neurotoxin A injections can be a very good alternative to punctal plugs in improving the clinical manifestations of dry eye disease They are associated with the development of fewer and milder complications and with higher levels of patient satisfaction.

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

    Science.gov (United States)

    Chatla, Kamalakar; Gaunt, Patricia; Petrie-Hanson, Lora; Hohn, Claudia; Ford, Lorelei; Hanson, Larry

    2014-03-01

    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 signs and mortality after sera from affected fish is administered to sentinel catfish. The diagnosis is confirmed if the toxicity is neutralized with BoNT/E antitoxin. Because small catfish are often unavailable, the utility of adult zebrafish (Danio rerio) was evaluated in BoNT/E and VTC bioassays. Channel catfish and zebrafish susceptibilities were compared using trypsin-activated BoNT/E in a 96-hr trial by intracoelomically administering 0, 1.87, 3.7, 7.5, 15, or 30 pg of toxin per gram of body weight (g-bw) of fish. All of the zebrafish died at the 7.5 pg/g-bw and higher, while the catfish died at the 15 pg/g-bw dose and higher. To test the bioassay, sera from VTC-affected fish or control sera were intracoelomically injected at a dose of 10 µl per zebrafish and 20 µl/g-bw for channel catfish. At 96 hr post-injection, 78% of the zebrafish and 50% of the catfish receiving VTC sera died, while no control fish died. When the VTC sera were preincubated with BoNT/E antitoxin, they became nontoxic to zebrafish. Histology of zebrafish injected with either VTC serum or BoNT/E demonstrated renal necrosis. Normal catfish serum was toxic to larval zebrafish in immersion exposures, abrogating their utility in VTC bioassays. The results demonstrate bioassays using adult zebrafish for detecting BoNT/E and VTC are sensitive and practical.

  4. A comparative study on three analytical methods for the determination of the neurotoxin BMAA in cyanobacteria.

    Directory of Open Access Journals (Sweden)

    Elisabeth J Faassen

    Full Text Available The cyanobacterial neurotoxin β-N-methylamino-L-alanine (BMAA has been considered a serious health threat because of its putative role in multiple neurodegenerative diseases. First reports on BMAA concentrations in cyanobacteria were alarming: nearly all cyanobacteria were assumed to contain high BMAA concentrations, implying ubiquitous exposure. Recent studies however question this presence of high BMAA concentrations in cyanobacteria. To assess the real risk of BMAA to human health, this discrepancy must be resolved. We therefore tested whether the differences found could be caused by the analytical methods used in different studies. Eight cyanobacterial samples and two control samples were analyzed by three commonly used methods: HPLC-FLD analysis and LC-MS/MS analysis of both derivatized and underivatized samples. In line with published results, HPLC-FLD detected relatively high BMAA concentrations in some cyanobacterial samples, while both LC-MS/MS methods only detected BMAA in the positive control (cycad seed sarcotesta. Because we could eliminate the use of different samples and treatments as causal factors, we demonstrate that the observed differences were caused by the analytical methods. We conclude that HPLC-FLD overestimated BMAA concentrations in some cyanobacterial samples due to its low selectivity and propose that BMAA might be present in (some cyanobacteria, but in the low µg/g or ng/g range instead of the high µg/g range as sometimes reported before. We therefore recommend to use only selective and sensitive analytical methods like LC-MS/MS for BMAA analysis. Although possibly present in low concentrations in cyanobacteria, BMAA can still form a health risk. Recent evidence on BMAA accumulation in aquatic food chains suggests human exposure through consumption of fish and shellfish which expectedly exceeds exposure through cyanobacteria.

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

    Directory of Open Access Journals (Sweden)

    Yun Jeong Kim

    Full Text Available 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.

  6. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library

    Science.gov (United States)

    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

  7. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library

    Directory of Open Access Journals (Sweden)

    Han Wang

    2016-09-01

    Full Text Available 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.

  8. Discovery of nuclear-encoded genes for the neurotoxin saxitoxin in dinoflagellates.

    Science.gov (United States)

    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 >1.2×10(6) 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.

  9. Epitopic Profiling of Antibody Response against Neurotoxins from the Black Mamba (Dendroaspis polylepis)

    DEFF Research Database (Denmark)

    Jespersen, Martin Closter; Engmark, Mikael; Laustsen, Andreas Hougaard

    The black mamba (Dendroaspis Polylepis) is among the most dangerous snakes in the world, with a venom dominated by three-finger toxins and dendrotoxins. Among the three-finger toxins, the α-neurotoxins (α-NT) are the most important, and these are conserved between snake species. Cross......-reactivity between threefinger toxins is known to occur, and understanding this phenomenon in depth may help guide future design of antivenoms to obtain optimal specificity against medically important toxins from different snake species. Using a bioinformatic approach, we investigated the cross-reactivity be- tween...

  10. Amino acid sequence of versutoxin, a lethal neurotoxin from the venom of the funnel-web spider Atrax versutus.

    Science.gov (United States)

    Brown, M R; Sheumack, D D; Tyler, M I; Howden, M E

    1988-03-01

    The complete amino acid sequence of versutoxin, a lethal neurotoxic polypeptide isolated from the venom of male and female funnel-web spiders of the species Atrax versutus, was determined. Sequencing was performed in a gas-phase protein sequencer by automated Edman degradation of the S-carboxymethylated toxin and fragments of it produced by reaction with CNBr. Versutoxin consisted of a single chain of 42 amino acid residues. It was found to have a high proportion of basic residues and of cystine. The primary structure showed marked homology with that of robustoxin, a novel neurotoxin recently isolated from the venom of another funnel-web-spider species, Atrax robustus.

  11. Toxicity evolution of Vipera aspis aspis venom: identification and molecular modeling of a novel phospholipase A(2) heterodimer neurotoxin.

    Science.gov (United States)

    Jan, Virginie; Maroun, R C; Robbe-Vincent, Annie; De Haro, Luc; Choumet, Valérie

    2002-09-11

    We report the simultaneous presence of two phospholipase A(2) (PLA(2)) neurotoxins in the venom of Vipera aspis aspis, the first such observation. One is monomeric and identical to ammodytoxin B of Vipera ammodytes ammodytes. Its presence may result from gene flux after interbreeding between V. aspis aspis and V. ammodytes ammodytes. The second, a novel heterodimer named vaspin, is very similar to vipoxin of Vipera ammodytes meridionalis and to PLA(2)-I of Vipera aspis zinnikeri. It may result from expression of preexisting genes, the acidic subunit evolving from an ancestor common to ammodytin I2 from V. ammodytes ammodytes, which we also found in V. aspis aspis.

  12. Researchers unmask secret to long-lasting effects of botulinum neurotoxin A in motor neurons | Center for Cancer Research

    Science.gov (United States)

    A team of scientists led by the Center for Cancer Research's Allan M. Weissman, M.D., and Yien Che Tsai, Ph.D., has discovered a molecular mechanism that explains the extreme toxicity of botulinum neurotoxin A (BoNT/A), the most potent BoNT strain. The discovery, published June 5 in PNAS, also identifies a molecular target that the researchers hope will eventually lead to improved therapies to treat exposure and severely undermine the potential use of BoNTs as bioweapons.  Read more...  

  13. Crystal structures of human pyridoxal kinase in complex with the neurotoxins, ginkgotoxin and theophylline: insights into pyridoxal kinase inhibition.

    Directory of Open Access Journals (Sweden)

    Amit K Gandhi

    Full Text Available Several drugs and natural compounds are known to be highly neurotoxic, triggering epileptic convulsions or seizures, and causing headaches, agitations, as well as other neuronal symptoms. The neurotoxic effects of some of these compounds, including theophylline and ginkgotoxin, have been traced to their inhibitory activity against human pyridoxal kinase (hPL kinase, resulting in deficiency of the active cofactor form of vitamin B₆, pyridoxal 5'-phosphate (PLP. Pyridoxal (PL, an inactive form of vitamin B₆ is converted to PLP by PL kinase. PLP is the B₆ vitamer required as a cofactor for over 160 enzymatic activities essential in primary and secondary metabolism. We have performed structural and kinetic studies on hPL kinase with several potential inhibitors, including ginkgotoxin and theophylline. The structural studies show ginkgotoxin and theophylline bound at the substrate site, and are involved in similar protein interactions as the natural substrate, PL. Interestingly, the phosphorylated product of ginkgotoxin is also observed bound at the active site. This work provides insights into the molecular basis of hPL kinase inhibition and may provide a working hypothesis to quickly screen or identify neurotoxic drugs as potential hPL kinase inhibitors. Such adverse effects may be prevented by administration of an appropriate form of vitamin B₆, or provide clues of how to modify these drugs to help reduce their hPL kinase inhibitory effects.

  14. Clostridium botulinum strain Af84 contains three neurotoxin gene clusters: bont/A2, bont/F4 and bont/F5.

    Directory of Open Access Journals (Sweden)

    Nir Dover

    Full Text Available Sanger and shotgun sequencing of Clostridium botulinum strain Af84 type Af and its botulinum neurotoxin gene (bont clusters identified the presence of three bont gene clusters rather than the expected two. The three toxin gene clusters consisted of bont subtypes A2, F4 and F5. The bont/A2 and bont/F4 gene clusters were located within the chromosome (the latter in a novel location, while the bont/F5 toxin gene cluster was located within a large 246 kb plasmid. These findings are the first identification of a C. botulinum strain that contains three botulinum neurotoxin gene clusters.

  15. Structural analysis of Clostridium botulinum neurotoxin type D as a platform for the development of targeted secretion inhibitors.

    Science.gov (United States)

    Masuyer, Geoffrey; Davies, Jonathan R; Moore, Kevin; Chaddock, John A; Ravi Acharya, K

    2015-09-01

    The botulinum neurotoxin type D is one of seven highly potent toxins produced by Clostridium botulinum which inhibit neurotransmission at cholinergic nerve terminals. A functional fragment derived from the toxin, LHn, consisting of the catalytic and translocation domains, has been heralded as a platform for the development of targeted secretion inhibitors. These secretion inhibitors are aimed at retargeting the toxin towards a specific cell type to inhibit vesicular secretion. Here we report crystal structures of LHn from serotype D at 2.3 Å, and that of SXN101959 at 3.1 Å resolution. SXN101959, a derivative that combines LHn from serotype D with a fragment of the growth hormone releasing hormone, has previously revealed promising results in inhibiting growth hormone release in pituitary somatotrophs. These structures offer for the first time insights into the translocation domain interaction with the catalytic domain in serotype D. Furthermore, structural information from small-angle X-ray scattering of LHn/D is compared among serotypes A, B, and D. Taken together, these results demonstrate the robustness of the 'LHn fold' across serotypes and its use in engineering additional polypeptide components with added functionality. Our study demonstrates the suitability of botulinum neurotoxin, and serotype D in particular, as a basis for engineering novel secretion inhibitors.

  16. A Label Free Colorimetric Assay for the Detection of Active Botulinum Neurotoxin Type A by SNAP-25 Conjugated Colloidal Gold

    Directory of Open Access Journals (Sweden)

    Christopher Gwenin

    2013-08-01

    Full Text Available Botulinum neurotoxins are one of the most potent toxins known to man. Current methods of detection involve the quantification of the toxin but do not take into account the percentage of the toxin that is active. At present the assay used for monitoring the activity of the toxin is the mouse bioassay, which is lengthy and has ethical issues due to the use of live animals. This report demonstrates a novel assay that utilises the endopeptidase activity of the toxin to detect Botulinum neurotoxin in a pharmaceutical sample. The cleaving of SNAP-25 is monitored via UV-Visible spectroscopy with a limit of detection of 373 fg/mL and has been further developed into a high throughput method using a microplate reader detecting down to 600 fg/mL of active toxin. The results show clear differences between the toxin product and the placebo, which contains the pharmaceutical excipients human serum albumin and lactose, showing that the assay detects the active form of the toxin.

  17. Development of a Highly Sensitive Cell-Based Assay for Detecting Botulinum Neurotoxin Type A through Neural Culture Media Optimization.

    Science.gov (United States)

    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.

  18. Molecular Characteristics of Four New Depressant Insect Neurotoxins Purified From Venom of Buthus martensi Karsch by HPLC

    Institute of Scientific and Technical Information of China (English)

    吉永华; H.Hattori; 徐科; S.Terakawa

    1994-01-01

    Suitable pattern and high yield were obtained when the reverse-phase performance liquidchromatography (RP-HPLC) was used to separate neurotoxins from venom of Chinese scorpion Buthusmartensi Karsch.Using this technique,the venom was first separated to two main regions.The toxicitytests show that the insect-selective neurotoxical components are concentrated in the latter region,from whichfive insect-selective neurotoxins designated by BmK IT1-IT5 were obtained.According to the results of thetoxicity test as well as the amino acid composition and N-terminal analyses,BmK IT1 is the excitatory insectneurotoxin as reported in a previous paper,and the others are the newly found depressant insect-selectiveneurotoxins.The molecules of all the four toxins are single-chain minipeptides of about 60 amino acids.Their isoelectric points (pI) are between 8.3 and 8.5.The fact that BmK IT2 loses completely its insect tox-icity after being modified by fluorochrome shows that the positive charges on the molecular surface of thiskind of toxins are important to maintaining the bioactivity of the molecules.

  19. Three-dimensional ordered macroporous (3DOM) composite for electrochemical study on acetylcholinesterase inhibition induced by endogenous neurotoxin.

    Science.gov (United States)

    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.

  20. NO DECREASE IN MUSCLE STRENGTH AFTER BOTULINUM NEUROTOXIN-A INJECTION IN CHILDREN WITH CEREBRAL PALSY

    Directory of Open Access Journals (Sweden)

    Meta Nyström Eek

    2016-10-01

    Full Text Available 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 cerebral palsy. A secondary aim was to relate the effect of BoNT-A to gait pattern and range of motion.Twenty children with spastic cerebral palsy were included in the study, eight 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 six weeks after at the peak effect of BoNT-A, and at six 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

  1. No Decrease in Muscle Strength after Botulinum Neurotoxin-A Injection in Children with Cerebral Palsy

    Science.gov (United States)

    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

  2. No Decrease in Muscle Strength after Botulinum Neurotoxin-A Injection in Children with Cerebral Palsy.

    Science.gov (United States)

    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

  3. Crystal Structure of the Botulinum Neurotoxin Type G Binding Domain: Insight into Cell Surface Binding

    Energy Technology Data Exchange (ETDEWEB)

    Stenmark, Pål; Dong, Min; Dupuy, Jérôme; Chapman, Edwin R.; Stevens, Raymond C. (Scripps); (UW)

    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.

  4. Grey Matter Microstructural Integrity Alterations in Blepharospasm Are Partially Reversed by Botulinum Neurotoxin Therapy

    Science.gov (United States)

    Chirumamilla, Venkata Chaitanya; Koirala, Nabin; Paktas, Burcu; Deuschl, Günther; Zeuner, Kirsten E.; Groppa, Sergiu

    2016-01-01

    Objective Benign Essential Blepharospasm (BEB) and hemifacial spasm (HFS) are the most common hyperkinetic movement disorders of facial muscles. Although similar in clinical presentation different pathophysiological mechanisms are assumed. Botulinum Neurotoxin (BoNT) is a standard evidence-based treatment for both conditions. In this study we aimed to assess grey matter microstructural differences between these two groups of patients and compared them with healthy controls. In patients we furthermore tracked the longitudinal morphometric changes associated with BoNT therapy. We hypothesized microstructural differences between the groups at the time point of maximum symptoms representation and distinct longitudinal grey matter dynamics with symptom improvement. Methods Cross-sectional and longitudinal analyses of 3T 3D-T1 MRI images from BEB, HFS patients prior to and one month after BoNT therapy and from a group of age and sex matched healthy controls. Cortical thickness as extracted from Freesurfer was assessed as parameter of microstructural integrity. Results BoNT therapy markedly improved motor symptoms in patients with BEB and HFS. Significant differences of grey matter integrity have been found between the two patients groups. The BEB group showed lower cortical thickness at baseline in the frontal-rostral, supramarginal and temporal regions compared to patients with HFS. In this group BoNT treatment was associated with a cortical thinning in the primary motor cortex and the pre-supplementary motor area (pre-SMA). Contrary patients with HFS showed no longitudinal CT changes. A decreased cortical thickness was attested bilaterally in the temporal poles and in the right superior frontal region in BEB patients in comparison to HC. Patients in the HFS group presented a decreased CT in the left lingual gyrus and temporal pole. Conclusions Although patients with BEB and HFS present clinically with involuntary movements of facial muscles, they exhibited differences

  5. Safety and administration of treatment with botulinum neurotoxin for sialorrhoea in ALS patients: Review of the litterature and a proposal for tailored treatment

    DEFF Research Database (Denmark)

    Stokholm, Morten; Bisgård, Carsten; Vilholm, Ole Jakob

    2013-01-01

    Botulinum neurotoxin (BoNT) is a second-line treatment of sialorrhoea in ALS (amyotrophic lateral sclerosis) patients. This article is a review of the published literature concerning safety and administration of this treatment to ALS patients. A PubMed search was performed. All original...

  6. 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

    Directory of Open Access Journals (Sweden)

    Edward H Hagen

    2013-11-01

    Full Text Available 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.

  7. Recombinant botulinum neurotoxin Hc subunit (BoNT Hc) and catalytically inactive Clostridium botulinum holoproteins (ciBoNT HPs) as vaccine candidates for the prevention of botulism

    Science.gov (United States)

    2017-09-03

    nontoxic accessory proteins were purchased from 502 Metabiologics (Madison, Wisconsin) and WAKO (Kyoto, Japan ), or produced at USAMRIID. 503...J.; Marks, J.D.; Nakagawa, T.; Montal, 631 M. Molecular architecture of botulinum neurotoxin E revealed by single particle electron 632 microscopy

  8. Reconstitution of high-affinity binding of a beta-scorpion toxin to neurotoxin receptor site 4 on purified sodium channels.

    Science.gov (United States)

    Thomsen, W; Martin-Eauclaire, M F; Rochat, H; Catterall, W A

    1995-09-01

    Reconstitution of purified sodium channels into phospholipid vesicles restores many aspects of sodium channel function including high-affinity neurotoxin binding and action at neurotoxin receptor sites 1-3 and 5, but neurotoxin binding and action at receptor site 4 has not previously been demonstrated in purified and reconstituted preparations. Toxin IV from the venom of the American scorpion Centruroides suffusus suffusus (Css IV), a beta-scorpion toxin, shifts the voltage dependence of sodium channel activation by binding with high affinity to neurotoxin receptor site 4. Sodium channels were purified from rat brain and reconstituted into phospholipid vesicles composed of phosphatidylcholine and phosphatidylethanolamine (65:35). 125I-Css IV, purified by reversed-phase HPLC, bound rapidly and specifically to reconstituted sodium channels. Dissociation of the bound toxin was biphasic with half-times of 0.22 min-1 and 0.015 min-1. At equilibrium, the toxin bound to two classes of specific high-affinity sites, a variable minor class with KD of approximately 0.1 nM and a major class with a KD of approximately 5 nM. Approximately 0.8 mol 125I-Css IV was bound per mole of reconstituted, right-side-out sodium channels, as assessed from comparison of binding of saxitoxin and Css IV. Binding of Css IV was unaffected by membrane potential or by neurotoxins that bind at sites 1-3 or 5, consistent with the characteristics of binding of beta-scorpion toxins to sodium channels in cells and membrane preparations.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. Quantitative real-time reverse transcription-PCR analysis reveals stable and prolonged neurotoxin cluster gene activity in a Clostridium botulinum type E strain at refrigeration temperature.

    Science.gov (United States)

    Chen, Ying; Korkeala, Hannu; Lindén, Jere; Lindström, Miia

    2008-10-01

    The relative expression levels of six botulinum neurotoxin cluster genes in a group II Clostridium botulinum type E strain grown at 10 or 30 degrees C were investigated using quantitative real-time reverse transcription-PCR. An enzyme-linked immunosorbent assay was used to confirm neurotoxin expression. Distinct mRNA and toxin production patterns were observed at the two temperatures. The average relative mRNA levels at 10 degrees C were higher than (ntnh and p47), similar to (botE), or lower than (orfx1, orfx2, orfx3) those at 30 degrees C. The maximum botE expression levels and average neurotoxin levels at 10 degrees C were 45 to 65% of those at 30 degrees C. The relative mRNA levels at 10 degrees C declined generally slowly within 8 days, as opposed to the rapid decline observed at 30 degrees C within 24 h. Distinct expression patterns of the six genes at the two temperatures suggest that the type E neurotoxin cluster genes are transcribed as two tricistronic operons at 30 degrees C, whereas at 10 degrees C monocistronic (botE or orfx1 alone) and bicistronic (ntnh-p47 and orfx2-orfx3) transcription may dominate. Thus, type E botulinum neurotoxin production may be involved with various temperature-dependent regulatory events. In light of group II C. botulinum type E being a dangerous food-borne pathogen, these findings may be important in terms of the safety of refrigerated packaged foods of extended durability.

  10. Cross-Neutralisation of In Vitro Neurotoxicity of Asian and Australian Snake Neurotoxins and Venoms by Different Antivenoms

    Science.gov (United States)

    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

  11. Botulinum neurotoxins for the treatment of focal dystonias: Review of rating tools used in clinical trials.

    Science.gov (United States)

    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

  12. Potent new small-molecule inhibitor of botulinum neurotoxin serotype A endopeptidase developed by synthesis-based computer-aided molecular design.

    Directory of Open Access Journals (Sweden)

    Yuan-Ping Pang

    Full Text Available Botulinum neurotoxin serotype A (BoNTA causes a life-threatening neuroparalytic disease known as botulism. Current treatment for post exposure of BoNTA uses antibodies that are effective in neutralizing the extracellular toxin to prevent further intoxication but generally cannot rescue already intoxicated neurons. Effective small-molecule inhibitors of BoNTA endopeptidase (BoNTAe are desirable because such inhibitors potentially can neutralize the intracellular BoNTA and offer complementary treatment for botulism. Previously we reported a serotype-selective, small-molecule BoNTAe inhibitor with a K(i (app value of 3.8+/-0.8 microM. This inhibitor was developed by lead identification using virtual screening followed by computer-aided optimization of a lead with an IC(50 value of 100 microM. However, it was difficult to further improve the lead from micromolar to even high nanomolar potency due to the unusually large enzyme-substrate interface of BoNTAe. The enzyme-substrate interface area of 4,840 A(2 for BoNTAe is about four times larger than the typical protein-protein interface area of 750-1,500 A(2. Inhibitors must carry several functional groups to block the unusually large interface of BoNTAe, and syntheses of such inhibitors are therefore time-consuming and expensive. Herein we report the development of a serotype-selective, small-molecule, and competitive inhibitor of BoNTAe with a K(i value of 760+/-170 nM using synthesis-based computer-aided molecular design (SBCAMD. This new approach accounts the practicality and efficiency of inhibitor synthesis in addition to binding affinity and selectivity. We also report a three-dimensional model of BoNTAe in complex with the new inhibitor and the dynamics of the complex predicted by multiple molecular dynamics simulations, and discuss further structural optimization to achieve better in vivo efficacy in neutralizing BoNTA than those of our early micromolar leads. This work provides new insight

  13. Functional analysis of the alpha-neurotoxin, BmalphaTX14, derived from the Chinese scorpion, Buthus martensii Karsch.

    Science.gov (United States)

    Wang, Kun; Yin, Shi-Jin; Lu, Meng; Yi, Hong; Dai, Chao; Xu, Xiu-Jing; Cao, Zhi-Jian; Wu, Ying-Liang; Li, Wen-Xin

    2006-11-01

    The gene encoding the BmalphaTX14 (alpha-neurotoxin TX14) protein, derived from the cDNA library of the Chinese scorpion Buthus martensii Karsch, was expressed in Pichia pastoris. The recombinant protein was purified by metal chelate affinity chromatography and gel filtration chromatography. Using patch-clamp technique, electrophysiological activity of rBmalphaTX14 was identified. In the neurons isolated from mice trigeminal root ganglion, the Na+ current amplitude was reduced by 80% under whole cell patch-clamp recording. There were no apparent modifications to the gating mechanism in the presence of rBmalphaTX14. Although BmalphaTX14 shared a high amino acid sequence similarity with other typical alpha-toxins, it has different effects on neurons. Further electrophysiological analysis suggested that rBmalphaTX14 selectively blocked Na+ channels and is a member of a new group of scorpion toxins.

  14. Structure-function relationships in scorpion neurotoxins. Identification of the supperreactive lysine residue in toxin I of Androctonus australis Hector.

    Science.gov (United States)

    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.

  15. Individual and bivalent vaccines against botulinum neurotoxin serotypes A and B using DNA-based Semliki Forest virus vectors.

    Science.gov (United States)

    Yu, Yunzhou; Yu, Jiyu; Li, Na; Wang, Shuang; Yu, Weiyuan; Sun, Zhiwei

    2009-10-19

    We evaluated individual and bivalent replicon vaccines against Clostridiumbotulinum neurotoxin serotypes A (BoNT/A) or B (BoNT/B). The DNA replicon vaccine (pSCARSBHc) encoding the Hc domain of BoNT/B (BHc) induced better responses and protection against BoNT/B mouse challenge than conventional DNA vaccine. The dual-expressing DNA vaccine (pSCARSA/BHc) protected similarly to a DNA replicon vaccine mixture (pSCARSAHc+pSCARSBHc). Additionally, recombinant SFV particles, VRP-AHc or VRP-BHc, protected mice from high-dose BoNT/A or BoNT/B challenge, respectively. Mice given either dual-expressing VRP-A/BHc or mixture of VRP-AHc and VRP-BHc were protected from challenge with serotype A/B mixtures. These data justify further testing in other animals or humans.

  16. Evidence that plasmid-borne botulinum neurotoxin type B genes are widespread among Clostridium botulinum serotype B strains.

    Directory of Open Access Journals (Sweden)

    Giovanna Franciosa

    Full Text Available BACKGROUND: Plasmids that encode certain subtypes of the botulinum neurotoxin type B have recently been detected in some Clostridium botulinum strains. The objective of the present study was to investigate the frequency with which plasmid carriage of the botulinum neurotoxin type B gene (bont/B occurs in strains of C. botulinum type B, Ab, and A(B, and whether plasmid carriage is bont/B subtype-related. METHODOLOGY/PRINCIPAL FINDINGS: PCR-Restriction fragment length polymorphism was employed to identify subtypes of the bont/B gene. Pulsed-field gel electrophoresis and Southern blot hybridization with specific probes were performed to analyze the genomic location of the bont/B subtype genes. All five known bont/B subtype genes were detected among the strains; the most frequently detected subtype genes were bont/B1 and /B2. Surprisingly, the bont/B subtype gene was shown to be plasmid-borne in >50% of the total strains. The same bont/B subtype gene was associated with the chromosome in some strains, whereas it was associated with a plasmid in others. All five known bont/B subtype genes were in some cases found to reside on plasmids, though with varying frequency (e.g., most of the bont/B1 subtype genes were located on plasmids, whereas all but one of the bont/B2 subtypes were chromosomally-located. Three bivalent isolates carried both bont/A and /B genes on the same plasmid. The plasmids carrying the bont gene were five different sizes, ranging from approximately 55 kb to approximately 245 kb. CONCLUSIONS/SIGNIFICANCE: The unexpected finding of the widespread distribution of plasmids harboring the bont/B gene among C. botulinum serotype B strains provides a chance to examine their contribution to the dissemination of the bont genes among heterogeneous clostridia, with potential implications on issues related to pathogenesis and food safety.

  17. Botulinum neurotoxin type A counteracts neuropathic pain and facilitates functional recovery after peripheral nerve injury in animal models.

    Science.gov (United States)

    Marinelli, S; Luvisetto, S; Cobianchi, S; Makuch, W; Obara, I; Mezzaroma, E; Caruso, M; Straface, E; Przewlocka, B; Pavone, F

    2010-11-24

    A growing interest was recently focused on the use of Botulinum neurotoxin serotype A (BoNT/A) for fighting pain. The aim of this study was to investigate the effects of BoNT/A on neuropathic pain. It was observed that BoNT/A is able to counteract neuropathic pain induced by chronic constriction injury (CCI) to the sciatic nerve both in mice and in rats. This effect is already present after a single intraplantar (i.pl.) or intrathecal (i.t.) neurotoxin administration that significantly reduces the sciatic nerve ligation-induced mechanical allodynia in mice and rats and thermal hyperalgesia in rats. This effect was evident starting 24 h after the administration of BoNT/A and it was long-lasting, being present 81 or 25 days after i.pl. injection of the higher dose in mice (15 pg/paw) and rats (75 pg/paw), respectively, and 35 days after i.t. injection in rats (75 pg/rat). Moreover, BoNT/A-injected mice showed a quicker recovery of the walking pattern and weight bearing compared to control groups. The behavioral improvement was accompanied by structural modifications, as revealed by the expression of cell division cycle 2 (Cdc2) and growth associated protein 43 (GAP-43) regeneration associated proteins, investigated by immunofluorescence and Western blotting in the sciatic nerve, and by the immunofluorescence expression of S100β and glial fibrillary acidic protein (GFAP) Schwann cells proteins. In conclusion, the present research demonstrate long-lasting anti-allodynic and anti-hyperalgesic effects of BoNT/A in animal models of neuropathic pain together with an acceleration of regenerative processes in the injured nerve, as evidenced by both behavioral and immunohistochemistry/blotting analysis. These results may have important implications in the therapy of neuropathic pain.

  18. A scorpion venom neurotoxin paralytic to insects that affects sodium current inactivation: Purification, primary structure, and mode of action

    Energy Technology Data Exchange (ETDEWEB)

    Eitan, M.; Fowler, E.; Herrmann, R.; Duval, A.; Pelhate, M.; Zlotkin, E. (Hebrew Univ., Jerusalem (Israel))

    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.

  19. Detection, differentiation, and identification of botulinum neurotoxin serotypes C, CD, D, and DC by highly specific immunoassays and mass spectrometry.

    Science.gov (United States)

    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.

  20. Increased levels of SV2A botulinum neurotoxin receptor in clinical sensory disorders and functional effects of botulinum toxins A and E in cultured human sensory neurons

    Directory of Open Access Journals (Sweden)

    Yiangou Y

    2011-10-01

    Full Text Available Yiangos Yiangou1 Uma Anand1,2, William R. Otto2, Marco Sinisi3, Michael Fox3, Rolfe Birch3 Keith A. Foster4, Gaurav Mukerji1,5, Ayesha Akbar1,6, Sanjiv K. Agarwal5, Praveen Anand11Department of Clinical Neuroscience, Imperial College London, Hammersmith Hospital, London; 2Histopathology Laboratory, Cancer Research UK, London Research Institute, London; 3Peripheral Nerve Injury Unit, Royal National Orthopaedic Hospital, Stanmore; 4Syntaxin Ltd, Oxford; 5Department of Urology; 6Department of Gastroenterology, Imperial College London, Hammersmith Hospital, London, United Kingdom Background: There is increasing evidence that botulinum neurotoxin A may affect sensory nociceptor fibers, but the expression of its receptors in clinical pain states, and its effects in human sensory neurons, are largely unknown.Methods: We studied synaptic vesicle protein subtype SV2A, a receptor for botulinum neurotoxin A, by immunostaining in a range of clinical tissues, including human dorsal root ganglion sensory neurons, peripheral nerves, the urinary bladder, and the colon. We also determined the effects of botulinum neurotoxins A and E on localization of the capsaicin receptor, TRPV1, and functional sensitivity to capsaicin stimuli in cultured human dorsal root ganglion neurons.Results: Image analysis showed that SV2A immunoreactive nerve fibers were increased in injured nerves proximal to the injury (P = 0.002, and in painful neuromas (P = 0.0027; the ratio of percentage area SV2A to neurofilaments (a structural marker was increased proximal to injury (P = 0.0022 and in neuromas (P = 0.0001, indicating increased SV2A levels in injured nerve fibers. In the urinary bladder, SV2A nerve fibers were found in detrusor muscle and associated with blood vessels, with a significant increase in idiopathic detrusor overactivity (P = 0.002 and painful bladder syndrome (P = 0.0087. Colon biopsies showed numerous SV2A-positive nerve fibers, which were increased in quiescent

  1. 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.

    Directory of Open Access Journals (Sweden)

    Kentaro Tsukamoto

    Full Text Available 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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. (UIUC); (Scripps); (UCSF)

    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.

  3. Complete amino acid sequence of a new type of lethal neurotoxin from the venom of the funnel-web spider Atrax robustus.

    Science.gov (United States)

    Sheumack, D D; Claassens, R; Whiteley, N M; Howden, M E

    1985-02-11

    Robustoxin, the lethal neurotoxin isolated from the venom of the male Sydney funnel-web spider, Atrax robustus, is of unique structural type and physiological mode of action. The primary structure of this 42-residue peptide was determined to be H2N-Cys-Ala-Lys-Lys-Arg-Asn-Trp-Cys-Gly-Lys-Asn-Glu-Asp-Cys-Cys-Cys-Pro- Met-Lys-Cys-Ile-Tyr-Ala-Trp-Tyr-Asn-Gln-Gln-Gly-Ser-Cys-Gln-Thr-Thr-Ile- Thr-Gly-Leu-Phe-Lys-Lys-Cys-H. The disposition of disulphide-bridged cysteine residues at both the amino- and carboxy-termini and as a triplet at residues 14-16 appears to have no precedent amongst neurotoxins.

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

    Directory of Open Access Journals (Sweden)

    Carmel M. Barber

    2016-02-01

    Full Text Available 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.

  5. Identification of Small Molecules against Botulinum Neurotoxin B Binding to Neuronal Cells at Ganglioside GT1b Binding Site with Low to Moderate Affinity

    Science.gov (United States)

    2014-10-01

    Diagnosis of the National Cancer Institute for the chemicals we received from the repository. iii ABSTRACT Clostridium botulinum bacteria produce...proteins. A recombinant bivalent vaccine (rBV A/B) expressed in Pichia pastoris entered Phase III clinical trials in 2013. Currently no FDA-licensed...intoxication inhibition of botulinum neurotoxin serotype A within neurons by small-molecule, non-peptidic inhibitors. Toxins ( Basel ) 3:207-217. Schiavo, G

  6. Label-Free (XIC) Quantification of Venom Procoagulant and Neurotoxin Expression in Related Australian Elapid Snakes Gives Insight into Venom Toxicity Evolution.

    Science.gov (United States)

    Skejic, Jure; Steer, David L; Dunstan, Nathan; Hodgson, Wayne C

    2015-11-06

    This study demonstrates a direct role of venom protein expression alteration in the evolution of snake venom toxicity. Avian skeletal muscle contractile response to exogenously administered acetylcholine is completely inhibited upon exposure to South Australian and largely preserved following exposure to Queensland eastern brown snake Pseudonaja textilis venom, indicating potent postsynaptic neurotoxicity of the former and lack thereof of the latter venom. Label-free quantitative proteomics reveals extremely large differences in the expression of postsynaptic three-finger α-neurotoxins in these venoms, explaining the difference in the muscle contractile response and suggesting that the type of toxicity induced by venom can be modified by altered expression of venom proteins. Furthermore, the onset of neuromuscular paralysis in the rat phrenic nerve-diaphragm preparation occurs sooner upon exposure to the venom (10 μg/mL) with high expression of α-neurotoxins than the venoms containing predominately presynaptic β-neurotoxins. The study also finds that the onset of rat plasma coagulation is faster following exposure to the venoms with higher expression of venom prothrombin activator subunits. This is the first quantitative proteomic study that uses extracted ion chromatogram peak areas (MS1 XIC) of distinct homologous tryptic peptides to directly show the differences in the expression of venom proteins.

  7. Endangered North Atlantic right whales (Eubalaena glacialis) experience repeated, concurrent exposure to multiple environmental neurotoxins produced by marine algae.

    Science.gov (United States)

    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

  8. 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.

    Science.gov (United States)

    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.

  9. Exogenous mRNA encoding tetanus or botulinum neurotoxins expressed in Aplysia neurons

    NARCIS (Netherlands)

    Mochida, Sumiko; Poulain, Bernard; Eisel, Ulrich; Binz, Thomas; Kurazono, Hisao; Niemann, Heiner; Tauc, Ladislav; Bullock, Theodore H.

    1990-01-01

    Injection of exogenous mRNA purified from various tissue preparations into cellular translation systems such as Xenopus oocytes has allowed expression of complex proteins (e.g., receptors for neurotransmitters). No evidence for expression of injected exogenous mRNA, however, has been reported in

  10. Exogenous mRNA encoding tetanus or botulinum neurotoxins expressed in Aplysia neurons

    NARCIS (Netherlands)

    Mochida, Sumiko; Poulain, Bernard; Eisel, Ulrich; Binz, Thomas; Kurazono, Hisao; Niemann, Heiner; Tauc, Ladislav; Bullock, Theodore H.

    1990-01-01

    Injection of exogenous mRNA purified from various tissue preparations into cellular translation systems such as Xenopus oocytes has allowed expression of complex proteins (e.g., receptors for neurotransmitters). No evidence for expression of injected exogenous mRNA, however, has been reported in ter

  11. Development of a Cell-Based Functional Assay for the Detection of Clostridium botulinum Neurotoxin Types A and E

    Directory of Open Access Journals (Sweden)

    Uma Basavanna

    2013-01-01

    Full Text Available The standard procedure for definitive detection of BoNT-producing Clostridia is a culture method combined with neurotoxin detection using a standard mouse bioassay (MBA. The mouse bioassay is highly sensitive and specific, but it is expensive and time-consuming, and there are ethical concerns due to use of laboratory animals. Cell-based assays provide an alternative to the MBA in screening for BoNT-producing Clostridia. Here, we describe a cell-based assay utilizing a fluorescence reporter construct expressed in a neuronal cell model to study toxin activity in situ. Our data indicates that the assay can detect as little as 100 pM BoNT/A activity within living cells, and the assay is currently being evaluated for the analysis of BoNT in food matrices. Among available in vitro assays, we believe that cell-based assays are widely applicable in high-throughput screenings and have the potential to at least reduce and refine animal assays if not replace it.

  12. DSP-4, a noradrenergic neurotoxin, produces sex-specific effects on pairing and courtship behavior in zebra finches.

    Science.gov (United States)

    Vahaba, Daniel M; Lacey, William H; Tomaszycki, Michelle L

    2013-09-01

    Norepinephrine (NE) is involved in a variety of behaviors across vertebrate species. In songbirds, NE is involved in singing and auditory perception, fundamental components of pair formation. Mechanisms of pairing remain poorly understood in avian species. NE is likely involved given its role in vocal communication and perception. Here, we tested the hypothesis that DSP-4 treatments (a noradrenergic neurotoxin that decreases NE) decreases singing in males, song perception in females and pairing in both sexes using a naturalistic paradigm. Females were tested for preferences of either control or DSP-4 males in a two-choice paradigm using live males. Both sexes were then tested for courtship and pair formation in aviaries. In the two-choice paradigm, control females showed a significant preference for control males over DSP-4 males, whereas DSP-4 females showed no such preference. In the aviary tests, DSP-4 males engaged in less courtship behavior, showed decreased pairing behaviors and increased pair latencies compared to control males. In females, DSP-4 treatments did not alter courtship or pairing behavior. Lower neural densities of noradrenergic fibers in song, auditory, and affiliative regions were observed in DSP-4 animals of both sexes. Furthermore, DBH-ir densities in these regions explained variations in courtship and pairing behaviors, as well as pairing status. Our results extend previous findings to naturalistic contexts, provide evidence that DBH-ir densities in specific regions correlate with pairing-related behaviors, and inform us of sex differences in the role of NE in pairing.

  13. Effects of the noradrenergic neurotoxin DSP-4 on the expression of α1-adrenoceptor subtypes after antidepressant treatment.

    Science.gov (United States)

    Kreiner, Grzegorz; Zelek-Molik, Agnieszka; Kowalska, Marta; Bielawski, Adam; Antkiewicz-Michaluk, Lucyna; Nalepa, Irena

    2011-01-01

    We have previously reported that chronic imipramine and electroconvulsive treatments increase the α(1A)-adrenoceptor (but not the α(1B) subtype) mRNA level and the receptor density in the rat cerebral cortex. Furthermore, we have also shown that chronic treatment with citalopram does not affect the expression of either the α(1A)- or the α(1B)-adrenoceptor, indicating that the previously observed up-regulation of α(1A)-adrenoceptor may depend on the noradrenergic component of the pharmacological mechanism of action of these antidepressants. Here, we report that previous noradrenergic depletion with DSP-4 (50 mg/kg) (a neurotoxin selective for the noradrenergic nerve terminals) significantly attenuated the increase of α(1A)-adrenoceptor mRNA induced by a 14-day treatment with imipramine (IMI, 20 mg/kg, ip) and abolished the effect of electroconvulsive shock (ECS, 150 mA, 0.5 s) in the prefrontal cortex of the rat brain. The changes in the receptor protein expression (as reflected by its density) that were induced by IMI and ECS treatments were differently modulated by DSP-4 lesioning, and only the ECS-induced increase in α(1A)-adrenoceptor level was abolished. This study provides further evidence corroborating our initial hypothesis that the noradrenergic component of the action of antidepressant agents plays an essential role in the modulation of α(1A)-adrenoceptor in the rat cerebral cortex.

  14. The neurotoxin 1-methyl-4-phenylpyridinium (MPP+ alters hippocampal excitatory synaptic transmission by modulation of the GABAergic system

    Directory of Open Access Journals (Sweden)

    YuYing eHuang

    2015-08-01

    Full Text Available The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP induces Parkinson’s disease (PD-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. Moreover, the hippocampus contains dopaminergic fibers, which are projecting from the ventral tegmental area, substantia nigra 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 (DAT did not prevent but increased the depression of excitatory postsynaptic 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 postsynaptic 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

  15. Cross talk between tetanus neurotoxin-insensitive vesicle-associated membrane protein-mediated transport and L1-mediated adhesion.

    Science.gov (United States)

    Alberts, Philipp; Rudge, Rachel; Hinners, Ina; Muzerelle, Aude; Martinez-Arca, Sonia; Irinopoulou, Theano; Marthiens, Veronique; Tooze, Sharon; Rathjen, Fritz; Gaspar, Patricia; Galli, Thierry

    2003-10-01

    The membrane-trafficking pathway mediated by tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) in neurons is still unknown. We show herein that TI-VAMP expression is necessary for neurite outgrowth in PC12 cells and hippocampal neurons in culture. TI-VAMP interacts with plasma membrane and endosomal target soluble N-ethylmaleimide-sensitive factor attachment protein receptors, suggesting that TI-VAMP mediates a recycling pathway. L1, a cell-cell adhesion molecule involved in axonal outgrowth, colocalized with TI-VAMP in the developing brain, neurons in culture, and PC12 cells. Plasma membrane L1 was internalized into the TI-VAMP-containing compartment. Silencing of TI-VAMP resulted in reduced expression of L1 at the plasma membrane. Finally, using the extracellular domain of L1 and N-cadherin immobilized on beads, we found that the silencing of TI-VAMP led to impaired L1- but not N-cadherin-mediated adhesion. Furthermore, TI-VAMP- but not synaptobrevin 2-containing vesicles accumulated at the site of the L1 bead-cell junction. We conclude that TI-VAMP mediates the intracellular transport of L1 and that L1-mediated adhesion controls this membrane trafficking, thereby suggesting an important cross talk between membrane trafficking and cell-cell adhesion.

  16. A venom-derived neurotoxin, CsTx-1, from the spider Cupiennius salei exhibits cytolytic activities.

    Science.gov (United States)

    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.

  17. Isolation and molecular cloning of beta-neurotoxins from the venom of the scorpion Centruroides suffusus suffusus.

    Science.gov (United States)

    Espino-Solis, Gerardo Pavel; Estrada, Georgina; Olamendi-Portugal, Timoteo; Villegas, Elba; Zamudio, Fernando; Cestele, Sandrine; Possani, Lourival D; Corzo, Gerardo

    2011-04-01

    This communication reports the identification and characterization of two new toxins from the venom of the scorpion Centruroides suffusus suffusus, named: CssVIII and CssIX, according to the original nomenclature of toxins previously described for this scorpion. The isolation was obtained by means of two chromatographic steps, and a cDNA library was used to fully identify their precursors. CssVIII and CssIX contain signal peptides of 19 and 17 amino acid residues, and mature peptides of 66 and 65 residues, respectively. Intracranial injections into mice of both purified toxins showed toxicity results similar to those found for toxins CssII and CssIV. Additionally, they compete with the parent toxin CssIV, in binding and displacement experiments, conducted with brain synaptosomes showing nanomolar affinities. These results strongly support the conclusion that they are new β-neurotoxins and certainly would be of the interest of researchers in the field of venomics for studying sodium channels.

  18. Comparative characterization of botulinum neurotoxin subtypes F1 and F7 featuring differential substrate recognition and cleavage mechanisms.

    Science.gov (United States)

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

    2016-03-01

    BoNT/F7, one of the seven subtypes of botulinum neurotoxin type F (F1 to F7), is the second-most divergent subtype of this group. Despite sharing >60% identity with BoNT/F1 at both holotoxin and enzymatic domain levels, it requires an N-terminal extended peptide substrate for efficient substrate cleavage, suggesting its unique substrate recognition and specificity mechanism. Substrate mapping and saturation mutagenesis analysis revealed that VAMP2 (20-65) was likely a minimally effective substrate for LC/F7 (light chain of BoNT/F7), and in addition, LC/F7 recognized VAMP2 in a unique way, which differed significantly from that of LC/F1, although both of them share similar substrate binding and hydrolysis mode. LC/F7 utilizes distinct pockets for specific substrate binding and recognition in particular for the B1, B2 and S2 sites recognitions. Our findings provide insights into the distinct substrate recognition features of BoNT subtypes and useful information for therapy development for BoNT/F. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Effect of Noradrenergic Neurotoxin DSP-4 and Maprotiline on Heart Rate Spectral Components in Stressed and Resting Rats.

    Science.gov (United States)

    Kur'yanova, E V; Zhukova, Yu D; Teplyi, D L

    2017-07-01

    The effects of intraperitoneal DSP-4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine, a noradrenergic neurotoxin) and maprotiline (an inhibitor of norepinephrine reuptake in synapses) on spectral components of heart rhythm variability were examined in outbred male and female rats treated with these agents in daily doses of 10 mg/kg for 3 days. At rest, DSP-4 elevated LF and VLF spectral components in male and female rats. Maprotiline elevated LF and VLF components in males at rest, increased HR and reduced all spectral components in resting females. Stress against the background of DSP-4 treatment sharply increased heart rate and reduced the powers of all spectral components (especially LF and VLF components). In maprotiline-treated rats, stress increased the powers of LF and VLF components. Thus, the central noradrenergic system participates in the formation of LF and VLF spectral components of heart rate variability at rest and especially during stressful stimulation, which can determine the phasic character of changes in the heart rate variability observed in stressed organism.

  20. Effect of nitrogen on cellular production and release of the neurotoxin anatoxin-a in a nitrogen-fixing cyanobacterium

    Directory of Open Access Journals (Sweden)

    Alexis eGagnon

    2012-06-01

    Full Text Available 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 on ANTX production and release in a toxic strain of the 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 nutrient stress as described recently for another cyanotoxin (microcystin.

  1. In vitro potency determination of botulinum neurotoxin B based on its receptor-binding and proteolytic characteristics.

    Science.gov (United States)

    Wild, Emina; Bonifas, Ursula; Klimek, Jolanta; Trösemeier, Jan-Hendrik; Krämer, Beate; Kegel, Birgit; Behrensdorf-Nicol, Heike A

    2016-08-01

    Botulinum neurotoxins (BoNTs) are the most potent toxins known. However, the paralytic effect caused by BoNT serotypes A and B is taken advantage of to treat different forms of dystonia and in cosmetic procedures. Due to the increasing areas of application, the demand for BoNTs A and B is rising steadily. Because of the high toxicity, it is mandatory to precisely determine the potency of every produced BoNT batch, which is usually accomplished by performing toxicity testing (LD50 test) in mice. Here we describe an alternative in vitro assay for the potency determination of the BoNT serotype B. In this assay, the toxin is first bound to its specific receptor molecules. After the proteolytic subunit of the toxin has been released and activated by chemical reduction, it is exposed to synaptobrevin, its substrate protein. Finally the proteolytic cleavage is quantified by an antibody-mediated detection of the neoepitope, reaching a detection limit below 0.1mouseLD50/ml. Thus, the assay, named BoNT/B binding and cleavage assay (BoNT/B BINACLE), takes into account the binding as well as the protease function of the toxin, thereby measuring its biological activity.

  2. [Production of recombinant fragments of the Clostridium tetani neurotoxin for the development of new immune-prophylaxis preparations against tetanus].

    Science.gov (United States)

    Varfolomeeva, N A; Makhotina, O A; Sergeeva, T I; Belyĭ, Iu F

    2003-01-01

    Tetanus belongs to dangerous infection diseases, whose effective prevention can be ensured by vaccines. The acting substance of tetanus vaccines, presently in use, is a partially purified and deprived-of-lethal-action Clostridium tetani neurotoxin. The construction of a subunit preparation on the basis of toxin fragments obtained through gene engineering could be a method aimed at promoting the quality of the used tetanus vaccines. With this goal in mind, we built, within the present case study, the expressing genetic constructions and obtained, in the pure form, an extensive tetanus-vaccine chain with its C-terminal (Hc) fragment, hydride peptides, containing the Hc-fragment and C-terminal fragment of toxin B C. difficile, as well as Hc-fragment and S3 collagen-binding domain of collagenase C. histolyticum. The thus obtained proteins can be used in testing their immunogenic and protective properties, while the conducted study could be a basis for further research of a new-generation vaccine against tetanus and other human infection diseases.

  3. Ethyl-eicosapentaenoate modulates changes in neurochemistry and brain lipids induced by parkinsonian neurotoxin 1-methyl-4-phenylpyridinium in mouse brain slices.

    Science.gov (United States)

    Meng, QingJia; Luchtman, Dirk W; El Bahh, Bouchaib; Zidichouski, Jeffrey A; Yang, Jun; Song, Cai

    2010-12-15

    Evidence suggests a link between Parkinson's disease and the dietary intake of omega (n)-3 and n-6 polyunsaturated fatty acids (PUFAs). Presently, we investigated whether an acute dose of parkinsonian neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) affects brain n-3 and n-6 PUFA content and expression of fatty acid metabolic enzymes cytosolic phospholipase A2 (cPLA2) and cyclooxygenase-2 (COX-2) in brain slices from C57Bl/6 mice. Furthermore, we investigated whether feeding a diet of n-3 PUFA ethyl-eicosapentaenoate (E-EPA) to these mice can attenuate the MPP(+) induced changes in brain PUFA content and expression of cPLA2 and COX-2, and attenuate MPP(+) induced changes in neurotransmitters and metabolites and apoptotic markers, bax, bcl-2 and caspase-3. MPP(+) increased brain content of n-6 PUFAs linoleic acid and arachidonic acid, and increased the mRNA expression of cPLA2. MPP(+) also depleted striatal dopamine levels and increased dopamine turnover, and depleted noradrenaline levels in the frontal cortex. The neurotoxin induced increases in bax, bcl-2 and caspase-3 mRNA expression that approached significance. E-EPA by itself increased brain n-3 content, including EPA and docosapentaenoic acid (C22:5, n-3), and increased cortical dopamine. More importantly, E-EPA attenuated the MPP(+) induced increase in n-6 fatty acids content, partially attenuated the striatal dopaminergic turnover, and prevented the increases of pro-apoptotic bax and caspase-3 mRNAs. In conclusion, increases in n-6 PUFAs in the acute stage of exposure to parkinsonian neurotoxins may promote pro-inflammatory conditions. EPA may provide modest beneficial effects in Parkinson's disease, but further investigation is warranted.

  4. Involvement of Cholinergic and Adrenergic Receptors in Pathogenesis and Inflammatory Response Induced by Alpha-Neurotoxin Bot III of Scorpion Venom.

    Science.gov (United States)

    Nakib, Imene; Martin-Eauclaire, Marie-France; Laraba-Djebari, Fatima

    2016-10-01

    Bot III neurotoxin is the most lethal α neurotoxin purified from Buthus occitanus tunetanus scorpion venom. This toxin binds to the voltage-gated sodium channel of excitable cells and blocks its inactivation, inducing an increased release of neurotransmitters (acetylcholine and catecholamines). This study aims to elucidate the involvement of cholinergic and adrenergic receptors in pathogenesis and inflammatory response triggered by this toxin. Injection of Bot III to animals induces an increase of peroxidase activities, an imbalance of oxidative status, tissue damages in lung parenchyma, and myocardium correlated with metabolic disorders. The pretreatment with nicotine (nicotinic receptor agonist) or atropine (muscarinic receptor antagonist) protected the animals from almost all disorders caused by Bot III toxin, especially the immunological alterations. Bisoprolol administration (selective β1 adrenergic receptor antagonist) was also efficient in the protection of animals, mainly on tissue damage. Propranolol (non-selective adrenergic receptor antagonist) showed less effect. These results suggest that both cholinergic and adrenergic receptors are activated in the cardiopulmonary manifestations induced by Bot III. Indeed, the muscarinic receptor appears to be more involved than the nicotinic one, and the β1 adrenergic receptor seems to dominate the β2 receptor. These results showed also that the activation of nicotinic receptor leads to a significant protection of animals against Bot III toxin effect. These findings supply a supplementary data leading to better understanding of the mechanism triggered by scorpionic neurotoxins and suggest the use of drugs targeting these receptors, especially the nicotinic one in order to counteract the inflammatory response observed in scorpion envenomation.

  5. Synthesis and characterization of delta-atracotoxin-Ar1a, the lethal neurotoxin from venom of the Sydney funnel-web spider (Atrax robustus).

    Science.gov (United States)

    Alewood, Dianne; Birinyi-Strachan, Liesl C; Pallaghy, Paul K; Norton, Raymond S; Nicholson, Graham M; Alewood, Paul F

    2003-11-11

    Delta-atracotoxin-Ar1a (delta-ACTX-Ar1a) is the major polypeptide neurotoxin isolated from the venom of the male Sydney funnel-web spider, Atrax robustus. This neurotoxin targets both insect and mammalian voltage-gated sodium channels, where it competes with scorpion alpha-toxins for neurotoxin receptor site-3 to slow sodium-channel inactivation. Progress in characterizing the structure and mechanism of action of this toxin has been hampered by the limited supply of pure toxin from natural sources. In this paper, we describe the first successful chemical synthesis and oxidative refolding of the four-disulfide bond containing delta-ACTX-Ar1a. This synthesis involved solid-phase Boc chemistry using double coupling, followed by oxidative folding of purified peptide using a buffer of 2 M GdnHCl and glutathione/glutathiol in a 1:1 mixture of 2-propanol (pH 8.5). Successful oxidation and refolding was confirmed using both chemical and pharmacological characterization. Ion spray mass spectrometry was employed to confirm the molecular weight. (1)H NMR analysis showed identical chemical shifts for native and synthetic toxins, indicating that the synthetic toxin adopts the native fold. Pharmacological studies employing whole-cell patch clamp recordings from rat dorsal root ganglion neurons confirmed that synthetic delta-ACTX-Ar1a produced a slowing of the sodium current inactivation and hyperpolarizing shifts in the voltage-dependence of activation and inactivation similar to native toxin. Under current clamp conditions, we show for the first time that delta-ACTX-Ar1a produces spontaneous repetitive plateau potentials underlying the clinical symptoms seen during envenomation. This successful oxidative refolding of synthetic delta-ACTX-Ar1a paves the way for future structure-activity studies to determine the toxin pharmacophore.

  6. The selective neurotoxin DSP-4 impairs the noradrenergic projections from the locus coeruleus to the inferior colliculus in rats.

    Directory of Open Access Journals (Sweden)

    Sebastián eHormigo

    2012-06-01

    Full Text Available The inferior colliculus (IC and the locus coeruleus (LC are two midbrain nuclei that integrate multimodal information and play a major role in novelty detection to elicit an orienting response. Despite the reciprocal connections between these two structures, the projection pattern and target areas of the LC within the subdivisions of the rat IC are still unknown. Here, we used tract-tracing approaches combined with immunohistochemistry, densitometry and confocal microscopy analysis to describe a projection from the LC to the IC. Biotinylated dextran amine (BDA injections into the LC showed that the LC-IC projection is mainly ipsilateral (90% and reaches, to a major extent, the dorsal and lateral part of the IC and the intercollicular commissure. Additionally, some LC fibers extend into the central nucleus of the IC. The neurochemical nature of this projection is noradrenergic, given that tyrosine hydroxylase (TH and dopamine beta hydroxylase (DBH colocalize with the BDA-labeled fibers from the LC. To determine the total field of the LC innervations in the IC, we destroyed the LC neurons and fibers using a highly selective neurotoxin, DSP-4, and then studied the distribution and density of TH- and DBH-immunolabeled axons in the IC. In the DSP-4 treated animals, the number of axonal fibers immunolabeled for TH and DBH were deeply decreased throughout the entire rostrocaudal extent of the IC and its subdivisions compared to controls. Our densitometry results showed that the IC receives up to 97% of its noradrenergic innervations from the LC neurons and only 3% from non-coeruleus neurons. Our results also indicate that TH immunoreactivity in the IC was less impaired than the immunoreactivity for DBH after DSP-4 administration. This is consistent with the existence of an important dopaminergic projection from the substantia nigra to the IC. In conclusion, our study demonstrates and quantifies the noradrenergic projection from the LC to the IC and its

  7. DSP4, a selective neurotoxin for the locus coeruleus noradrenergic system. A review of its mode of action.

    Science.gov (United States)

    Ross, Svante B; Stenfors, Carina

    2015-01-01

    DSP4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride) is a selective neurotoxin for the locus coeruleus noradrenergic system in the rodent and bird brain. It readily passes the blood-brain barrier and cyclizes to a reactive aziridinium derivative that is accumulated into the noradrenergic nerve terminals via the noradrenaline transporter. DSP4 is also an irreversible inhibitor of this transporter. Within the nerve terminals the aziridinium derivative reacts with unknown vital cellular components, destroying the terminals. At the dose 50 mg/kg i.p. this is characterized by a rapid and long-lasting loss of noradrenaline and a slower decrease in the dopamine-β-hydroxylase enzyme activity and immunoreactivity in the regions innervated from locus coeruleus. The tissue level of noradrenaline is reduced to 10-30% of the normal value. The extraneuronal concentration is, on the other hand, increased due to inflow from non-lesioned regions. Like the peripheral sympathetic nerves the non-locus coeruleus noradrenergic systems in the rodent brain is resistant to the neurotoxic action of DSP4. Serotoninergic and dopaminergic nerves are only slightly or not at all affected by DSP4. The neurotoxic effect is counteracted by pretreatment with noradrenaline uptake inhibitors (e.g., desipramine). MAO-B inhibitors of the N-propargylamine type (e.g., selegiline) also counteract the DSP4-induced neurotoxicity with another, yet unknown mechanism. Because of its selectivity for the locus coeruleus system DSP4 is a useful tool in studies of the functional role of this noradrenergic system in the brain.

  8. Reduced Neck Muscle Strength and Altered Muscle Mechanical Properties in Cervical Dystonia Following Botulinum Neurotoxin Injections: A Prospective Study

    Directory of Open Access Journals (Sweden)

    Sirpa Mustalampi

    2016-01-01

    Full Text Available Objective To evaluate changes in the strength and mechanical properties of neck muscles and disability in patients with cervical dystonia (CD during a 12-week period following botulinum neurotoxin (BoNT injections. Methods Eight patients with CD volunteered for this prospective clinical cohort study. Patients had received BoNT injections regularly in neck muscles at three-month intervals for several years. Maximal isometric neck strength was measured by a dynamometer, and the mechanical properties of the splenius capitis were evaluated using two myotonometers. Clinical assessment was performed using the Toronto Western Spasmodic Torticollis Rating Scale (TWSTRS before and at 2, 4, 8, and 12 weeks after the BoNT injections. Results Mean maximal isometric neck strength at two weeks after the BoNT injections decreased by 28% in extension, 25% in rotation of the affected side and 17% in flexion. At four weeks, muscle stiffness of the affected side decreased by 17% and tension decreased by 6%. At eight weeks, the muscle elasticity on the affected side increased by 12%. At two weeks after the BoNT injections, the TWSTRS-severity and TWSTRS-total scores decreased by 4.3 and 6.4, respectively. The strength, muscle mechanical properties and TWSTRS scores returned to baseline values at 12 weeks. Conclusions Although maximal neck strength and muscle tone decreased after BoNT injections, the disability improved. The changes observed after BoNT injections were temporary and returned to pre-injection levels within twelve weeks. Despite having a possible negative effect on function and decreasing neck strength, the BoNT injections improved the patients reported disability.

  9. Vocal training, levodopa, and environment effects on ultrasonic vocalizations in a rat neurotoxin model of Parkinson disease

    Science.gov (United States)

    Kelm-Nelson, Cynthia A.; Brauer, Alexander F.L.; Ciucci, Michelle R.

    2016-01-01

    Levodopa does not improve dysarthria in patients with Parkinson Disease (PD), although vocal exercise therapy, such as “LSVT/LOUD®”, does improve vocal communication. Most patients receive vocal exercise therapy while concurrently being treated with levodopa, although the interaction between levodopa and vocal exercise therapy on communication in PD is relatively unknown. Further, carryover of vocal exercise therapy to novel situations is critical for successful outcomes, but the influence of novel situations on rehabilitated vocal communication is not well understood. To address the influence of exercise, medications, and environment on vocal communication with precise experimental control, we employed the widely used 6-OHDA rat neurotoxin model of PD (infusion to the medial forebrain bundle), and assessed ultrasonic vocalizations after: vocal exercise, vocal exercise with levodopa, levodopa alone, and control conditions. We tested USVs in the familiar training environment of the home cage and a novel cage. We hypothesized that parkinsonian rats that undergo vocal exercise would demonstrate significant improvement of ultrasonic vocalization (USV) acoustic parameters as compared to the control exercise and levodopa-only treatment groups. We further hypothesized that vocal exercise in combination with levodopa administration, similar to what is common in humans, would lead to improvement in USV outcomes, particularly when tested in a familiar versus a novel environment. We found that the combination of exercise and levodopa lead to some improvement in USV acoustic parameters and these effects were stronger in a familiar vs. a novel environment. Our results suggest that although treatment can improve aspects of communication, environment can influence the benefits of these effects. PMID:27025445

  10. Crystal Structure of the Receptor-Binding Domain of Botulinum Neurotoxin Type HA, Also Known as Type FA or H

    Directory of Open Access Journals (Sweden)

    Guorui Yao

    2017-03-01

    Full Text Available Botulinum neurotoxins (BoNTs, which have been exploited as cosmetics and muscle-disorder treatment medicines for decades, are well known for their extreme neurotoxicity to humans. They pose a potential bioterrorism threat because they cause botulism, a flaccid muscular paralysis-associated disease that requires immediate antitoxin treatment and intensive care over a long period of time. In addition to the existing seven established BoNT serotypes (BoNT/A–G, a new mosaic toxin type termed BoNT/HA (aka type FA or H was reported recently. Sequence analyses indicate that the receptor-binding domain (HC of BoNT/HA is ~84% identical to that of BoNT/A1. However, BoNT/HA responds differently to some potent BoNT/A-neutralizing antibodies (e.g., CR2 that target the HC. Therefore, it raises a serious concern as to whether BoNT/HA poses a new threat to our biosecurity. In this study, we report the first high-resolution crystal structure of BoNT/HA-HC at 1.8 Å. Sequence and structure analyses reveal that BoNT/HA and BoNT/A1 are different regarding their binding to cell-surface receptors including both polysialoganglioside (PSG and synaptic vesicle glycoprotein 2 (SV2. Furthermore, the new structure also provides explanations for the ~540-fold decreased affinity of antibody CR2 towards BoNT/HA compared to BoNT/A1. Taken together, these new findings advance our understanding of the structure and function of this newly identified toxin at the molecular level, and pave the way for the future development of more effective countermeasures.

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

    Directory of Open Access Journals (Sweden)

    Edwin J Vazquez-Cintron

    Full Text Available 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 ad, which was rendered atoxic by introducing two amino acid mutations to the light chain (LC of wt BoNT/A, and which is being developed as a molecular vehicle for delivering drugs to the neuronal cytoplasm. The neuronal binding, internalization, and intracellular trafficking of BoNT/A ad in primary hippocampal cultures was evaluated using three complimentary techniques: flow cytometry, immunohistochemistry, and Western blotting. Neuronal binding of BoNT ad was significantly increased when neurons were incubated in depolarizing medium. Flow cytometry demonstrated that BoNT/A ad internalized into neurons but not glia. After 24 hours, the majority of the neuron-bound BoNT/A ad became internalized, as determined by its resistance to pronase E-induced proteolytic degradation of proteins associated with the plasma membrane of intact cells. Significant amounts of the atoxic LC accumulated in a Triton X-100-extractable fraction of the neurons, and persisted as such for at least 11 days with no evidence of degradation. Immunocytochemical analysis demonstrated that the LC of BoNT/A ad was translocated to the neuronal cytoplasm after uptake and was specifically targeted to SNARE proteins. The atoxic LC consistently co-localized with synaptic markers SNAP-25 and VAMP-2, but was rarely co-localized with markers for early or late endosomes. These data demonstrate that BoNT/A ad mimics the trafficking properties of wt BoNT/A, confirming that our platform for designing and expressing BoNT derivatives provides an accessible system for elucidating the molecular details of BoNT trafficking, and can potentially be used to address multiple medical and biodefense needs.

  12. Neuronal entry and high neurotoxicity of botulinum neurotoxin A require its N-terminal binding sub-domain.

    Science.gov (United States)

    Wang, Jiafu; Meng, Jianghui; Nugent, Marc; Tang, Minhong; Dolly, J Oliver

    2017-03-15

    Botulinum neurotoxins (BoNTs) are the most toxic proteins known, due to inhibiting the neuronal release of acetylcholine and causing flaccid paralysis. Most BoNT serotypes target neurons by binding to synaptic vesicle proteins and gangliosides via a C-terminal binding sub-domain (HCC). However, the role of their conserved N-terminal sub-domain (HCN) has not been established. Herein, we created a mutant form of recombinant BoNT/A lacking HCN (rAΔHCN) and showed that the lethality of this mutant is reduced 3.3 × 10(4)-fold compared to wild-type BoNT/A. Accordingly, low concentrations of rAΔHCN failed to bind either synaptic vesicle protein 2C or neurons, unlike the high-affinity neuronal binding obtained with (125)I-BoNT/A (Kd = 0.46 nM). At a higher concentration, rAΔHCN did bind to cultured sensory neurons and cluster on the surface, even after 24 h exposure. In contrast, BoNT/A became internalised and its light chain appeared associated with the plasmalemma, and partially co-localised with vesicle-associated membrane protein 2 in some vesicular compartments. We further found that a point mutation (W985L) within HCN reduced the toxicity over 10-fold, while this mutant maintained the same level of binding to neurons as wild type BoNT/A, suggesting that HCN makes additional contributions to productive internalization/translocation steps beyond binding to neurons.

  13. Reduced Neck Muscle Strength and Altered Muscle Mechanical Properties in Cervical Dystonia Following Botulinum Neurotoxin Injections: A Prospective Study

    Science.gov (United States)

    Mustalampi, Sirpa; Ylinen, Jari; Korniloff, Katariina; Weir, Adam; Häkkinen, Arja

    2016-01-01

    Objective To evaluate changes in the strength and mechanical properties of neck muscles and disability in patients with cervical dystonia (CD) during a 12-week period following botulinum neurotoxin (BoNT) injections. Methods Eight patients with CD volunteered for this prospective clinical cohort study. Patients had received BoNT injections regularly in neck muscles at three-month intervals for several years. Maximal isometric neck strength was measured by a dynamometer, and the mechanical properties of the splenius capitis were evaluated using two myotonometers. Clinical assessment was performed using the Toronto Western Spasmodic Torticollis Rating Scale (TWSTRS) before and at 2, 4, 8, and 12 weeks after the BoNT injections. Results Mean maximal isometric neck strength at two weeks after the BoNT injections decreased by 28% in extension, 25% in rotation of the affected side and 17% in flexion. At four weeks, muscle stiffness of the affected side decreased by 17% and tension decreased by 6%. At eight weeks, the muscle elasticity on the affected side increased by 12%. At two weeks after the BoNT injections, the TWSTRS-severity and TWSTRS-total scores decreased by 4.3 and 6.4, respectively. The strength, muscle mechanical properties and TWSTRS scores returned to baseline values at 12 weeks. Conclusions Although maximal neck strength and muscle tone decreased after BoNT injections, the disability improved. The changes observed after BoNT injections were temporary and returned to pre-injection levels within twelve weeks. Despite having a possible negative effect on function and decreasing neck strength, the BoNT injections improved the patients reported disability. PMID:26828215

  14. Nuclear magnetic resonance study of the solution properties of the polypeptide neurotoxin I from the sea anemone Stichodactyla helianthus

    Energy Technology Data Exchange (ETDEWEB)

    Norton, R.S.; Cossins, A.I.; Kem, W.R. (Univ. of New South Wales, Kensington (Australia))

    1989-02-21

    The solution properties of the polypeptide neurotoxin I from the sea anemone Stichodactyla helianthus (Sh I) have been investigated by high-resolution H nuclear magnetic resonance (NMR) spectroscopy at 300 MHz. The pH dependence of the spectra has been examined over the range 1.1-12.2 at 27{degree}C. Individual pK{sub a} values have been obtained for the {alpha}-ammonium group of Ala-1 (8.6) and the side chains of Glu-8 (3.7), Tyr-36 (10.9), and Tyr-37 (10.8). For the remaining seven carboxyl groups in the molecule, four pK{sub a} values can be clearly identified. The five Lys residues titrate in the range 10.5-11, but individual pK{sub a} values could not be obtained because of peak overlap. Conformational changes associated with the protonation of carboxylates occur below pH 4, while in the alkaline pH range major unfolding occurs above pH 10. The molecule also unfolds at elevated temperatures. Exchange of the backbone amide protons has been monitored at various values of pH and temperature in the ranges pH 4-5 and 12-27{degree}C. Comparison of these properties of Sh I in solution with those of the related polypeptides anthopleurin A and Anemonia sulcata toxins I and II indicates that Sh I is less stable thermally and that there are some significant differences in the ionic interactions that maintain the tertiary structure. The solvent accessibility of aromatic residues has been probed with photochemically induced dynamic nuclear polarization NMR at 360 MHz.

  15. Crystal Structure of the Receptor-Binding Domain of Botulinum Neurotoxin Type HA, Also Known as Type FA or H

    Energy Technology Data Exchange (ETDEWEB)

    Yao, Guorui; Lam, Kwok-ho; Perry, Kay; Weisemann, Jasmin; Rummel, Andreas; Jin, Rongsheng (Cornell); (Dusseldorf); (UCI)

    2017-03-01

    Botulinum neurotoxins (BoNTs), which have been exploited as cosmetics and muscle-disorder treatment medicines for decades, are well known for their extreme neurotoxicity to humans. They pose a potential bioterrorism threat because they cause botulism, a flaccid muscular paralysis-associated disease that requires immediate antitoxin treatment and intensive care over a long period of time. In addition to the existing seven established BoNT serotypes (BoNT/A–G), a new mosaic toxin type termed BoNT/HA (aka type FA or H) was reported recently. Sequence analyses indicate that the receptor-binding domain (HC) of BoNT/HA is ~84% identical to that of BoNT/A1. However, BoNT/HA responds differently to some potent BoNT/A-neutralizing antibodies (e.g., CR2) that target the HC. Therefore, it raises a serious concern as to whether BoNT/HA poses a new threat to our biosecurity. In this study, we report the first high-resolution crystal structure of BoNT/HA-HC at 1.8 Å. Sequence and structure analyses reveal that BoNT/HA and BoNT/A1 are different regarding their binding to cell-surface receptors including both polysialoganglioside (PSG) and synaptic vesicle glycoprotein 2 (SV2). Furthermore, the new structure also provides explanations for the ~540-fold decreased affinity of antibody CR2 towards BoNT/HA compared to BoNT/A1. Taken together, these new findings advance our understanding of the structure and function of this newly identified toxin at the molecular level, and pave the way for the future development of more effective countermeasures

  16. THE PARKINSONIAN NEUROTOXIN ROTENONE ACTIVATES CALPAIN AND CASPASE-3 LEADING TO MOTONEURON DEGENERATION IN SPINAL CORD OF LEWIS RATS

    Science.gov (United States)

    SAMANTARAY, S.; KNARYAN, V. H.; GUYTON, M. K.; MATZELLE, D. D.; RAY, S. K.; BANIK, N. L.

    2007-01-01

    Exposure to environmental toxins increases the risk of neurodegenerative diseases including Parkinson’s disease (PD). Rotenone is a neurotoxin that has been used to induce experimental parkinsonism in rats. We used the rotenone model of experimental parkinsonism to explore a novel aspect of extra-nigral degeneration, the neurodegeneration of spinal cord (SC), in PD. Rotenone administration to male Lewis rats caused significant neuronal cell death in cervical and lumbar SC as compared to control animals. Dying neurons were motoneurons as identified by double immunofluorescent labeling for TUNEL+ cells and ChAT-immunoreactivity. Neuronal death was accompanied by abundant astrogliosis and microgliosis as evidenced from GFAP-immunoreactivity and OX-42-immunoreactivity, respectively, implicating an inflammatory component during neurodegeneration in SC. However, the integrity of the white matter in SC was not affected by rotenone administration as evidenced from the non co-localization of any TUNEL+ cells with GFAP-immunoreactivity and MBP-immunoreactivity, the selective markers for astrocytes and oligodendrocytes, respectively. Increased activities of 76 kD active m-calpain and 17/19 kD active caspase-3 further demonstrated involvement of these enzymes in cell death in SC. The finding of ChAT+ cell death also suggested degeneration of SC motoneurons in rotenone-induced experimental parkinsonism. Thus, this is the first report of its kind in which the selective vulnerability of a putative parkinsonian target outside of nigrostriatal system has been tested using an environmental toxin to understand the pathophysiology of PD. Moreover, rotenone-induced degeneration of SC motoneuron in this model of experimental parkinsonism progressed with upregulation of calpain and caspase-3. PMID:17367952

  17. Effects of Megaplasmid Loss on Growth of Neurotoxigenic Clostridium butyricum Strains and Botulinum Neurotoxin Type E Expression.

    Science.gov (United States)

    Scalfaro, Concetta; Iacobino, Angelo; Grande, Laura; Morabito, Stefano; Franciosa, Giovanna

    2016-01-01

    Clostridium butyricum strains that atypically produce the botulinum neurotoxin type E (BoNT/E) possess a megaplasmid of unknown functions in their genome. In this study, we cured two botulinum neurotoxigenic C. butyricum type E strains of their megaplasmids, and compared the obtained megaplasmid-cured strains to their respective wild-type parental strains. Our results showed that the megaplasmids do not confer beta-lactam resistance on the neurotoxigenic C. butyricum type E strains, although they carry several putative beta-lactamase genes. Instead, we found that the megaplasmids are essential for growth of the neurotoxigenic C. butyricum type E strains at the relatively low temperature of 15°C, and are also relevant for growth of strains under limiting pH and salinity conditions, as well as under favorable environmental conditions. Moreover, the presence of the megaplasmids was associated with increased transcript levels of the gene encoding BoNT/E in the C. butyricum type E strains, indicating that the megaplasmids likely contain transcriptional regulators. However, the levels of BoNT/E in the supernatants of the cured and uncured strains were similar after 24 and 48 h culture, suggesting that expression of BoNT/E in the C. butyricum type E strains is not ultimately controlled by the megaplasmids. Together, our results reveal that the C. butyricum type E megaplasmids exert pleiotropic effects on the growth of their microbial hosts under optimal and limiting environmental conditions, and also highlight the possibility of original regulatory mechanisms controlling the expression of BoNT/E.

  18. A Novel Neurotoxin Gene ar1b Recombination Enhances the Efficiency of Helicoverpa armigera Nucleopolyhedrovirus as a Pesticide by Inhibiting the Host Larvae Ability to Feed and Grow

    OpenAIRE

    Huan Yu; Jiao Meng; Jian Xu; Tong-Xian Liu; Dun Wang

    2015-01-01

    A recombinant Helicoverpa armigera nucleopolyhedrovirus (HearNPV), Ar1b-HearNPV, was constructed and identified as an improved bio-control agent of Helicoverpa armigera larvae. The HearNPV polyhedrin promoter was used to express the insect-specific neurotoxin gene, ar1b, which was originally isolated from the Australian funnel-web spider (Atrax robustus). RT-PCR and Western blotting analysis showed that both the ar1b transcript and protein were produced successfully in Ar1b-HearNPV-infected H...

  19. Structure-function relationships in the receptor for urokinase-type plasminogen activator. Comparison to other members of the Ly-6 family and snake venom alpha-neurotoxins

    DEFF Research Database (Denmark)

    Ploug, M; Ellis, V

    1994-01-01

    Plasminogen activation is regulated by the interaction between urokinase-type plasminogen activator (uPA) and its specific glycolipid-anchored cell surface receptor (uPAR). uPAR is composed of three homologous domains and is the only multi-domain member of the Ly-6 family of glycolipid-anchored m......Plasminogen activation is regulated by the interaction between urokinase-type plasminogen activator (uPA) and its specific glycolipid-anchored cell surface receptor (uPAR). uPAR is composed of three homologous domains and is the only multi-domain member of the Ly-6 family of glycolipid......-anchored membrane proteins. Recent evidence has highlighted similarities between the individual domains of uPAR and the large family of secreted, single domain snake venom alpha-neurotoxins, suggesting that uPAR may adopt the same gross folding pattern as these structurally well characterized proteins. Structural...... aspects of the binding between alpha-neurotoxins and the acetylcholine receptor may have a major influence on future studies of the interaction between uPA and uPAR....

  20. The first venomous crustacean revealed by transcriptomics and functional morphology: remipede venom glands express a unique toxin cocktail dominated by enzymes and a neurotoxin.

    Science.gov (United States)

    von Reumont, Björn M; Blanke, Alexander; Richter, Sandy; Alvarez, Fernando; Bleidorn, Christoph; Jenner, Ronald A

    2014-01-01

    Animal venoms have evolved many times. Venomous species are especially common in three of the four main groups of arthropods (Chelicerata, Myriapoda, and Hexapoda), which together represent tens of thousands of species of venomous spiders, scorpions, centipedes, and hymenopterans. Surprisingly, despite their great diversity of body plans, there is no unambiguous evidence that any crustacean is venomous. We provide the first conclusive evidence that the aquatic, blind, and cave-dwelling remipede crustaceans are venomous and that venoms evolved in all four major arthropod groups. We produced a three-dimensional reconstruction of the venom delivery apparatus of the remipede Speleonectes tulumensis, showing that remipedes can inject venom in a controlled manner. A transcriptomic profile of its venom glands shows that they express a unique cocktail of transcripts coding for known venom toxins, including a diversity of enzymes and a probable paralytic neurotoxin very similar to one described from spider venom. We screened a transcriptomic library obtained from whole animals and identified a nontoxin paralog of the remipede neurotoxin that is not expressed in the venom glands. This allowed us to reconstruct its probable evolutionary origin and underlines the importance of incorporating data derived from nonvenom gland tissue to elucidate the evolution of candidate venom proteins. This first glimpse into the venom of a crustacean and primitively aquatic arthropod reveals conspicuous differences from the venoms of other predatory arthropods such as centipedes, scorpions, and spiders and contributes valuable information for ultimately disentangling the many factors shaping the biology and evolution of venoms and venomous species.

  1. A Conformational Change of C Fragment of Tetanus Neurotoxin Reduces Its Ganglioside-Binding Activity but Does Not Destroy Its Immunogenicity ▿

    Science.gov (United States)

    Yu, Rui; Yi, Shaoqiong; Yu, Changming; Fang, Ting; Liu, Shuling; Yu, Ting; Song, Xiaohong; Fu, Ling; Hou, Lihua; Chen, Wei

    2011-01-01

    The C fragment of tetanus neurotoxin (TeNT-Hc) with different conformations was observed due to the four cysteine residues within it which could form different intramolecular disulfide bonds. In this study, we prepared and compared three types of monomeric TeNT-Hc with different conformational components: free sulfhydryls (50 kDa), bound sulfhydryls (44 kDa), and a mixture of the two conformational proteins (half 50 kDa and half 44 kDa). TeNT-Hc with bound sulfhydryls reduced its binding activity to ganglioside GT1b and neuronal PC-12 cells compared to what was seen for TeNT-Hc with free sulfhydryls. However, there was no significant difference among their immunogenicities in mice, including induction of antitetanus toxoid IgG titers, antibody types, and protective capacities against tetanus neurotoxin challenge. Our results showed that the conformational changes of TeNT-Hc resulting from disulfide bond formation reduced its ganglioside-binding activity but did not destroy its immunogenicity, and the protein still retained continuous B cell and T cell epitopes; that is, the presence of the ganglioside-binding site within TeNT-Hc may be not essential for the induction of a fully protective antitetanus response. TeNT-Hc with bound sulfhydryls may be developed into an ideal human vaccine with a lower potential for side effects. PMID:21813664

  2. Purification, Characterization and 1H NMR Resonance Assignment of an α-Like Neurotoxin BmK 16 from the Venom of Chinese Scorpion Buthus martensii Karsch

    Institute of Scientific and Technical Information of China (English)

    ZHANG, Nai-Xia; WU, Gong; WANG, Zhong-Hua; WU, Hou-Ming

    2003-01-01

    A natural scorpion toxin BmK 16 was purified for the first time from the venom of the Chinese scorpion Buthus martensii Karsch (BmK) by using combined gel-filtration, ion exchange and reversed phase chromatography. The sequence of the Nterminal 8 amino acid residues was determined by Edman degradation. Using the N-terminal sequence as a tag, the database searching revealed a hit in the scorpion cDNA Bank.The sequence for N-terminal 8 amino acid residues, molecular weight and amino acid compositions of BmK 16 were identical with the calculated values according to the first 64 residues' sequence of the precursor peptide alpha-neurotoxin TX16 derived from the sequence of the cDNA AF156597 (EMBL). The sequence-specific resonance assignment of BmK 16 was achieved and the intact sequence of BmK 16 was determined as followings: VRDAY IAKPH NCVYE CARNE YCNDL CTKNGAKSGY CQWVG KYGNG CWCKE LPDNV PIRVP GKCH.Furthermore, the results from the sequence homology analysis and the toxicity assays indicated that BmK 16 was an -like scorpion neurotoxin.

  3. Improvement in laboratory diagnosis of wound botulism and tetanus among injecting illicit-drug users by use of real-time PCR assays for neurotoxin gene fragments.

    Science.gov (United States)

    Akbulut, D; Grant, K A; McLauchlin, J

    2005-09-01

    An upsurge in wound infections due to Clostridium botulinum and Clostridium tetani among users of illegal injected drugs (IDUs) occurred in the United Kingdom during 2003 and 2004. A real-time PCR assay was developed to detect a fragment of the neurotoxin gene of C. tetani (TeNT) and was used in conjunction with previously described assays for C. botulinum neurotoxin types A, B, and E (BoNTA, -B, and -E). The assays were sensitive, specific, rapid to perform, and applicable to investigating infections among IDUs using DNA extracted directly from wound tissue, as well as bacteria growing among mixed microflora in enrichment cultures and in pure culture on solid media. A combination of bioassay and PCR test results confirmed the clinical diagnosis in 10 of 25 cases of suspected botulism and two of five suspected cases of tetanus among IDUs. The PCR assays were in almost complete agreement with the conventional bioassays when considering results from different samples collected from the same patient. The replacement of bioassays by real-time PCR for the isolation and identification of both C. botulinum and C. tetani demonstrates a sensitivity and specificity similar to those of conventional approaches. However, the real-time PCR assays substantially improves the diagnostic process in terms of the speed of results and by the replacement of experimental animals. Recommendations are given for an improved strategy for the laboratory investigation of suspected wound botulism and tetanus among IDUs.

  4. The selective neurotoxin DSP-4 impairs the noradrenergic projections from the locus coeruleus to the inferior colliculus in rats.

    Science.gov (United States)

    Hormigo, Sebastián; Horta Júnior, José de Anchieta de Castro E; Gómez-Nieto, Ricardo; López, Dolores E

    2012-01-01

    The inferior colliculus (IC) and the locus coeruleus (LC) are two midbrain nuclei that integrate multimodal information and play a major role in novelty detection to elicit an orienting response. Despite the reciprocal connections between these two structures, the projection pattern and target areas of the LC within the subdivisions of the rat IC are still unknown. Here, we used tract-tracing approaches combined with immunohistochemistry, densitometry, and confocal microscopy (CM) analysis to describe a projection from the LC to the IC. Biotinylated dextran amine (BDA) injections into the LC showed that the LC-IC projection is mainly ipsilateral (90%) and reaches, to a major extent, the dorsal and lateral part of the IC and the intercollicular commissure. Additionally, some LC fibers extend into the central nucleus of the IC. The neurochemical nature of this projection is noradrenergic, given that tyrosine hydroxylase (TH) and dopamine beta hydroxylase (DBH) colocalize with the BDA-labeled fibers from the LC. To determine the total field of the LC innervations in the IC, we destroyed the LC neurons and fibers using a highly selective neurotoxin, DSP-4, and then studied the distribution and density of TH- and DBH-immunolabeled axons in the IC. In the DSP-4 treated animals, the number of axonal fibers immunolabeled for TH and DBH were deeply decreased throughout the entire rostrocaudal extent of the IC and its subdivisions compared to controls. Our densitometry results showed that the IC receives up to 97% of its noradrenergic innervations from the LC neurons and only 3% from non-coeruleus neurons. Our results also indicate that TH immunoreactivity in the IC was less impaired than the immunoreactivity for DBH after DSP-4 administration. This is consistent with the existence of an important dopaminergic projection from the substantia nigra to the IC. In conclusion, our study demonstrates and quantifies the noradrenergic projection from the LC to the IC and its

  5. Design and construction of immune phage antibody library against Tetanus neurotoxin: Production of single chain antibody fragments.

    Science.gov (United States)

    Sadreddini, Sanam; Seifi-Najmi, Mehrnosh; Ghasemi, Babollah; Kafil, Hossein Samadi; Alinejad, Vahideh; Sadreddini, Sevil; Younesi, Vahid; Jadidi-Niaragh, Farhad; Yousefi, Mehdi

    2015-12-23

    Tetanus neurotoxin (TeNT) is composed of a light (LC) and heavy chain (HC) polypeptides, released by anaerobic bacterium Clostridium tetani and can cause fatal life-threatening infectious disease. Toxin HC and LC modules represents receptor binding and zinc metalloprotease activity, respectively. The passive administration of animal-derived antibodies against tetanus toxin has been considered as the mainstay therapy for years. However, this treatment is associated with several adverse effects due to the presence of anti-isotype antibodies. In the present study, we have produced the fully human single chain antibody fragments (HuScFv) from two human antibody phage display libraries. Twenty-four different HuscFvs were isolated from two anti TeNT immune libraries. Our produced human ScFv (HuScFv) were converted to IgG platform and analyzed regarding their specific reactivity to TeNT. All of the selected scFvs have the same VL but different VH. Three HuscFvs from the first library (TTX15, 51, 75) and two HuscFvs from the second library (TTX16, 20) were chosen to convert to IgG1 using pOptiVEC and pcDNA3.3 systems. Production of IgG1 from transfected DG44 and binding capacity of them to tetanus toxin and toxoid were measured by ELISA. ELISA results showed no detectable production of TTX16 and TTX20 IgG1. Although, TTX51 and TTX75 were converted and produced as IgG1, no reactivity to tetanus toxin and toxoid was observed. However, TTX15 was successfully produced as whole IgG1 platform with reactivity to both tetanus toxin and toxoid. The latter would be an appropriate replacement for conventional polyclonal antibodies if would meet the further characterization including specificity determination, affinity measurement and toxin neutralizing assays. Our results demonstrated production of functional IgG1 derived from TTX15 scFv and might be an appropriate replacement for polyclonal Tetabulin but it needs further characterization.

  6. Heat shock protein 70 overexpression does not attenuate atrophy in botulinum neurotoxin type A-treated skeletal muscle.

    Science.gov (United States)

    Houston, Fraser E; Hain, Brian A; Adams, Thomas J; Houston, Kati L; O'Keeffe, Roderic; Dodd, Stephen L

    2015-07-01

    Botulinum neurotoxin type A (BoNT/A) is used clinically to induce therapeutic chemical denervation of spastically contracted skeletal muscles. However, BoNT/A administration can also cause atrophy. We sought to determine whether a major proteolytic pathway contributing to atrophy in multiple models of muscle wasting, the ubiquitin proteasome system (UPS), is involved in BoNT/A-induced atrophy. Three and ten days following BoNT/A injection of rat hindlimb, soleus muscle fiber cross-sectional area was reduced 25 and 65%, respectively. The transcriptional activity of NF-κB and Foxo was significantly elevated at 3 days (2- to 4-fold) and 10 days (5- to 6-fold). Muscle RING-finger protein-1 (MuRF1) activity was elevated (2-fold) after 3 days but not 10 days, while atrogin-1 activity was not elevated at any time point. BoNT/A-induced polyubiquitination occurred after 3 days (3-fold increase) but was totally absent after 10 days. Proteasome activity was elevated (1.5- to 2-fold) after 3 and 10 days. We employed the use of heat shock protein 70 (Hsp70) to inhibit NF-κB and Foxo transcriptional activity. Electrotransfer of Hsp70 into rat soleus, before BoNT/A administration, was insufficient to attenuate atrophy. It was also insufficient to decrease BoNT/A-induced Foxo activity at 3 days, although NF-κB activity was abolished. By 10 days both NF-κB and Foxo activation were abolished by Hsp70. Hsp70-overexpression was unable to alter the levels of BoNT/A-induced effects on MuRF1/atrogin-1, polyubiquitination, or proteasome activity. In conclusion, Hsp70 overexpression is insufficient to attenuate BoNT/A-induced atrophy. It remains unclear what proteolytic mechanism/s are contributing to BoNT/A-induced atrophy, although a Foxo-MuRF1-ubiquitin-proteasome contribution may exist, at least in early BoNT/A-induced atrophy. Further clarification of UPS involvement in BoNT/A-induced atrophy is warranted. Copyright © 2015 the American Physiological Society.

  7. Carney Complex

    Science.gov (United States)

    ... Types of Cancer > Carney Complex Request Permissions Carney Complex Approved by the Cancer.Net Editorial Board , 11/2015 What is Carney complex? Carney complex is a hereditary condition associated with: ...

  8. Use of a Recombinant Fluorescent Substrate with Cleavage Sites for All Botulinum Neurotoxins in High-Throughput Screening of Natural Product Extracts for Inhibitors of Serotypes A, B, and E7

    Science.gov (United States)

    2007-12-14

    inhibitory molecules in the extracts, as exemplified by Fig. 4. Figure 4A shows the fractionation of Terminalia brownii root bark aque - ous extract (Table...from toxins. CRC Press, Boca Raton, FL. 31. Schmidt, J. J., and K. A. Bostian. 1997 . Endoproteinase activity of type A botulinum neurotoxin: substrate

  9. α-Elapitoxin-Aa2a, a long-chain snake α-neurotoxin with potent actions on muscle (α1)(2)βγδ nicotinic receptors, lacks the classical high affinity for neuronal α7 nicotinic receptors.

    Science.gov (United States)

    Blacklow, Benjamin; Kornhauser, Rachelle; Hains, Peter G; Loiacono, Richard; Escoubas, Pierre; Graudins, Andis; Nicholson, Graham M

    2011-01-15

    In contrast to all classical long-chain α-neurotoxins possessing the critical fifth disulfide bond, α-elapitoxin-Aa2a (α-EPTX-Aa2a), a novel long-chain α-neurotoxin from the common death adder Acanthophis antarcticus, lacks affinity for neuronal α7-type nicotinic acetylcholine receptors (nAChRs). α-EPTX-Aa2a (8850Da; 0.1-1μM) caused a concentration-dependent inhibition of indirect twitches, and blocked contractures to cholinergic agonists in the isolated chick biventer cervicis nerve-muscle preparation, consistent with a postsynaptic curaremimetic mode of action. α-EPTX-Aa2a (1-10nM) produced a potent pseudo-irreversible antagonism of chick muscle nAChRs, with an estimated pA(2) value of 8.311±0.031, which was not reversed by monovalent death adder antivenom. This is only 2.5-fold less potent than the prototypical long-chain α-neurotoxin, α-bungarotoxin. In contrast, α-EPTX-Aa2a produced complete, but weak, inhibition of (125)I-α-bungarotoxin binding to rat hippocampal α7 nAChRs (pK(I)=3.670), despite high sequence homology and similar mass to a wide range of long-chain α-neurotoxins. The mostly likely cause for the loss of α7 binding affinity is a leucine substitution, in loop II of α-EPTX-Aa2a, for the highly conserved Arg(33) in long-chain α-neurotoxins. Arg(33) has been shown to be critical for both neuronal and muscle activity. Despite this substitution, α-EPTX-Aa2a retains high affinity for muscle (α1)(2)βγδ nAChRs. This is probably as a result of an Arg(29) residue, previously shown to be critical for muscle (α1)(2)βγδ nAChR affinity, and highly conserved across all short-chain, but not long-chain, α-neurotoxins. α-EPTX-Aa2a therefore represents a novel atypical long-chain α-neurotoxin that includes a fifth disulfide but exhibits differential affinity for nAChR subtypes.

  10. Complex Beauty

    OpenAIRE

    Franceschet, Massimo

    2014-01-01

    Complex systems and their underlying convoluted networks are ubiquitous, all we need is an eye for them. They pose problems of organized complexity which cannot be approached with a reductionist method. Complexity science and its emergent sister network science both come to grips with the inherent complexity of complex systems with an holistic strategy. The relevance of complexity, however, transcends the sciences. Complex systems and networks are the focal point of a philosophical, cultural ...

  11. Recombination and Insertion Events Involving the Botulinum Neurotoxin Complex Genes in Clostridium botulinum Types A, B, E and F and Clostridium butyricum Type E Strains

    Science.gov (United States)

    2009-10-05

    Bruggemann H, Baumer S, Fricke WF, Wiezer A, Liesegang H, Decker I, et al.: The genome sequence of Clostridium tetani , the caus- ative agent of... Clostridium botulinum types A, B, E and F and Clostridium butyricum type E strains Karen K Hill*1, Gary Xie2, Brian T Foley3, Theresa J Smith4, Amy C Munk2...ornl.gov; John C Detter - cdetter@lanl.gov * Corresponding author Abstract Background: Clostridium botulinum is a taxonomic designation for at least

  12. Eosinophil protein X/eosinophil derived neurotoxin (EPX/EDN). Detection by enzyme-linked immunosorbent assay and purification from normal human urine

    DEFF Research Database (Denmark)

    Reimert, C M; Minuva, U; Kharazmi, A

    1991-01-01

    subsequently raised in rabbits. The anti-EPX-antibodies raised in rabbits showed no reactivity with other proteins in the granule extract. The sandwich ELISA utilized the biotin/avidin amplification system and measured EPX over the range of 60-2000 pg/ml. The intra- and interassay coefficients of variation......Eosinophil protein X/eosinophil derived neurotoxin (EPX/EDN) is one of the cationic proteins found in the granules of the human eosinophilic granulocytes. EPX was purified from extracts of granules isolated from blood buffy coat cells of healthy donors. Polyclonal anti-EPX antibodies were...... procedure involving affinity chromatography on heparin Sepharose and size exclusion chromatography on Sephadex G-50 superfine. Extracted EPX and U-EPX had ribonuclease activity and comigrated on agarose electrophoresis. They also showed immunological identity when evaluated with rabbit anti-EPX antibodies...

  13. The role of Ser54 in the antinociceptive activity of BmK9, a neurotoxin from the scorpion Buthus martensii Karsch.

    Science.gov (United States)

    Wang, Yueqiu; Hao, Zhihui; Shao, Jianhua; Song, Yongbo; Li, Chunli; Li, Chuan; Zhao, Yongshan; Liu, Yanfeng; Wei, Tingting; Wu, Chunfu; Zhang, Jinghai

    2011-11-01

    Residue 54 has been shown to be important for bioactivity in several toxins. However, its role in the antinociceptive activity of toxins has not been evaluated yet. In this study, site-directed mutagenesis and mouse acetic acid writhing test were used to investigate the role of Ser54 in the antinociceptive activity of BmK9 neurotoxin from the Buthus martensii Karsch scorpion. Detailed mutagenesis analysis revealed that substitution of Ser54 by various polar amino acids produced no significant change in the antinociceptive activity, while all substitutions of nonpolar amino acid for Ser54 led to a significant loss of antinociceptive activity. Following the conformational analysis, it was suggested that Ser54 in BmK9 plays a functional role in the antinociceptive activity, the residue exerts its effect by means of a side-chain hydrogen bond.

  14. Functional influence of botulinum neurotoxin type A treatment (Xeomin® of multifocal upper and lower limb spasticity on chronic hemiparetic gait

    Directory of Open Access Journals (Sweden)

    Maurizio Falso

    2012-05-01

    Full Text Available This report describes the modification of hemiplegic shoulder pain and walking velocity through injections of Xeomin®, a new botulinum neurotoxin type A formulation, in a 67-year-old woman with chronic residual left hemiparesis and hemiparetic gait attributable to stroke. Clinical evaluation included upper and lower limb spasticity, upper and lower limb pain, trunk control, upper and lower limb motricity index, visual gait analysis, and gait velocity. Assessments were performed before, 1 week after, and 1 month after treatment. Improvement was observed in all clinical parameters assessed. Amelioration of spasticity of the upper and lower limbs and shoulder pain was observed after 1 month. Trunk postural attitude and paraxial muscle recruitment recovered. No adverse events were observed and the patient shows significant improvement of functional impairment derived from chronic spasticity after treatment with Xeomin®. We also provide a simple and useful protocol for clinical evaluation of the treatment.

  15. Binding sites and actions of Tx1, a neurotoxin from the venom of the spider Phoneutria nigriventer, in guinea pig ileum

    Directory of Open Access Journals (Sweden)

    R.G. Santos

    1999-12-01

    Full Text Available Tx1, a neurotoxin isolated from the venom of the South American spider Phoneutria nigriventer, produces tail elevation, behavioral excitation and spastic paralysis of the hind limbs after intracerebroventricular injection in mice. Since Tx1 contracts isolated guinea pig ileum, we have investigated the effect of this toxin on acetylcholine release, as well as its binding to myenteric plexus-longitudinal muscle membranes from the guinea pig ileum. [125I]-Tx1 binds specifically and with high affinity (Kd = 0.36 ± 0.02 nM to a single, non-interacting (nH = 1.1, low capacity (Bmax 1.1 pmol/mg protein binding site. In competition experiments using several compounds (including ion channel ligands, only PhTx2 and PhTx3 competed with [125I]-Tx1 for specific binding sites (K0.5 apparent = 7.50 x 10-4 g/l and 1.85 x 10-5 g/l, respectively. PhTx2 and PhTx3, fractions from P. nigriventer venom, contain toxins acting on sodium and calcium channels, respectively. However, the neurotoxin PhTx2-6, one of the isoforms found in the PhTx2 pool, did not affect [125I]-Tx1 binding. Tx1 reduced the [3H]-ACh release evoked by the PhTx2 pool by 33%, but did not affect basal or KCl-induced [3H]-ACh release. Based on these results, as well as on the homology of Tx1 with toxins acting on calcium channels (w-Aga IA and IB and its competition with [125I]-w-Cono GVIA in the central nervous system, we suggest that the target site for Tx1 may be calcium channels.

  16. Neurotoxins from snake venoms and α-conotoxin ImI inhibit functionally active ionotropic γ-aminobutyric acid (GABA) receptors.

    Science.gov (United States)

    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

    2015-09-11

    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. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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

    Science.gov (United States)

    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

  18. Complexity explained

    CERN Document Server

    Erdi, Peter

    2008-01-01

    This book explains why complex systems research is important in understanding the structure, function and dynamics of complex natural and social phenomena. Readers will learn the basic concepts and methods of complex system research.

  19. Attomolar detection of botulinum toxin type A in complex biological matrices.

    Directory of Open Access Journals (Sweden)

    Karine Bagramyan

    Full Text Available BACKGROUND: A highly sensitive, rapid and cost efficient method that can detect active botulinum neurotoxin (BoNT in complex biological samples such as foods or serum is desired in order to 1 counter the potential bioterrorist threat 2 enhance food safety 3 enable future pharmacokinetic studies in medical applications that utilize BoNTs. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe a botulinum neurotoxin serotype A assay with a large immuno-sorbent surface area (BoNT/A ALISSA that captures a low number of toxin molecules and measures their intrinsic metalloprotease activity with a fluorogenic substrate. In direct comparison with the "gold standard" mouse bioassay, the ALISSA is four to five orders of magnitudes more sensitive and considerably faster. Our method reaches attomolar sensitivities in serum, milk, carrot juice, and in the diluent fluid used in the mouse assay. ALISSA has high specificity for the targeted type A toxin when tested against alternative proteases including other BoNT serotypes and trypsin, and it detects the holotoxin as well as the multi-protein complex form of BoNT/A. The assay was optimized for temperature, substrate concentration, size and volume proportions of the immuno-sorbent matrix, enrichment and reaction times. Finally, a kinetic model is presented that is consistent with the observed improvement in sensitivity. CONCLUSIONS/SIGNIFICANCE: The sensitivity, specificity, speed and simplicity of the BoNT ALISSA should make this method attractive for diagnostic, biodefense and pharmacological applications.

  20. Bucolic Complexes

    CERN Document Server

    Brešar, Bostjan; Chepoi, Victor; Gologranc, Tanja; Osajda, Damian

    2012-01-01

    In this article, we introduce and investigate bucolic complexes, a common generalization of systolic complexes and of CAT(0) cubical complexes. This class of complexes is closed under Cartesian products and amalgamations over some convex subcomplexes. We study various approaches to bucolic complexes: from graph-theoretic and topological viewpoints, as well as from the point of view of geometric group theory. Bucolic complexes can be defined as locally-finite simply connected prism complexes satisfying some local combinatorial conditions. We show that bucolic complexes are contractible, and satisfy some nonpositive-curvature-like properties. In particular, we prove a version of the Cartan-Hadamard theorem, the fixed point theorem for finite group actions, and establish some results on groups acting geometrically on such complexes. We also characterize the 1-skeletons (which we call bucolic graphs) and the 2-skeletons of bucolic complexes. In particular, we prove that bucolic graphs are precisely retracts of Ca...

  1. Extraction and inhibition of enzymatic activity of botulinum neurotoxins /B1, /B2, /B3, /B4, and /B5 by a panel of monoclonal anti-BoNT/B antibodies

    Directory of Open Access Journals (Sweden)

    Kalb Suzanne R

    2011-11-01

    Full Text Available Abstract Background Botulism is caused by botulinum neurotoxins (BoNTs, extremely toxic proteins which can induce respiratory failure leading to long-term intensive care or death. Treatment for botulism includes administration of antitoxins, which must be administered early in the course of the intoxication; therefore, rapid determination of human exposure to BoNT is an important public health goal. In previous work, our laboratory reported on Endopep-MS, a mass spectrometry-based activity method for detecting and differentiating BoNT/A, /B, /E, and /F in clinical samples. We also demonstrated that antibody-capture is effective for purification and concentration of BoNTs from complex matrices such as clinical samples. However, some antibodies inhibit or neutralize the enzymatic activity of BoNT, so the choice of antibody for toxin extraction is critical. Results In this work, we evaluated 24 anti-BoNT/B monoclonal antibodies (mAbs for their ability to inhibit the in vitro activity of BoNT/B1, /B2, /B3, /B4, and /B5 and to extract those toxins. Among the mAbs, there were significant differences in ability to extract BoNT/B subtypes and inhibitory effect on BoNT catalytic activity. Some of the mAbs tested enhanced the in vitro light chain activity of BoNT/B, suggesting that BoNT/B may undergo conformational change upon binding some mAbs. Conclusions In addition to determining in vitro inhibition abilities of a panel of mAbs against BoNT/B1-/B5, this work has determined B12.2 and 2B18.2 to be the best mAbs for sample preparation before Endopep-MS. These mAb characterizations also have the potential to assist with mechanistic studies of BoNT/B protection and treatment, which is important for studying alternative therapeutics for botulism.

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

    Science.gov (United States)

    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

  3. Detection of botulinum neurotoxin serotype B at sub mouse LD(50 levels by a sandwich immunoassay and its application to toxin detection in milk.

    Directory of Open Access Journals (Sweden)

    Miles C Scotcher

    Full Text Available BACKGROUND: Botulinum neurotoxin (BoNT, the causative agent of botulism, a serious neuroparylatic disease, is produced by the anaerobic bacterium Clostridium botulinum and consists of a family of seven serotypes (A-H. We previously reported production of high-affinity monoclonal antibodies to BoNT serotype A. METHODS AND FINDINGS: Recombinant peptide fragments of the light chain, the transmembrane and receptor-binding domains of the heavy chain of botulinum neurotoxin type B (BoNT/B were expressed in Escherichia coli as GST-fusion proteins and purified. These proteins were used to immunize BALB/cJ mice for the generation of monoclonal antibodies (mAbs. Antibody-producing hybridomas were detected using either a direct binding ELISA binding to plate-immobilized BoNT/B, or with a capture-capture ELISA whereby the capacity of the antibody to capture BoNT/B from solution was tested. A total of five mAbs were selected, two of which bound the toxin light chain and three bound the receptor-binding domain of BoNT/B heavy chain. MAb MCS6-27 was identified via capture-capture ELISA and was the only mAb able to bind BoNT/B in solution under physiological conditions. MAbs F24-1, F26-16, F27-33 and F29-40 were identified via direct binding ELISA, and were able to capture BoNT/B in solution only in the presence of 0.5-0.9 mM sodium dodecyl sulphate (SDS. MAb MCS6-27 and an anti-BoNT/B polyclonal antibody were incorporated into a sandwich ELISA that did not require SDS. CONCLUSIONS: We report here the generation of monoclonal antibodies to serotype B and the subsequent development of a sensitive sandwich immunoassay. This immunoassay has a detection limit of 100 fg BoNT/B, fifty times more sensitive than the mouse bioassay detection limit of 5 pg BoNT/B. Additionally, this assay detected as little as 39 pg/mL of toxin in skim, 2% and whole milk.

  4. Communication complexity and information complexity

    Science.gov (United States)

    Pankratov, Denis

    Information complexity enables the use of information-theoretic tools in communication complexity theory. Prior to the results presented in this thesis, information complexity was mainly used for proving lower bounds and direct-sum theorems in the setting of communication complexity. We present three results that demonstrate new connections between information complexity and communication complexity. In the first contribution we thoroughly study the information complexity of the smallest nontrivial two-party function: the AND function. While computing the communication complexity of AND is trivial, computing its exact information complexity presents a major technical challenge. In overcoming this challenge, we reveal that information complexity gives rise to rich geometrical structures. Our analysis of information complexity relies on new analytic techniques and new characterizations of communication protocols. We also uncover a connection of information complexity to the theory of elliptic partial differential equations. Once we compute the exact information complexity of AND, we can compute exact communication complexity of several related functions on n-bit inputs with some additional technical work. Previous combinatorial and algebraic techniques could only prove bounds of the form theta( n). Interestingly, this level of precision is typical in the area of information theory, so our result demonstrates that this meta-property of precise bounds carries over to information complexity and in certain cases even to communication complexity. Our result does not only strengthen the lower bound on communication complexity of disjointness by making it more exact, but it also shows that information complexity provides the exact upper bound on communication complexity. In fact, this result is more general and applies to a whole class of communication problems. In the second contribution, we use self-reduction methods to prove strong lower bounds on the information

  5. Application of Purified Botulinum Type A Neurotoxin to Treat Experimental Trigeminal Neuropathy in Rats and Patients with Urinary Incontinence and Prostatic Hyperplasia

    Directory of Open Access Journals (Sweden)

    Yoshizo Matsuka

    2012-01-01

    Full Text Available Type A neurotoxin (NTX of Clostridium botulinum was purified by a simple procedure using a lactose gel column. The toxicity of this purified toxin preparation was retained for at least 1 year at −30°C by supplementation with either 0.1% albumin or 0.05% albumin plus 1% trehalose. When purified NTX was used to treat 49 patients with urinary incontinence caused by either refractory idiopathic or neurogenic detrusor overactivity, 36 patients showed significant improvement in symptoms. These beneficial effects were also observed in cases of prostatic hyperplasia. The results obtained with NTX were similar to that of Botox. The effects of NTX on trigeminal neuralgia induced by infraorbital nerve constriction (IoNC in rats were also studied. Trigeminal ganglion neurons from ipsilateral to IoNC exhibited significantly faster onset of FM4-64 release than sham-operated contralateral neurons. Intradermal injection of NTX in the area of IoNC alleviated IoNC-induced pain behavior and reduced the exaggerated FM4-64 release in trigeminal ganglion neurons.

  6. Oxyopinins, large amphipathic peptides isolated from the venom of the wolf spider Oxyopes kitabensis with cytolytic properties and positive insecticidal cooperativity with spider neurotoxins.

    Science.gov (United States)

    Corzo, Gerardo; Villegas, Elba; Gómez-Lagunas, Froylan; Possani, Lourival D; Belokoneva, Olga S; Nakajima, Terumi

    2002-06-28

    Five amphipathic peptides with antimicrobial, hemolytic, and insecticidal activity were isolated from the crude venom of the wolf spider Oxyopes kitabensis. The peptides, named oxyopinins, are the largest linear cationic amphipathic peptides from the venom of a spider that have been chemically characterized at present. According to their primary structure Oxyopinin 1 is composed of 48 amino acid residues showing extended sequence similarity to the ant insecticidal peptide ponericinL2 and to the frog antimicrobial peptide dermaseptin. Oxyopinins 2a, 2b, 2c, and 2d have highly similar sequences. At least 27 out of 37 amino acid residues are conserved. They also show a segment of sequence similar to ponericinL2. Circular dichroism analyses showed that the secondary structure of the five peptides is essentially alpha-helical. Oxyopinins showed disrupting activities toward both biological membranes and artificial vesicles, particularly to those rich in phosphatidylcholine. Electrophysiological recordings performed on insect cells (Sf9) showed that the oxyopinins produce a drastic reduction of cell membrane resistance by opening non-selective ion channels. Additionally, a new paralytic neurotoxin named Oxytoxin 1 was purified from the same spider venom. It contains 69 amino acid residue cross-linked by five disulfide bridges. Application of mixtures containing oxyopinins and Oxytoxin 1 to insect larvae showed a potentiation phenomenon, by which an increase lethality effect is observed. These results suggest that the linear amphipathic peptides in spider venoms and neuropeptides cooperate to capture insects efficiently.

  7. Reconstitution of neurotoxin-modulated ion transport by the voltage-regulated sodium channel isolated from the electroplax of Electrophorus electricus.

    Science.gov (United States)

    Rosenberg, R L; Tomiko, S A; Agnew, W S

    1984-01-01

    The functional reconstitution of the voltage-regulated Na channel purified from the electroplax of Electrophorus electricus is described. Reconstitution was achieved by removing detergent with Bio-Beads SM-2 followed by freeze-thaw-sonication in the presence of added liposomes. This preparation displayed heat-stable binding of 3H-labeled tetrodotoxin (TTX) (Kd = 33 nM). 22Na+ influx was stimulated 2- to 5-fold by alkaloid neurotoxins and blocked by TTX. Veratridine activated Na+ influx with a K1/2 of 18 microM, and this activation was blocked by TTX precisely in parallel with specific [3H]TTX binding. Batrachotoxin stimulated 22Na+ flux more effectively than did veratridine. No effect of the peptide anemone toxin II was found. Insertion of the Na channel into membranes resulted in 60-70% of the TTX-binding sites facing the vesicle exterior. Thus, external TTX partially inhibited flux, whereas blockade was complete when TTX was also equilibrated with the vesicle interior. The lipid-soluble local anesthetics tetracaine and dibucaine inhibited flux completely. QX-222, a charged derivative of lidocaine, blocked only a fraction of the channels, apparently those oriented inside-out. Purified samples were predominantly composed of the Mr 260,000-300,000 glycopeptide but contained variable quantities of smaller peptides. Veratridine-dependent flux and peptide compositions were determined on fractions across a gel filtration column profile. Stimulated flux codistributed only with the large peptide. Images PMID:6322191

  8. Botulinum Neurotoxin A Injections Influence Stretching of the Gastrocnemius Muscle-Tendon Unit in an Animal Model

    Directory of Open Access Journals (Sweden)

    Christopher J. Tuohy

    2012-08-01

    Full Text Available Botulinum Neurotoxin A (BoNT-A injections have been used for the treatment of muscle contractures and spasticity. This study assessed the influence of (BoNT-A injections on passive biomechanical properties of the muscle-tendon unit. Mouse gastrocnemius muscle (GC was injected with BoNT-A (n = 18 or normal saline (n = 18 and passive, non-destructive, in vivo load relaxation experimentation was performed to examine how the muscle-tendon unit behaves after chemical denervation with BoNT-A. Injection of BoNT-A impaired passive muscle recovery (15% vs. 35% recovery to pre-stretching baseline, p < 0.05 and decreased GC stiffness (0.531 ± 0.061 N/mm vs. 0.780 ± 0.037 N/mm, p < 0.05 compared to saline controls. The successful use of BoNT-A injections as an adjunct to physical therapy may be in part attributed to the disruption of the stretch reflex; thereby modulating in vivo passive muscle properties. However, it is also possible that BoNT-A injection may alter the structure of skeletal muscle; thus modulating the in vivo passive biomechanical properties of the muscle-tendon unit.

  9. High-level expression, purification, crystallization and preliminary X-ray crystallographic studies of the receptor binding domain of botulinum neurotoxin serotype D

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yanfeng; Gao, Xiaoli; Qin, Lin; Buchko, Garry W.; Robinson, Howard; Varnum, Susan M.

    2010-12-01

    Botulinum neurotoxins (BoNTs) are highly toxic proteins for humans and can cause neuroparalytic disease botulism. Due to the limitations of production and manipulation of holoenzymes, expressing non-toxic heavy chain receptor binding domains (HCR) has become a common strategy for vaccine and antibody development. Meanwhile, large quantities and highly purified soluble proteins are required for research areas such as antibody maturation and structural biology. We present high level expression and purification of the BoNT serotype D HCR in E. coli using a codon-optimized cDNA. By varying expression conditions, especially at low temperature, the protein was expressed at a high level with high solubility. About 150-200 mg protein was purified to >90% purity from 1 L cell culture. The recombinant D_HCR was crystallized and the crystals diffracted to 1.65 Å resolution. The crystals belong to space group P212121 with unit cell dimensions a = 60.8 Å, b = 89.7 Å, c = 93.9 Å. Preliminary crystallographic data analysis revealed one molecule in asymmetric unit.

  10. The Translocation Domain of Botulinum Neurotoxin A Moderates the Propensity of the Catalytic Domain to Interact with Membranes at Acidic pH

    Science.gov (United States)

    Araye, Anne; Goudet, Amélie; Barbier, Julien; Pichard, Sylvain; Baron, Bruno; England, Patrick; Pérez, Javier; Zinn-Justin, Sophie; Chenal, Alexandre; Gillet, Daniel

    2016-01-01

    Botulinum neurotoxin A (BoNT/A) is composed of three domains: a catalytic domain (LC), a translocation domain (HN) and a receptor-binding domain (HC). Like most bacterial toxins BoNT/A is an amphitropic protein, produced in a soluble form that is able to interact, penetrate and/or cross a membrane to achieve its toxic function. During intoxication BoNT/A is internalized by the cell by receptor-mediated endocytosis. Then, LC crosses the membrane of the endocytic compartment and reaches the cytosol. This translocation is initiated by the low pH found in this compartment. It has been suggested that LC passes in an unfolded state through a transmembrane passage formed by HN. We report here that acidification induces no major conformational change in either secondary or tertiary structures of LC and HN of BoNT/A in solution. GdnHCl-induced denaturation experiments showed that the stability of LC and HN increases as pH drops, and that HN further stabilizes LC. Unexpectedly we found that LC has a high propensity to interact with and permeabilize anionic lipid bilayers upon acidification without the help of HN. This property is downplayed when LC is linked to HN. HN thus acts as a chaperone for LC by enhancing its stability but also as a moderator of the membrane interaction of LC. PMID:27070312

  11. Crystal structure determination of a neutral neurotoxin BmK M4 from Buthus martensii Karsch at 0.20 nm

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    BmK M4 is a neutral neurotoxin in the BmK toxin series.It is medially toxic and belongs to group III α-toxins.The purified sample was crystallized in rhombic space group P61.Using an X-ray diffraction technique,the crystal structure of BmK M4 was revealed by molecular replacement at 0.20 nm resolution.The model was refined.The final crystallographic R factor was 0.142 and the free R factor was 0.173.The root mean square deviation is 0.001 5 nm for the bond length and 1.753°for the bond angles.64 water molecules were added to the asymmetric unit.The refined structure showed an unusual non-prolyl cis peptide bond at residue 10.The structure was compared with group II α-toxin BmK M8 (an acidic,weak toxin).The potential structural implications of the cis peptide bond were discussed.

  12. Crystal structure determination of a neutral neurotoxin BmK M4 from Buthus martensii Karsch at 0.20 nm

    Institute of Scientific and Technical Information of China (English)

    何小林; 刘新奇; 曾宗浩; 李宏民; 王淼; 张英; 王大成

    2000-01-01

    BmK M4 is a neutral neurotoxin in the BmK toxin series. It is medially toxic and belongs to group III cc-toxins. The purified sample was crystallized in rhombic space group P6 Using an X-ray diffraction technique, the crystal structure of BmK M4 was revealed by molecular replacement at 0.20 nm resolution. The model was refined. The final crystallographic R factor was 0.142 and the free R factor was 0.173. The root mean square deviation is 0.001 5 nm for the bond length and 1.753° for the bond angles. 64 water molecules were added to the asymmetric unit. The refined structure showed an unusual non-prolyl cis peptide bond at residue 10. The structure was compared with group II a-toxin BmK M8 (an acidic, weak toxin). The potential structural implications of the cis peptide bond were discussed.

  13. Response of Human T Cells to Tetanus Neurotoxin HCC Sub-Domain: T Cell Cytokine Production and Activation Marker Induced by HCC

    Directory of Open Access Journals (Sweden)

    Maryam Ghafari-Khamene

    2015-10-01

    Full Text Available Tetanus is caused by the tetanus neurotoxin (TeNT, a 150 kDa single polypeptide molecule which is cleaved into active two-chain molecules composed of a 50 kDa N-terminal light (L and a 100 kDa C-terminal heavy (H chains. Fragment C is further subdivided into two subdomains: the proximal HCN  subdomain and the extreme carboxy subdomain, HCC. HCC is considered as an immunodominant part of TeNT and is responsible for TeNT binding activity to neurons.In the present study, we investigated the ability of recombinant HCC(r HCC to induce Tcell activation. Our results showed that recombinant HCC has a stimulatory effect on IFN-γ secretion by T cells after 48h co-incubation in the presence of anti-TLR-2 Ab. Also, Hcc can induce the expression of CD69 on T cells.Our finding indicated that stimulatory effects of HCC on T cells are TLR-2 independentand anti-TLR-2 inhibitory antibody fails to neutralize HCC stimulatory effects on T cells.Furthermore, HCC  is critical for immunogenic activity of TeNT and is able to induce Tcells through TLR-2 independent pathway.

  14. The venom of the fishing spider Dolomedes sulfurous contains various neurotoxins acting on voltage-activated ion channels in rat dorsal root ganglion neurons.

    Science.gov (United States)

    Wang, Hengyun; Zhang, Fan; Li, Dan; Xu, Shiyan; He, Juan; Yu, Hai; Li, Jiayan; Liu, Zhonghua; Liang, Songping

    2013-04-01

    Dolomedes sulfurous is a venomous spider distributed in the south of China and characterized with feeding on fish. The venom exhibits great diversity and contains hundreds of peptides as revealed by off-line RP-HPLC/MALDI-TOF-MS analysis. The venom peptides followed a triple-modal distribution, with 40.7% of peptides falling in the mass range of 1000-3000 Da, 25.6% peptides in the 7000-9000 Da range and 23.5% peptides in the 3000-5000 Da range. This distribution modal is rather different from these of peptides from other spider venoms analyzed. The venom could inhibit voltage-activated Na(+), K(+) and Ca(2+) channels in rat DRG neurons as revealed by voltage-clamp analysis. Significantly, the venom exhibited inhibitory effects on TTX-R Na(+) and T-type Ca(2+) currents, suggesting that there exist both channel antagonists which might be valuable tools for investigation of both channels and drug development. Additionally, intrathoracically injection of venom could cause serve neurotoxic effects on zebrafish and death at higher concentrations. The LD50 value was calculated to be 28.8 μg/g body weight. Our results indicated that the venom of D. sulfurous contain diverse neurotoxins which serve to capture prey. Intensive studies will be necessary to investigate the structures and functions of specific peptides of the venom in the future.

  15. Rapid Multiplexed Flow Cytometric Assay for Botulinum Neurotoxin Detection Using an Automated Fluidic Microbead-Trapping Flow Cell for Enhanced Sensitivity

    Energy Technology Data Exchange (ETDEWEB)

    Ozanich, Richard M.; Bruckner-Lea, Cindy J.; Warner, Marvin G.; Miller, Keith D.; Antolick, Kathryn C.; Marks, James D.; Lou, Jianlong; Grate, Jay W.

    2009-07-15

    A bead-based sandwich immunoassay for botulinum neurotoxin serotype A (BoNT/A) has been developed and demonstrated using a recombinant 50 kDa fragment (BoNT/A-HC-fragment) of the BoNT/A heavy chain (BoNT/A-HC) as a structurally valid simulant. Three different anti-BoNT/A antibodies were attached to three different fluorescent dye encoded flow cytometry beads for multiplexing. The assay was conducted in two formats: a manual microcentrifuge tube format and an automated fluidic system format. Flow cytometry detection was used for both formats. The fluidic system used a novel microbead-trapping flow cell to capture antibody-coupled beads with subsequent sequential perfusion of sample, wash, dye-labeled reporter antibody, and final wash solutions. After the reaction period, the beads were collected for analysis by flow cytometry. Sandwich assays performed on the fluidic system gave median fluorescence intensity signals on the flow cytometer that were 2-4 times higher than assays performed manually in the same amount of time. Limits of detection were estimated at 1 pM (~50 pg/mL for BoNT/A-HC-fragment) for the 15 minute fluidic assay.

  16. Preferential entry of botulinum neurotoxin A Hc domain through intestinal crypt cells and targeting to cholinergic neurons of the mouse intestine.

    Directory of Open Access Journals (Sweden)

    Aurélie Couesnon

    Full Text Available Botulism, characterized by flaccid paralysis, commonly results from botulinum neurotoxin (BoNT absorption across the epithelial barrier from the digestive tract and then dissemination through the blood circulation to target autonomic and motor nerve terminals. The trafficking pathway of BoNT/A passage through the intestinal barrier is not yet fully understood. We report that intralumenal administration of purified BoNT/A into mouse ileum segment impaired spontaneous muscle contractions and abolished the smooth muscle contractions evoked by electric field stimulation. Entry of BoNT/A into the mouse upper small intestine was monitored with fluorescent HcA (half C-terminal domain of heavy chain which interacts with cell surface receptor(s. We show that HcA preferentially recognizes a subset of neuroendocrine intestinal crypt cells, which probably represent the entry site of the toxin through the intestinal barrier, then targets specific neurons in the submucosa and later (90-120 min in the musculosa. HcA mainly binds to certain cholinergic neurons of both submucosal and myenteric plexuses, but also recognizes, although to a lower extent, other neuronal cells including glutamatergic and serotoninergic neurons in the submucosa. Intestinal cholinergic neuron targeting by HcA could account for the inhibition of intestinal peristaltism and secretion observed in botulism, but the consequences of the targeting to non-cholinergic neurons remains to be determined.

  17. Src Family Kinase Inhibitors Antagonize the Toxicity of Multiple Serotypes of Botulinum Neurotoxin in Human Embryonic Stem Cell-Derived Motor Neurons

    Science.gov (United States)

    Burnett, James C.; Nuss, Jonathan E.; Wanner, Laura M.; Peyser, Brian D.; Du, Hao T.; Gomba, Glenn Y.; Kota, Krishna P.; Panchal, Rekha G.; Gussio, Rick; Kane, Christopher D.; Tessarollo, Lino

    2015-01-01

    Botulinum neurotoxins (BoNTs), the causative agents of botulism, are potent inhibitors of neurotransmitter release from motor neurons. There are currently no drugs to treat BoNT intoxication after the onset of the disease symptoms. In this study, we explored how modulation of key host pathways affects the process of BoNT intoxication in human motor neurons, focusing on Src family kinase (SFK) signaling. Motor neurons derived from human embryonic stem (hES) cells were treated with a panel of SFK inhibitors and intoxicated with BoNT serotypes A, B, or E (which are responsible for >95 % of human botulism cases). Subsequently, it was found that bosutinib, dasatinib, KX2-391, PP1, PP2, Src inhibitor-1, and SU6656 significantly antagonized all three of the serotypes. Furthermore, the data indicated that the treatment of hES-derived motor neurons with multiple SFK inhibitors increased the antagonistic effect synergistically. Mechanistically, the small molecules appear to inhibit BoNTs by targeting host pathways necessary for intoxication and not by directly inhibiting the toxins’ proteolytic activity. Importantly, the identified inhibitors are all well-studied with some in clinical trials while others are FDA-approved drugs. Overall, this study emphasizes the importance of targeting host neuronal pathways, rather than the toxin’s enzymatic components, to antagonize multiple BoNT serotypes in motor neurons. PMID:25782580

  18. The Translocation Domain of Botulinum Neurotoxin A Moderates the Propensity of the Catalytic Domain to Interact with Membranes at Acidic pH.

    Science.gov (United States)

    Araye, Anne; Goudet, Amélie; Barbier, Julien; Pichard, Sylvain; Baron, Bruno; England, Patrick; Pérez, Javier; Zinn-Justin, Sophie; Chenal, Alexandre; Gillet, Daniel

    2016-01-01

    Botulinum neurotoxin A (BoNT/A) is composed of three domains: a catalytic domain (LC), a translocation domain (HN) and a receptor-binding domain (HC). Like most bacterial toxins BoNT/A is an amphitropic protein, produced in a soluble form that is able to interact, penetrate and/or cross a membrane to achieve its toxic function. During intoxication BoNT/A is internalized by the cell by receptor-mediated endocytosis. Then, LC crosses the membrane of the endocytic compartment and reaches the cytosol. This translocation is initiated by the low pH found in this compartment. It has been suggested that LC passes in an unfolded state through a transmembrane passage formed by HN. We report here that acidification induces no major conformational change in either secondary or tertiary structures of LC and HN of BoNT/A in solution. GdnHCl-induced denaturation experiments showed that the stability of LC and HN increases as pH drops, and that HN further stabilizes LC. Unexpectedly we found that LC has a high propensity to interact with and permeabilize anionic lipid bilayers upon acidification without the help of HN. This property is downplayed when LC is linked to HN. HN thus acts as a chaperone for LC by enhancing its stability but also as a moderator of the membrane interaction of LC.

  19. Crystal structure determination of an acidic neurotoxin (BmK M8) from scorpion Buthm martensii Karsch at 0.25nm resolution

    Institute of Scientific and Technical Information of China (English)

    李宏民; 金雷; 曾宗浩; 王淼; 张英; 王大成

    1996-01-01

    The crystal structure of an acidic neurotoxin, BmK M8, from Chinese scorpion Buthus martensii Karsch was determined at 0.25 nm resolution. The X-ray diffraction data of BmK M8 crystals at 0.25nm resolution were collected on a Siemens area detector. Using molecular replacement method with a basic scorpion toxin AaH II in a search model, the cross-rotation function, PC-refinement and translation function were calculated by X-PLOR program package. The correct orientation and position of BmK M8 molecule in crystal were determined in a resolution range of 1.5 - 0.35nm, The oystallographic refinement was further performed by stereo-chemical restrict least-square technique, followed by simulated annealing, slow-cooling protocols. The final crystallographic R-factor at 0.8-0.25 nm is 0.171. The standard deviations of bond length and bond angle from ideality are 0.001 7nm and 2.24° , respectively. The final model of BmK M8 structure is composed of a dense core of secondary structure elements by a stretch of α-

  20. Functional impact of different muscle localization techniques for Botulinum neurotoxin A injections in clinical routine management of post-stroke spasticity.

    Science.gov (United States)

    Zeuner, Kirsten E; Knutzen, Arne; Kühl, Carina; Möller, Bettina; Hellriegel, Helge; Margraf, Nils G; Deuschl, Günther; Stolze, Henning

    2017-01-01

    Treatment options for spasticity include intramuscular botulinum neurotoxin A (BoNT-A) injections. Both ultrasound (US) or electromyographic (EMG) guided BoNT-A injections are employed to isolate muscles. To date, most studies have included patients naïve to BoNT-A or following a prolonged wash out phase. To determine the impact of US/EMG guided BoNT-A injections on function in outpatients with spasticity receiving an established re-injection regime. Thirty patients post-stroke were investigated in a single-blinded, randomized controlled trial using a cross-over design for the EMG and US and a parallel design for the control group. The Modified Ashworth (MAS), Disability Assessment (DAS), Quality of Life (EQ-5D), self-rating scale and Barthel Index were assessed pre- and post-BoNT-A injections of upper limb muscles by a to the injection technique blinded person. MAS improved in arm, finger and upper limb 4 weeks after BoNT-A treatment. The improvement showed no significant differences between the three injection techniques. Barthel Index, DAS and EQ-5D remained unchanged in all groups. This pilot study questions the impact of the instrumental guided injection techniques on everyday functionality in a routine clinical setting with established re-injection intervals. Larger trials are warranted with patients who are under long-term treatment on a regular basis.

  1. The effect of pH on recombinant C-terminal domain of Botulinum Neurotoxin type E (rBoNT/E-HCC

    Directory of Open Access Journals (Sweden)

    Seyed Jafar Mousavy

    2013-12-01

    Full Text Available Recombinant proteins are tending to be the most favorable vaccine-candidates against botulism. Recombinant Carboxy-terminal of botulinum neurotoxin serotype E (rBoNT/E-HCC has been introduced as an efficient vaccine against botulism type E. In this report, we made an effort to investigate the effect of different pH on protein structure to assess if rBoNT/E-HCC could be used as a vaccine for oral administration. Initially, rBoNT/E-HCC was expressed and purified. Structural changes of rBoNT/E-HCC at several pH conditions were studied by various techniques including circular dichroism (CD, fluorescence, aggregation and UV-Vis spectroscopy. The results showed the more compact and more stable structure for rBoNT/E-HCC at acidic pH, and loosely folded structure at alkaline pH. Our finding as the first step of rBoNT/E-HCC evaluation, hopefully introduce it as a suitable vaccine candidate for oral administration.

  2. Isolation, characterization, cloning and expression of an alpha-neurotoxin from the venom of the Mexican coral snake Micrurus laticollaris (Squamata: Elapidae).

    Science.gov (United States)

    Carbajal-Saucedo, Alejandro; López-Vera, Estuardo; Bénard-Valle, Melisa; Smith, Eric N; Zamudio, Fernando; de Roodt, Adolfo R; Olvera-Rodríguez, Alejandro

    2013-05-01

    A new member of short chain α-neurotoxic protein family from venom of the Mexican coral snake, Micrurus laticollaris, was characterized. This protein, named MlatA1, possesses 61 amino acids with 8 conserved cysteine residues, sharing 30-91% sequence identity with other fully sequenced Micrurus toxins. MlatA1 (LD50i.v. = 0.064 mg/kg) antagonizes with both fetal and adult nicotinic acetylcholine receptor (nAChR) as well as α-7 neuronal nAChR in a dose-dependent way. Specific rabbit anti-Mlat serum (titer higher than 18,000) does not show any protective ability against this toxin, nevertheless it was able to recognize protein bands in six out of twelve Micrurus venoms showing the existence of two distinct antigenic groups for α-neurotoxins in North American coral snakes species. The MlatA1 gene was cloned and used to produce recombinant toxin (rMlatA1) that was recognized by rabbit anti-native toxin but was depleted of toxic activity.

  3. Botulinum neurotoxin type A injection of the pelvic floor muscle in pain due to spasticity: a review of the current literature.

    Science.gov (United States)

    Bhide, Alka A; Puccini, Federica; Khullar, Vik; Elneil, Suzy; Digesu, G Alessandro

    2013-09-01

    The role of muscle spasm is not a new concept in the genesis of pain. Botulinum neurotoxin type A (BoNTA) has been successfully employed in a variety of muscular and inflammatory conditions. The aim of our study was to review the published literature on the role of BoNTA injection of the pelvic floor muscle in the management of women with chronic pelvic pain (CPP). A systematic search of the literature published up to June 2012 on the use of BoNTA in the treatment of female pelvic floor muscle spasm was carried out using relevant search terms in MEDLINE and EMBASE databases. The results were limited to full-text English language articles. Relevant trials as well as relevant reviews were selected and analyzed by two independent reviewers. Five studies (2 case reports, 1 prospective pilot study, 1 retrospective study and 1 randomised double-blind placebo controlled study) were included in this systematic review. Overall, BoNTA has shown to be beneficial in relieving CPP related to pelvic floor spasm. The role of BoNTA as a treatment of CPP has been recognized for more than 10 years. Although data are still scarce preliminary results are encouraging. BoNTA is an attractive option for refractory CPP related to pelvic floor muscle spasm, but further studies using validated and reproducible outcome measures are needed, to establish its effectiveness, safeness, technique, optimal dosage, and duration of symptom relief.

  4. Complexity Plots

    KAUST Repository

    Thiyagalingam, Jeyarajan

    2013-06-01

    In this paper, we present a novel visualization technique for assisting the observation and analysis of algorithmic complexity. In comparison with conventional line graphs, this new technique is not sensitive to the units of measurement, allowing multivariate data series of different physical qualities (e.g., time, space and energy) to be juxtaposed together conveniently and consistently. It supports multivariate visualization as well as uncertainty visualization. It enables users to focus on algorithm categorization by complexity classes, while reducing visual impact caused by constants and algorithmic components that are insignificant to complexity analysis. It provides an effective means for observing the algorithmic complexity of programs with a mixture of algorithms and black-box software through visualization. Through two case studies, we demonstrate the effectiveness of complexity plots in complexity analysis in research, education and application. © 2013 The Author(s) Computer Graphics Forum © 2013 The Eurographics Association and Blackwell Publishing Ltd.

  5. Engaging complexity

    Directory of Open Access Journals (Sweden)

    Gys M. Loubser

    2014-01-01

    Full Text Available In this article, I discuss studies in complexity and its epistemological implications for systematic and practical theology. I argue that engagement with complexity does not necessarily assurea non-reductionist approach. However, if complexity is engaged transversally, it becomes possible to transcend reductionist approaches. Moreover, systematic and practical the ologians can draw on complexity in developing new ways of understanding and, therefore, new ways of describing the focus, epistemic scope and heuristic structures of systematic and practical theology. Firstly, Edgar Morin draws a distinction between restricted and general complexity based on the epistemology drawn upon in studies in complexity. Moving away from foundationalist approaches to epistemology, Morin argues for a paradigm of systems. Secondly,I discuss Kees van Kooten Niekerk�s distinction between epistemology, methodology andontology in studies in complexity and offer an example of a theological argument that drawson complexity. Thirdly, I argue for the importance of transversality in engaging complexity by drawing on the work of Wentzel van Huyssteen and Paul Cilliers. In conclusion, I argue that theologians have to be conscious of the epistemic foundations of each study in complexity, and these studies illuminate the heart of Reformed theology.Intradisciplinary and/or interdisciplinary implications: Therefore, this article has both intradisciplinary and interdisciplinary implications. When theologians engage studies incomplexity, the epistemological roots of these studies need to be considered seeing thatresearchers in complexity draw on different epistemologies. Drawing on transversality wouldenhance such considerations. Furthermore, Edgar Morin�s and Paul Cilliers� approach tocomplexity will inform practical and theoretical considerations in church polity and unity.

  6. Computational Complexity

    Directory of Open Access Journals (Sweden)

    J. A. Tenreiro Machado

    2017-02-01

    Full Text Available Complex systems (CS involve many elements that interact at different scales in time and space. The challenges in modeling CS led to the development of novel computational tools with applications in a wide range of scientific areas. The computational problems posed by CS exhibit intrinsic difficulties that are a major concern in Computational Complexity Theory. [...

  7. Complex narratives

    NARCIS (Netherlands)

    Simons, J.

    2008-01-01

    This paper brings together narratology, game theory, and complexity theory to untangle the intricate nature of complex narratives in contemporary cinema. It interrogates the different terms - forking-path narratives, mind-game films, modular narratives, multiple-draft films, database narratives, puz

  8. Complex odontoma.

    Science.gov (United States)

    Preetha, A; Balikai, Bharati S; Sujatha, D; Pai, Anuradha; Ganapathy, K S

    2010-01-01

    Odontomas are hamartomatous lesions or malformations composed of mature enamel, dentin, and pulp. They may be compound or complex, depending on the extent of morphodifferentiation or their resemblance to normal teeth. The etiology of odontoma is unknown, although several theories have been proposed. This article describes a case of a large infected complex odontoma in the residual mandibular ridge, resulting in considerable mandibular expansion.

  9. Simplifying complexity

    NARCIS (Netherlands)

    Leemput, van de I.A.

    2016-01-01

    In this thesis I use mathematical models to explore the properties of complex systems ranging from microbial nitrogen pathways and coral reefs to the human state of mind. All are examples of complex systems, defined as systems composed of a number of interconnected parts, where the systemic behavior

  10. Structure of the heterodimeric neurotoxic complex viperotoxin F (RV-4/RV-7) from the venom of Vipera russelli formosensis at 1.9 A resolution.

    Science.gov (United States)

    Perbandt, Markus; Tsai, Inn-Ho; Fuchs, Annemarie; Banumathi, Sankaran; Rajashankar, Kanagalaghata R; Georgieva, Dessislava; Kalkura, Narayana; Singh, Tej P; Genov, Nicolay; Betzel, Christian

    2003-10-01

    The presynaptic viperotoxin F is the major lethal component of the venom of Vipera russelli formosensis (Taiwan viper). It is a heterodimer of two highly homologous (65% identity) but oppositely charged subunits: a basic and neurotoxic PLA(2) (RV-4) and an acidic non-toxic component with a very low enzymatic activity (RV-7). The crystal structure of the complex has been determined by molecular replacement and refined to 1.9 A resolution and an R factor of 22.3% with four RV-4/RV-7 complexes in the asymmetric unit, which do not exhibit any local point-group symmetry. The complex formation decreases the accessible surface area of the two subunits by approximately 1425 A(2). Both PLA(2)s are predicted to have very low, if any, anticoagulant activity. The structure of viperotoxin F is compared with that of the heterodimeric neurotoxin vipoxin from the venom of another viper, V. ammodytes meridionalis. The structural basis for the differences between the pharmacological activities of the two toxins is discussed. The neutralization of the negative charge of the major ligand for Ca(2+), Asp49, by intersubunit salt bridges is probably a common mechanism of self-stabilization of heterodimeric Viperinae snake-venom neurotoxins in the absence of bound calcium.

  11. Complex variables

    CERN Document Server

    Fisher, Stephen D

    1999-01-01

    The most important topics in the theory and application of complex variables receive a thorough, coherent treatment in this introductory text. Intended for undergraduates or graduate students in science, mathematics, and engineering, this volume features hundreds of solved examples, exercises, and applications designed to foster a complete understanding of complex variables as well as an appreciation of their mathematical beauty and elegance. Prerequisites are minimal; a three-semester course in calculus will suffice to prepare students for discussions of these topics: the complex plane, basic

  12. Managing Complexity

    DEFF Research Database (Denmark)

    Maylath, Bruce; Vandepitte, Sonia; Minacori, Patricia

    2013-01-01

    This article discusses the largest and most complex international learning-by-doing project to date- a project involving translation from Danish and Dutch into English and editing into American English alongside a project involving writing, usability testing, and translation from English into Dut...... and into French. The complexity of the undertaking proved to be a central element in the students' learning, as the collaboration closely resembles the complexity of international documentation workplaces of language service providers. © Association of Teachers of Technical Writing....

  13. Lecithin Complex

    African Journals Online (AJOL)

    yellow power was collected as polydatin-lecithin complex. ... performed on an Agilent 1260 HPLC system. The injection volume .... rabbits. Biomed. Pharmacother 2009; 63: 457-462. 4. Liu B, Du J, Zeng J, Chen C, Niu S. Characterization and.

  14. BotR/A and TetR are alternative RNA polymerase sigma factors controlling the expression of the neurotoxin and associated protein genes in Clostridium botulinum type A and Clostridium tetani.

    Science.gov (United States)

    Raffestin, Stéphanie; Dupuy, Bruno; Marvaud, Jean Christophe; Popoff, Michel R

    2005-01-01

    Clostridium botulinum and Clostridium tetani, respectively, produce potent toxins, botulinum neurotoxin (BoNT) and tetanus neurotoxin (TeTx), which are responsible for severe diseases, botulism and tetanus. Neurotoxin synthesis is a regulated process in Clostridium. The genes botR/A in C. botulinum A and tetR in C. tetani positively regulate expression of BoNT/A and associated non-toxic proteins (ANTPs), as well as TeTx respectively. The botR/A gene lies in close vicinity of the two operons which contain bont/A and antps genes in C. botulinum A, and tetR immediately precedes the tetX gene in C. tetani. We show that BotR/A and TetR function as specific alternative sigma factors rather than positive regulators based on the following results: (i) BotR/A and TetR associated with target DNAs only in the presence of the RNA polymerase core enzyme (Core), (ii) BotR/A and TetR directly bound with the core enzyme, (iii) BotR/A-Core recognized -35 and -10 regions of ntnh-bont/A promoter and (iv) BotR/A and TetR triggered in vitro transcription from the target promoters. In C. botulinum A, bont/A and antps genes are transcribed as bi- and tricistronic operons controlled by BotR/A. BotR/A and TetR are seemingly related to a new subgroup of the sigma70 family that includes TcdR and UviA, which, respectively, regulate production of toxins A and B in C. difficile and bacteriocin in C. perfringens. Sequences of -35 region are highly conserved in the promoter of target toxin genes in C. botulinum, C. tetani, C. difficile and C. perfringens. Overall, a common regulation mechanism probably controls toxin gene expression in these four toxigenic clostridial species.

  15. Detection of Clostridium botulinum neurotoxin genes (A-F) in dairy farms from Northern Germany using PCR: A case-control study.

    Science.gov (United States)

    Fohler, Svenja; Discher, Sabrina; Jordan, Eva; Seyboldt, Christian; Klein, Guenter; Neubauer, Heinrich; Hoedemaker, Martina; Scheu, Theresa; Campe, Amely; Charlotte Jensen, Katharina; Abdulmawjood, Amir

    2016-06-01

    Classical botulism in cattle mainly occurs after ingestion of feed contaminated with preformed toxin. In 2001 a form of botulism ("visceral botulism") was postulated to occur after ingestion of Clostridium (C.) botulinum cells or spores, followed by colonization of the intestine, and local production of botulinum neurotoxin (BoNT) causing chronic generalized disease. To verify the potential role of C. botulinum in the described syndrome, a case-control study was conducted, including 139 farms. Fecal samples, rumen content, water and silage samples were collected on each farm. Real time BoNT gene PCR assays were conducted after enrichment in RCM (Reinforced Clostridial Medium) at 37 °C and conventional PCRs after enrichment in MCM (Modified Cooked Meat Medium) at 30 °C. Furthermore, a direct detection of BoNT genes without prior enrichment was attempted. BoNT A, B, C, D, E and F genes were detected in animal samples from 25 (17.99%), 3 (2.16%), 0 (0.0%), 2 (1.44%), 1 (0.72%), and 3 (2.16%) farms, respectively. Eleven feed samples were positive for BoNT A gene. By enrichment a significant increase in sensitivity was achieved. Therefore, this should be an essential part of any protocol. No significant differences regarding BoNT gene occurrence could be observed between Case and Control farms or chronically diseased and clinically healthy animals within the particular category. Thus, the postulated form of chronic botulism in cows could not be confirmed. This study supports the general opinion that C. botulinum can occasionally be found in the rumen and intestine of cows without causing disease.

  16. Embryonic Stem Cell-Derived Neurons Grown on Multi-Electrode Arrays as a Novel In vitro Bioassay for the Detection of Clostridium botulinum Neurotoxins

    Science.gov (United States)

    Jenkinson, Stephen P.; Grandgirard, Denis; Heidemann, Martina; Tscherter, Anne; Avondet, Marc-André; Leib, Stephen L.

    2017-01-01

    Clostridium botulinum neurotoxins (BoNTs) are the most poisonous naturally occurring protein toxins known to mankind and are the causative agents of the severe and potentially life-threatening disease botulism. They are also known for their application as cosmetics and as unique bio-pharmaceuticals to treat an increasing number of neurological and non-neurological disorders. Currently, the potency of biologically active BoNT for therapeutic use is mainly monitored by the murine LD50-assay, an ethically disputable test causing suffering and death of a considerable number of mice. The aim of this study was to establish an in vitro assay as an alternative to the widely used in vivo mouse bioassay. We report a novel BoNT detection assay using mouse embryonic stem cell-derived neurons (mESN) cultured on multi-electrode arrays (MEAs). After 21 days in culture, the mESN formed a neuronal network showing spontaneous bursting activity based on functional synapses and express the necessary target proteins for BoNTs. Treating cultures for 6 h with 16.6 pM of BoNT serotype A and incubation with 1.66 pM BoNT/A or 33 Units/ml of Botox® for 24 h lead to a significant reduction of both spontaneous network bursts and average spike rate. This data suggests that mESN cultured on MEAs pose a novel, biologically relevant model that can be used to detect and quantify functional BoNT effects, thus accelerating BoNT research while decreasing animal use. PMID:28280466

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

    Science.gov (United States)

    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.

  18. Time Course Analysis of the Effects of Botulinum Neurotoxin Type A on Pain and Vasomotor Responses Evoked by Glutamate Injection into Human Temporalis Muscles

    Directory of Open Access Journals (Sweden)

    Larissa Bittencourt da Silva

    2014-02-01

    Full Text Available The effect of botulinum neurotoxin type A (BoNTA on glutamate-evoked temporalis muscle pain and vasomotor responses was investigated in healthy men and women over a 60 day time course. Subjects participated in a pre-BoNTA session where their responses to injection of glutamate (1 M, 0.2 mL and saline (0.2 mL into the temporalis muscles were assessed. On Day 1, BoNTA (5 U was injected into one temporalis muscle and saline into the contralateral temporalis muscle, in a randomized order. Subjects then received intramuscular injections of glutamate (1 M, 0.2 mL into the left and right temporalis muscles at 3 h and subsequently 7, 30 and 60 days post-injection of BoNTA. Pain intensity, pain area, and neurogenic inflammation (skin temperature and skin blood perfusion were recorded. Prior to BoNTA treatment, glutamate evoked significantly greater pain and vasomotor reactions (P < 0.001 than saline. BoNTA significantly reduced glutamate-evoked pain intensity (P < 0.05, pain area (P < 0.01, skin blood perfusion (P < 0.05, and skin temperature (P < 0.001. The inhibitory effect of BoNTA was present at 3 h after injection, peaked after 7 days and returned to baseline by 60 days. Findings from the present study demonstrated a rapid action of BoNTA on glutamate-evoked pain and neurogenic inflammation, which is in line with animal studies.

  19. Three-dimensional structure of the neurotoxin ATX Ia from Anemonia sulcata in aqueous solution determined by nuclear magnetic resonance spectroscopy.

    Science.gov (United States)

    Widmer, H; Billeter, M; Wüthrich, K

    1989-01-01

    With the aid of 1H nuclear magnetic resonance (NMR) spectroscopy, the three-dimensional structure in aqueous solution was determined for ATX Ia, which is a 46 residue polypeptide neurotoxin of the sea anemone Anemonia sulcata. The input for the structure calculations consisted of 263 distance constraints from nuclear Overhauser effects (NOE) and 76 vicinal coupling constants. For the structure calculation several new or ammended programs were used in a revised strategy consisting of five successive computational steps. First, the program HABAS was used for a complete search of all backbone and chi 1 conformations that are compatible with the intraresidual and sequential NMR constraints. Second, using the program DISMAN, we extended this approach to pentapeptides by extensive sampling of all conformations that are consistent with the local and medium-range NMR constraints. Both steps resulted in the definition of additional dihedral angle constraints and in stereospecific assignments for a number of beta-methylene groups. In the next two steps DISMAN was used to obtain a group of eight conformers that contain no significant residual violations of the NMR constraints or van der Waals contacts. Finally, these structures were subjected to restrained energy refinement with a modified version of the molecular mechanics module of AMBER, which in addition to the energy force field includes potentials for the NOE distance constraints and the dihedral angle constraints. The average of the pairwise minimal RMS distances between the resulting refined conformers calculated for the well defined molecular core, which contains the backbone atoms of 35 residues and 20 interior side chains, is 1.5 +/- 0.3 A. This core is formed by a four-stranded beta-sheet connected by two well-defined loops, and there is an additional flexible loop consisting of the eleven residues 8-18. The core of the protein is stabilized by three disulfide bridges, which are surrounded by hydrophobic residues

  20. Purification of a neurotoxin from the venom of the 'armed spider' and synthesis of a radioactive probe

    Energy Technology Data Exchange (ETDEWEB)

    Santos, Raquel Gouvea [INSERM, Marseille (France). Lab. Canaux Ioniques]|[Centro de Desenvolvimento da Tecnologia Nuclear (CDTN), Belo Horizonte, MG (Brazil); Renterghem, Catherine Van; Mori, Yasuo; Martin-Moutot, Nicole; Mansuelle, Pascal; Sampieri, Francois; Seagar, Michel [INSERM, Marseille (France). Lab. Canaux Ioniques; Cordeiro, Marta Nascimento; Diniz, Carlos Ribeiro [FUNED - Fundacao Ezequiel Dias, Belo Horizonte, MG (Brazil); Lima, Maria Elena de [Minas Gerais Univ., Belo Horizonte, MG (Brazil). Inst. de Ciencias Biologicas. Lab. de Venenos e Toxinas Animais

    2002-07-01

    The venom of the 'armed spider', a South American spider from the genus Phoneutria contains several toxins that exert important biological effects. {omega}-Phonetoxin IIA ({omega}-PtxIIA) is a potent neurotoxin from this venom evoking flaccid paralysis and death after intracerebro-ventricular injection in mouse. This toxin blocks HVA calcium channels. Calcium channels have been shown to be important targets in neurological pathophysiology like ataxia and migraine. In order to shed more light on the mechanism of action of w-PtxIIA, we have purified this toxin to synthesize a radioactive probe. Crude venom was fractionated in three steps of reversed-phase liquid chromatography (RP-HPLC). Spectrometric analysis of the purified fraction (causing flaccid paralysis) was recorded on a MALDI-TOF Perseptive Voyager Elite spectrometer and a 8363 kDa peptide was detected. After amino acid sequencing the purity of the peptide was confirmed and it was identified as {omega}-PtxIIA. {omega}-PtxIIA was radiolabeled with {sup 125} I using the lactoperoxidase as a oxidizing agent. The stability of the probe synthesized was verified by the binding studies on rat brain synaptosomal membranes. The specific and saturable binding of the {sup 125} I-{omega}-PtxIIA to the membranes indicate that the iodine introduced in the toxin molecule did not interfere with its activity. Native {omega}-PtxIIA competed with the {sup 125} I-{omega}-PtxIIA bound to the specific sites on synaptosomes (IC{sub 50}= 0.54 n M). In this paper we described a new and simpler purification protocol of the {omega}-PtxIIA and synthesized a probe that proved to be useful to study the mechanism of action of {sup 125} I-{omega}-PtxIIA. Selective toxins for calcium channel are very useful tools to the study of some neuronal pathophysiology. (author)

  1. Method development for quantification of the environmental neurotoxin annonacin in Rat plasma by UPLC-MS/MS and application to a pharmacokinetic study.

    Science.gov (United States)

    Bonneau, Natacha; Schmitz-Afonso, Isabelle; Brunelle, Alain; Touboul, David; Champy, Pierre

    2015-11-01

    Annonacin is an environmental neurotoxin identified in the pulp of several fruits of the Annonaceae family (for example in Annona muricata, Asimina triloba), whose consumption was linked with the occurrence of sporadic atypical Parkinsonism with dementia. Pharmacokinetic parameters of this molecule are unknown. A method for its quantification in Rat plasma was developed, using its analogue annonacinone as an internal standard. Extraction from plasma was performed using ethylacetate with a good recovery. Quantification was performed by UPLC-MS/MS in SRM mode, based on the loss of the γ-methyl-γ-lactone (-112amu) from the sodium-cationized species [M+Na](+) of both annonacin and internal standard. The limit of quantification was 0.25ng/mL. Despite strong matrix effects, a good linearity was obtained over two distinct ranges 0.25-10ng/mL and 10-100ng/mL. The intra- and inter-day precisions (RSD) were lower than 10%, while accuracy was within ±10%. This method was applied to a pharmacokinetic study in the Rat. After oral administration of 10mg/kg annonacin, a Cmax of 7.9±1.5ng/mL was reached at Tmax 0.25h; T1/2 was 4.8±0.7h and apparent distribution volume was 387.9±64.6L. The bioavailability of annonacin was estimated to be 3.2±0.3% of the ingested dose.

  2. A Novel Neurotoxin Gene ar1b Recombination Enhances the Efficiency of Helicoverpa armigera Nucleopolyhedrovirus as a Pesticide by Inhibiting the Host Larvae Ability to Feed and Grow.

    Directory of Open Access Journals (Sweden)

    Huan Yu

    Full Text Available A recombinant Helicoverpa armigera nucleopolyhedrovirus (HearNPV, Ar1b-HearNPV, was constructed and identified as an improved bio-control agent of Helicoverpa armigera larvae. The HearNPV polyhedrin promoter was used to express the insect-specific neurotoxin gene, ar1b, which was originally isolated from the Australian funnel-web spider (Atrax robustus. RT-PCR and Western blotting analysis showed that both the ar1b transcript and protein were produced successfully in Ar1b-HearNPV-infected HzAM1 cells. In order to investigate the influence of foreign gene insertion in HearNPV, including the ar1b gene, chloramphenicol resistance gene, lacZ, kanamycin resistance gene, and the gentamicin resistance gene, two virus strains (HZ8-HearNPV and wt-HearNPV were used as controls in the cell transfection analysis. As expected, foreign gene insertion had no impact on budded virus production and viral DNA replication. Both optical microscopy and electron microscopy observations indicated that the formation of the occlusion bodies of recombinant virus was similar to wild type virus. The Ar1b-HearNPV-infected H. armigera larvae exhibited paralysis and weight loss before dying. This recombinant virus also showed a 32.87% decrease in LT50 assays compared with the wild type virus. Besides, Ar1b-HearNPV also inhibited host larval growth and diet consumption. This inhibition was still significant in the older instar larvae treated with the recombinant virus. All of these positive properties of this novel recombinant HearNPV provide a further opportunity to develop this virus strain into a commercial product to control the cotton bollworm.

  3. An Invisible Lethal Killer: Clostridium Botulinum and Botulinum Neurotoxins%一种致命的隐形杀手:肉毒杆菌与肉毒毒素

    Institute of Scientific and Technical Information of China (English)

    王景林

    2013-01-01

    2013年8月,因肉毒杆菌污染,问题奶粉再次进入公众视野.肉毒杆菌(Clostridium botulinum)是一种革兰阳性粗短杆菌,严格厌氧,有A~G7个亚型,每个亚型都可产生一种剧毒的大分子外毒素,即肉毒毒素(botulinum toxin).这种毒素可引起人和动物发生以松弛性麻痹为主症的肉毒中毒(botulism),虽然并不常见,但却是一种致命的中毒性疾病.本文综述了肉毒杆菌生物学特征、肉毒毒素结构与毒性、肉毒中毒临床表现以及检验鉴定等,并介绍了食品安全生产中肉毒杆菌与肉毒毒素的安全风险与控制措施.%The milkpowder contaminated by Clostridium botulinum once again go into the public view in August 2013.Clostridium botulinum,which is a strictly anaerobic gram-positive bacillus,is classified into seven main types from A through G according to exotoxins and they can produce antigenically distinct botulinum neurotoxins.It is a potent toxin that causes the most severe form of food poisoning with pronounced flaccid paralysis,called botulism.The review covers biological features of C.botulinum,structure and toxicity of toxin,clinical symptoms and identification of botulism as well as the risk and control of C.botulinum and toxins during food processing and preservation.

  4. A Novel Neurotoxin Gene ar1b Recombination Enhances the Efficiency of Helicoverpa armigera Nucleopolyhedrovirus as a Pesticide by Inhibiting the Host Larvae Ability to Feed and Grow.

    Science.gov (United States)

    Yu, Huan; Meng, Jiao; Xu, Jian; Liu, Tong-Xian; Wang, Dun

    2015-01-01

    A recombinant Helicoverpa armigera nucleopolyhedrovirus (HearNPV), Ar1b-HearNPV, was constructed and identified as an improved bio-control agent of Helicoverpa armigera larvae. The HearNPV polyhedrin promoter was used to express the insect-specific neurotoxin gene, ar1b, which was originally isolated from the Australian funnel-web spider (Atrax robustus). RT-PCR and Western blotting analysis showed that both the ar1b transcript and protein were produced successfully in Ar1b-HearNPV-infected HzAM1 cells. In order to investigate the influence of foreign gene insertion in HearNPV, including the ar1b gene, chloramphenicol resistance gene, lacZ, kanamycin resistance gene, and the gentamicin resistance gene, two virus strains (HZ8-HearNPV and wt-HearNPV) were used as controls in the cell transfection analysis. As expected, foreign gene insertion had no impact on budded virus production and viral DNA replication. Both optical microscopy and electron microscopy observations indicated that the formation of the occlusion bodies of recombinant virus was similar to wild type virus. The Ar1b-HearNPV-infected H. armigera larvae exhibited paralysis and weight loss before dying. This recombinant virus also showed a 32.87% decrease in LT50 assays compared with the wild type virus. Besides, Ar1b-HearNPV also inhibited host larval growth and diet consumption. This inhibition was still significant in the older instar larvae treated with the recombinant virus. All of these positive properties of this novel recombinant HearNPV provide a further opportunity to develop this virus strain into a commercial product to control the cotton bollworm.

  5. C terminal half fragment (50 kDa) of heavy chain components of Clostridium botulinum type C and D neurotoxins can be used as an effective vaccine.

    Science.gov (United States)

    Lee, Jae-Chul; Hwang, Hyun-Jung; Sakaguchi, Yoshihiko; Yamamoto, Yumiko; Arimitsu, Hideyuki; Tsuji, Takao; Watanabe, Toshihiro; Ohyama, Tohru; Tsuchiya, Tomofusa; Oguma, Keiji

    2007-01-01

    Recombinant whole heavy chains (H, 100 kDa) and their N-terminal (Hn, 50 kDa) and C-terminal (Hc, 50 kDa) half fragments of Clostridium botulinum type C and D neurotoxins were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. GST eliminated-preparations of H (10 microg), Hn (5 microg), Hc (5 microg), or a mixture of Hn (5 microg) and Hc (5 microg) of types C and D were mixed with an equal volume of adjuvant, and then were twice injected into mice subcutaneously. After immunization, the mice were challenged with up to 10(6) the minimum lethal doses (MLD)/0.5 ml of C or D toxin, the type of which was same as that of the immunogens. All of the mice immunized with antigens except for Hn survived against 10(5) to 10(6) MLD/0.5 ml of the toxins, but the mice immunized with Hn were killed by 100 MLD/0.5 ml. The mice immunized with a mixture of C-Hc and D-Hc, each 5 microg, also showed a high level of resistance against both C and D toxins. Antibody levels immunized with GST fused-or GST eliminatedpreparation were quite similar. These results indicate that recombinant GST-fused Hc can be used as a safe and effective vaccine for type C and D botulism in animals. It also became clear that one time inoculation with a large amount of C-Hc or D-Hc, 100 microg, is useful for vaccine trials in mice.

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

    Science.gov (United States)

    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. Copyright © 2012 Elsevier Ltd. All rights reserved.

  7. The Regions on the Light Chain of Botulinum Neurotoxin Type A Recognized by T Cells from Toxin-Treated Cervical Dystonia Patients. The Complete Human T-Cell Recognition Map of the Toxin Molecule.

    Science.gov (United States)

    Oshima, Minako; Deitiker, Philip; Jankovic, Joseph; Atassi, M Zouhair

    2017-09-11

    We have recently mapped the in vitro proliferative responses of T cells from botulinum neurotoxin type A (BoNT/A)-treated cervical dystonia (CD) patients with overlapping peptides encompassing BoNT/A heavy chain (residues 449-1296). In the present study, we determined the recognition profiles, by peripheral blood lymphocytes (PBL) from the same set of patients, of BoNT/A light (L) chain (residues 1-453) by using 32 synthetic overlapping peptides that encompassed the entire L chain. Profiles of the T-cell responses (expressed in stimulation index, SI; Z score based on transformed SI) to the peptides varied among the patients. Samples from 14 patients treated solely with BoNT/A recognized 3-13 (average 7.2) peptides/sample at Z > 3.0 level. Two peptide regions representing residues 113-131 and 225-243 were recognized by around 40% of these patients. Regarding treatment parameters, treatment history with current BOTOX(®) only group produced significantly lower average T-cell responses to the 32 L-chain peptides compared to treatments with mix of type A including original and current BOTOX(®). Influence of other treatment parameters on T-cell recognition of the L-chain peptides was also observed. Results of the submolecular T-cell recognition of the L chain are compared to those of the H chain and the T-cell recognition profile of the entire BoNT/A molecule is discussed. Abbreviations used: BoNT/A, botulinum neurotoxin type A; BoNT/Ai, inactivated BoNT/A; BoNT/B, botulinum neurotoxin type B; CD, cervical dystonia; L chain, the light chain (residues 1-448) of BoNT/A; LNC, lymph node cells; H chain, the heavy chain (residues 449-1296) of BoNT/A; HC, C-terminal domain (residues 855-1296) of H chain; HN, N-terminal domain (residues 449-859) of H chain; MPA, mouse protection assay; SI, stimulation index (SI = cpm of (3)H-thymidine incorporated by antigen-stimulated T cells/cpm incorporated by unstimulated cells); TeNT, tetanus neurotoxin; TeNTi, inactivated TeNT.

  8. Exercise-induced rescue of tongue function without striatal dopamine sparing in a rat neurotoxin model of Parkinson disease.

    Science.gov (United States)

    Ciucci, Michelle R; Schaser, Allison J; Russell, John A

    2013-09-01

    Unilateral lesions to the medial forebrain bundle with 6-hydroxydopamine (6-OHDA) lead to force and timing deficits during a complex licking task. We hypothesized that training targeting tongue force generation during licking would improve timing and force measures and also lead to striatal dopamine sparing. Nine month-old male Fisher344/Brown Norway rats were used in this experiment. Sixteen rats were in the control condition and received tongue exercise (n=8) or no exercise (n=8). Fourteen rats were in the 6-OHDA lesion condition and underwent tongue exercise (n=7) and or no exercise (n=7). Following 4 weeks of training and post-training measures, all animals underwent bilateral stimulation of the hypoglossal nerves to measure muscle contractile properties and were then transcardially perfused and brain tissues collected for immunohistochemistry to examine striatal dopamine content. Results demonstrated that exercise animals performed better for maximal force, average force, and press rate than their no-exercise counterparts, and the 6-OHDA animals that underwent exercise performed as well as the Control No Exercise group. Interestingly, there were no group differences for tetanic muscle force, despite behavioral recovery of forces. Additionally, behavioral and neurochemical analyses indicate that there were no differences in striatal dopamine. Thus, targeted exercise can improve tongue force and timing deficits related to 6-OHDA lesions and this exercise likely has a central, versus peripheral (muscle strength) mechanism. However, this mechanism is not related to sparing of striatal dopamine content. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. Complex analysis

    CERN Document Server

    Freitag, Eberhard

    2005-01-01

    The guiding principle of this presentation of ``Classical Complex Analysis'' is to proceed as quickly as possible to the central results while using a small number of notions and concepts from other fields. Thus the prerequisites for understanding this book are minimal; only elementary facts of calculus and algebra are required. The first four chapters cover the essential core of complex analysis: - differentiation in C (including elementary facts about conformal mappings) - integration in C (including complex line integrals, Cauchy's Integral Theorem, and the Integral Formulas) - sequences and series of analytic functions, (isolated) singularities, Laurent series, calculus of residues - construction of analytic functions: the gamma function, Weierstrass' Factorization Theorem, Mittag-Leffler Partial Fraction Decomposition, and -as a particular highlight- the Riemann Mapping Theorem, which characterizes the simply connected domains in C. Further topics included are: - the theory of elliptic functions based on...

  10. A Polychaete's powerful punch: venom gland transcriptomics of Glycera reveals a complex cocktail of toxin homologs.

    Science.gov (United States)

    von Reumont, Björn M; Campbell, Lahcen I; Richter, Sandy; Hering, Lars; Sykes, Dan; Hetmank, Jörg; Jenner, Ronald A; Bleidorn, Christoph

    2014-09-05

    Glycerids are marine annelids commonly known as bloodworms. Bloodworms have an eversible proboscis adorned with jaws connected to venom glands. Bloodworms prey on invertebrates, and it is known that the venom glands produce compounds that can induce toxic effects in animals. Yet, none of these putative toxins has been characterized on a molecular basis. Here we present the transcriptomic profiles of the venom glands of three species of bloodworm, Glycera dibranchiata, Glycera fallax and Glycera tridactyla, as well as the body tissue of G. tridactyla. The venom glands express a complex mixture of transcripts coding for putative toxin precursors. These transcripts represent 20 known toxin classes that have been convergently recruited into animal venoms, as well as transcripts potentially coding for Glycera-specific toxins. The toxins represent five functional categories: Pore-forming and membrane-disrupting toxins, neurotoxins, protease inhibitors, other enzymes, and CAP domain toxins. Many of the transcripts coding for putative Glycera toxins belong to classes that have been widely recruited into venoms, but some are homologs of toxins previously only known from the venoms of scorpaeniform fish and monotremes (stonustoxin-like toxin), turrid gastropods (turripeptide-like peptides), and sea anemones (gigantoxin I-like neurotoxin). This complex mixture of toxin homologs suggests that bloodworms employ venom while predating on macroscopic prey, casting doubt on the previously widespread opinion that G. dibranchiata is a detritivore. Our results further show that researchers should be aware that different assembly methods, as well as different methods of homology prediction, can influence the transcriptomic profiling of venom glands. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  11. Crotoxin: Structural Studies, Mechanism of Action and Cloning of Its gene

    Science.gov (United States)

    1989-12-01

    basic subunit clones and the acidic subunit clones that were partially sequenced, had multiple sequence differences. New cONA and genomic libraries from...were partially sequenced, had multiple sequence differences. New cDNA and genomic libraries from C. s. scutulatus are in being prepared and screened...unsuccessful in identifying any clones that contained basic subunit cDNA. Work is continuing with new cDNA and genomic libraries from . t , in collaboration

  12. Complex Networks

    CERN Document Server

    Evsukoff, Alexandre; González, Marta

    2013-01-01

    In the last decade we have seen the emergence of a new inter-disciplinary field focusing on the understanding of networks which are dynamic, large, open, and have a structure sometimes called random-biased. The field of Complex Networks is helping us better understand many complex phenomena such as the spread of  deseases, protein interactions, social relationships, to name but a few. Studies in Complex Networks are gaining attention due to some major scientific breakthroughs proposed by network scientists helping us understand and model interactions contained in large datasets. In fact, if we could point to one event leading to the widespread use of complex network analysis is the availability of online databases. Theories of Random Graphs from Erdös and Rényi from the late 1950s led us to believe that most networks had random characteristics. The work on large online datasets told us otherwise. Starting with the work of Barabási and Albert as well as Watts and Strogatz in the late 1990s, we now know th...

  13. Spectroscopic properties and stability of the neurotoxic complex Vipoxin and its components

    Science.gov (United States)

    Genov, Nicolay; Dolashka, Pavlina; Aleksiev, Boris; Mancheva, Ivanka; Rajashankar, Kanagalaghatta R.; Betzel, Christian

    1998-08-01

    The neurotoxin Vipoxin from the venom of Vipera ammodytes meridionalis is a complex between a toxic basic phospholipase A 2 (PLA 2) and a non-toxic acidic protein inhibitor (Inh). Tryptophan fluorescence parameters are determined for the complex and for its components. Iodide, caesium and acrylamide are not efficient quenchers of the Vipoxin indole emission. Increased accessibilities of tryptophans to ionic and neutral quenchers are found after the dissociation of the complex. Trp 20 and Trp 31 became more 'exposed' in the separated individual proteins. The indole rings of the complex are located in a positively charged environment. Inspection of the Vipoxin X-ray model showed that the three tryptophyl side chains are located in the interface region between the enzyme and the inhibitor and are completely 'exposed' in the separated components of the complex. In Vipoxin an efficient 'interchain' energy transfer between tyrosyl and tryptophyl residues from different polypeptide chains occurs. Static quenching with acrylamide is also detected in PLA 2 and Inh. The free energy changes Δ GD for the unfolding reactions of Vipoxin, PLA 2 and Inh are determined by circular dichroism spectroscopy. The complex formation between the toxic PLA 2 and the inhibitor increases Δ GDH 2O to 23.5 kJ mol -1.

  14. Complex chemistry with complex compounds

    Directory of Open Access Journals (Sweden)

    Eichler Robert

    2016-01-01

    Full Text Available In recent years gas-phase chemical studies assisted by physical pre-separation allowed for the investigation of fragile single molecular species by gas-phase chromatography. The latest success with the heaviest group 6 transactinide seaborgium is highlighted. The formation of a very volatile hexacarbonyl compound Sg(CO6 was observed similarly to its lighter homologues molybdenum and tungsten. The interactions of these gaseous carbonyl complex compounds with quartz surfaces were investigated by thermochromatography. Second-generation experiments are under way to investigate the intramolecular bond between the central metal atom of the complexes and the ligands addressing the influence of relativistic effects in the heaviest compounds. Our contribution comprises some aspects of the ongoing challenging experiments as well as an outlook towards other interesting compounds related to volatile complex compounds in the gas phase.

  15. Managing Complexity

    Energy Technology Data Exchange (ETDEWEB)

    Chassin, David P.; Posse, Christian; Malard, Joel M.

    2004-08-01

    Physical analogs have shown considerable promise for understanding the behavior of complex adaptive systems, including macroeconomics, biological systems, social networks, and electric power markets. Many of today’s most challenging technical and policy questions can be reduced to a distributed economic control problem. Indeed, economically-based control of large-scale systems is founded on the conjecture that the price-based regulation (e.g., auctions, markets) results in an optimal allocation of resources and emergent optimal system control. This paper explores the state of the art in the use physical analogs for understanding the behavior of some econophysical systems and to deriving stable and robust control strategies for them. In particular we review and discussion applications of some analytic methods based on the thermodynamic metaphor according to which the interplay between system entropy and conservation laws gives rise to intuitive and governing global properties of complex systems that cannot be otherwise understood.

  16. Crystal structure of the receptor binding domain of the botulinum C-D mosaic neurotoxin reveals potential roles of lysines 1118 and 1136 in membrane interactions

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yanfeng; Buchko, Garry W.; Qin, Ling; Robinson, Howard; Varnum, Susan M.

    2011-01-07

    The botulinum neurotoxins (BoNTs) produced by different strains of the bacterium Clostridium botulinum are responsible for the disease botulism and include a group of immunologically distinct serotypes (A, B, E, and F) that are considered to be the most lethal natural proteins known for humans. Two BoNT serotypes, C and D, while rarely associated with human infection, are responsible for deadly botulism outbreaks afflicting animals. Also associated with animal infections is the BoNT C-D mosaic protein (BoNT/CD), a BoNT subtype that is essentially a hybrid of the BoNT/C (~two-thirds) and BoNT/D (~one-third) serotypes. While the amino acid sequence of the heavy chain receptor binding (HCR) domain of BoNT/CD (BoNT/CD-HCR) is very similar to the corresponding amino acid sequence of BoNT/D, BoNT/CD-HCR binds synaptosome membranes better than BoNT/D-HCR. To obtain structural insights for the different membrane binding properties, the crystal structure of BoNT/CD-HCR (S867-E1280) was determined at 1.56 Å resolution and compared to previously reported structures for BoNT/D-HCR. Overall, the BoNT/CD-HCR structure is similar to the two sub-domain organization observed for other BoNT HCRs: an N-terminal jellyroll barrel motif and a C-terminal β-trefoil fold. Comparison of the structure of BoNT/CD-HCR with BoNT/D-HCR indicates that K1118 has a similar structural role as the equivalent residue, E1114, in BoNT/D-HCR, while K1136 has a structurally different role than the equivalent residue, G1132, in BoNT/D-HCR. Lysine-1118 forms a salt bridge with E1247 and may enhance membrane interactions by stabilizing the putative membrane binding loop (K1240-N1248). Lysine-1136 is observed on the surface of the protein. A sulfate ion bound to K1136 may mimic a natural interaction with the negatively changed phospholipid membrane surface. Liposome-binding experiments demonstrate that BoNT/CD-HCR binds phosphatidylethanolamine liposomes more tightly than BoNT/D-HCR

  17. Crystal Structure of the Receptor Binding Domain of the botulinum C-D Mosiac Neurotoxin Reveals Potential Roles of Lysines 1118 and 1136 in Membrane Interactions

    Energy Technology Data Exchange (ETDEWEB)

    Y Zhang; G Buchko; L Qin; H Robinson; S Varnum

    2011-12-31

    The botulinum neurotoxins (BoNTs) produced by different strains of the bacterium Clostridium botulinum are responsible for the disease botulism and include a group of immunologically distinct serotypes (A, B, E, and F) that are considered to be the most lethal natural proteins known for humans. Two BoNT serotypes, C and D, while rarely associated with human infection, are responsible for deadly botulism outbreaks afflicting animals. Also associated with animal infections is the BoNT C-D mosaic protein (BoNT/CD), a BoNT subtype that is essentially a hybrid of the BoNT/C ({approx}two-third) and BoNT/D ({approx}one-third) serotypes. While the amino acid sequence of the heavy chain receptor binding (HCR) domain of BoNT/CD (BoNT/CD-HCR) is very similar to the corresponding amino acid sequence of BoNT/D, BoNT/CD-HCR binds synaptosome membranes better than BoNT/D-HCR. To obtain structural insights for the different membrane binding properties, the crystal structure of BoNT/CD-HCR (S867-E1280) was determined at 1.56 {angstrom} resolution and compared to previously reported structures for BoNT/D-HCR. Overall, the BoNT/CD-HCR structure is similar to the two sub-domain organization observed for other BoNT HCRs: an N-terminal jellyroll barrel motif and a C-terminal {beta}-trefoil fold. Comparison of the structure of BoNT/CD-HCR with BoNT/D-HCR indicates that K1118 has a similar structural role as the equivalent residue, E1114, in BoNT/D-HCR, while K1136 has a structurally different role than the equivalent residue, G1132, in BoNT/D-HCR. Lysine-1118 forms a salt bridge with E1247 and may enhance membrane interactions by stabilizing the putative membrane binding loop (K1240-N1248). Lysine-1136 is observed on the surface of the protein. A sulfate ion bound to K1136 may mimic a natural interaction with the negatively changed phospholipid membrane surface. Liposome-binding experiments demonstrate that BoNT/CD-HCR binds phosphatidylethanolamine liposomes more tightly than BoNT/D-HCR.

  18. Epitope characterization and variable region sequence of f1-40, a high-affinity monoclonal antibody to botulinum neurotoxin type a (Hall strain.

    Directory of Open Access Journals (Sweden)

    Miles C Scotcher

    Full Text Available BACKGROUND: Botulism, an often fatal neuroparalytic disease, is caused by botulinum neurotoxins (BoNT which consist of a family of seven serotypes (A-H produced by the anaerobic bacterium Clostridium botulinum. BoNT, considered the most potent biological toxin known, is a 150 kDa protein consisting of a 100 kDa heavy-chain (Hc and a 50 kDa light-chain (Lc. F1-40 is a mouse-derived, IgG1 monoclonal antibody that binds the light chain of BoNT serotype A (BoNT/A and is used in a sensitive immunoassay for toxin detection. We report the fine epitope mapping of F1-40 and the deduced amino acid sequence of the variable regions of the heavy and light chains of the antibody. METHODS AND FINDINGS: To characterize the binding epitope of F1-40, three complementary experimental approaches were selected. Firstly, recombinant peptide fragments of BoNT/A light-chain were used in Western blots to identify the epitope domains. Secondly, a peptide phage-display library was used to identify the specific amino acid sequences. Thirdly, the three-dimensional structure of BoNT/A was examined in silico, and the amino acid sequences determined from the phage-display studies were mapped onto the three-dimensional structure in order to visualize the epitope. F1-40 was found to bind a peptide fragment of BoNT/A, designated L1-3, which spans from T125 to L200. The motif QPDRS was identified by phage-display, and was mapped to a region within L1-3. When the three amino acids Q138, P139 and D140 were all mutated to glycine, binding of F1-40 to the recombinant BoNT/A light chain peptide was abolished. Q-138, P-139 and D-140 form a loop on the external surface of BoNT/A, exposed to solvent and accessible to F1-40 binding. CONCLUSIONS: The epitope of F1-40 was localized to a single exposed loop (ss4, ss5 on the Lc of BoNT. Furthermore amino acids Q138, P139 and D140 forming the tip of the loop appear critical for binding.

  19. Structural Insights into the Functional Role of the Hcn Sub-domain of the Receptor-Binding Domain of the Botulinum Neurotoxin Mosaic Serotype C/D

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yanfeng; Gardberg, Anna; Edwards, Tom E.; Sankaran, Banumathi; Robinson, Howard; Varnum, Susan M.; Buchko, Garry W.

    2013-07-01

    Botulinum neurotoxin (BoNT), the causative agent of the deadly neuroparalytic disease botulism, is the most poisonous protein known for humans. Produced by different strains of the anaerobic bacterium Clostridium botulinum, BoNT effects cellular intoxication via a multistep mechanism executed by the three modules of the activated protein. Endocytosis, the first step of cellular intoxication, is triggered by the ~50 kDa, heavy-chain receptor-binding module (HCR) that is specific for a ganglioside and a protein receptor on neuronal cell surfaces. This dual receptor recognition mechanism between BoNT and the host cell’s membrane is well documented and occurs via specific intermolecular interactions with the C-terminal sub-domain, Hcc, of BoNT-HCR. The N-terminal sub-domain of BoNT-HCR, Hcn, comprises ~50% of BoNT-HCR and adopts a B-sheet jelly roll fold. While suspected in assisting cell surface recognition, no unambiguous function for the Hcn sub-domain in BoNT has been indentified. To obtain insights into the potential function of the Hcn sub-domain in BoNT, the first crystal structure of a BoNT with an organic ligand bound to the Hcn sub-domain has been obtained. Here, we describe the crystal structure of BoNT/CD-HCR determined at 1.70 Å resolution with a tetraethylene glycol (PG4) molecule bound in an hydrophobic cleft between B-strands in the B-sheet jelly fold roll of the Hcn sub-domain. The molecule is completely engulfed in the cleft, making numerous hydrophobic (Y932, S959, W966, and D1042) and hydrophilic (S935, W977, L979, N1013, and I1066) contacts with the protein’s side chain and backbone that may mimic in vivo interactions with the phospholipid membranes on neuronal cell surfaces. A sulfate ion was also observed bound to residues T1176, D1177, K1196, and R1243 in the Hcc sub-domain of BoNT/CD-HCR. In the crystal structure of a similar protein, BoNT/D-HCR, a sialic acid

  20. The effect of botulinum neurotoxin A on sciatic nerve injury-induced neuroimmunological changes in rat dorsal root ganglia and spinal cord.

    Science.gov (United States)

    Mika, J; Rojewska, E; Makuch, W; Korostynski, M; Luvisetto, S; Marinelli, S; Pavone, F; Przewlocka, B

    2011-02-23

    Botulinum neurotoxin serotype A (BoNT/A) acts by cleaving synaptosome-associated-protein-25 (SNAP-25) in nerve terminals to inhibit neuronal release and shows long-lasting antinociceptive action in neuropathic pain. However, its precise mechanism of action remains unclear. Our study aimed to characterize BoNT/A-induced neuroimmunological changes after chronic constriction injury (CCI) of the sciatic nerve. In the ipsilateral lumbar spinal cords of CCI-exposed rats, the mRNA of microglial marker (complement component 1q, C1q), astroglial marker (glial fibrillary acidic protein, GFAP), and prodynorphin were upregulated, as measured by reverse transcription-polymerase chain reaction (RT-PCR). No changes appeared in mRNA for proenkephalin, pronociceptin, or neuronal and inducible nitric oxide synthase (NOS1 and NOS2, respectively). In the dorsal root ganglia (DRG), an ipsilateral upregulation of prodynorphin, pronociceptin, C1q, GFAP, NOS1 and NOS2 mRNA and a downregulation of proenkephalin mRNA were observed. A single intraplantar BoNT/A (75 pg/paw) injection induced long-lasting antinociception in this model. BoNT/A diminished the injury-induced ipsilateral spinal upregulation of C1q mRNA. In the ipsilateral DRG a significant decrease of C1q-positive cell activation and of the upregulation of prodynorphin, pronociceptin and NOS1 mRNA was also observed following BoNT/A admistration. BoNT/A also diminished the injury-induced upregulation of SNAP-25 expression in both structures. We provide evidence that BoNT/A impedes injury-activated neuronal function in structures distant from the injection site, which is demonstrated by its influence on NOS1, prodynorphin and pronociceptin mRNA levels in the DRG. Moreover, the silence of microglia/macrophages after BoNT/A administration could be secondary to the inhibition of neuronal activity, but this decrease in neuroimmune interactions could be the key to the long-lasting BoNT/A effect on neuropathic pain.

  1. Expression and Characterization of Two Kinds of Recombinant Snake Neurotoxins%蛇神经毒素的表达和鉴定

    Institute of Scientific and Technical Information of China (English)

    钱友存; 沈雁; 范春阳; 胡太山; 杨胜利; 龚毅

    2000-01-01

    The cDNA encoding the precursor of cobrotoxin was cloned from the venom gland of the Chinese continental cobra ( Naja naja atra ) by RT-PCR. Its deduced amino acid sequence analysis showed that the mature protein was identical to that identified from the Taiwan cobra ( Naja naja atra ) by protein sequencing technique. The cDNA encoding the mature protein was then subcloned into the expression vector pMAL-P2. The gene of CMl 1, which was formed by ligation of the fragments of the synthetic oligonucleotides, was also cloned into the expression vector pMAL-P2. After induction of IPTG, both of the neurotoxins were overexpressed as soluble fusion proteins which were confirmed by SDS-PAGE and western blotting. The expressed fusion proteins was purified by sepharose 6B-amylose affinity chromatography and DEAE-sepharose FF chromatography. Both of the recombinant proteins achieved after digestion by factor Xa showed the in vivo toxicity.%抽提中华眼镜蛇毒腺总RNA,通过反转录PCR扩增cobrotoxin cDNA,克隆并测序。该cDNA编码83个氨基酸,包括21个氨基酸的信号肽和62个氨基酸的成熟蛋白。该成熟蛋白的氨基酸序列和通过蛋白测序从台湾眼镜蛇鉴定的Cobrotoxin完全一致。PCR扩增编码Cobrotoxin的DNA,并亚克隆到表达载体pMAL-P2。此外,通过合成寡核苷酸片段,拼接成完整的CM-11基因,并将其克隆至pMAL-P2。经IPTG诱导,两种神经毒素基因在大肠杆菌中都得到高效的可溶性融合表达。表达产物通过SDS-PAGE和蛋白印迹杂交加以鉴定。表达的融合蛋白经过Sepharose 6B-amylose亲和色谱和DEAE-Sepharose FF离子交换色谱得到有效纯化。经Xa因子酶切后得到的两种重组神经毒素都具有小白鼠体内毒性。

  2. Time-dependent alterations in growth, photosynthetic pigments and enzymatic defense systems of submerged Ceratophyllum demersum during exposure to the cyanobacterial neurotoxin anatoxin-a

    Energy Technology Data Exchange (ETDEWEB)

    Ha, Mi-Hee; Pflugmacher, Stephan, E-mail: stephan.pflugmacher@tu-berlin.de

    2013-08-15

    Highlights: •We examined time-dependent metabolic changes in C. demersum exposed to anatoxin-a. •Biotransformation and antioxidative defense mechanisms responded positively to anatoxin-a. •Decline in chlorophylls contents was detected in company with irreversible plant growth inhibition during exposure to anatoxin-a. •Anatoxin-a exhibits phytotoxic allelopathy by provoking oxidative stress. •Macrophytes may have interactions with anatoxin-a in aquatic environments. -- Abstract: Recently, aquatic macrophytes have been considered as promising tools for eco-friendly water management with a low running cost. However, only little information is available thus far regarding the metabolic capacity of macrophytes for coping with cyanobacterial toxins (cyanotoxins) in the aquatic environment. Cyanotoxins have become emerging contaminants of great concern due to the high proliferation of cyanobacteria (cyanobacterial bloom) accelerated by eutrophication and climate change. Anatoxin-a, one of the common and major cyanotoxins, is suggested as a high priority water pollutant for regulatory consideration owing to its notoriously rapid mode of action as a neurotoxin. In this study, the time-course metabolic regulation of the submerged macrophyte Ceratophyllum demersum (C. demersum) was investigated during exposure to anatoxin-a at an environmentally relevant concentration (15 μg/L). Biotransformation and antioxidative systems in C. demersum responded positively to anatoxin-a through the promoted synthesis of most of the involved enzymes within 8 h. Maximum enzyme activities were exhibited after 24 or 48 h of exposure to anatoxin-a. However, an apparent decline in enzyme activities was also observed at longer exposure duration (168 and 336 h) in company with high steady-state levels of cell internal H{sub 2}O{sub 2}, which showed its highest level after 48 h. Meanwhile, irreversible inhibitory influence on chlorophyll content (vitality) was noticed, whereas the ratio of

  3. Characterisation of the effects of robustoxin, the lethal neurotoxin from the Sydney funnel-web spider Atrax robustus, on sodium channel activation and inactivation.

    Science.gov (United States)

    Nicholson, G M; Walsh, R; Little, M J; Tyler, M I

    1998-06-01

    reclassification of funnel-web spider toxins as atracotoxins, robustoxin should henceforth be known as delta-atracotoxin-Ar1 to reflect this main action on sodium channel inactivation. These present results further support the hypothesis that funnel-web spider toxins interact with neurotoxin receptor site 3 to slow channel inactivation in a manner similar to that of alpha-scorpion and sea anemone toxins.

  4. Gene expression profiling in brain of mice exposed to the marine neurotoxin ciguatoxin reveals an acute anti-inflammatory, neuroprotective response

    Directory of Open Access Journals (Sweden)

    Ryan James C

    2010-08-01

    Full Text Available Abstract Background Ciguatoxins (CTXs are polyether marine neurotoxins and potent activators of voltage-gated sodium channels. This toxin is carried by multiple reef-fish species and human consumption of ciguatoxins can result in an explosive gastrointestinal/neurologic illness. This study characterizes the global transcriptional response in mouse brain to a symptomatic dose of the highly toxic Pacific ciguatoxin P-CTX-1 and additionally compares this data to transcriptional profiles from liver and whole blood examined previously. Adult male C57/BL6 mice were injected with 0.26 ng/g P-CTX-1 while controls received only vehicle. Animals were sacrificed at 1, 4 and 24 hrs and transcriptional profiling was performed on brain RNA with Agilent whole genome microarrays. RT-PCR was used to independently validate gene expression and the web tool DAVID was used to analyze gene ontology (GO and molecular pathway enrichment of the gene expression data. Results A pronounced 4°C hypothermic response was recorded in these mice, reaching a minimum at 1 hr and lasting for 8 hrs post toxin exposure. Ratio expression data were filtered by intensity, fold change and p-value, with the resulting data used for time course analysis, K-means clustering, ontology classification and KEGG pathway enrichment. Top GO hits for this gene set included acute phase response and mono-oxygenase activity. Molecular pathway analysis showed enrichment for complement/coagulation cascades and metabolism of xenobiotics. Many immediate early genes such as Fos, Jun and Early Growth Response isoforms were down-regulated although others associated with stress such as glucocorticoid responsive genes were up-regulated. Real time PCR confirmation was performed on 22 differentially expressed genes with a correlation of 0.9 (Spearman's Rho, p Conclusions Many of the genes differentially expressed in this study, in parallel with the hypothermia, figure prominently in protection against

  5. Complex variables

    CERN Document Server

    Taylor, Joseph L

    2011-01-01

    The text covers a broad spectrum between basic and advanced complex variables on the one hand and between theoretical and applied or computational material on the other hand. With careful selection of the emphasis put on the various sections, examples, and exercises, the book can be used in a one- or two-semester course for undergraduate mathematics majors, a one-semester course for engineering or physics majors, or a one-semester course for first-year mathematics graduate students. It has been tested in all three settings at the University of Utah. The exposition is clear, concise, and lively

  6. Complex variables

    CERN Document Server

    Flanigan, Francis J

    2010-01-01

    A caution to mathematics professors: Complex Variables does not follow conventional outlines of course material. One reviewer noting its originality wrote: ""A standard text is often preferred [to a superior text like this] because the professor knows the order of topics and the problems, and doesn't really have to pay attention to the text. He can go to class without preparation."" Not so here-Dr. Flanigan treats this most important field of contemporary mathematics in a most unusual way. While all the material for an advanced undergraduate or first-year graduate course is covered, discussion

  7. Complex dynamics

    CERN Document Server

    Carleson, Lennart

    1993-01-01

    Complex dynamics is today very much a focus of interest. Though several fine expository articles were available, by P. Blanchard and by M. Yu. Lyubich in particular, until recently there was no single source where students could find the material with proofs. For anyone in our position, gathering and organizing the material required a great deal of work going through preprints and papers and in some cases even finding a proof. We hope that the results of our efforts will be of help to others who plan to learn about complex dynamics and perhaps even lecture. Meanwhile books in the field a. re beginning to appear. The Stony Brook course notes of J. Milnor were particularly welcome and useful. Still we hope that our special emphasis on the analytic side will satisfy a need. This book is a revised and expanded version of notes based on lectures of the first author at UCLA over several \\Vinter Quarters, particularly 1986 and 1990. We owe Chris Bishop a great deal of gratitude for supervising the production of cour...

  8. Cosmic Complexity

    Science.gov (United States)

    Mather, John C.

    2012-01-01

    What explains the extraordinary complexity of the observed universe, on all scales from quarks to the accelerating universe? My favorite explanation (which I certainty did not invent) ls that the fundamental laws of physics produce natural instability, energy flows, and chaos. Some call the result the Life Force, some note that the Earth is a living system itself (Gaia, a "tough bitch" according to Margulis), and some conclude that the observed complexity requires a supernatural explanation (of which we have many). But my dad was a statistician (of dairy cows) and he told me about cells and genes and evolution and chance when I was very small. So a scientist must look for me explanation of how nature's laws and statistics brought us into conscious existence. And how is that seemll"!gly Improbable events are actually happening a!1 the time? Well, the physicists have countless examples of natural instability, in which energy is released to power change from simplicity to complexity. One of the most common to see is that cooling water vapor below the freezing point produces snowflakes, no two alike, and all complex and beautiful. We see it often so we are not amazed. But physlc!sts have observed so many kinds of these changes from one structure to another (we call them phase transitions) that the Nobel Prize in 1992 could be awarded for understanding the mathematics of their common features. Now for a few examples of how the laws of nature produce the instabilities that lead to our own existence. First, the Big Bang (what an insufficient name!) apparently came from an instability, in which the "false vacuum" eventually decayed into the ordinary vacuum we have today, plus the most fundamental particles we know, the quarks and leptons. So the universe as a whole started with an instability. Then, a great expansion and cooling happened, and the loose quarks, finding themselves unstable too, bound themselves together into today's less elementary particles like protons and

  9. Cosmic Complexity

    Science.gov (United States)

    Mather, John C.

    2012-01-01

    What explains the extraordinary complexity of the observed universe, on all scales from quarks to the accelerating universe? My favorite explanation (which I certainty did not invent) ls that the fundamental laws of physics produce natural instability, energy flows, and chaos. Some call the result the Life Force, some note that the Earth is a living system itself (Gaia, a "tough bitch" according to Margulis), and some conclude that the observed complexity requires a supernatural explanation (of which we have many). But my dad was a statistician (of dairy cows) and he told me about cells and genes and evolution and chance when I was very small. So a scientist must look for me explanation of how nature's laws and statistics brought us into conscious existence. And how is that seemll"!gly Improbable events are actually happening a!1 the time? Well, the physicists have countless examples of natural instability, in which energy is released to power change from simplicity to complexity. One of the most common to see is that cooling water vapor below the freezing poin