Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.69 (botulinum neurotoxin)
 1,901 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The pharmacologic activity of several clostridial neurotoxins was assayed on the mouse phrenic nerve-hemidiaphragm preparation. The substances that were assayed included botulinum neurotoxin types A, B, C and E and tetanus toxin. Experiments were done in the presence or absence of antagonists that inhibit either the internalization of toxins or intracellular expression of toxicity. Ammonium chloride and methylamine hydrochloride, agents that inhibit toxins that enter cells by receptor-mediated endocytosis, antagonized botulinum and tetanus neurotoxins. The magnitude of antagonism was substantial for all toxins. Calcium, 3,4-diaminopyridine and guanidine, agents that alter the intracellular expression of toxicity, produced a variable result. They were effective antagonists of botulinum neurotoxin type A, but they were less effective or inactive against the other neurotoxins. The ability of 3,4-diaminopyridine and guanidine to antagonize botulinum neurotoxin type A was highly calcium dependent. When ambient levels of the cation were reduced from 1.8 to 1.0 mM, the activity of the drugs was substantially reduced. The ability of these drugs to produce antagonism was also time dependent. When added simultaneously with toxin, they were maximally active; when added at later times, activity was diminished. A host of agents that alter intracellular levels of cyclic AMP, including theophylline, forskolin, isobutylmethylxanthine and cholera toxin, were evaluated as potential neurotoxin antagonists. Theophylline and isobutylmethylxanthine produced a transient increase in nerve-evoked muscle twitch. None of the drugs that alter tissue levels of cyclic AMP had a universal effect in antagonizing clostridial toxins. The data here have been compared with published data on drugs that antagonize binding of botulinum toxin and tetanus toxin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Use of pharmacologic antagonists to deduce commonalities of biologic activity among clostridial neurotoxins. 245 38

We recently reported that type D botulinum neurotoxin ADP-ribosylates a specific protein of Mr 21,000 in membrane fractions of various tissues (Ohashi, Y. and Narumiya, S. (1987) J. Biol. Chem. in press). We examined similar enzyme activities in other types (types A, B, C1 and E) of botulinum neurotoxins. Of these, only type C1 toxin showed the activity similar to type D toxin and ADP-ribosylated the same Mr 21,000 protein in membranes of mouse brain. No enzyme activities were detected in type A, B and E toxins under the present experimental conditions. GTP stimulated ADP-ribosylation by the two toxins in a concentration dependent manner from 10 nM to 100 microM. The maximum stimulation was about 6 fold. GDP was 10 times less potent than GTP and achieved similar maximum at 1 mM, while GMP, ADP and ATP had little effect. Several guanidino-containing compounds dose-dependently inhibited the activities of both toxins. The IC50 values were 8.5, 14.5 and 45 mM for agmatine, L-arginine methyl ester and guanidine, respectively.
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PMID:ADP-ribosylation by type C1 and D botulinum neurotoxins: stimulation by guanine nucleotides and inhibition by guanidino-containing compounds. 382 91

Clostridium botulinum may produce any of seven known serotypes of neurotoxin (BoNT/A-/G), which are the most toxic bacterial proteins known. Efforts to develop a second-generation vaccine to these toxins would benefit from the isolation of hybridomas producing neutralizing monoclonal antibodies (MAbs). We hypothesized that previous efforts to isolate neutralizing MAbs against various BoNTs failed due to use of toxoided, chemically altered antigens. We employed a novel vaccination regimen employing native, active, single-chain BoNT/E (scBoNT/E). A number of the BoNT/E immunized mice were further vaccinated with lethal doses of fully active BoNT/F. MAb 7F8 consistently neutralized BoNT/F in three different assays: in vivo neutralization, passive neutralization, and neutralization of regional paralysis. There was no detectable recognition and essentially no neutralization of scBoNT/E. The epitope recognized by this MAb was denatured when treated with formalin, urea, guanidine chloride, or sodium dodecyl sulfate. Preliminary epitope mapping studies indicate that the MAb bound to a conformational epitope.
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PMID:Identification and characterization of a neutralizing monoclonal antibody against botulinum neurotoxin serotype F, following vaccination with active toxin. 938 28

Clostridium botulinum neurotoxin (NT) serotypes A, B, and E have 9, 10, and 8 Cys residues, respectively, as deduced from nucleotide sequences [Whelan et al. (1992), Appl. Environ. Microbiol. 48, 2345-2354]. Each of the 150-kDa NTs has at least one disulfide; but type B, like types A and E, may have two disulfides. Using two different chemical reagents, we studied the status of the Cys residues in these three proteins after (i) the final anion exchange chromatographic step in their purification (fresh NT), (ii) 24 hr storage at 8 degrees C, (iii) precipitation with ammonium sulfate (precipitated NT), and (iv) dissolving the precipitated NT in 6 M guanidine HCl. In all three NT serotypes the number of Cys residues titrated with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) as free -SH groups varied, depending upon the absence or presence of EDTA added to the chromatography buffer, storage condition, age, and presence of the denaturant. Titration of 9.5-10 and 5.4-6.0 -SH groups in fresh NTs type B and E, respectively, indicated total and partial absence of disulfide bonds. Fewer titratable -SH groups in the precipitated NT than in the fresh NT suggested formation of disulfide and/or inaccessibility of the -SH groups due to protein's conformational change(s). When the precipitated NTs were dissolved in 6 M guanidine HCl, in the absence of any added reducing agent, all Cys residues of types B and E, and 6.4-8.3 Cys in type A NT were titratable with DTNB. Iodoacetamide modification of precipitated NT types A, B, and E carboxymethylated 4, 2, and 2 Cys residues, respectively; these numbers rose to 6, 9.4, and 8 when these proteins were carboxymethylated after dissolving in 6 M guanidine HCl in the absence of any added reducing agent. We propose that S-S- cleavage mediated by the -SH/-S-S- exchange observed in vitro after unfolding the NTs (also unfolded by 2 M guanidine HCl or urea) possibly mimicks a similar exchange process inside the endosomes, where the NTs are thought to undergo conformational changes, resulting in the reductive cleavage of the interchain disulfide between the 50-kDa light and 100-kDa heavy chain, which in turn releases the light chain and allows its egress out of the endosomes into the cytosol.
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PMID:Status of Cys residues in the covalent structure of botulinum neurotoxin types A, B, and E. 958 42

A rapid, quantitative PCR assay (TaqMan assay) which quantifies Clostridium botulinum type E by amplifying a 280-bp sequence from the botulinum neurotoxin type E (BoNT/E) gene is described. With this method, which uses the hydrolysis of an internal fluoregenic probe and monitors in real time the increase in the intensity of fluorescence during PCR by using the ABI Prism 7700 sequence detection system, it was possible to perform accurate and reproducible quantification of the C. botulinum type E toxin gene. The sensitivity and specificity of the assay were verified by using 6 strains of C. botulinum type E and 18 genera of 42 non-C. botulinum type E strains, including strains of C. botulinum types A, B, C, D, F, and G. In both pure cultures and modified-atmosphere-packaged fish samples (jack mackerel), the increase in amounts of C. botulinum DNA could be monitored (the quantifiable range was 10(2) to 10(8) CFU/ml or g) much earlier than toxin could be detected by mouse assay. The method was applied to a variety of seafood samples with a DNA extraction protocol using guanidine isothiocyanate. Overall, an efficient recovery of C. botulinum cells was obtained from all of the samples tested. These results suggested that quantification of BoNT/E DNA by the rapid, quantitative PCR method was a good method for the sensitive assessment of botulinal risk in the seafood samples tested.
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PMID:Rapid, quantitative PCR monitoring of growth of Clostridium botulinum type E in modified-atmosphere-packaged fish. 1113 47

Botulinum neurotoxins (BoNTs) are highly potent toxins that inhibit neurotransmitter release from peripheral cholinergic synapses and associate with infant botulism. BoNT is a approximately 150kDa protein, consisting of a binding/translocating heavy chain (HC; 100kDa) and a toxifying light chain (LC; 50kDa) linked through a disulfide bond. C-terminal half of the heavy chain is binding domain, and N-terminal half of the heavy chain is translocation domain that includes transmembrane domain. A functional botulinum neurotoxin type B heavy chain transmembrane and binding domain (Ile 624-Glu 1291) has been cloned into a bacterial expression vector pET 15b and produced as an N-terminally six-histidine-tagged fusion protein (BoNT/B HC TBD). (His(6))-BoNT/B HC TBD was highly expressed in Escherichia coli BL21-CodonPlus (DE3)-RIL and isolated from the E. coli inclusion bodies. After solubilizing the purified inclusion bodies with 6M guanidine-HCl in the presence of 10mM beta-mercaptoethanol, the protein was purified and refolded in a single step on Ni(2+) affinity column by removing beta-mercaptoethanol first, followed by the removal of urea. The purified protein was determined to be 98% pure as assessed by SDS-polyacrylamide gel. (His(6))-BoNT/B HC TBD retained binding to synaptotagmin II, the receptor of BoNT/B, which was confirmed by immunological dot blot assay, also to ganglioside, which was investigated using enzyme-linked immunosorbent assay.
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PMID:Cloning, high-level expression, single-step purification, and binding activity of His6-tagged recombinant type B botulinum neurotoxin heavy chain transmembrane and binding domain. 1476 96