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

Botulinum neurotoxin (BoNT) is one of the most toxic substances known to produce severe neuromuscular paralysis. The currently used vaccine is prepared mainly from biohazardous toxins. Thus, we studied an alternative method and demonstrated that DNA immunization provided sufficient protection against botulism in a murine model. A plasmid of pBoNT/A-Hc, which encodes the fragment C gene of type A botulinum neurotoxin, was constructed and fused with an Igkappa leader sequence under the control of a human cytomegalovirus promoter. After 10 cycles of DNA inoculation with this plasmid, mice survived lethal doses of type A botulinum neurotoxin challenges. Immunized mice also elicited cross-protection to the challenges of type E botulinum neurotoxin. This is the first study demonstrating the potential use of DNA vaccination for botulinum neurotoxins.
 ...
PMID:DNA vaccination using the fragment C of botulinum neurotoxin type A provided protective immunity in mice. 1064 89

A synthetic gene encoding the Hc (binding) domain of Clostridium botulinum neurotoxin F (FHc) was expressed in Escherichia coli fused to maltose binding protein (MBP). The purified MBP-FHc and FHc isolated after removal of MBP were evaluated in mice for their ability to protect against toxin challenge. Balb/c mice developed a protective immune response following administration of either protein via the intraperitoneal or intramuscular routes. A comparison of antibody titres and protection following single and multiple vaccinations and the effects of dosage are shown. The long term protection afforded by the vaccines was also investigated. Ten months following vaccination mice were still protected when challenged with 10(4) MLD(50) doses of botulinum toxin F.
 ...
PMID:Cloning, expression and evaluation of a recombinant sub-unit vaccine against Clostridium botulinum type F toxin. 1093 Jun 84

Effective expression of the HIV-1 core protein Gag-p55 was obtained in Saccharomyces cerevisiae under control of the inducible UASgal/CYC1 promoter as a translational fusion with the prion-forming NM domain of the translation terminator Sup35p (eRF3) of S. cerevisiae. where only poor expression of the original-type Gag-p55 was observed. A deletion within the Sup35NM prion-forming domain altering Sup35-associated [PSI] inheritance did not compromise expression of the Sup35NM Gag-p55 fusion protein. Therefore, either the mechanism of this phenomenon is not directly related to the effect of Sup35p prion-formation or the modified protein maintains residual prion-forming abilities. The recombinant Sup35p-Gag-p55 protein was quite stable under boiling in an alkali/sodium dodecyl sulfate (SDS) solution and completely retained its antigenic properties. Moreover, 10-min boiling of the native yeast cells in this solution allowed immediate inhibition of lysosomal and other yeast proteases, responsible for autolysis of many natural and recombinant proteins. The use of this method of preliminary enrichment for the recombinant fusion protein Sup35p-Gag-p55 with the SDS-alkaline extraction could be useful for yeast heterologous expression and purification of other of insoluble and unstable proteins. A translational fusion with the NM domain of Sup35p was also used to produce another poorly soluble protein, the L-chain of botulinum exotoxin A, in S. cerevisiae. When the Sup35p fragment was removed from the recombinant construct encoding a fused Sup35/BoNT protein, a dramatic drop in both transformation efficiency and growth rate of transformants was shown.
 ...
PMID:Sup35p yeast prion-like protein as an adapter for production of the Gag-p55 antigen of HIV-1 and the L-chain of botulinum neurotoxin in Saccharomyces cerevisiae. 1128 22

A DNA vaccine was constructed which expressed the binding domain of Clostridium botulinum neurotoxin serotype F fused to a signal peptide. Three intra-muscular doses fully protected Balb/c mice against 10(4) MLD of serotype F toxin. Priming of the immune response by DNA vaccination followed by a single booster with type F binding domain protein resulted in high levels of antibody against the binding domain. This study demonstrates the utility of DNA vaccination for protection against botulinum neurotoxin type F and indicates that a prime-boost regimen could be an efficient method of generating antibody for passive immune therapy in cases of botulism involving serotype F toxin.
 ...
PMID:DNA vaccination protects against botulinum neurotoxin type F. 1280 37

DNA vaccines which expressed the Hc fragment of the Clostridium botulinum type F neurotoxin (BoNT/F Hc) fused to a signal peptide downstream of four different eukaryotic promoters were prepared. Subsequently, the immunogenicity of the DNA vaccines and protection afforded in mice against challenge with 10(4) MLD of type F botulinum toxin was evaluated. The DNA vaccine containing the human ubiquitin gene (UbC) promoter induced the highest BoNT/F Hc-specific antibody concentration following two intramuscular immunisations and afforded 90% protection against challenge. The results from this study indicate that the selection of promoter used in DNA vaccination studies may be of importance in designing optimised vaccines.
 ...
PMID:Efficacy of DNA vaccines expressing the type F botulinum toxin Hc fragment using different promoters. 1536 42

The seven functionally distinct serotypes (A-G) of botulinum neurotoxin (BoNT) are dichains consisting of light chain (LC) with zinc-dependent endoprotease activity connected by one disulfide bond to heavy chain with neuronal-cell translocation and receptor-binding domains. LC-mediated proteolysis of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and consequent inhibition of synaptic vesicle fusion to the presynaptic membrane of human motor neurons are responsible for flaccid paralysis associated with botulism. LC endoproteolysis is complex, requiring highly extended SNARE sequences at the surface of intracellular membranes and prompting our development of a genetically amenable assay to monitor the interaction between BoNT/LC and its SNARE substrate. Using BoNT serotype B as a model, the assay employs a chimeric SNARE protein where a portion of neuronal synaptobrevin (Sb) is fused to Snc2p, a Sb ortholog required for protein secretion from yeast cells. Regulated expression of serotype B-LC in yeast leads to cleavage of the chimera and a conditional growth defect. To assess utility of this assay for monitoring SNARE protein cleavage, we growth-selected chimeric SNARE mutations that inhibited proteolysis. When these mutations were introduced into Sb and examined for cleavage, substrate residues located near and distal to the cleavage site were important, including residues positioned near the Sb transmembrane domain, an unexplored aspect of BoNT cell intoxication. Additional mutations were positioned in a nine-residue SNARE motif, supporting a previously assigned role for this motif in LC recognition and providing proof of principle for the application of yeast-based technology to study intracellular BoNT/LC endoproteases.
 ...
PMID:A yeast assay probes the interaction between botulinum neurotoxin serotype B and its SNARE substrate. 1663 86

Botulinum neurotoxin serotype A (BoNT/A) is a proteolytic enzyme that induces muscle paralysis. It is a cause of food poisoning, a potential bioterrorist threat and, in low doses an emerging pharmaceutical product. No effective treatment is currently available for BoNT intoxication. Previously we developed a BoNT/A light chain enzyme assay using a peptide substrate based on the SNAP-25 protein target, with HPLC separation and UV detection of assay products, and applied the method to screen combinatorial peptide libraries for inhibitory activity to BoNT/A. We now report on development of a capillary electrophoresis laser-induced fluorescence (CE-LIF) method for measuring BoNT/A activity. The enzyme assay products were labeled with CBQCA dye followed by CE separation on a bare fused silica column in a HEPES-based buffer and LIF detection. All assay products were separated in CE within 8 min compared to incomplete separation of assay products within 1h by HPLC. The labeled products showed linear dependence of intensity versus concentration, and quantitative mole-fraction assignments. We used the CE-LIF method to screen combinatorial peptide libraries for potential modulating effects on BoNT/A peptidase activity. With some of the libraries, peptides co-migrated with assay products and interfered with quantitation. In such cases, interference was reduced by substituting sodium dodecyl sulfate (SDS) for Tween-20 in the running buffer. Separation in the capillaries then occurred by micellar electrokinetic chromatography (MEKC). The CE-LIF method is quick and lends itself to high-throughput or microfluidic formats.
 ...
PMID:Capillary electrophoresis laser-induced fluorescence for screening combinatorial peptide libraries in assays of botulinum neurotoxin A. 1682 48

Monoclonal antibodies have demonstrated significant potential as therapeutics for botulinum neurotoxin exposures. We previously described a hybridoma method for cloning native human antibodies that uses a murine myeloma cell line that ectopically expresses the human telomerase catalytic subunit gene (hTERT) and the murine interleukin-6 gene (mIL-6). Here we describe a heterohybridoma cell line that ectopically expresses mIL-6 and hTERT and has improved stability of hTERT expression. We fused this cell line to human peripheral blood B cells from a subject who had received the botulinum toxoid vaccine, cloning a high-affinity antibody (13A) specific for serotype A botulinum neurotoxin (BoNT/A). The 13A antibody is an affinity-matured, post-germinal center IgG(1) lambda antibody that has partial neutralization activity in vivo. 13A binds an epitope on BoNT/A that overlaps the binding epitope of an IgG antibody previously shown to fully neutralize a lethal dose of BoNT/A in vivo. The 13A antibody may be useful for diagnostic testing or for incorporation into an oligoclonal therapeutic to counteract BoNT/A exposure.
 ...
PMID:A human monoclonal antibody that binds serotype A botulinum neurotoxin. 1829 71

The expression of SNAP-25 fused to green fluorescent protein (GFP) has been instrumental in demonstrating SNARE role in exocytosis. The wild-type GFP-SNAP-25 and a Delta9 form, product of botulinum neurotoxin A activity, the main ingredient in the BOTOX preparation, were employed here to study SNARE implication in vesicle mobility and fusion in cultured bovine chromaffin cells, a neuroendocrine exocytotic model. Using total internal reflection fluorescent microscopy, we have identified membrane microdomains of 500-600 nm diameter that contain both SNAP-25 and syntaxin-1 and associate with synaptobrevin-2. Interestingly, while the SNAP-25 Delta9 formed similar clusters, they displayed increased mobility both laterally and in the axis perpendicular to the plasmalemma, and this correlates with the enhanced dynamics of associated chromaffin granules. SNARE cluster-enhanced motion is reversed by elevation of the intracellular calcium level. Furthermore, single vesicle fusion was unlikely in the highly mobile vesicles present in the cells expressing SNAP-25 Delta9, which, in addition, displayed in average slower fusion kinetics. Consequently, SNARE cluster dynamics is a new aspect to consider when determining the factors contributing to the mobility of the vesicles in close vicinity to the plasma membrane and also the probability of exocytosis of this granule population.
 ...
PMID:Vesicle motion and fusion are altered in chromaffin cells with increased SNARE cluster dynamics. 1905 86

Antitoxins for botulinum neurotoxins (BoNTs) and other toxins are needed that can be produced economically with improved safety and shelf-life properties compared to conventional therapeutics with large-animal antisera. Here we show that protection from BoNT lethality and rapid BoNT clearance through the liver can be elicited in mice by administration of a pool of epitope-tagged small protein binding agents together with a single anti-tag monoclonal antibody (MAb). The protein binding agents used in this study were single-chain Fv domains (scFvs) with high affinity for BoNT serotype A (BoNT/A). The addition of increasing numbers of differently tagged scFvs synergistically increased the level of protection against BoNT/A. It was not necessary that any of the BoNT/A binding agents possess toxin-neutralizing activity. Mice were protected from a dose equivalent to 1,000 to 10,000 50% lethal doses (LD(50)) of BoNT/A when given three or four different anti-BoNT scFvs, each fused to an E-tag peptide, and an anti-E-tag IgG1 MAb. Toxin protection was enhanced when an scFv contained two copies of the E tag. Pharmacokinetic studies demonstrated that BoNT/A was rapidly cleared from the sera of mice given a pool of anti-BoNT/A scFvs and an anti-tag MAb but not from the sera of mice given scFvs alone or anti-tag MAb alone. The scFv pool and anti-tag MAb protected mice from lethality when administered up to 2 h following exposure of mice to a dose equivalent to 10 LD(50) of BoNT/A. These results suggest that it will be possible to rapidly and economically develop and produce therapeutic antitoxins consisting of pools of tagged binding agents that are administered with a single, stockpiled anti-tag MAb.
 ...
PMID:Efficient serum clearance of botulinum neurotoxin achieved using a pool of small antitoxin binding agents. 1991 18


1 2 3 Next >>