The transmembrane domain of syntaxin 1A negatively regulates voltage-sensitive Ca(2+) channels.

Syntaxin 1A has a pronounced inhibitory effect on the activation kinetics and current amplitude of voltage-gated Ca(2+) channels. This study explores the molecular basis of syntaxin interaction with N- and Lc-type Ca(2+) channels by way of functional assays of channel gating in a Xenopus oocytes expression system. A chimera of syntaxin 1A and syntaxin 2 in which the transmembrane domain of syntaxin 2 replaced the transmembrane of syntaxin 1A (Sx1-2), significantly reduced the rate of activation of N- and Lc-channels. This shows a similar effect to that demonstrated by syntaxin 1A, though the current was not inhibited. The major sequence differences at the transmembrane of the syntaxin isoforms are that the two highly conserved cysteines Cys 271 and Cys 272 in syntaxin 1A correspond to the valines Val 272 and Val 273 in syntaxin 2 transmembrane. Mutating either cysteines in Sx1-1 (syntaxin 1A) to valines, did not affect modulation of the channel while a double mutant C271/272V was unable to regulate inward current. Transfer of these two cysteines to the transmembrane of syntaxin 2 by mutating Val 272 and Val 273 to Cys 272 and Cys 273 led to channel inhibition. When cleaved by botulinum toxin, the syntaxin 1A fragments, amino acids 1-253 and 254-288, which includes the transmembrane domain, were both unable to inhibit current amplitude but retained the ability to modify the activation kinetics of the channel. A full-length syntaxin 1A and the integrity of the two cysteines within the transmembrane are crucial for coordinating Ca(2+) entry through the N- and Lc-channels. These results suggest that upon membrane depolarization, the voltage-gated N- and Lc-type Ca(2+)-channels signal the exocytotic machinery by interacting with syntaxin 1A at the transmembrane and the cytosolic domains. Cleavage with botulinum toxin disrupts the coupling of the N- and Lc-type channels with syntaxin 1A and abolishes exocytosis, supporting the hypothesis that these channels actively participate in Ca(2+) regulated secretion.

Pure botulinum neurotoxin is absorbed from the stomach and small intestine and produces peripheral neuromuscular blockade.

Clostridium botulinum serotype A produces a neurotoxin composed of a 100-kDa heavy chain and a 50-kDa light chain linked by a disulfide bond. This neurotoxin is part of a ca. 900-kDa complex, formed by noncovalent association with a single nontoxin, nonhemagglutinin subunit and a family of hemagglutinating proteins. Previous work has suggested, although never conclusively demonstrated, that neurotoxin alone cannot survive passage through the stomach and/or cannot be absorbed from the gut without the involvement of auxiliary proteins in the complex. Therefore, this study compared the relative absorption and toxicity of three preparations of neurotoxin in an in vivo mouse model. Equimolar amounts of serotype A complex with hemagglutinins, complex without hemagglutinins, and purified neurotoxin were surgically introduced into the stomach or into the small intestine. In some experiments, movement of neurotoxin from the site of administration was restricted by ligation of the pylorus. Comparison of relative toxicities demonstrated that at adequate doses, complex with hemagglutinins, complex without hemagglutinins, and pure neurotoxin can be absorbed from the stomach. The potency of neurotoxin in complex was greater than that of pure neurotoxin, but the magnitude of this difference diminished as the dosage of neurotoxin increased. Qualitatively similar results were obtained when complex with hemagglutinins, complex without hemagglutinins, and pure neurotoxin were placed directly into the intestine. This work establishes that pure botulinum neurotoxin serotype A is toxic when administered orally. This means that pure neurotoxin does not require hemagglutinins or other auxiliary proteins for absorption from the gastrointestinal system into the general circulation.

Conjugative transfer of the Escherichia coli-Clostridium perfringens shuttle vector pJIR1457 to Clostridium botulinum type A strains.

An RP4-oriT shuttle vector pJIR1457 originally developed for Clostridium perfringens was successfully transferred by conjugation from Escherichia coli to Clostridium botulinum type A strains and to a nontoxigenic C. botulinum type A-transposon Tn916 mutant strain lacking the entire toxin gene cluster. The light chain (LC) of botulinum toxin was highly expressed in the toxin deletion mutant strain from a pJIR1457 construct containing the recombinant botulinal gene for LC. This shuttle vector system will be valuable for genetic analysis of C. botulinum and will enable genetic manipulation and recombinant expression studies of botulinum neurotoxins as pharmaceutical agents.

Colony immunoblot assay of botulinal toxin.

Botulinal neurotoxin in and around colonies of Clostridium botulinum types A, B, and E and of toxigenic Clostridium butyricum was detected by an enzyme-linked immunoassay procedure whereby the toxin was transferred from the agar medium to a nitrocellulose support and the immobilized toxin was probed with type-specific antibodies. The method identified the toxin types of the colonies grown from a mixed inoculum of C. botulinum serotypes. The specificity of the antitoxins for type A and B toxins was improved by adsorption of the antitoxins with the antigens of heterologous type cultures.

Stabilization of botulinum toxin type A during lyophilization.

Botulinum toxin for medical use is diluted to very low concentrations (nanograms per milliliter); when it is preserved by lyophilization, considerable loss of activity can occur. In the present study, conditions that gave > 90% recovery of the toxicity after lyophilization of solutions containing 20 to 1,000 mouse 50% lethal doses per ml were found. Toxicity was recovered upon drying 0.1 ml of toxin solution when the pH was maintained below 7 and bovine or human serum albumins were used as stabilizers. Various other substances tested with albumin, including glucose, sucrose, trehalose, mannitol, glycine, and cellibiose, did not increase recovery on drying.

Control of Salmonella enteritidis infections in poultry by polymyxin B and trimethoprim.

Antimicrobial compounds were screened in vitro in Trypticase soy broth for antimicrobial activity against a virulent strain of Salmonella enteritidis. Of the several compounds tested, polymyxin B showed the strongest inhibition in vitro, preventing growth at a concentration of less than or equal to 10 micrograms/ml. Polymyxin B administered in the drinking water was effective in vivo for preventing infections in 1-day-old chickens but did not remove established infections in 1-week-old chickens. It was found that trimethoprim, which was not active in vitro, prevented colonization and removed existing infections in 1-day-old chickens when it was administered together with polymyxin B sulfate. Enrichment cultures in which selenite-cystine and tetrathionate broth media were used showed that chickens given a combination of 100 micrograms of polymyxin B sulfate per ml and 250 micrograms of trimethoprim per ml 24 h prior to oral inoculation with 10(8) to 10(9) CFU were negative for S. enteritidis after 7 days. Established infections (10(5) to 10(6) CFU/g of feces) in 1-week-old chickens were eliminated by treatment with the polymyxin-trimethoprim system. This antimicrobial agent treatment may be useful for preventing colonization in poultry and for eliminating S. enteritidis from infected flocks.