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L L Simpson

Publications and source records attributed to L L Simpson.

At least 73 records · Page 4Linked to original sources
PubMed1988

Partial characterization of the enzymatic activity associated with the binary toxin (type C2) produced by Clostridium botulinum.

Clostridium botulinum produces a binary toxin that possesses a heavy chain (approximately 100,000 daltons) and a light chain (approximately 50,000 daltons). The heavy chain is a binding component that directs the toxin to vulnerable cells, and the light chain is an enzyme that has mono(ADP-ribosyl)ating activity. A number of experiments have been done to help characterize the enzymatic activity of the toxin. The data reveal that the enzyme has a pH optimum within the range of 7.0 to 8.0. It is not inhibited or stimulated by physiological concentrations of sodium, potassium, calcium, or magnesium. The enzyme is inhibited by high concentrations of salt, however, as well as high concentrations of nicotinamide, thymidine, theophylline, and histamine; and it is stimulated by histone and lysolecithin. Boiling irreversibly denatures the light chain of the toxin, but denaturation caused by guanidine and urea is substantially reversible. Enzymatic activity is not altered by short exposure to lysosomal proteases, including cathepsin B, cathepsin H, dipeptidyl aminopeptidase, and catheptic carboxypeptidase B.

ADP Ribose Transferases↗
PubMed1988

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

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)

1-Methyl-3-isobutylxanthine↗
PubMed1988

Actions of the Clostridium botulinum binary toxin on the structure and function of Y-1 adrenal cells.

The binary toxin produced by Clostridium botulinum, also known as type C2 toxin, was examined for its ability to alter the structure and function of Y-1 cells, a murine adrenocortical tumor line. The toxin produced time- and concentration-dependent changes in morphology, characterized by retraction of cell extensions and by rounding of the cell body. These changes were not accompanied by increases or decreases in tissue levels of c-AMP or c-GMP, although there was an increase in the release of total steroids. When cells were exposed to toxin for 24 hr there was no evidence of cell death or lysis. Total nucleic acid content and the rate of incorporation of 14C-leucine into protein were comparable in control cells and intoxicated cells. The toxin has been shown to possess mono(ADP-ribosyl)ating activity, and actin is the presumed substrate. When Y-1 cells were ruptured and then exposed to the toxin in the presence of 32P-NAD, actin was ADP-ribosylated. When cells were exposed to the toxin before being ruptured, there was a subsequent loss in the amount of actin that was available for ADP-ribosylation (32P-NAD) in the broken cell assay. The data suggest that Y-1 cells can survive challenge with the botulinum binary toxin, and thus they are a suitable tissue in which to use the toxin as a pharmacological tool.

Actins↗
PubMed1987

Molecular basis for the pathological actions of Clostridium perfringens iota toxin.

Clostridium perfringens type E iota toxin is composed of two separate and independent polypeptide chains that act synergistically in mouse lethal assays. The light chain is an enzyme that mono(ADP-ribosyl)ates certain amino acids. The enzyme displays substantial activity when homopoly-L-arginine is used as a substrate, but it shows little activity when polyasparagine, polylysine or polyglutamic acid are used. In keeping with the properties of an ADP-ribosylating enzyme, the toxin possesses the following characteristics. It produces incorporation of radioactivity into polyarginine when adenine-labeled NAD is used, but radioactivity is not incorporated when nicotinamide-labeled NAD is used. Irrespective of labeling, enzymatic activity is accompanied by the release of free nicotinamide. After incorporation of ADP-ribose groups into polyarginine, enzymatic and chemical techniques can be used to release the incorporated material. Snake venom phosphodiesterase releases mainly AMP; hydroxylamine releases AMP and ADP-ribose. The heavy chain of iota toxin has little or no enzyme activity, and it does not substantially affect the enzyme activity of the light chain. The heavy chain may be a binding component that directs the toxin to vulnerable cells. The data suggest that iota toxin is a representative of a novel class of ADP-ribosylating toxins.

ADP Ribose Transferases↗
PubMed1986

A preclinical evaluation of aminopyridines as putative therapeutic agents in the treatment of botulism.

4-Aminopyridine and 3,4-diaminopyridine were evaluated for their abilities to delay the onset of paralysis due to botulinum neurotoxin types A, B, and E. Experiments were done on phrenic nerve-hemidiaphragm preparations excised from mice. At a concentration that produced an enhancement in muscle twitch amplitude, 4-aminopyridine and 3,4-diaminopyridine delayed the onset of paralysis due to botulinum toxin type A. Under the same conditions, the drugs did little to protect tissues against botulinum toxin types B and E. 3,4-Diaminopyridine was also evaluated for its ability to reverse the paralysis due to botulinum toxin. Experiments were done on rat phrenic nerve-hemidiaphragm preparations that had previously been poisoned in vivo. The drug produced transient increases in neuromuscular transmission, with the effect being greater for botulinum neurotoxin type A than for botulinum neurotoxin types B and E. Equivalent types of experiments were done with tetanus toxin. The results with 3,4-diaminopyridine showed that tetanus toxin resembled botulinum toxin types B and E. The data help to clarify the role of aminopyridines as therapeutic agents in the treatment of botulism. They also provide insights into the mechanism of action of clostridial neurotoxins.

4-Aminopyridine↗
PubMed1985

Epithelial cell differentiation in organotypic cultures of fetal rat lung.

The purpose of this investigation was to examine the suitability of an organotypic lung-cell culture model for the study of factors influencing fetal lung-cell differentiation. It has been reported that the use of carbon-stripped (hormone-depleted) bovine fetal calf serum in monolayer cell cultures of fetal rat lung prevents continued epithelial cell differentiation in vitro. In this study, organotypic cultures of fetal rat lung cells taken at day 20 of gestation (late canalicular stage) were prepared with a carbon-stripped medium. These organotypic cultures were examined by light, scanning, and transmission electron microscopy for comparison with controls prepared with unstripped bovine fetal calf serum. Highly organized three-dimensional tubular epithelial structures resembling saccules of immature lung were observed within the gelatin sponge matrix. Morphometric analysis of day 20 carbon-stripped samples revealed that 74.6% of the epithelial cells in the tubular structures contained osmiophilic lamellar bodies characteristic of type II pneumonocytes. Control specimens had 71.2% cells with lamellar bodies and did not differ significantly from the experimental group. These data are similar to those obtained with organ cultures of fetal rat lung but are in contrast to findings with monolayer culture systems. The observations of this study suggest that 1) the hormones extracted from bovine fetal calf serum by carbon-stripping are not solely responsible for the continued fetal lung cell differentiation observed in vitro, and 2) that spatial relationships between lung cells in vitro may be a significant factor in the control of differentiation.

Animals↗
PubMed1985

Channels formed by botulinum, tetanus, and diphtheria toxins in planar lipid bilayers: relevance to translocation of proteins across membranes.

The heavy chains of both botulinum neurotoxin type B and tetanus toxin form channels in planar bilayer membranes. These channels have pH-dependent and voltage-dependent properties that are remarkably similar to those previously described for diphtheria toxin. Selectivity experiments with anions and cations show that the channels formed by the heavy chains of all three toxins are large; thus, these channels could serve as "tunnel proteins" for translocation of active peptide fragments. These findings support the hypothesis that the active fragments of botulinum neurotoxin and tetanus toxin, like that of diphtheria toxin, are translocated across the membranes of acidic vesicles.

Botulinum Toxins↗
PubMed1985

Pharmacological experiments on the binding and internalization of the 50,000 dalton carboxyterminus of tetanus toxin at the cholinergic neuromuscular junction.

Experiments were done to compare the binding and subsequent internalization of the intact tetanus toxin molecule and of the isolated binding component from the molecule (i.e., 50,000 dalton carboxyterminus). In all respects, the isolated component behaved like the intact molecule. Both the holotoxin and the isolated carboxyterminus bind to neuromuscular preparations in a way that is only poorly dissociable. At low temperature (4 degrees C), the holotoxin remains at or near the cell surface and thus remains accessible to the neutralizing effects of polyclonal antibody. At the same temperature, the isolated binding component remains associated with receptors and thus antagonizes binding of the intact toxin. Warming tissues (35 degrees C) slowly promotes internalization of the toxin and of the isolated binding component, and nerve stimulation more rapidly promotes internalization. Ammonium chloride and methylamine hydrochloride antagonize the process by which nerve stimulation promotes internalization of bound protein. Data from chase experiments suggest that the receptor for tetanus toxin either has a relatively rapid turnover rate or exists in relative excess. Data from monoclonal antibody experiments show that, even when the toxin is bound to nerve endings, antibodies directed against epitopes in the carboxyterminus can cause appreciable neutralization. The most important implication of the data is that the isolated carboxyterminus of tetanus toxin can be used as a pharmacological tool to target drugs of interest to nerve endings.

Ammonium Chloride↗
PubMed1985

Neuropharmacological characterization of fragment B from tetanus toxin.

Tetanus toxin and Fragment B from tetanus toxin were assayed for activity on the mouse phrenic nerve-hemidiaphragm preparation. Both molecules produced blockade of neuromuscular transmission, but the parent molecule was at least two orders of magnitude more potent than the fragment. Experiments were done to determine whether the toxicity attributed to Fragment B was authentic or due to contamination with the parent molecule. Analysis of the fragment by high-performance liquid chromatography and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed trace contamination. Removal of the major contaminant (1-2%) did not abolish toxicity of the material. However, in pharmacological experiments with native toxin and its fragment, the latter behaved indistinguishably from the former. At equiactive concentrations, both were antagonized by Fragment C and both were antagonized by lysosomotropic agents (ammonium chloride and methylamine hydrochloride). In addition, monoclonal antibodies directed against epitopes in Fragment C neutralized both native toxin and the material presumed to be Fragment B. The studies with antagonists and antibodies suggest that the toxicity apparently associated with Fragment B was in fact due to trace contamination with the parent molecule. In experiments on planar lipid bilayers, Fragment B formed pH-dependent channels. This activity was not abolished by monoclonal antibodies directed against epitopes in Fragment C. The data indicate that Fragment B retains the ability to form channels in membranes, but in the absence of Fragment C it retains little ability to paralyze neuromuscular transmission.

Animals↗
PubMed1984

Botulinum toxin and tetanus toxin recognize similar membrane determinants.

The binding fragment of tetanus toxin (50,000 dalton carboxy-terminus of heavy chain) does not block neuromuscular transmission, but it does antagonize the ability of native tetanus toxin to block neuromuscular transmission. The binding fragment of tetanus toxin also antagonizes certain botulinum toxins, including types C and E. Antagonism is at the cell surface, suggesting that the various molecules compete for a similar membrane binding site. The data indicate that the binding site is specific for botulinum toxin rather than tetanus toxin.

Animals↗
PubMed1984

Molecular basis for the pharmacological actions of Clostridium botulinum type C2 toxin.

The light chain of type C2 toxin produced by Clostridium botulinum was isolated by high-performance liquid chromatography. The protein eluted as a single peak; as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, it had an apparent molecular weight of 51,000 daltons. The light chain was an enzyme that possessed ADP-ribosylating activity. In experiments with synthetic substrates (homo-poly-L-amino acids; alanine, arginine, asparagine, aspartic acid, histidine, leucine, lysine, methionine, phenylalanine, proline, serine and tryptophan), only poly-L-arginine was ADP-ribosylated by the enzyme. In experiments with endogenous substrates (50,000 X g pellet and 50,000 X g supernatant from homogenates of mouse brain, liver and lung), the enzyme ADP-ribosylated proteins or polypeptides in both the particulate and soluble fractions. ADP-ribosylation of the soluble substrate was antagonized by adenine (K1 approximately 2.1 X 10(-5) M) and by adenosine (K1 approximately 2.7 X 10(-4) M); the reaction was reversed by a large molar excess of nicotinamide (0.1 M). ADP-ribosylation of soluble substrate was diminished when the substrate had been pretreated with 1,2-cyclohexane-dione (0.1 M), a site reactive reagent that modified selectively arginine residues. Neither the light chain nor the heavy chain of the binary toxin possessed adenylate cyclase activity. Tissue fractions did possess endogenous adenylate cyclase activity, but the toxin did not stimulate this activity. The data indicate that the binary toxin produced by Clostridium botulinum resembles other protein toxins.

Adenine↗
PubMed1984

Fragment C of tetanus toxin antagonizes the neuromuscular blocking properties of native tetanus toxin.

Tetanus toxin, fragment B and fragment C were assayed for toxicity on the mouse phrenic nerve-hemidiaphragm preparation. The native toxin was a potent blocker of neuromuscular transmission; fragment B possessed little toxicity and fragment C was atoxic. Pretreatment of tissues with fragment C antagonized the neuromuscular blocking properties of tetanus toxin, but not those of type A botulinum toxin or beta-bungarotoxin. Agents that antagonize cholera toxin (B subunit) and diphtheria toxin (CRM197) did not antagonize tetanus toxin and did not alter the ability of fragment C to antagonize tetanus toxin. Fragment C exerted its effect by competing with unbound toxin for receptor sites on the nerve membrane. The fragment did not: 1) displace bound toxin; 2) inhibit internalization of toxin; or 3) inhibit intracellular expression of toxicity. In assays on intact cells, under conditions in which toxin binding was not dissociable, fragment C binding to phrenic nerves had an apparent KD of approximately 1.4 X 10(-7) M. Homogenates of mouse cerebral cortex adsorbed tetanus toxin and these homogenates competed with phrenic nerves for unbound toxin. Homogenized cortex did not displace or promote desorption of toxin already bound to phrenic nerves. Homogenates of eel and torpedo electric organ were not very effective in adsorbing toxin.

Adsorption↗
PubMed1984

The binding fragment from tetanus toxin antagonizes the neuromuscular blocking actions of botulinum toxin.

A chromatographically homogenous preparation of the binding fragment from tetanus toxin was tested for its ability to antagonize the neuromuscular blocking properties of native tetanus toxin. At a concentration of 1 X 10(-6) M, the binding fragment antagonized the paralytic effects of native toxin (1 X 10(-9) M) on mouse phrenic nerve-hemidiaphragms. The binding fragment of tetanus toxin (1 X 10(-6) M) also was tested for its ability to antagonize types A to G botulinum neurotoxin. The fragment did not produce statistically significant antagonism of types A, B, D, F and G neurotoxins, but it did produce highly significant antagonism of types C and E neurotoxin. A series of experiments involving column chromatography, dialysis and high-performance liquid chromatography confirmed that the binding fragment rather than a contaminant was responsible for antagonism. Experiments with type C neurotoxin showed that antagonism between the binding fragment and the toxin occurred extracellularly at the level of the cell membrane. The fragment did not act directly on the toxin to produce inactivation, but instead competed with the toxin for a binding site on the membrane. The fact that the binding moiety of tetanus toxin and the binding moiety of botulinum toxin compete for a similar membrane site suggests that the molecules have areas of structural homology. Such homologies, if confirmed, would have important therapeutic implications.

Animals↗
PubMed1983

Botulinum neurotoxin type E: studies on mechanism of action and on structure-activity relationships.

Single chain type E botulinum neurotoxin was isolated from culture fluids of Clostridium botulinum (strain Alaska E-43). The neurotoxin, which migrated as a single band in polyacrylamide gel electrophoresis with sodium dodecylsulfate, had a molecular weight of approximately 147,000. Single chain type E neurotoxin that was exposed to trypsin was converted to a dichain molecule. Pretreatment of the single chain molecule with 1,2-cyclohexanedione, a reagent that selectively modifies arginine residues, inhibited trypsin-induced generation of the dichain molecule. In dose-response experiments (10(-13) to 10(-9) M) on the isolated neuromuscular junction (phrenic nerve-hemidiaphragm preparation), the dichain neurotoxin was approximately two orders of magnitude more potent than the single chain neurotoxin. Neither specie of neurotoxin (1 pmol/mouse, in vivo; 1 X 10(-11) M, in vitro) was very effective in blocking autonomic transmission (vagus nerve-atrium preparation). The neuromuscular blocking action of the dichain molecule was divided into a sequence of three steps. There was an initial binding step that was relatively rapid, little influenced by temperature and which left the neurotoxin partially accessible to the neutralizing effects of antitoxin. There was a translocation step that was temperature dependent, antagonized by ammonium chloride and methylamine hydrochloride and which caused the neurotoxin to become inaccessible to the neutralizing effects of antitoxin. Finally, there was an intracellular lytic step, during which the toxin blocked excitation-secretion coupling.

Animals↗
PubMed1983

Ammonium chloride and methylamine hydrochloride antagonize clostridial neurotoxins.

Ammonium chloride (1-8 mM) and methylamine hydrochloride (1-16 mM) produce concentration-dependent antagonism of the onset of neuromuscular blockade caused by botulinum toxin types A, B and C (all at 1 X 10(-11) M) and by tetanus toxin (3 X 10(-10) M). Neither drug antagonizes the onset of paralysis caused by beta-bungarotoxin (1 X 10(-7) M) or by taipoxin (1 X 10(-8) M). At concentrations that produce antagonism of clostridial neurotoxins, ammonium chloride and methylamine hydrochloride (8-10 mM) do not inactivate toxin molecules, nor do they produce irreversible changes in tissue function. When studied under conditions that impose partial synchrony on the mechanism of clostridial neurotoxin action, ammonium chloride and methylamine hydrochloride do not inhibit ligand binding and do not reverse neuromuscular blockade. The drugs act solely to antagonize internalization of toxins by cholinergic nerve endings. As a result of inhibiting the process of internalization, the drugs trap the toxins at an antitoxin sensitive site.

Ammonium Chloride↗
PubMed1982

The interaction between aminoquinolines and presynaptically acting neurotoxins.

Chloroquine and hydroxychloroquine block neuromuscular transmission in isolated tissues from mouse, rat, guinea pig and chick. Blockade is associated with depressed muscle responses to potassium and abolished muscle responses to nicotinic cholinergic agonists. Within certain time and concentration limits, the blocking effects of chloroquine and hydroxychloroquine are reversible. Both drugs antagonize the onset of paralysis caused by botulinum neurotoxin types A and B, but neither drug antagonizes tetanus toxin or beta-bungarotoxin. The ability of chloroquine and hydroxychloroquine to antagonize botulinum toxin is not due to blockade of nicotinic cholinergic receptors. At concentrations that produce neuromuscular blockade, d-tubocurarine does not antagonize botulinum toxin types A and B. Chloroquine causes botulinum toxin to remain at an antitoxin sensitive site. These data could mean that chloroquine acts at the cell membrane to inhibit toxin binding or internalization, or that it acts in the cell interior to inhibit lysosomal processing of toxin. Whatever its action, chloroquine is the most effective antagonist of botulinum toxin yet described.

Aminoquinolines↗