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Biomedical subjects

J Melling

Publications and source records attributed to J Melling.

At least 55 records · Page 3Linked to original sources
PubMed1986

Actions of beta-bungarotoxin on spontaneous release of transmitter at muscle end-plates treated with botulinum toxin.

Rat leg muscles were injected subcutaneously with sublethal doses of type A botulinum neurotoxin, and the extensor digitorum longus muscle removed three days later. Intracellular microelectrode recordings were then made of miniature end-plate potentials (mepps). The mepp frequency was reduced by botulinum toxin, while mepp rise times were slowed. Mepp amplitude distributions became characteristically skew. beta-Bungarotoxin (140 nM) was applied to normal muscles in vitro and recordings were made 10-30 min later. The main effect was an increase in mepp frequency during this period. Mepp rise times were unaffected. When beta-bungarotoxin was applied in vitro to muscles treated with botulinum toxin there was also an increase in mepp frequency, although to a value less than in normal muscles. The mepp rise times were speeded up to normal values. The mepp amplitude and rise time distributions showed no obvious evidence for the addition of a second component to the distribution. The data appear to support the hypothesis that the sites for spontaneous release in botulinised muscle may be located at or near the usual release sites at the active zones.

Animals↗
PubMed1986

Development of antibodies to protective antigen and lethal factor components of anthrax toxin in humans and guinea pigs and their relevance to protective immunity.

A competitive inhibition enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies in serum to the protective antigen (PA) and lethal factor (LF) components of anthrax toxin. Current human vaccination schedules with an acellular vaccine induce predictable and lasting antibody titers to PA and, when present in the vaccine, to LF. Live spore vaccine administered to guinea pigs in a single dose conferred significantly better protection than the human vaccines (P less than 0.001), although they elicited significantly lower (P less than 0.0005) anti-PA and anti-LF titers at time of challenge with virulent Bacillus anthracis. Substantial anti-PA and anti-LF titers may not, therefore, indicate solid protective immunity against anthrax infection. The ELISA system was also shown to be capable of detecting anti-PA and anti-LF antibodies in the sera of individuals with histories of clinical anthrax. The advantage of ELISA over the Ouchterlony gel diffusion test and indirect microhemagglutination assay are demonstrated. There was a highly significant degree of correlation between ELISA and the indirect microhemagglutination assay (P less than 0.0005); but ELISA was markedly superior in terms of reproducibility, reliability, specificity, and simplicity in performance and stability of the bound antigen.

Animals↗
PubMed1986

In vitro assays for botulinum toxin and antitoxins.

Clostridium botulinum produces several powerful neuroparalytic toxins which, although rare in food-poisoning instances, are generally fatal. A considerable amount of effort has therefore been made by the food industry to ensure that food treatment processes adequate to prevent growth and toxin production by Cl. botulinum. Laboratory mice and guinea-pigs are presently used extensively both for the assay of botulinum toxins and for the development and assessment of vaccines used to protect laboratory workers. An amplified ELISA, using a monoclonal antibody, has been developed for botulinum type A toxin with a sensitivity similar to that of the mouse acute toxicity test. The immunoassay has been found to be applicable to the detection of toxin in foodstuffs and could replace the currently used mouse bioassay in many laboratories. Immunoassays have also been developed for the detection of antibodies to botulinum toxins. A preliminary study has shown that antibody titres to botulinum types A and B toxins in sera taken from immunised personnel, as measured by ELISA, showed limited correlation with those measured by the toxin neutralisation test in mice. A more extensive study should determine whether the latter test can be replaced by the ELISA.

Animal Testing Alternatives↗
PubMed1985

Inactivation of Clostridium botulinum type A neurotoxin by trypsin and purification of two tryptic fragments. Proteolytic action near the COOH-terminus of the heavy subunit destroys toxin-binding activity.

Limited treatment of Clostridium botulinum type A neurotoxin with trypsin resulted in the cleavage of the heavy (95000 Da) subunit at approximately the mid-position and a loss of toxic activity. The rate of toxicity loss was considerably faster than that of mid-chain cleavage; thus a loss of toxicity in excess of 90% was accompanied by only 30-35% mid-chain cleavage of the heavy subunit. A study of the binding of 125I-labelled neurotoxin to rat brain synaptosomes showed the loss of toxicity on trypsin treatment to be paralleled by a loss of toxin binding to rat brain synaptosomes suggesting the presence of at least two sites of tryptic action on the 95000-Da binding subunit. Prolonged treatment of the neurotoxin with trypsin resulted in the complete digestion of a 46000-Da fragment of the heavy subunit, leaving intact a soluble fragment of approximately 105000 Da containing the light subunit linked to the remaining (49000-Da) portion of the heavy subunit. This fragment exhibited less than 0.01% of the original toxicity and gave immunoprecipitation reactions indistinguishable from the native toxin. The 49000-Da portion of the heavy chain was purified from the 105000-Da fragment of the toxin and the sequence of the first 35 amino acids determined. The sequence of the first 10 residues was found to be identical to that previously reported for the heavy subunit showing that the 49000-Da fragment represents the NH2-terminal portion of the heavy chain and that this region is resistant to tryptic action. It is suggested that the primary site(s) of tryptic action on the heavy subunit of botulinum type A neurotoxin is close to the COOH terminus and that cleavage of the polypeptide chain in this region results in a loss of toxic activity mediated by the destruction of the neurotoxin-binding site.

Amino Acid Sequence↗
PubMed1985

Control of herpes simplex virus infections of the genital tract by vaccination.

The apparent increasing incidence of herpes simplex virus infections of the genital tract has focused attention on the efficacy of vaccination in preventing infection or modifying established disease. Results of an 'open trial' using a DNA-free inactivated virus subunit vaccine have shown that vaccination of subjects at risk of contracting infection from their sexual partner reduced the transmission rate from 34% in unvaccinated controls to 0.5%. In a separate study, vaccination of patients who had experienced their first overt attack of herpes genitalis (the initial clinical episode) had significantly fewer recurrences over the follow-up period of 12 months than the unvaccinated control group. The results, we feel, justify a placebo controlled trial.

Adolescent↗
PubMed1985

Identification of Bacillus anthracis by API tests.

API and morphological tests were examined for their ability to distinguish between 37 Bacillus anthracis strains (virulent and avirulent) and 194 strains of closely related Bacillus species (B. cereus, B. mycoides and B. thuringiensis). In addition, 34 strains of B. anthracis and four of B. cereus were tested by several other methods that included capsule formation, ability to grow on a selective medium, and sensitivity to phage. It was found that virulent strains of B. anthracis were easily separated from the closely related Bacillus species by most of the test methods; but separation of slightly virulent and avirulent strains of B. anthracis from the closely related species could be done only by API and phage-sensitivity tests.

Animals↗
PubMed1985

Monoclonal antibody-based immunoassay for type A Clostridium botulinum toxin is comparable to the mouse bioassay.

A monoclonal antibody (BA11) has been produced against Clostridium botulinum type A neurotoxin by the fusion of myeloma cells (P3 NS1/1-Ag4-1) with spleen cells from BALB/c mice immunized with botulinum type A neurotoxoid. The antibody bound specifically to botulinum type A neurotoxin, showing no cross-reactivity with types B and E botulinum toxins or with any of several other bacterial toxins tested. The monoclonal antibody did not bind to botulinum type A neurotoxin which had been denatured with sodium dodecyl sulfate and bound only weakly to each of the separated heavy and light subunits of the neurotoxin, suggesting a conformational requirement for the antigenic determinant of the antibody. A sensitive immunoassay for C. botulinum type A toxin with monoclonal antibody BA11 in conjunction with an enzyme amplication system has been developed which allows detection of 5 to 10 mouse 50% lethal doses ml-1 of purified neurotoxin. The assay was equally sensitive when applied to the detection of crude toxin in food stuffs; the average value for the minimum level of detectable toxin in extracts of tinned salmon or corned beef was 9 +/- 3.1 mouse 50% lethal doses ml-1.

Animals↗
PubMed1985

In vivo evidence that bacteria in urinary tract infection grow under iron-restricted conditions.

The outer membrane protein composition of bacteria isolated directly and without subculturing from the urine of two patients with urinary tract infections was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results indicated that the bacteria grew under iron-restricted conditions, as revealed by the expression of several high-molecular-weight outer membrane proteins which could also be observed when the same isolates were grown under iron-depleted conditions in laboratory media. The antigenicity of outer membrane components of the bacteria isolated was studied by immunoblotting with serum samples from the patients. The results indicated that the sera from the patients contained antibodies against major outer membrane components of the bacteria present in the urine, including the iron-regulated membrane proteins.

Aged↗
PubMed1984

Anthrax: the disease in relation to vaccines.

The authors trace the origins and history of anthrax and anthrax vaccines. They describe the aetiology and pathogenesis of the disease and the variety of symptoms which result from infection. The authors relate the early work performed by Pasteur, the development of existing vaccines and the efficacy of these vaccines, and predict the type of non-living vaccines which may be used to combat anthrax in the future.

Animals↗
PubMed1983

The effect of naloxone and morphine on convulsions in mice following withdrawal from nitrous oxide.

The incidence of withdrawal convulsions was determined in mice following removal from a 70 per cent nitrous oxide environment. Groups of 20 mice received saline (control), naloxone or morphine subcutaneous injections five minutes prior to withdrawal. The observer was blind to the treatments. In comparison to the control group, the proportion convulsing was significantly (p less than 0.05) increased following naloxone 0.125 mg (n = 40), 0.25 mg, but not 0.5 mg. The proportion convulsing was significantly decreased following morphine 0.4 mg. Overall proportions of mice convulsing was 0.55 for the saline control group; 0.73 for naloxone 0.125 mg; 0.80 for naloxone 0.25 mg; 0.60 for naloxone 0.50 mg; and 0.38 for morphine 0.4 mg. Modification of this phenomenon by both an opiate antagonist and agonist suggests endorphin withdrawal as a possible mechanism. However, this should be regarded as indirect evidence pending further study of this area.

Animals↗
PubMed1982

Anthrax island.

Explore the source record for details and available documents.

Animals↗
PubMed1982

Preparation and characterisation of homogeneous neurotoxin type A from Clostridium botulinum. Its inhibitory action on neuronal release of acetylcholine in the absence and presence of beta-bungarotoxin.

1. Large-scale production and purification of complexes between Clostridium botulinum neurotoxin and haemagglutinin have been achieved. 2. Haemagglutinin-free neurotoxic protein of the complexes was purified to high specific neurotoxicity by affinity chromatography, on p-aminophenyl beta-D-thiogalactopyranoside coupled to Sepharose 4B, followed by chromatography on DEAE-Sephacel. 3. The resultant neurotoxin was homogeneous on isoelectric focussing (pI = 6.3) and on dodecylsulphate/polyacrylamide gel electrophoresis under non-reducing conditions when its Mr was 1.4 X 10(5); after reduction two polypeptides (Mr = 9.9 and 5.5 X 10(4) were present. 4. On double-immunodiffusion gels, using antiserum against neurotoxin-haemagglutinin complex, the neurotoxin showed a single, sharp precipitin line that was immunologically distinct from a relatively non-toxic protein (Mr = 1.3 X 10(5), which co-purifies with the neurotoxin but is removed by the ion-exchange chromatography step. 5. Application of the neurotoxin to animals in vitro or in vivo produced near complete and irreversible blockade of neurotransmission. Botulinisation of rat leg muscles reduced spontaneous transmitter release; the amplitude of miniature end-plate potentials was altered from the normal 'bell-shaped" to a skewed distribution. 6. In normal muscle, a large transient increase in frequency of the miniatures was produced by beta-bungarotoxin. In contrast, with botulinised muscle the latter induced a much smaller increase in the absolute frequency; in addition, the mean amplitude was increased somewhat but the distribution remained skewed. The results show botulinisation of muscle modifies the action of beta-bungarotoxin.

Acetylcholine↗
PubMed1982

Localization of sites for 125I-labelled botulinum neurotoxin at murine neuromuscular junction and its binding to rat brain synaptosomes.

Botulinum neurotoxin, purified to homogeneity from Clostridium botulinum (Type A), was found to be highly neurotoxic (greater than 8 X 10(7) mouse LD50/mg protein). Labelling of this pure neurotoxin with 125I-iodine to high specific radioactivity was achieved without appreciable loss of biological activity. This was used to demonstrate saturable binding sites for this toxin at the neuromuscular junction, following in vivo administration into mice. A demonstrable inhibitory effect of the neurotoxin on release of acetylcholine from rat cerebrocortical synaptosomes indicates that it affects synapses in the central nervous system. Kinetic studies on the binding of 125I-labelled neurotoxin to brain synaptosomes yielded an association rate constant of 2.3 x 10(5)M-1s-1; dissociation plots were biphasic and the predominant species showed a rate constant of 1.2 X 10(-4)s-1. The saturable binding component is heat-sensitive and inactivated by trypsin. Preliminary studies showed that botulinum neurotoxin associates with plasma membrane fractions of synaptosomes and that binding does not result in any gross structural changes, at least in the majority of the toxin molecules.

Animals↗
PubMed1982

Preparation and immunogenicity of vaccine Ac NFU1 (S-) MRC towards the prevention of herpes genitalis.

A subunit antigenoid vaccine, Ac NFU1 (S-) MRC, was used to prevent primary herpes genitalis in 60 subjects considered to be at risk of this infection. There was no evidence of serious local or general side effects. Neutralising antibody responses were detected in 59% and 90% of subjects receiving the low and high doses of vaccine respectively; immunoprecipitating antibody was detected at a lower frequency, namely in 23% and 43% of subjects receiving the low and high doses respectively. After a mean follow-up period of 18 months none of the vaccinated subjects contracted herpes genitalis after completing the vaccination course.

Adult↗
PubMed1982

Early experience with "antigenoid" vaccine Ac NFU1(S-) MRC towards prevention or modification of herpes genitalis.

The preparation and early clinical experience with "antigenoid" vaccine Ac NFU1(S-) MRC is described. A neutralising antibody response against both type 1 and type 2 herpes simplex virus was stimulated in 19 of 21 and 25 of 60 patients who received the high and low dosage of vaccine, respectively: immunoprecipitating antibody against type-common virus antigen "band II", a virus antigen of major importance in neutralization of herpes simplex virus, was detected in 12 of 19 and 2 of consorts who received the high and low dosage vaccine, respectively. To date, after a mean follow-up period of one year, none of the 42 consorts have contracted herpes genitalis: following a mean follow-up period of 6 months, only 1 patient vaccinated after the initial clinical episode has reported a recurrence of herpetic disease. These results compare favourably with the rates of consort transmission and first year recurrences in unvaccinated subjects. Seventeen of 24 patients with recurrent herpetic disease reported an "improvement" in terms of modification of the frequency, severity or extent of herpetic lesions. There were no important side-effects from vaccination: most patients experienced a degree of minor local reaction at the vaccination site for 24-72 hours and 3 patients reported a mild transient "flu-like" syndrome which disappeared within 48 hours of vaccination. It is intended to subject these data to the scrutiny of a placebo-controlled trial with an objective system of clinical assessment.

Animals↗
PubMed1980

A comparison of chemically defined and complex media for the production of Bacillus subtilis spores having reproducible resistance and germination characteristics.

Spores of Bacillus subtilis were produced on five batches of Antibiotic Assay Medium No. 1 obtained from different suppliers, five batches obtained from a single supplier and five batches of a chemically defined liquid medium. The magnitude of the variation within each of the three groups was compared in terms of heat resistance, glutaraldehyde resistance, germination rate and sensitivity of germinated spores to neomycin. For all these parameters the degree of reproducibility achieved by using a chemically defined liquid medium was substantially better than that using batches of complex medium from a single supplier. Even larger variations in these parameters resulted when different suppliers were used. The value of defined media for the production of spore inocula to be used in sterilization control and similar procedures is discussed.

Bacillus subtilis↗