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Biomedical subjects

J B Ward

Publications and source records attributed to J B Ward.

At least 91 records · Page 5Linked to original sources
PubMed1980

The evaluation of the epoxide diluent, n-butylglycidyl ether, in a series of mutagenicity assays.

The epoxide diluent, n-butylglycidyl ether (n-BGE), was tested in vitro for mutagenicity using Salmonella typhimurium and for transforming ability in mouse embryo cells (BAlb/3T3-A31-1-13). The n-BGE produced base-pair type mutations in S typhimurium strains TA1535 and TA100. However, it failed to transform mouse embryo cells when assayed up to toxic levels. The mutagenic activity could not be detected in the urine of mice, although several doses, routes of administrations, and lengths of exposure were examined. Orally administered n-BGE did not produce any increase in micronuclei in mice, but when the diluent was administered intraperitoneally, either one or two times, dose-related increases in the number of micronuclei were observed.

Animals↗
PubMed1979

Characterization of the autolytic enzymes of Clostridium perfringens.

Clostridium perfringens and isolated walls of this organism autolysed rapidly when incubated in buffer at pH 7.0 with the release of free-reducing groups but no N-terminal amino acids. The predominant autolytic enzyme was an endo-beta-N-acetylglucosaminidase, and an endo-beta-N-acetylmuramidase was also present. The autolytic enzymes could be solubilized by extraction of the organisms with 5 M-LiCl and would then subsequently bind to and rapidly lyse walls of Micrococcus luteus and, more slowly, formamide-extracted walls of C. perfringens and walls of Bacillus subtilis. Lysis of C. perfringens walls by these extracted enzymes could not be demonstrated.

Acetylglucosaminidase↗
PubMed1978

Purification and characterization of the penicillin-binding protein that is the lethal target of penicillin in Bacillus megaterium and Bacillus licheniformis. Protein exchange and complex stability.

The penicillin-binding protein that is thought to be the lethal target of penicillin in Bacillus megaterium (protein 1) has been purified to greater than 95% homogeneity. The membrane-bound penicillin-binding proteins were solubilized with a non-ionic detergent and partially separated from each other by ion-exchange chromatography on DEAE-Sepharose CL-6B. Protein 1 was subsequently purified by covalent affinity chromatography on ampicillin-affinose. Bacillus licheniformis contains an equivalent penicillin-binding protein (protein 1) that can be more readily purified to virtual homogeneity in a one-step procedure. It was separated from the other penicillin-binding proteins by utilizing the observation that in this organism, this particular protein is the only one whose covalent complex with benzylpenicillin subsequently breaks down. Membranes were treated with saturating concentrations of benzylpenicillin followed by the removal of free penicillin and further incubation to allow the complex between benzylpenicillin and protein 1 to break down. The penicillin-binding proteins were then solubilized and applied to a column of ampicillin-affinose to which only protein 1 was bound as the other penicillin-binding proteins still had benzylpenicillin bound to them. Pure protein 1 was eluted from the affinity resin with hydroxylamine. The interaction of benzylpenicillin with purified protein 1 has been studied by separating unbound antibiotic from the benzylpenicillin . protein complex by paper electrophoresis. Benzylpenicillin reacts with the protein rapidly to form a covalent complex and the fully saturated complex has a molar ratio of bound [14C] benzylpenicillin: protein of 0.7:1. The complex breaks down, obeying first-order kinetics, with a half-life of 16 min at 35 degrees C, a value identical to that obtained with the membrane-bound protein. The concentration of benzylpenicillin that results in the formation of 50% of the maximum amount of benzylpenicillin . protein complex is that at which the molar amount of benzylpenicillin present is equal to 50% of the molar amount of penicillin-binding protein, rather than being a measure of any of the kinetic parameters of the binding reaction. This observation may be significant in the interpretation of previous results where the amounts of penicillins needed to kill cells or to inhibit penicillin-sensitive reactions have been expressed as concentrations. The possible importance of the breakdown of beta-lactam . protein complexes in the clinical use of these antibiotics is discussed.

Bacillus↗
PubMed1978

Detection of alkylating agents by the analysis of amino acid residues in hemoglobin and urine. 1. The in vivo and in vitro effects of ethyl methanesulfonate, methyl methanesulfonate, hycanthone methanesulfonate, and naltrexone.

The effect of alkylating agents on the amino acid composition of rat and human hemoglobin has been examined. Because the amino acid compositions of these proteins are well established, the changes in the molar ratios of specific amino acids could be monitored in purified hemoglobin samples. Ratios of histidine, which is readily attacked by alkylating agents, to proline, which is not, were consistent in hemoglobin purified from untreated blood samples from 14 rats and 25 human subjects. Treatment of hemoglobin in vitro or in vivo with methyl methanesulfonate, ethyl methanesulfonate, or hycanthone methanesulfonate resulted in a loss of histidine residues relative to proline or phenylalanine. The decrease in histidine content increased with treatment dose and time and reached a maximum at about 15% of total histidine. Treatment of rats with methyl methanesulfonate or hycanthone methanesulfonate resulted in increased urinary excretion of methyl histidines.

Alkylating Agents↗
PubMed1978

Hypoxia with a third flowmeter tube on the anaesthetic machine.

Hypoxic mixtures of oxygen and nitrous oxide were demonstrated with three anaesthesia machines which had cyclopropane flowmeters. In one machine the bobbin did not prevent back flow and the hypoxic mixture occurred when the cyclopropane flow control was left open. Two other machines only delivered hypoxic mixtures if the cyclopropane bobbins were removed from their seats and the flow controls opened. The vaporizer on one machine increased the hypoxia when turned on.

Anesthesia, Inhalation↗
PubMed1977

Biosynthesis of wall polymers in Bacillus subtilis.

Preparations of membrane plus wall derived from Bacillus subtilis W23 were used to study the in vitro synthesis of peptidoglycan and teichoic acid and their linkage to the preexisting cell wall. The teichoic acid synthesis showed an ordered requirement for the incorporation of N-acetylglucosamine from uridine 5'-diphosphate (UDP)-N-acetylglucosamine followed by addition of glycerol phosphate from cytidine 5'-diphosphate (CDP)-glycerol and finally by addition of ribitol phosphate from CDP-ribitol. UDP-N-acetylglucosamine was not only required for the synthesis of the teichoic acid, but N-acetylglucosamine residues formed an integral part of the linkage unit attaching polyribitol phosphate to the cell wall. Synthesis of the teichoic acid was exquisitely sensitive to the antibiotic tunicamycin, and this was shown to be due to the inhibition of incorporation of N-acetylglucosamine units from UDP-N-acetylglucosamine.

Anti-Bacterial Agents↗
PubMed1976

Cultured cells in cystic fibrosis research: A review.

Cystic fibrosis (CF) is a common inherited disorder which is characterized by the production of exocrine secretions with elevated ion content and abnormally viscous mucus. Over the last few years cells obtained from the peripheral blood or cultured from tissues of cystic fibrosis patients have been used increasingly in the study of the disease. Investigations of the following properties of cystic fibrosis cells are reviewed: morphology, ultrastructure, growth kinetics, cellular metachromasia, the production of ciliary inhibitors, cellular composition, plasma membrane composition, the transport of inorganic ions and small organic molecules, lysosomal enzyme content, and RNA methylation. Studies of the effects on cultured cells and erythrocyte membranes of factors in CF cell culture medium and biological fluids from CF patients are discussed.

Animals↗
PubMed1976

Surface enzymes in cultured fibroblasts from cystic fibrosis patients.

Membrane function was examined in cultured cells from cystic fibrosis patients by assaying several enzymes on intact skin fibroblasts attached to culture dishes. This technique required few cells and minimized disruption of cellular organization. Comparison of enzyme activities of intact and broken cells showed that 12% of total glucose-6-phosphate dehydrogenase, a cytoplasmic enzyme, was measurable using intact cells, while all adenosine monophosphatase was measurable using intact cells. Alkaline paranitrophenylphosphatase activity was divided between the cell surface and interior. Substrate competition experiments indicated that substrate specificities for adenosine monophosphatase and paranitrophenylphosphatase activities were different. Adenosine monophosphatase activities of 2 control and 2 cystic fibrosis strains fluctuated similarly during the cell culture cycle. The apparent Km values relative to adenosine monophosphate were similar in all strains. A chromatographic fraction of serum from a cystic fibrosis patient that was inhibitory to oyster ciliary activity had no effect on adenosine monophosphatase activity of normal fibroblasts. Furthermore, fractions of media from cystic fibrosis homozygote and heterozygote fibroblast cultures were not inhibitory to adenosine monophosphatase activities of intact normal fibroblasts or of part iculate fractions prepared from them. In light of previous studies that showed that factors from cystic fibrosis serum of culture medium disrupted specific membrane activities, it is proposed that the cystic fibrosis factor interacts with the plasma membrane, interfering most conspicuously with the protein functions that are sensitive to changes in their membrane environment.

4-Nitrophenylphosphatase↗
PubMed1975

Peptidoglycan synthesis in Bacillus licheniformis. The inhibition of cross-linking by benzylpenicillin and cephaloridine in vivo accompanied by the formation of soluble peptidoglycan.

The synthesis of peptidoglycan by an autolysin-deficient beta-lactamase-negative mutant of Bacillus licheniformis was studied in vivo in the absence of protein synthesis. Benzylpenicillin and cephaloridine inhibited the formation of cross-bridges between newly synthesized peptidoglycan and the pre-existing cell wall. This inhibition, detected by measurement of the incorporation of N-acetyl[14C]glucosamine into the glycan fraction of the cell wall, was reversed by treatment with beta-lactamase and washing. Inhibition of D-alanine carboxypeptidase by benzylpenicillin was not reversed under similar conditions. Cells in which the initial penicillin inhibition of transpeptidation had been reversed showed an increased sensitivity to a subsequent addition of the antibiotic. Chemical analysis of peptidoglycan synthesized after reversal of penicillin inhibition revealed the presence of excess of alanine resulting from the continued inhibition of D-alanine carboxypeptidase. When the cell walls were digested to yield muropeptides so that the degree of cross-linking could be measured, the product after reversal of penicillin inhibition contained fewer cross-links than did the control preparation. Cultures treated with benzylpenicillin and cephaloridine continued to synthesize uncross-linked soluble peptidoglycan, which accumulated in the medium. This soluble material was all newly synthesized peptidoglycan and did not result from autolysis of the bacteria. The average chain lengths of the glycan synthesized in vivo and released as soluble peptidoglycan in the presence of both benzylpenicillin and cephaloridine were similar to those found previously in this organism.

Acetylglucosamine↗
PubMed1975

Formation of cell wall polymers by reverting protoplasts of Bacillus licheniformis.

The biosynthesis of peptidoglycan and teichoic acid by reverting protoplasts of Bacillus licheniformis 6346 His-, in cubated at 35 C on medium containing 2.5% agar, is detectable after 40 min. The amount of N-acetyl-[1-14C]glucosamine incorporated into peptidoglycan and teichoic acid on continued incubation doubles at the same rate as the incorporation of [3H]tryptophan into protein. At the early stages of reversion the average glycan chain length, measured by the ratio of free reducing groups of muramic acid and glucosamine to total muramic acid present, is very short. As reversion proceeds, the average chain length increases to a value similar to the found in the wall of the parent bacillus. The extent of cross-linkage found in the peptide side chains of the peptidoglycan also increases as reversion proceeds. At the completion of reversion the wall material synthesized has similar characteristics to those of the walls of the parent bacilli, containing peptidoglycan and teichoic and teichuronic acids in about the same proportions. Soluble peptidoglycan can be isolated from the reversion medium, amounting to 30% of the total formed after 3 h of incubation and 8% after 12 h. This amount was reduced by the presence in the medium of the walls of an autolysin-deficient mutant; they were not formed at all by reverting protoplasts of the autolysin-deficient mutant itself. Analysis of the soluble material provided additional evidence for their being autolytic products rather than small unchanged molecules. When protoplasts were incubated on medium containing only 0.8% agar, 53 to 67% of the peptidoglycan formed after 3 h of incubation was soluble, and 21% after 12 h. Fibers that appeared to be sheared from the protoplasts at intermediate stages of reversion on medium containing 2.5% agar were similar in composition to the bacillary walls.

Alanine↗
PubMed1975

Peptidoglycan synthesis in L-phase variants of Bacillus licheniformis and Bacillus subtilis.

Stable L-phase variants isolated from Bacillus licheniformis and Bacillus subtilis, when grown in osmotically stabilized media, do not synthesize peptidoglycan but have been found to accumulate the nucleotide precursors of this polymer. The enzymes involved in the synthesis of these precursors and the later membrane-bound stages of peptidoglycan synthesis have been investigated, and the L-phase variants have been shown to contain lesions, which provide a rational explanation for the absence of peptidoglycan and for the nature of the precursor accumulated. The majority of the L-phase variants contained a single enzymic defect, but two strains were isolated with double lesions. Five out of seven strains examined accumulated uridine 5'-diphosphate (UDP)-MurAc-L-ala-D-glu and were unable to synthesize diaminopimelic acid as a consequence of a defect in aspartate-beta-semialdehyde dehydrogenase activity. Two strains were deficient in UDP-MurAc: L-alanine ligase and accumulated UDP-MurAc. One strain accumulated the complete nucleotide precursor UDP-MurAc-L-ala-D-glu-mA2pm-D-ala-D-ala and was deficient in phospho-N-acetylmuramyl pentapeptide translocase. A second strain also had this lesion, together with defective aspartate-beta-semialdehyde dehydrogenase activity. The other enzymes of peptidoglycan synthesis were present in the L-phase variants, with activities similar to those found in the parent bacilli grown under identical conditions. Membrane preparations from certain of the L-phase variants were also capable of synthesizing the secondary polymers poly(glycerol phosphate) teichoic acid and teichuronic acid and also a polymer of N-acetylglucosamine.

Alanine↗
PubMed1975

Ultrastructural study of the reversion of protoplasts of Bacillus licheniformis to bacilli.

The reversion of protoplasts of Bacillus licheniformis 6346 His- on a medium containing 2.5% agar has been studied in sectioned material after reaction with a ferritin-conjugated antibody specific to the peptidoglycan isolated from the walls of the bacilli. Freeze etching has also been used. Fibrils of material reacting with the antibody have been detected emerging from isolated areas of the protoplasts after 3 h of incubation. This material gradually covers the cell and can eventually (at 6 h) be seen in freeze-etched preparations as a fringe of up to 400 nm around the cells and covering the surfaces with particles that can be removed by lysozyme. At later stages the wall begins to take on a compact, well-defined appearance that can be seen in sections; however, the cells are still grossly deformed. A transitory emergence, beyond the wall of long fibers of 6 nm in diameter, takes place after about 12 h of incubation. These fibers react with the conjugated antibody and after freeze etching show a regular banded structure. They are probably indentical with the fibers isolated elsewhere (Elliott et al., 1975) and shown to contain all the wall constituents (i.e., peptidoglycan, teichoic acid, and teichuronic acid). These fibers are not detectable in the final stages of reversion.

Antibodies, Bacterial↗