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Biomedical subjects

E F Gale

Publications and source records attributed to E F Gale.

At least 19 recordsLinked to original sources
PubMed1984

The action of 2-deoxy-D-glucose on the incorporation of glucose into (1----3)-beta-glucan in stationary phase cultures of Candida albicans.

2-Deoxy-D-glucose added to cultures of Candida albicans in the stationary phase of growth inhibited the incorporation of glucose into the (1----3)-beta-glucan fraction of the organisms. In the presence of ATP and cell extracts it was converted to 2-deoxy-D-glucose phosphate and when UTP was also present, material with the electrophoretic properties of UDP-2-deoxy-D-glucose was formed. In similar conditions glucose formed glucose phosphates, UDP-glucose and other products. Evidence was obtained that the analogue, after conversion to a phosphate derivative, was an inhibitor of phosphoglucomutase. When C. albicans was grown in the presence of 2-deoxy-D-glucose for 24 h, analogue residues became incorporated into the (1----3)-beta-glucan fraction and the subsequent rate of incorporation of glucose into that fraction was enhanced. The rate of turnover of glucose in this beta-glucan fraction was greater than in controls. Pretreatment of cultures with beta-glucanase, or incubation under conditions known to stimulate endogenous beta-glucanases, increased the subsequent rate of glucose incorporation and this increase was enhanced by growth in the presence of 2-deoxy-D-glucose. The analogue thus had the effect of altering the stability and glucose-acceptor function of (1----3)-beta-glucan chains. This could affect the properties of the polymer network leading to the known effect of the analogue in delaying the onset of phenotypic resistance to amphotericin methyl ester in stationary phase cultures of C. albicans.

Adenosine Triphosphate↗
PubMed1982

Phenotypic resistance to amphotericin B in Candida albicans: relationship to glucan metabolism.

The phenotypic resistance to amphotericin methyl ester (AME) of stationary phase cultures of Candida albicans was decreased by alkaline pH values and by treatment with 2-mercaptoethanol or glucanase preparations, and was increased by acid pH values, increased aeration, treatment with N-ethylmaleimide, or the presence of inhibitors of protein synthesis such as trichodermin. The effects of such treatments on endogenous glucanase activity and on the incorporation of glucose residues into the 'glucan fraction' of the organism were studied. The changes in the endogenous levels of lytic activities on laminarin [as a measure of the total (1 leads to 3)-beta-D-glucanase] and on p-nitrophenyl-beta-D-glucoside [reflecting the exo-(1 leads to 3)-beta-D-glucanase] were followed in C. albicans cells under a variety of conditions. Treatments which increased AME sensitivity stimulated both total and exo-(1 leads to 3)-beta-D-glucanase activities, while treatments which promoted resistance decreased the levels of both (1 leads to 3)-beta-D-glucanases. Changes in the 'glucan fraction' were followed by incubating suspensions of organisms in the presence of trace amounts of [U-14C]glucose. The rate of incorporation of radioactivity fell during the first 2-3 d of stationary phase culture and then rose to high values by 7-8 d; AME resistance increased throughout this period. The rate of incorporation was markedly stimulated by prior treatment of the organisms with 2-mercaptoethanol or glucanase and inhibited by trichodermin or treatment with N-ethylmaleimide. The addition in the concentration range 0.3-3 mM of the glucose analogues beta-D-allose, 3-O-methyl-D-glucose, 2-deoxy-D-glucose or 5-thio-D-glucose to cultures 24 h after inoculation prevented any further increase in AME resistance for the next 2-3 d and resulted in a decrease in the level of resistance established at the time of addition. Radioactivity from 14C- or 3H-labelled analogues added, 24 h after inoculation, to stationary phase cultures was incorporated into the 'glucan fraction' of the organisms. The incorporation of glucose residues into the 'glucan fraction' is controlled by the activity of glucanases in producing glucose acceptor sites. The results reported confirm that there is a correlation between glucan metabolism, glucanase activity and resistance to AME, in that any factor leading to increased glucanase action also results in decreased resistance and vice versa, while incorporation of certain glucose analogues into the 'glucan fraction' delays the further increase in resistance.

Amphotericin B↗
PubMed1980

Reduction of amphotericin resistance in stationary phase cultures of Candida albicans by treatment with enzymes.

The resistance of Candida albicans to amphotericin B methyl ester increases rapidly as cultures enter the stationary phase of growth; organisms harvested after several days in the stationary phase may have a resistance two or three orders of magnitude greater than that of exponentially growing organisms. This resistance is decreased by incubation of the organisms with enzymes which attack components of the cell wall. Of the enzymes tested, (1 leads to 3)-beta-D-glucanases are the most effective; incubation of 7 d batch cultures with exo-(1 leads to 3)-beta-D-glucanase at a concentration of 10 microgram enzyme protein (mg dry wt organisms)-1 for 24 h at 37 degrees C and pH 6.5 reduces the resistance of the organisms to a value approximating to that of exponentially growing organisms. Resistance is also decreased by treatment with chitinase, lipase, trypsin, alpha-mannosidase and (1 leads to 6)-beta-D-glucanases but, on a specific activity basis, none of these enzymes is as effective as (1 leads to 3)-beta-D-glucanase. The action of (1 leads to 3)-beta-D-glucanase is markedly enhanced by the addition during incubation of chitinase, trypsin or lipase.

Amphotericin B↗
PubMed1980

Phenotypic resistance to miconazole and amphotericin B in Candida albicans.

Phenotypic resistance to both amphotericin B and miconazole develops in stationary phase cultures of Candida albicans and this resistance lies in changes in the cell wall. Study of the effects of growth conditions, treatment with SH-reactive agents and treatment with enzymes indicates that the nature of the changes leading to resistance must be different for the two drugs.

Amphotericin B↗
PubMed1979

Ultrastructural changes in the cell wall of Candida albicans following cessation of growth and their possible relationship to the development of polyene resistance.

The ultrastructure of the wall of Candida albicans strain 6406 was examined in polyeneresistant organisms obtained by continued incubation after the cessation of growth. The walls of organisms harvested either during the exponential phase of growth or after 24 h starvation, when examined in situ, showed the typical layered appearance. After 72 h starvation, when the resistance to amphotericin B methyl ester (AME) was 60 times greater than that of exponentially growing organisms, both the periplasmic material and the distinct electron-dense layers were absent from the wall. At this stage there was no increase in the thickness of the wall. After 144 h starvation the thickness of the wall had increased from 143 +/-22 nm (exponential phase organisms) to 211+/-58 nm. If after 144 h starvation the organisms were incubated for 1 h in fresh nutrient medium they regained their sensitivity to AME and the wall regained the periplasmic material and its characteristic multilayered appearance. During the first 24 h starvation there was a considerable fall in the soluble glucan fraction, but on continued incubation there was little change in the relative proportions of the major carbohydrate constituents of the cell. Thin sections of purified walls isolated from organisms harvested either during exponential growth or after 144 h starvation were identical in appearance and characterized by the absence of the electrondense layers observed in sections of intact cells and by a reduction in thickness to 100+/-20nm.

Amphotericin B↗
PubMed1977

Growth characteristics and polyene sensitivity of a fatty acid auxotroph of Candida albicans.

A fatty acid auxotroph of Candida albicans 6406, designated A' 44 and originally isolated as an oleic acid requiring strain, has been shown to be a delta9 desaturase mutant. Although lacking this step in fatty acid biosynthesis, it appears to retain the ability to desaturate monounsaturated fatty acids. The polyene sensitivity of the organism grown on different fatty acid supplements varied between 0-08 +/- 0-02 and 1-20 +/- 0-30 microgram amphotericin B methyl ester ml-1 for exponentially growing cells. In spite of this variation, the sterol composition remained fairly constant, the major differences lying in fatty acid composition. Stationary-phase cells were more resistant to amphotericin B methyl ester, although again this change was not associated with changes in sterol content. The organism was most resistant when grown in the presence of oleic or linoleic acid. Protoplasts derived from resistant organisms grown on these two fatty acids were also resistant, indicating that the structure of the cell wall was less important than that of the plasma membrane in determining polyene sensitivity under these conditions.

Candida albicans↗
PubMed1977

The effect of aeration and metabolic inhibitors on resistance to amphotericin in starved cultures of Candida albicans.

The development of resistance to amphotericin methyl ester, measured in terms of the amount of drug required to induce a standard rate of release of K+ from suspensions of washed organisms, has been followed in Candida albicans in starved cultures under controlled conditions of aeration, stirring and temperature. Resistance develops at a rate which increases with the rate of aeration, limited by the onset of damage due to turbulence. Resistance decreases rapidly if gassing with N2 is substituted for aeration, but sensitivity does not reach that of exponentially growing cells. Resumption of aeration is followed by a slow recovery of resistance. The addition of inhibitors of protein synthesis (trichodermin, verrucarin) or uncoupling agents (2,4-dinitrophenol, sodium azide) at the beginning of starvation results in an increased rate of development of resistance. Adding inhibitors at a later stage, when resistance has developed after 72 h aeration, does not affect the decrease in resistance produced by gassing with N2 but the presence of trichodermin or verrucarin delays the recovery of resistance o

Amphotericin B↗
PubMed1975

Factors affecting the changes in amphotericin sensitivity of Candida albicans during growth.

The sensitivity of Candida albicans, grown in batch culture at 37 degrees C, to amphotericin methyl ester (AME), judged by the concentration of AME required to induce a standard rate of leakage of K+ from suspensions of organisms, decreased with the time of growth. Organisms in exponential growth were sensitive to 0-I to 0-2 mug AME/ml while organisms in the stationary phase were resistant to 4 to 60 mug AME/ml, depending on the initial concentration of glucose in the medium and the length of time for which incubation had been continued. When the initial concentration of glucose was low (0-I%, w/v), the AME resistance rose during the early stationary phase and then, after 40 h incubation at 37 degrees C, decrease again. Sphaeroplasts were prepared from organisms at different phases of growth and did not show these changes in AME sensitivity, but remained highly sensitive for growth up to 40 h. Sphaeroplasts were prepared by treating suspensions of organisms with mercaptoethanol and then digesting with Streptomyces enzyme preparation. Addition of the material extracted by the digestion to suspensions of exponential-phase organisms or sphaeroplasts increased their AME resistance. Fractionation of the digest showed that the antagonistic material was contained in the neutral lipid fraction. Pure lipids fell into the following order of decreasing antagonism to AME when added together with the antibiotic to suspensions of exponential-phase organisms: sterol esters (ergosterol esters greater than cholesterol esters; unsaturated fatty acid esters greater than saturated fatty acid esters), sterols, triglycerides, unsaturated fatty acids, saturated fatty acids. The amount of antagonistic material released from stationary organisms was not markedly greater than that from exponential-phase organisms and analysis of the lipid content of wall preparations showed that the content of total lipid, neutral lipid and triglyceride of 40 h organisms was not more than 75, 25 and 30%, respectively, greater than that of exponential-phase organisms. The AME resistance of stationary-phase organisms decreased rapidly if suspensions were incubated with glucose or mercaptoethanol. The decrease in the presence of glucose was prevented by metabolic inhibitors, especially SH binding agents. Treatment of organisms with either iodoacetamide or N-ethylmaleimide gave a rapid increase in AME resistance, amounting in some cases to 5- to 15-fold. The effect of iodoacetamide decreased as the organisms passed into the stationary phase and their intrinsic resistance increased. Evidence is presented which suggests that the degree of reduction of SH groups in the cell surface is an important factor in determining AME resistance.

Amphotericin B↗
PubMed1975

Polyene sensitivity during germination of conidia of Aspergillus fumigatus.

A system for the rapid and relatively synchromous germination of conidia from a clinical isolate of Aspergillus fumigatus is described. The polyene-mediated release of K plus from germinating conidia has been determined. Ungerminated conidia were insensitive to amphotericin B methyl ester (AME) at concentrations greater than 50 mug/ml, but rapidly became sensitive to 1 to 2 mug AME/ml during the intitial stages of germination. These findings have been correlated with minimum inhibitory concentration values obtained in studies of conidial germination and hyphal outgrowth using a variety of growth tests.

Amphotericin B↗