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Biomedical subjects

D Kerridge

Publications and source records attributed to D Kerridge.

At least 37 records · Page 2Linked to original sources
PubMed1986

A 19F nuclear magnetic resonance study of uptake and metabolism of 5-fluorocytosine in susceptible and resistant strains of Candida albicans.

The metabolism of the antifungal drug 5-fluorocytosine (5-FC) was studied in intact viable cells of Candida albicans by 19F nuclear magnetic resonance (NMR). The uptake of the drug and its conversion to the deaminated product 5-fluorouracil (5-FU) were easily observed by NMR analysis of both the cells and the supernatants of the incubation mixture. In the 5-FC-resistant mutant D14 of C. albicans, which lacked cytosine deaminase activity, the resonance peak of 5-FU was not observed. In intact cells of all 5-FC-susceptible strains the metabolism of 5-FU progressed to the formation of other fluorinated derivatives which were visualized as a single, broad resonance band at a lower field with respect to 5-FC and 5-FU. This band was resolved into three distinct peaks in the acid extract of treated cells, one of these peaks being attributable to 5-fluoro-dUMP (5-FdUMP). In strain 72R of C. albicans, which is 5-FC resistant because of a low level of UMP-pyrophosphorylase activity, the broad, low-field resonance band was detected later and with much less intensity than in the 5-FC-sensitive strains. This suggests that, besides 5-FdUMP, this band is also contributed to by 5-FUMP and possibly other phosphorylated derivatives. 19F NMR analysis also revealed that a significant amount of 5-FU is secreted into the external medium, the rate of secretion being higher in 5-FC-resistant strain 72R than in 5-FC-sensitive strain 72S. Although not all resonances were definitely identified, this study shows that 19F NMR spectroscopy may be an important tool for noninvasive analysis of the metabolism of fluorinated drugs in yeasts.

Candida albicans↗
PubMed1985

Present status of antimycotics.

The four principal classes of antimycotic drugs used in clinical practice are the polyene macrolide antibiotics, the synthetic imidazole drugs, griseofulvin and 5-fluorocytosine. Drugs in the first two groups interfere with the synthesis and functioning of cell membranes and the others interfere with nuclear metabolism. The mode of action of 5-fluorocytosine is well understood, but the detailed molecular mechanisms by which the other compounds work have yet to be elucidated.

Antifungal Agents↗
PubMed1984

Echinocandin inhibition of 1,3-beta-D-glucan synthase from Candida albicans.

The cyclic peptide antibiotic echinocandin was found to inhibit 1,3-beta-D-glucan synthase activity present in a mixed membrane fraction from Candida albicans. Addition of antibiotic reduced the Vmax of the enzyme, but the Km was unaffected. GTP stimulated enzyme activity approximately 4-fold, but did not affect the percentage inhibition of the enzyme by echinocandin. Treatment of the reaction products with alpha-amylase and beta-glucanase confirmed that the polymer synthesised was 1,3-beta-D-glucan, not glycogen.

Anti-Bacterial Agents↗
PubMed1984

Decreased activity of UMP pyrophosphorylase associated with resistance to 5-fluorocytosine in Candida albicans.

UMP pyrophosphorylase activity was assayed in crude lysates prepared from Candida albicans. Specific activity of UMP pyrophosphorylase was high in clinical isolates which were susceptible to 5-fluorocytosine. Resistant clinical isolates displayed low activity, and partially resistant (heterozygous) isolates displayed intermediate activity. Segregation from the heterozygous state resulted in a homozygous susceptible segregant with high UMP pyrophosphorylase activity and a homozygous resistant segregant with low activity. The observed specific activities were consistent with the hypothesis that specific activity was determined by the sum of the activities due to the dominant (FCY) and recessive (fcy) alleles of the resistance gene. Strains which possessed little UMP pyrophosphorylase activity released uracil into the medium when grown in the absence of 5-fluorocytosine; this result suggested that recycling of intracellular uracil is a normal function of this enzyme.

Candida albicans↗
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↗
PubMed1981

Lysis of growing yeast-form cells of Candida albicans by echinocandin: a cytological study.

Yeast form cells of Candida albicans 6406 were treated with echinocandin, a new antifungal agent, which, in the absence of osmotic protection, provoked the lysis of exponentially growing cells. Lysis did not occur in stationary-phase cells and when protein synthesis was blocked. In intact cells, the synthesis of glucan, but not other important wall components, was partially inhibited. A cytological study of the effects of echinocandin at lytic doses (3.0 microgram ml-1) on osmotically protected yeast cells revealed a substantial thinning of the bud cell wall and derangement of its constitutive layers within 5-10 min, showing that the balance of wall growth was quickly and critically affected by the drug. Associated with this effect, a number of membranous bodies of myelin-like appearance were often seen in close proximity to the plasmamembrane of the emerging bud. Later during treatment (15 min onwards) membranous, convoluted bodies were detected in the nuclear and other intracytoplasmic membranes. Subsequent lytic events, unevenly distributed in cell population, eventually brought about complete lysis of the cell cytoplasmic structure. These results suggest that echinocandin may block a biosynthetic step during wall construction, or that it could alter wall metabolism as a result of a primary interaction with membranes.

Anti-Bacterial Agents↗
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↗
PubMed1979

The polyene macrolide antibiotics.

The mode of action of the polyene antibiotics is reviewed together with the effect of genetic and environmental factors on sensitive organisms. The future prospects of polyenes in the treatment of systemic mycoses are considered.

Amphotericin B↗
PubMed1977

Sterol metabolism during germination of conidia of Aspergillus fumigatus.

The sterol content of germinating conidia of the opportunistic pathogenic fungus Aspergillus fumigatus has been correlated with germination phase and sensitivity to polyene antibiotics. The sterol and sterol ester contents of walls did not change during germination. The sterol ester content of membranes and cell sap remained constant during germination, whereas the sterol content increased during the outgrowth of germ tubes. On the basis of differential extraction studies it was concluded that the loss of resistance to polyenes that occurred in the early stages of swelling of conidia during germination was not due to a movement of sterol or sterol ester out of the wall. Radioactive-labelling experiments demonstrated that, although the amounts of conidial wall sterol and sterol ester did not change during germination, they were metabolically active. Changes in the turnover rate of wall and membrane sterol and sterol ester during germination were investigated and their relationship to a possible mechanism for the change from resistance to sensitivity to polyene antibiotics is discussed.

Antifungal Agents↗
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↗
PubMed1976

The interaction of amphotericin B methyl ester with protoplasts of Candida albicans.

The interaction of amphotericin B methyl ester (AME) with protoplasts of Candida albicans was measured indirectly by following the incorporation of [U-14C]phenylalanine into the acid-insoluble material. The inhibitory effects of AME at the minimum inhibitory concentration were prevented by the addition of 85 mM-KCl and 45 mM-MgCl2, as shown by Liras & Lampen (1974) for Saccharomyces cerevisiae. In C. albicans, pretreatment of the yeast before anti-biotic addition was unnecessary. KCl and MgCl2 did not prevent AME from binding to the protoplast membrane. This interaction was reversed by incubating the protoplasts in the presence of the protecting salts.

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↗