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M G Shepherd

Publications and source records attributed to M G Shepherd.

At least 73 records · Page 4Linked to original sources
PubMed1984

The control of morphogenesis in Candida albicans.

Morphogenesis (germ-tube formation) in Candida albicans was induced gratuitously by N-acetylhexosamine derivatives (N-acetyl-glucosamine covalently linked to agarose, N-acetylmannosamine, hyaluronic acid, colloidal chitin, and mucin). These compounds were not taken up by the yeast cells and did not support growth. 2-Deoxyglucose was a potent inhibitor of germ-tube formation (50 microM), but did not affect the yeast growth yield at a concentration of 2.5 mM. 2-Deoxyglucose covalently linked to agarose did not affect germ-tube formation, and the inhibition by free 2-deoxyglucose was overcome by the addition of glucose to the germ-tube-forming cells. Glucose competitively inhibited 2-deoxyglucose uptake (Ki = 0.14 mM), and these data indicate that 2-deoxyglucose acted intracellularly.

Acetylglucosamine↗
PubMed1983

Candidosis.

Explore the source record for details and available documents.

Antifungal Agents↗
PubMed1983

Chemical modification of L-lactate 2-monooxygenase with fluorodinitrobenzene: evidence for two essential histidine residues.

The modification of L-lactate 2-monooxygenase (lactate oxidase) with radioactively labeled fluorodinitrobenzene in 50 mM tris(hydroxymethyl)aminomethane (Tris)-acetate, pH 6.0, resulted in complete inactivation with the incorporation of 1 mol of dinitrophenyl per catalytic site. Analysis of acid hydrolysates using high-pressure liquid chromatography and an amino acid analyzer showed that Nim-(dinitrophenyl)histidine accounted for 95% of the incorporated label. Approximately 50% of the flavin in the modified enzyme was reduced by lactate, formed a sulfite complex, and underwent a photochemical reaction with oxalate. These results suggest that each active site in lactate oxidase contains two essential histidine residues that are modified in a mutually exclusive manner.

Amino Acids↗
PubMed1983

The in situ assay of Candida albicans enzymes during yeast growth and germ-tube formation.

Conditions are described for the preparation of permeabilized cells of Candida albicans. This method has been used for the in situ assay of enzymes in both yeast cells and germ-tube forming cells. A mixture of toluene/ethanol/Triton X-100 (1:4:0.2, by vol.) at 15% (v/v) and 8% (v/v) was optimal for the in situ assay of glucose-6-phosphate dehydrogenase in yeast and germ-tube forming cells, respectively. The concentration of toluene/ethanol/Triton X-100 required for optimal in situ activity of other enzymes was influenced by the cellular location of the enzyme, growth phase and morphology. The membrane-bound enzymes (chitin synthase, glucan synthase, ATPase), cytosolic enzymes (glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, pyruvate kinase, phosphofructokinase, alkaline phosphatase, glucosamine-6-phosphate deaminase and N-acetylglucosamine kinase) and wall enzymes (beta-glucosidase and acid phosphatase) were measured and compared to the activity obtained in cell extracts. The pattern of enzyme induction and the properties of the allosteric enzymes phosphofructokinase and pyruvate kinase were measured in situ. Pyruvate kinase in situ was homotropic for phosphoenolpyruvate with a Hill coefficient of 1.9 and a S0.5 of 0.6 mM, whereas in cell extracts, it had a Hill coefficient of 1.9 and a S0.5 of 1.0 mM. The Km for ATP was 1.6 mM in cell extracts and 1.8 mM in permeabilized cells. In situ phosphofructokinase was homotropic for fructose 6-phosphate (S0.5 of 2.3 mM, Hill coefficient of 4.0). The kinetic properties of pyruvate kinase and phosphofructokinase measured in situ or in vitro were similar for both yeast cells and germ-tube forming cells.

Aldose-Ketose Isomerases↗
PubMed1983

An analysis of the metabolism and cell wall composition of Candida albicans during germ-tube formation.

The uptake of nutrients (glucose, glutamine, and N-acetylglucosamine), the intracellular concentrations of metabolites (glucose-6-phosphate, cyclic AMP, amino acids, trehalose, and glycogen) and cell wall composition were studied in Candida albicans. These analyses were carried out with exponential-phase, stationary-phase, and starved yeast cells, and during germ-tube formation. Germ tubes formed during a 3-h incubation of starved yeast cells (0.8 X 10(8) cells/mL) at 37 degrees C during which time the nutrients glucose plus glutamine or N-acetylglucosamine (2.5 mM of each) were completely utilized. Control incubations with these nutrients at 28 degrees C did not form germ tubes. Uptake of N-acetylglucosamine and glutamine was inhibited by cycloheximide which suggests that de novo protein synthesis was required for the induction of these uptake systems. The glucose-6-phosphate content varied from 0.4 nmol/mg dry weight for starved cells to 2-3 nmol/mg dry weight for growing yeast cells and germ tube forming cells. Trehalose content varied from 85 nmol/mg dry weight (growing yeast cells and germ tube forming cells) to 165 nmol/mg weight (stationary-phase cells). The glycogen content decreased during germ-tube formation (from 800 to 600 nmol glucose equivalent/mg dry weight) but increased (to 1000 nmol glucose equivalent/mg dry weight) in the control incubation of yeast cells. Cyclic AMP remained constant throughout germ-tube formation at 4-6 pmol/mg dry weight. The total amino acid pool was similar in exponential, starved, and germ tube forming cells but there were changes in the amounts of individual amino acids. The overall cell wall composition of yeast cells and germ tube forming cells were similar: lipid (2%, w/w); protein (3-6%), and carbohydrate (77-85%). The total carbohydrates were accounted for as the following fractions: alkali-soluble glucan (3-8%), mannan (20-23%), acid-soluble glucan (24-27%), and acid-insoluble glucan (18-26%). The relative amounts of the alkali-soluble and insoluble glucan changed during starvation of yeast cells, reinitiation of yeast-phase growth, and germ-tube formation. Analysis of the insoluble glucan fraction from cells labelled with [14C]glucose during germ-tube formation showed that the chitin content of the cell wall increased from 0.6% to 2.7% (w/w).

Acetylglucosamine↗
PubMed1982

Enzymes of N-acetylglucosamine metabolism during germ-tube formation in Candida albicans.

The enzymes of N-acetyl-D-glucosamine (GlcNAc) metabolism, GlcNAc-6-phosphate deacetylase and GlcN-6-phosphate deaminase were found to be inducible in Candida albicans. The pattern of induction for these enzymes was the same under conditions of germ-tube formation (37 degrees C) and where yeast cells metabolized GlcNAc with no change in morphology (28 degrees C); this indicates that these enzymes are not control points in the dimorphic development of C. albicans. During induction there was a 40-and 25-fold increase in specific activity for the deacetylase and the deaminase, respectively, and the maximum specific activity correspond to the time when all the GlcNAc had been metabolized. The presence of lomofungin (an inhibitor of transcription) or trichodermin (an inhibitor of translation) in cell suspensions of C. albicans containing GlcNAc prevented the increase in specific activity of these enzymes. 2-Deoxyglucose inhibited germ-tube formation, partially inhibited the induction of the deacetylase (43%) and the deaminase (60%), but did not affect the growth of C. albicans on either Glc or GlcNAc. GlcN-6-phosphate was a competitive inhibitor of the deacetylase with a Ki of 1.4 mM while the other product of the reaction, acetate, did not inhibit the enzyme. The Km value for GlcN-6-phosphate on GlcN-6-phosphate deaminase was 0.24 mM. Incubation of starved yeast cells with GlcNAc produced a four-fold increase in the specific activity of UDP-GlcNAc-pyrophosphorylase at either 28 degrees C or 37 degrees C.

Acetylglucosamine↗
PubMed1982

Gratuitous induction by N-acetylmannosamine of germ tube formation and enzymes for N-acetylglucosamine utilization in Candida albicans.

N-Acetylmannosamine did not support the growth of Candida albicans, and this sugar was not accumulated by cells. Incubation of starved yeast cells at 37 degrees C with N-acetylmannosamine plus glucose resulted in germ tube formation. Furthermore, N-acetylmannosamine alone induced the uptake system for N-acetylglucosamine and the enzymes of the N-acetylglucosamine catabolic pathway to the same extent as the natural substrate. Induction of the uptake system and the enzymes was observed at 28 degrees C without germ tube formation and at 37 degrees C with germ tube formation. N-Acetylmannosamine is thus a gratuitous inducer for enzymes of the N-acetylglucosamine pathway and germ tube formation in C. albicans.

Acetylglucosamine↗
PubMed1982

Recombination analysis of naturally diploid Candida albicans.

A multiply auxotrophic strain, hOG45, was derived from Candida albicans ATCC 10261. Prototrophic revertants of this multiple auxotroph were selected after mutagenesis. These prototrophic revertants were distinguishable from the original prototroph, ATCC 10261, because of their mitotic instability. They gave rise to auxotrophic derivatives which displayed one or more of the auxotrophic requirements characteristic of hOG45. Two of the auxotrophic requirements, those for adenine and methionine, frequently reappeared together in the auxotrophic derivatives of the prototrophic revertants. This apparent linkage of ade and met was confirmed by protoplast fusion analysis of the original auxotroph. These data indicate that C. albicans ATCC 10261 is diploid, the multiple auxotroph h0G45 is homozygous for recessive auxotrophic alleles, the prototrophic revertants are multiple heterozygotes, the auxotrophic derivatives are homozygotes produced by mitotic crossing-over, and the association between the ade and met alleles is due to linkage.

Adenine↗
PubMed1982

The effects of ergosterol and alcohols on germ-tube formation and chitin synthase in Candida albicans.

Ergosterol inhibited germ-tube formation and the membrane-bound enzyme chitin synthase in Candida albicans. The sterol solvents methanol and ethanol stimulated chitin synthase activity but inhibited germination. Methanol increased both the rate of protease activation of the chitin synthase proenzyme and the Vmax of the enzyme. The activation was not due to a change in the protease action on the proenzyme. The methanol activation was reversible and therefore the extraction of inhibitory components from the membrane was not causing the activation. Methanol had no effect on the N-acetylglucosamine activation sites. Analysis of the N-acetylglucosamine activation by an iteration programme gave K1 values of 0.3 mM for the high affinity sites and 20 mM for the low affinity sites. The degree of cooperativity with respect to the substrate uridine diphosphate N-acetylglucosamine was not changed by the presence of methanol and therefore the solvent did not affect the subunit-subunit interactions of the enzyme. Arrhenius plots of chitin synthase have discontinuities and methanol did not affect the transition temperature indicating that the structure of the membrane in the immediate vicinity of the enzyme was unchanged.

Alcohols↗
PubMed1981

Substrate specificity and mode of action of the cellulases from the thermophilic fungus Thermoascus aurantiacus.

The substrate specificities of three cellulases and a beta-glucosidase purified from Thermoascus aurantiacus were examined. All three cellulases partially degraded native cellulose. Cellulase I, but not cellulase II and cellulase III, readily hydrolyzed the mixed beta-1,3; beta-1,6-polysaccharides such as carboxymethyl-pachyman, yeast glucan and laminarin. Both cellulase I and the beta-glucosidase degraded xylan, and it is proposed that the xylanase activity is an inherent feature of these two enzymes. Lichenin (beta-1,4; beta-1,3) was degraded by all three cellulases. Cellulase II cannot degrade carboxymethyl-cellulose, and with filter paper as substrate the end product was cellobiose, which indicates that cellulase II is an exo-beta-1,4-glucan cellobiosylhydrolase. Degradation of cellulose (filter paper) can be catalysed independently by each of the three cellulases; there was no synergistic effect between any of the cellulases, and cellobiose was the principal product of degradation. The mode of action of one cellulase (cellulase III) was examined by using reduced cellulodextrins. The central linkages of the cellulodextrins were the preferred points of cleavage, which, with the rapid decrease in viscosity of carboxymethyl-cellulose, confirmed that cellulase III was an endocellulase. The rate of hydrolysis increased with chain length of the reduced cellulodextrins, and these kinetic data indicated that the specificity region of cellulase III was five or six glucose units in length.

Ascomycota↗
PubMed1981

Parasexual genetic analysis of Candida albicans by spheroplast fusion.

Doubly auxotrophic strains of Candida albicans were selected from mutagenized cultures. Spheroplasts prepared from the auxotrophic strains were fused with polyethylene glycol. Prototrophic derivatives formed by this fusion protocol from auxotrophic strains were selected by complementation on minimal medium. These prototrophs had a cell volume twice that of the original strain and were shown to be heterozygous at four loci. Prototrophs obtained by this procedure infrequently gave rise to auxotrophic recombinants whose cell volume remained twice that of the original strain. It is suggested that these auxotrophic recombinants arise from mitotic crossing-over. This paper is the first report of a parasexual cycle in C. albicans.

Candida albicans↗
PubMed1981

Intracellular mineralization of Bacterionema matruchotii.

Intracellular calcium phosphate mineral formation in Bacterionema matruchotii has been examined relative to two established points: the product of calcium and phosphorus levels at which spontaneous precipitation occurs (Ca x P), 50 (mg%)2, and the product at which calcium hydroxyapatite itself induces crystal growth, 35 (mg%) 2. The extent of intracellular mineralization in the defined calcifying medium was determined analytically after washing the cells. Electron micrographs of B. matruchotii cells revealed needlelike crystals of mineral, typical of calcium hydroxyapatite. Bacterionema matruchotii was found to be an efficient nucleator and can bring about the formation of solid phase at a Ca x P product as low as 35 (mg%)2.

Actinomycetaceae↗
PubMed1981

The electron transport chain of Bacterionema matruchotii.

The membrane fraction of Bacterionema matruchotii contains an electron transport chain with oxidizing activity for NADH and succinate. Respiration was inhibited by KCN, 2-heptyl-4-hydroxyquinoline-N-oxide, UV light irradiation and CO. UV light irradiation, analysis of membrane extracts, and reconstitution of respiration in UV light treated membranes suggested that respiration is mediated by a menaquinone derivative. The membranes contained cytochromes a, b, and c. Inhibition studies and the effect of KCN and CO on the cytochrome spectrum indicated the presence of an a+a3 cytochrome oxidase and cytochrome o. The membrane fraction from cells grown under O2-limiting conditions contained nitrate reductase activity. In B. matruchotii, electron transport is coupled to oxidative phosphorylation as judged by the effects of substrates and inhibitors on the intracellular ATP concentration.

Actinomycetaceae↗
PubMed1980

Purification and properties of the cellulases from the thermophilic fungus Thermoascus aurantiacus.

Three cellulases and a beta-glucosidase were purified from the culture filtrate of the thermophilic fungus Thermoascus aurantiacus. The isolated enzymes were all homogeneous on polyacrylamide-disc-gel electrophoresis. Data from chromatography on Bio-Gel P-60 and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated mol.wts. of 87000 (beta-glucosidase), 78000 (cellulase I), 49000 (cellulase II) and 34000 (cellulase III); the carbohydrate contents of the enzymes were 33.0, 5.5, 2.6 and 1.8% (w/w) respectively. Although the three purified cellulases were active towards filter paper, only cellulases I and III were active towards CM(carboxymethyl)-cellulose. Cellulase I was also active towards yeast glucan. The Km and catalytic-centre-activity values for the enzymes were as follows; 0.52 mumol/ml and 6.5 X 10(4) for beta-glucosidase on p-nitrophenyl beta-D-glucoside, 3.9 mg/ml and 6.3 for cellulase I on CM-cellulose, 1.2 mg/ml and 1.1 for cellulase I on yeast glucan, 35.5 mg/ml and 0.34 for cellulase II on filter paper, and 1.9 mg/ml and 33 for cellulase III on CM-cellulose.

Ascomycota↗
PubMed1980

Regulation of chitin synthesis during germ-tube formation in Candida albicans.

The synthesis of chitin during germ-tube formation in Candida albicans may be regulated by the first and last steps in the chitin pathway: namely L-glutamine-D-fructose-6-phosphate aminotransferase and chitin synthase. Induction of germ-tube formation with either glucose and glutamine or serum was accompanied by a 4-fold increase in the specific activity of the aminotransferase. Chitin synthase in C. albicans is synthesized as a proenzyme. N-acetyl glucosamine increased the enzymic activity of the activated enzyme 3-fold and the enzyme exhibited positive co-operativity with the substrate. UDP-N-acetylglucosamine. Although chitin synthase was inhibited by polyoxin D (Ki = 1.2 microM) this antibiotic did not affect germination. During germ-tube formation the total chitin synthase activity increased 1.4-fold and the expressed activity (in vivo activated proenxyme) increased 5-fold. These results could account for the reported 5-fold increase in chitin content observed during the yeast to mycelial transformation.

Candida albicans↗