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Biomedical subjects

L W Parks

Publications and source records attributed to L W Parks.

At least 73 records · Page 4Linked to original sources
PubMed1983

Relationship between antifungal activity and inhibition of sterol biosynthesis in miconazole, clotrimazole, and 15-azasterol.

The availability of Saccharomyces cerevisiae mutants which are defective in sterol biosynthesis makes it possible to determine whether the ability of several antifungal agents to inhibit cell growth is due to their effect on sterol production. 15-Aza-24-methylene-8,14-cholestadien-3 beta-ol (15-azasterol) is known to block the reduction of the sterol delta 14 bond following C-14 demethylation. This agent inhibits the growth of wild-type S. cerevisiae but does not inhibit the growth of a strain that is defective in the removal of the C-14 methyl group of lanosterol and in the introduction of the 5,6 double bond. 15-Azasterol does not inhibit the growth of a sterol auxotrophic strain growing on an exogenous supply of sterol. Therefore, the effect of 15-azasterol on sterol biosynthesis is clearly the cause of its ability to inhibit growth. On the other hand, growth inhibition by two imidazole antifungal agents, clotrimazole and miconazole, cannot be ascribed to their ability to prevent the removal of the C-14 methyl group of lanosterol, because they inhibit the growth of the sterol auxotrophic strain as well as that of the demethylase mutant.

Antifungal Agents↗
PubMed1983

Requirement for a second sterol biosynthetic mutation for viability of a sterol C-14 demethylation defect in Saccharomyces cerevisiae.

Genetic analysis of a nystatin-resistant sterol mutant (strain JR4) of Saccharomyces cerevisiae defective in C-14 demethylation revealed the presence of a second mutation in 5,6-desaturation. It appeared from complementation tests that a defect in delta 5-desaturase enzyme activity was required for the viability of the C-14 demethylation mutant. Growth studies with a sterol auxotrophic strain indicated that the major sterol of strain JR4, 14 alpha-methyl-ergosta-8,24(28)-dien-3 beta-ol, could satisfy "bulk" membrane requirements but not the second, structurally specific, sterol function that we defined previously (Rodriguez et al., Biochem. Biophys. Res. Commun. 106:435-441, 1982). Leakiness in the sterol mutations in strain JR4 provided a small amount of ergosterol which could satisfy this second function.

Cytochrome P-450 Enzyme System↗
PubMed1982

Lipid synthesis in inositol-starved Saccharomyces cerevisiae.

Lipid synthesis was analyzed in an inositol-requiring mutant of Saccharomyces cerevisiae (MC13). Both rates and cellular amounts of [U-14C]acetate incorporation into phospholipids, triacylglycerols, free sterols and steryl esters were elevated in an inositol-starved culture compared to the supplemented control at a time when the deprived culture was losing viability (inositol-less death). The rates at a later time were greatly reduced. During the period when de novo lipid synthesis was high in the starved culture, phospholipid turnover and presumed conversion to triacylglycerols was also accelerated; no differences were apparent in the turnover of the sterol fractions between the two cultures. No change in the fractional percent of ergosterol or of the sterol precursors could be attributed to inositol starvation. The synthesis and maintenance of membrane lipids (phospholipids and free sterols) and their coupling in cellular metabolism are discussed in light of these results.

Culture Media↗
PubMed1982

Enrichment for auxotrophic mutants in Saccharomyces cerevisiae using the cell wall inhibitor, echinocandin B.

Echinocandin B has been shown to inhibit fungal cell wall synthesis. This report describes the use of echinocandin B to enrich for nutritional auxotrophs in a mutagenized strain of Saccharomyces cerevisiae. Up to 20-fold enrichment levels for total auxotrophs were achieved after a single round of treatment with echinocandin; this level of enrichment is the highest of all procedures for which a specific mutant strain is not required.

Anti-Bacterial Agents↗
PubMed1982

Effect of altered sterol composition on the osmotic behavior of sphaeroplasts and mitochondria of Saccharomyces cerevisiae.

The effect of sterols on the osmotic stability of mitochondrial and plasma membranes of yeast wild-types and mutants that are defective in ergosterol biosynthesis has been studied. Incorporation of the nonfungal sterol, cholesterol, into yeast membranes reduces membrane elasticity which is observed as an increased susceptibility to osmotic lysis. However, the wild-type and nystatin-resistant strains which were examined indicate that qualitative alterations in endogenously generated sterols do not affect resistance to swelling. Although these strains exhibit differences in membrane fluidity, which is influenced by the sterol accumulated by the organisms, the membrane stretching capacity shows no distinct dependence on sterol structure or bilayer fluidity.

Mitochondria↗
PubMed1981

An assessment of the specificity of sterol uptake and esterification in Saccharomyces cerevisiae.

By growing a sterol-requiring strain of Saccharomyces cerevisiae in the presence of pairs of sterols differing by a single structural change, the in vivo specificity of sterol uptake and esterification was measured. Uptake specificity was demonstrated for the delta 5-, delta 7-, and delta 22- bonds as well as the 24 beta-methyl. Sterol uptake was shown to depend on the metabolic state of the cell, and the apparent Km of uptake for ergosterol (11.1 microM) was lower than that of cholesterol (66.7 microM). This difference in apparent Km can explain the preferential utilization of ergosterol. The selectivity for esterification showed that sterols lacking the delta 7- or delta 22- bond or the 24 beta-methyl were preferentially esterified. However, sterols lacking the delta 5-bond were not preferentially esterified. This specificity of uptake and esterification did not change significantly with alterations in the fatty acid source. These results suggest that both uptake and esterification are used to control the types of sterols in the free sterol fraction, resulting in the enrichment of ergosterol-like sterols in cellular membranes. An additional finding was that cells supplemented with sterols which have a delta 5,7-diene (7-dehydrocholesterol and ergosterol) had much reduced levels of steryl ester. This may be attributable to inhibition by a breakdown product(s) of these sterols.

Biological Transport↗
PubMed1981

Physiological response of Saccharomyces cerevisiae to 15-azasterol-mediated growth inhibition.

We studied 15-aza-24-methylene-8,14-cholestadiene-3 beta-ol (15-azasterol) inhibition of Saccharomyces cerevisiae growth. Exposure to sublethal concentrations of this drug caused S. cerevisiae cells to undergo a transient period of inhibition at midlog phase. During growth inhibition the turbidity of each culture remained constant, as did the total cell number. Although the proportion of viable cells in cultures decreased from 90 to 12% during inhibition, methylene blue staining showed that less than 40% of the cells underwent metabolic inactivation. We monitored adenosine triphosphate levels throughout the inhibition cycle, and these levels followed kinetics identical to cell growth kinetics. After overcoming inhibition, cellular lipid extracts revealed the presence of a modified form of 15-azasterol. It appeared that the yeast cells were able to overcome 15-azasterol inhibition by an inactivating transmethylation reaction involving S-adenosylmethionine.

Adenosine Triphosphate↗
PubMed1981

Inhibition of sterol transmethylation by S-adenosylhomocysteine analogs.

Structural analogs of S-adenosylhomocysteine were tested in vitro for inhibition of the yeast S-adenosylmethionine:delta 24-sterol-C-methyltransferase enzyme. A wide inhibitory range by these compounds was observed, suggesting which structural features of the parent compound are important for binding to the enzyme. No analog tested had inhibitory activity specific only for this enzyme. The most active compound was sinefungin, a metabolite of Streptomyces griseolus, which was also able to inhibit growth of yeast cultures. Sterol extracts of cells grown in the presence of sinefungin revealed a dramatic increase in the levels of zymosterol, the sterol substrate in the transmethylation under study, and a concomitant decrease in the levels of ergosterol. Evidence is presented that sinefungin is transported inside the cell by the same permease as S-adenosylmethionine. We conclude that sinefungin is blocking the in vivo methylation of sterols in yeast. The implications of this finding are discussed.

Adenosine↗
PubMed1981

Corresponding changes in kynurenine hydroxylase activity, membrane fluidity, and sterol composition in Saccharomyces cerevisiae mitochondria.

The effect of sterol composition on the properties of the mitochondrial membrane of Saccharomyces cerevisiae was investigated. The physical state of mitochondrial membranes from wild-type strains and sterol mutants was compared, using a fluorescence polarization technique with 1,6-diphenyl-1,3-5-hexatriene. Changes in the rate of depolarization of the probe molecule as a function of temperature suggest the occurrence of a phase transition in the mitochondrial membranes isolated from the sterol mutants but not in the membranes isolated from the wild types. Arrhenius kinetics of the mitochondrial membrane-bound enzyme L-kynurenine-3-hydroxylase exhibited changes in activation energy at temperatures similar to those observed in the fluorescence polarization study. The ratio of mitochondrial sterol to phospholipid and the phospholipid fatty acid composition of the organisms were characterized.

Enzyme Activation↗
PubMed1980

Growth and antifungal homoazasterol production in Geotrichum flavo-brunneum.

The growth cycle and production of 15-aza-24-methylene-8, 14-cholestadiene-3 beta-ol (15-azasterol) in Geotrichum flavo-brunneum strain NRRL28804 have been studied. During the growth cycle of this organism, morphological changes were noted which corresponded to changes in the pH of the culture medium. A physiological shift from acid to base production also occurred during the growth cycle. Concomitant with this physiological shift was the synthesis of 15-azasterol. Upon synthesis of this azasterol, variations in the sterol pool were observed. These variations are identical to sterol alterations in susceptible yeast cells exposed to this drug (P. R. Hays, W. D. Neal, and L. W. Parks, Antimicrob. Agents Chemother. 12: 185-191, 1977.) It appears that NRRL28804 avoids growth inhibition from 15-azasterol by confining its production to late in the growth cycle.

Antifungal Agents↗
PubMed1980

Some effects of douglas fir terpenes on certain microorganisms.

The Douglas fir terpene alpha-pinene was shown to inhibit the growth of a variety of bacteria and a yeast. Other terpenes of the Douglas fir, including limonene, camphene, and isobornyl acetate, were also inhibitory to Bacillus thuringiensis. All terpenes were inhibitory at concentrations normally present in the fir needle diet of Douglas fir tussock moth larvae. The presence of such terpenes in the diet of these insects was found to strongly influence the infectivity of B. thuringiensis spores for the Douglas fir tussock moth larvae. The terpene alpha-pinene destroyed the cellular integrity and modified mitochondrial activity in certain microorganisms.

Journal Article↗
PubMed1979

Triaglycerol metabolism in Saccharomyces cerevisiae. Relation to phospholipid synthesis.

The acylglycerol content of Saccharomyces cerevisiae has been examined during cellular growth. The cells maintained a constant amount of phospholipid and diacylglycerol throughout growth. Triacylglycerol content fell in the early exponential phase of growth and then increased sharply upon entry of the culture into the stationary growth phase. Pulse-chase experiments with [1-14C]oleic acid and [2-3H]- and [1-14C]glycerol indicated that the triacylglycerol molecule was utilized for phospholipid synthesis in early exponential phase probably through a diacylglycerol intermediate. A substantial turnover of phospholipid during growth was also apparent. No role for the triacylglycerol could be found in regulating the fatty acid species of the phospholipid nor in the storage of fatty acid for energy metabolism.

Diglycerides↗
PubMed1978

Delta14-sterol reductase in Saccharomyces cerevisiae.

An in vitro assay for delta14-sterol reductase from yeast was developed, using ergosta-8,14-dien-3beta-ol as the substrate. The kinetics and localization of the enzyme were examined. The inhibition of the enzyme by the antimycotic agent, 15-azasterol, was verified.

Cholestadienols↗
PubMed1978

Sterols in yeast subcellular fractions.

Yeast is the most primitive organism synthesizing substantial amounts of sterols. Because of this eucaryotic organism's versatility in growth conditions, ease of culture, well-defined genetic mechanism, and characteristic subcellar architecture, it is readily applied to studies of the role of sterols in the general economy of the cell. Sterols exist in two major forms, as the free sterol, or esterified with long chain fatty acids. The importance of sterols for this organism can be demonstrated using a naturally occurring antimycotic azasterol. This agent inhibits yeast growth. Three effects are seen on sterol synthesis: inhibition of the enzymes delta14-reductase, sterol methyltransferase, and methylene reductase. Cells cultured on respiratory substrates are more sensitive to inhibition than are cells growing on glucose. We have demonstrated a relationship between respiratory competency and sterol biosynthesis in this organism. Many mutants altered in sterol synthesis are respirationally defective and must grow fermentatively. One clone has temperature conditional respiration. Experiments with purified mitochondria, prepared from this mutant and its isogenic wildtype, show that the mutant organism is able to respire at the higher temperature but lacks the ability to couple respiration to phosphorylation. No similar loss is seen in the wild-type clones. Data are given which support the proposal that, for inclusion in mitochondrial structures, yeast cells may discriminate among sterols available from the total sterol pool in favor of ergosterol.

Azasteroids↗