Biomedical subjects

L W Parks

Publications and source records attributed to L W Parks.

At least 37 records · Page 2Linked to original sources

PubMed1993

The ERG3 gene in Saccharomyces cerevisiae is required for the utilization of respiratory substrates and in heme-deficient cells.

ERG3 is the structural gene in Saccharomyces cerevisiae for the sterol delta 5 desaturase that introduces the C5 = 6 unsaturation in ergosterol biosynthesis. The ERG3 gene has been mapped on chromosome XII, 13.7 centimorgans from GAL2 toward SPT8. The essentiality of the gene is dependent on the conditions used for the cultivation of the mutants. Insertionally inactivated mutants of ERG3 fail to grow without 'sparking' levels of delta 5 sterols in heme-deficient cells, and are unable to grow on the respiratory substrates glycerol and ethanol.

Cloning, Molecular↗

PubMed1992

Stimulation by heme of steryl ester synthase and aerobic sterol exclusion in the yeast Saccharomyces cerevisiae.

Saccharomyces cerevisiae sterol and heme auxotrophs were used to elucidate a role for hemes in sterol esterification. Steryl ester synthase (SES) activity was stimulated on average fourfold in cells supplemented with 50 micrograms/ml delta-aminolevulinic acid (ALA). This stimulation was not dependent on ALA per se, but on the ability of this precursor to effect heme competency. The addition of ALA stimulated SES activity of yeast on either fermentative or respiratory carbon sources. The elevation of SES activity was independent of intracellular free sterol, unsaturated fatty acid, or methionine levels. SES activity increases as the cells enter stationary phase, and this increase is enhanced by heme competency. SES was directly inhibited by the hypocholesterolemic drug lovastatin (mevinolin). The inhibition of SES activity by lovastatin was enhanced in heme-competent cells.

Acyltransferases↗

PubMed1992

A defect in the sterol:steryl ester interconversion in a mutant of the yeast, Saccharomyces cerevisiae.

A culture cycle dependent interconversion of sterols and steryl esters is disturbed in a mutant of Saccharomyces cerevisiae. Independent extragenic suppressors to this mutant return the mutant's pleiotropic phenotype to that of the parental wild type. Concomitant with the alterations in interconversion, modifications were found in the yeast proteins that antigenically react with antibodies elicited against mammalian apolipoproteins. Suppressor mutations returned the aberrant immunoblot banding pattern of the mutant to that of the wild type in apolipoprotein B.

Apolipoproteins↗

PubMed1992

Effect of sterol alterations on conjugation in Saccharomyces cerevisiae.

Sterol auxotrophic strains of Saccharomyces cerevisiae were grown and allowed to conjugate on media supplemented with various sterols. The mating efficiency of the auxotrophs is perturbed by the replacement of the normal yeast sterol, ergosterol, with other sterols. After 4 h of mating, cells grown on ergosterol exhibited a 30-fold higher productive mating efficiency than those cells grown in stigmasterol. Aberrant budding by the conjugants was enhanced following incubation on stigmasterol and other non-ergosterol sterols. Using light and electron microscopy, we demonstrated that there is a reduced ability for stigmasterol-grown cells to undergo cytoplasmic fusion during conjugation. Many of the mated pairs remained adherent but prezygotic even after 12 h of incubation. The addition of ergosterol to cells previously grown on stigmasterol rescued the organisms, allowing for zygote formation and normal budding.

Cell Division↗

PubMed1992

Cloning, sequencing, and disruption of the gene encoding sterol C-14 reductase in Saccharomyces cerevisiae.

A sterol C-14 reductase (erg24-1) mutant of Saccharomyces cerevisiae was selected in a fen1, fen2, suppressor background on the basis of nystatin resistance and ignosterol (ergosta-8,14-dienol) production. The erg24-1 allele segregated genetically as a single, recessive gene. The wild-type ERG24 gene was cloned by complementation onto a 12-kb fragment from a yeast genomic library, and subsequently subcloned onto a 2.4-kb fragment. This was sequenced and found to contain an open reading frame of 1,314 bp, predicting a polypeptide of 438 amino acids (M(r) 50,612). A 1,088-bp internal region of the ERG24 gene was excised, replaced with a LEU2 gene, and integrated into the chromosome of the parental strain, FP13D (fen1, fen2) by gene replacement. The ERG24 null mutant produced ergosta-8,14-dienol as the major sterol, indicating that the delta 8-7 isomerase, delta 5-desaturase and the delta 22-desaturase were inactive on sterols with the C14 = 15 double bond.

Amino Acid Sequence↗

PubMed1992

Regulation of partitioned sterol biosynthesis in Saccharomyces cerevisiae.

Using yeast strains with null mutations in structural genes which encode delta-aminolevulinic acid synthetase (HEM1), isozymes of 3-hydroxy-3-methylglutaryl coenzyme A (HMG1 and HMG2), squalene epoxidase (ERG1), and fatty acid delta 9-desaturase (OLE1), we were able to determine the effect of hemes, sterols, and unsaturated fatty acids on both sterol production and the specific activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) in Saccharomyces cerevisiae. We found that the HMGR isozymes direct essentially equal amounts of carbon to the biosynthesis of sterols under heme-competent conditions, despite a huge disparity (57-fold) in the specific activities of the reductases. Our results demonstrate that palmitoleic acid (16:1) acts as a rate-limiting positive regulator and that ergosterol acts as a potent inhibitor of sterol production in strains which possess only the HMGR1 isozyme (HMG1 hmg2). In strains which contain only the HMGR2 isozyme (hmg1 HMG2), sterol production was inhibited by oleic acid (18:1) and to a lesser degree by ergosterol. The specific activities of the two reductases (HMGR1 and HMGR2) were found to be differentially regulated by hemes but not by ergosterol, palmitoleic acid, or oleic acid. The disparate effects of unsaturated fatty acids and sterols on these strains lead us to consider the possibility of separate, compartmentalized isoprenoid pathways in S. cerevisiae.

5-Aminolevulinate Synthetase↗

PubMed1991

Effect of sterol side-chain structure on the feed-back control of sterol biosynthesis in yeast.

We measured the incorporation of radiolabeled methionine and acetate into the sterol component of G204, a Saccharomyces cerevisiae mutant strain which is partially heme competent. By comparing the amount of label incorporated into the sterol pool of a control culture, to which no exogenous sterol was added, with a culture which had various sterols added to the growth medium, we were able to determine the specific structural features of ergosterol which facilitate its ability to restrict the sterol biosynthetic pathway. These experiments demonstrate that sterols which contain both a C22 unsaturation and a C24 methyl group are capable of reducing sterol biosynthesis by approx. 50%, regardless of B-ring structure. We examined the regulatory properties of various oxysterols; 24,25-epoxylanosterol reduced endogenous biosynthesis by 49%, whereas all cholesterol derivatives tested, including 25-hydroxycholesterol, had little effect. A new procedure for the synthesis of ergosterol peroxides is also described.

Genes, Fungal↗

PubMed1991

Yeast proteins with reactivity to antibodies elicited against mammalian apolipoproteins.

Hyperimmunized rabbit anti-rat apolipoprotein antibodies showed reactivity to proteins in Saccharomyces cerevisiae. Antiapolipoproteins A1, B and E reacted with proteins in both a crude extract and a lipid enriched fraction. Protein reactivity was dependent on antisera dilution. Furthermore, the different antiapolipoproteins tested reacted with individually distinct yeast proteins, possibly suggesting the presence of a variety of yeast apolipoproteins with distinct cellular functions as is the case with mammalian apolipoproteins. The specificity of the antibody was directed toward the yeast protein and not a lipid moiety.

Animals↗

PubMed1991

Involvement of heme components in sterol metabolism of Saccharomyces cerevisiae.

There is an intimate association between sterol biosynthesis in yeast and aerobicity. Besides the requirement for molecular oxygen for the epoxidation of squalene, cytochrome hemoproteins are involved in demethylation and desaturation steps. Regulatory effects of hemes on sterol formation have been demonstrated using specifically defective mutants of yeast. Heme competency participates in a mechanism whereby wild-type cells are prevented from taking exogenous sterols from the growth media. The multiple interactions of hemes and sterols appear to be associated with the variously defined functions for sterols in the yeast cells.

Esterification↗

PubMed1991

Physiological effects of fenpropimorph on wild-type Saccharomyces cerevisiae and fenpropimorph-resistant mutants.

Fenpropimorph-resistant mutants of Saccharomyces cerevisiae were isolated by a gradient selection procedure. The mutants were cross-resistant to other morpholines (fenpropidin, dodemorph, tridemorph) and 15-azasterol, but were susceptible to azoles (miconazole, clotrimazole, ketoconazole) and nystatin. In the absence of fenpropimorph, the major sterol produced by the mutants and the parental strain was ergosterol. In the presence of fenpropimorph, ignosterol (ergosta-8,14-dien-3 beta-ol) was the major sterol produced by the mutants and the parental strain. The resistance to fenpropimorph involves two recessive genes, each of which allows a semiresistance, when they are isolated apart from one another. Strain JR4 (erg3 erg11), which produces 14-methylfecosterol [14 alpha-methyl-ergosta-8,24(28)-dien- 3-beta-ol) as the major sterol in the presence or absence of fenpropimorph, was also found to be resistant to the drug. The growth inhibitory effect of fenpropimorph on wild-type cells appears to be linked to the production of ignosterol. The uptake of exogenous sterol by wild-type cells was greatly enhanced in the presence of fenpropimorph. The growth inhibition caused by fenpropimorph could only be overcome with bulk levels of exogenous C-5,6-unsaturated sterols.

Cell Division↗

PubMed1990

Effects of lovastatin (mevinolin) on sterol levels and on activity of azoles in Saccharomyces cerevisiae.

The hypocholesterolemic drug lovastatin (mevinolin) was found to be very effective in lowering the sterol levels of the wild-type yeast Saccharomyces cerevisiae. Lovastatin dramatically decreased the steryl ester content from 2.62 to 0.8 micrograms/mg (dry weight), whereas the free sterol content decreased only from 2.79 to 2.24 micrograms/mg (dry weight) when lovastatin was present in the medium at 10 micrograms/ml. At higher concentrations (100 micrograms/ml), lovastatin nearly abolished the accumulation of steryl esters and decreased the free sterol concentration to less than 1.3 micrograms/mg (dry weight). As a result of the lowered sterol levels, proportional amounts of exogenous sterol were taken up from the medium during aerobic, respiratory conditions. Nearly all of the exogenous sterol taken up was partitioned into the free sterol fraction. The inhibition of sterol esterification in the presence of lovastatin was dependent on heme synthesis. The result of these combined effects caused the MICs of three azole antifungal drugs (ketoconazole, clotrimazole, and miconazole) to be lowered from 6- to 32-fold when lovastatin was present in the medium at 10 micrograms/ml.

Azoles↗

PubMed1990

Saccharomyces cerevisiae membrane sterol modifications in response to growth in the presence of ethanol.

Membranes isolated from yeasts grown in the presence of ethanol do not display the thermally induced transition in diphenylhexatriene anisotropy that is seen in control cells when they are exposed to ethanol in vitro. The total sterol content of the cells that were exposed to ethanol during growth is reduced, with no steryl esters being detected. A greater proportion of the total sterol pool is ergosterol in cells grown in the presence of alcohol. The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase is reduced by ethanol in vitro. Ethanol-exposed cells take up more exogenous sterol under aerobic conditions than do control cells. The presence of ethanol during growth reduces the activity of the plasma membrane enzyme, chitin synthase, as well as increasing the thermosensitivity of this enzyme.

Chitin Synthase↗

PubMed1989

Gas chromatographic analysis of intact steryl esters in wild type Saccharomyces cerevisiae and in an ester accumulating mutant.

The steryl ester faction from wild type and mutant strains of the yeast Saccharomyces cerevisiae was analyzed without saponification by a non-polar capillary gas chromatographic column. When expressed as microgram ester/mg dry wt, the total ester fraction remained constant or declined slightly from log to stationary phase in the wild type. In the mutant the decrease was more dramatic. No individual ergosteryl ester species was dominant throughout the culture cycle in the wild type. A compound tentatively identified as zymosteryl palmitate was the most prevalent ester in wild type log phase cells, ergosta-5,7-dienyl palmitate and ergosta-5,7-dienyl palmitoleate were the major esters in stationary cells. In the mutant strain, ergosteryl esters of palmitate, palmitoleate, oleate, and stearate were the major ester components throughout the culture cycle. Like the wild type, however, the mutant showed an increase in the proportion of ergosta-5,7-dienyl esters in the stationary phase of the culture cycle. The data did not indicate a sterol/fatty acid specificity during the culture cycle.

Chromatography, Gas↗

PubMed1989

A role for sterols in the porphyrin mediated photosensitization of yeast.

The yeast Saccharomyces cerevisiae was used as a model system to determine the role of sterols in the porphyrin mediated photosensitization of yeast. A sterol auxotroph, RD5-R, was grown on sterols with different levels of unsaturation and assayed for photosensitivity in the presence of either protoporphyrin IX or hematoporphyrin (both at 100 micrograms/ml). Cells grown on the completely saturated sterol (stanol), cholestanol, were substantially more resistant to the photosensizing effects of the porphyrin. We hypothesize that this resistance arises from the inability of the porphyrin to mediate the oxidation of the membrane sterol. Our results indicate that photodegradation of the native yeast sterol, ergosterol, can account for substantial losses of cell viability.

Oxidation-Reduction↗

PubMed1989

Antibodies to nystatin demonstrate polyene sterol specificity and allow immunolabeling of sterols in Saccharomyces cerevisiae.

Polyclonal antibodies elicited by injection into rabbits of a nystatin-bovine serum albumin conjugate were reactive with both nystatin and amphotericin B. Upon labeling of polyene-treated Saccharomyces cerevisiae sterol auxotrophs grown on various sterols, nystatin reacted specifically with ergosterol, while amphotericin B did not react preferentially with ergosterol, cholesterol, or cholestanol. Time course labeling experiments demonstrated the rate of ergosterol transport into cholesterol-grown cells.

Animals↗

PubMed1989

Structural discrimination in the sparking function of sterols in the yeast Saccharomyces cerevisiae.

A Saccharomyces cerevisiae sterol auxotroph, SPK14 (a hem1 erg6 erg7 ura), was constructed to test the ability of selected C-5,6 unsaturated sterols at growth-limiting concentrations to spark growth on bulk cholestanol. The native sterol, ergosterol, initiated growth faster and allowed a greater cell yield than did other sterols selectively altered in one or more features of the sterol. Although the C-5,6 unsaturation is required for the sparking function, the presence of the C-22 unsaturation was found to facilitate sparking far better than did the C-7 unsaturation, whereas the C-24 methyl was the least important group. The addition of delta-aminolevulinic acid to the medium allowed the sparking of FY3 (hem1 erg7 ura) on bulk cholestanol due to the derepression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and the production of endogenous ergosterol. The optimal concentration of delta-aminolevulinic acid to spark growth was 800 ng/ml, whereas higher concentrations caused a growth inhibition. The growth yield of FY3 reached a plateau maximum at about 5 micrograms/ml when the bulk cholestanol was varied in the presence of 10 ng of sparking erogosterol per ml.

Cholestanols↗

PubMed1989

Temporal expression of transcription and relative copy number of plasmid pSFB-1 in Scytalidium flavo-brunneum.

Wild-type cultures of Scytalidium flavo-brunneum produce a 15-azasterol antifungal agent and a reddish brown pigment as secondary metabolites. Spontaneous mutants of S. flavo-brunneum that had simultaneously lost the ability to produce the 15-azasterol and the pigment were transformed with plasmid pSFB-1, which was obtained from wild-type S. flavo-brunneum. Each transformant possessed the plasmid and coincidentally reacquired azasterol and pigment production. Regulation of transcription and relative plasmid copy number was determined as a function of the culture cycle of the organism. Twenty-five-fold amplification of the plasmid was observed in the fungus during the stationary phase. RNA transcripts of 0.9, 1.0, 1.5, 2.0, and 2.5 kilobases were expressed from the plasmid by the organism. While differences in the temporal regulation of the transcripts were seen, all except the 1.0-kilobase transcript increased in abundance on entry of the culture into the stationary phase of growth.

Blotting, Northern↗

PubMed1989

Effects of sterol alterations on nystatin sensitivity in Saccharomyces cerevisiae.

A systematic study of the qualitative and quantitative effects of sterol on nystatin sensitivity has been made in a single organism. The use of a sterol auxotroph of Saccharomyces cerevisiae offered a convenient way to control the sterol content of the yeast cell. There was a correlation between the ergosterol content of the cell and sensitivity to nystatin, as monitored by both potassium leakage from the cell and viability. When the sterol auxotroph contained high levels of ergosterol, the cells were sensitive to the effects of nystatin. When the ergosterol content was low or when ergosterol was replaced by cholesterol or cholestanol, sensitivity to nystatin was markedly decreased. Although resistant to nystatin, cholestanol enriched cells showed an enhanced background of potassium ion loss.

Cholesterol↗