Resistance of yeasts to polyene antibiotics

The strains of Saccharomyces cerevisiae yeast with mutations in two genes NYS3 NYS4 were obtained by tetrad analysis. Sterol fraction of these mutants contains two sterols: ergosta-7-en-3 beta-ol (fungisterol) and ergosta-7,24-dien-3 beta-ol (episterol). The findings allowed to testify the sequence of the ergosterol biosynthesis reactions. Dehydrogenization of the sterol nucleus in C5(6) which is controlled by gene NYS3 occurs simultaneously with the introduction of double bond in C22(23) site of the side chain regulated by gene NYS4.     

Genetico-ecological aspects of the use of nystatin-resistant yeast mutants in the yeast-Drosophila system

Nystatin-resistant strains of Saccharomyces cerevisiae with mutations in final steps of ergosterol biosynthesis have been studied in the ecologo-genetic yeast--drosophila system. It has been shown that yeast strains which belong to the Petersghoff genetic yeast stock collection, with mutations in NYSX, NYS2 and NYS3 genes, provide the development of Drosophila melanogaster. In the process of nutrition with yeasts having mutations in the NYS2 gene, the development of drosophila larvae takes place, due to ergosterol accumulated in the yeast cells. Drosophila melanogaster was shown to be unable to utilize the sterols with 8(9) and 24(25) double bonds.     

特比萘芬与其他抗真菌药物对外阴阴道念珠菌病病原菌的体外相互作用研究

目的 了解特比萘芬( TEB)分别与氟康唑(FLU)、伊曲康唑(ITR)、咪康唑(MCZ)、克霉唑(CMZ)及制霉菌素(NYS)联合对外阴阴道念珠菌病患者分离的念珠菌体外相互作用.方法 受试念珠菌株46株,包括白念珠菌(20株)、热带念珠菌(15株)、光滑念珠菌(4株)、克柔念珠菌(3株)、近平滑念珠菌(3株)和季也蒙念珠菌(1株).方法参考CLSIM27-A3方案中的微量液基稀释法,观察各药单独和联合后的MIC,并计算FICI.结果 TEB、FLU、ITR、MCZ、CMZ和NYS 的单药MIC范围分别为0.5～256μg/mL、0.5～ 128 μg/mL、0.5 ～ 32.0 μg/mL,0.125 ～ 16μg/mL、0.03 ～ 4.0 μg/mL和2～8μg/mL.当TEB与FLU、ITR、MCZ和CMZ联合时,对白念株菌表现为协同作用的分别为100％ (20/20),80％ (16/20),55％ (11/20)和90％ (18/20);对1株季也蒙念珠菌均显示为协同作用;对热带念珠菌,TEB分别与上述4种唑类药物联合时,显示有协同作用的分别为67％ (10/15),60％ (9/15),20％(3/15)和20％ (3/15);TEB与唑类药物联合对光滑念珠菌、克柔念珠菌和近平滑念珠菌均无协同作用;TEB与NYS联合时对所有菌株均无协同作用.结论 TEB与唑类药物联合对不同种的念珠菌显示有不同的效应,但对外阴阴道念珠菌病主要的致病菌白念珠菌显示有良好的协同作用.TEB与NYS联合对念株菌均无协同作用.     

Cloning and functional expression of UGT genes encoding sterol glucosyltransferases from Saccharomyces cerevisiae, Candida albicans, Pichia pastoris, and Dictyostelium discoideum

Sterol glucosides, typical membrane-bound lipids of many eukaryotes, are biosynthesized by a UDP-glucose:sterol glucosyltransferase (EC 2. 4.1.173). We cloned genes from three different yeasts and from Dictyostelium discoideum, the deduced amino acid sequences of which all showed similarities with plant sterol glucosyltransferases (Ugt80A1, Ugt80A2). These genes from Saccharomyces cerevisiae (UGT51 = YLR189C), Pichia pastoris (UGT51B1), Candida albicans (UGT51C1), and Dictyostelium discoideum (ugt52) were expressed in Escherichia coli. In vitro enzyme assays with cell-free extracts of the transgenic E. coli strains showed that the genes encode UDP-glucose:sterol glucosyltransferases which can use different sterols such as cholesterol, sitosterol, and ergosterol as sugar acceptors. An S. cerevisiae null mutant of UGT51 had lost its ability to synthesize sterol glucoside but exhibited normal growth under various culture conditions. Expression of either UGT51 or UGT51B1 in this null mutant under the control of a galactose-induced promoter restored sterol glucoside synthesis in vitro. Lipid extracts of these cells contained a novel glycolipid. This lipid was purified and identified as ergosterol-beta-D-glucopyranoside by nuclear magnetic resonance spectroscopy. These data prove that the cloned genes encode sterol-beta-D-glucosyltransferases and that sterol glucoside synthesis is an inherent feature of eukaryotic microorganisms.     

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.     

Sterol level in Saccharomyces cerevisiae mutants with altered ergosterol biosynthesis

The sterol content in Saccharomyces cerevisiae mutants defective in the synthesis of cyclic ergosterol precursors has been studied. It was found that strains with mutational blocks involving the stages of zymosterol side chain methylation at C24 and delta 8----delta 7 isomerization accumulated twice more sterols as compared to parent strains. Regulation of the ergosterol biosynthesis is discussed.     

Sterol methylation in Saccharomyces cerevisiae.

Various nystatin-resistant mutants defective in S-adenosylmethionine: delta 24-sterol-C-methyltransferase (EC 2.1.1.41) were shown to possess alleles of the same gene, erg6. The genetic map location of erg6 was shown to be close to trp1 on chromosome 4. Despite the single locus for erg6, S-adenosylmethionine: delta 24-sterol-C-methyltransferase enzyme activity was found in three separate fractions: mitochondria, microsomes, and the "floating lipid layer." The amount of activity in each fraction could be manipulated by assay conditions. The lipids and lipid synthesis of mutants of Saccharomyces cerevisiae defective in the delta 24-sterol-C-methyltransferase were compared with a C5(6) desaturase mutant and parental wild types. No ergosterol (C28 sterol) could be detected in whole-cell sterol extracts of the erg6 mutants, the limits of detection being less than 10(-11) mol of ergosterol per 10(8) cells. The distribution of accumulated sterols by these mutants varied with growth phase and between free and esterified fractions. The steryl ester concentrations of the mutants were eight times higher than those of the wild type from exponential growth samples. However, the concentration of the ester accumulated by the mutants was not as great in stationary-phase cells. Whereas the head group phospholipid composition was the same between parental and mutant strains, strain-dependent changes in fatty acids were observed, most notably a 40% increase in the oleic acid content of phosphatidylethanolamine of one erg6 mutant, JR5.     

Yeast resistance to polyene antibiotics. VII. The interaction of the mutant alleles of the nystatin resistance genes in Saccharomyces cerevisiae

The data on allele interactions of nystatin resistance genes are presented. It has been shown that the mutant phenotype of heteroallelic hybrids NYS1, NYS4 and, probably, NYS3 is strengthened. The intragenic complementation has been found in NYS2 gene, allowing to imply the multimeric structure of delta 8----delta 7 isomerase which is controlled by this gene.     

A complex plasma plant sterol locus on mouse chromosome 14 has at least two genes regulating intestinal sterol absorption

We previously identified two inbred mouse strains, C57BL/6J and CASA/Rk, with different plasma plant sterol levels. An intercross between these strains revealed a broad plasma plant sterol locus on chromosome 14, which peaked at 17 centimorgan (cM) with a maximum logarithm of the odds score of 9.9. Studies in a chromosome 14 congenic strain, 14KK, with a 4-60 cM CASA/Rk interval on the C57BL/6J background revealed that males, but not females, had decreased plasma plant sterol levels and intestinal cholesterol absorption. In two subcongenic strains, 14PKK and 14DKK, with 4-19.5 and 19.5-60 cM CASA/Rk intervals, respectively, both males and females had decreased plasma plant sterol levels and decreased intestinal cholesterol absorption. Compatible with the decreased plasma plant sterol phenotype, 14PKK mice had increased biliary plant sterol excretion, whereas 14DKK mice did not. Therefore, gender-dependent interactions of genes at the 14PKK and 14DKK intervals are likely to underlie the 14KK interval effect on plasma plant sterol levels and sterol absorption from the intestine. These studies confirm the plasma plant sterol locus on mouse chromosome 14 and provide evidence that there are at least two sets of genes operating: one set affecting intestinal sterol absorption and biliary excretion, and the other set mainly affecting intestinal sterol absorption.     

妇科住院患者酵母样真菌感染的菌株分布与药敏研究

目的对妇科住院患者阴道分泌物酵母样真菌隐性感染的菌群分布与药敏结果进行回顾性分析,为临床围手术期合理使用抗真菌药物提供依据。方法妇科住院患者5813例阴道分泌物中分离到396株酵母样真菌,采用API-20Aux和ATBFUNGUS进行鉴定和药敏试验。结果396株酵母样真菌中,分离率最高的前3位是白色假丝酵母菌（59.6%）、光滑假丝酵母菌（21.2%）和接合假丝酵母菌（6.6%）。酵母样真菌对5-氟胞嘧啶（5-FC）、两性霉素B（AMB）和制霉菌素（NYS）敏感率最高,达90.7%、92.7%和96.2%,咪康唑（MIC）、益康唑（ECO）和酮康唑（KET）分别为74.2%、61.6%和69.4%。结论住院妇科患者阴道分泌物酵母样真菌隐性感染主要以白色念珠菌和光滑念珠菌为主,感染的酵母样真菌对5-FC、AMB和NYS的药物敏感性较高,对唑类抗真菌药则有不同程度的耐药率。     

Characterization of nystatin-resistant mutants of Saccharomyces cerevisiae and preparation of sterol intermediates using the mutants

Analysis of sterols of Saccharomyces cerevisiae mutants N3, N15, N26, and N3H, defective in sterol biosynthesis, was performed. Strains N3, N15, and N26 were isolated from their mother strain, M10, by screening with nystatin (Nagai et al. (1980) Mie Med. J. 30, 215-224), and strain N3H was isolated from N3 as a doubly-mutated strain. The main sterols of N3, N15, N26, and N3H were ergosta-7,22-dienol, ergost-8-enol, cholesta-5,7,24-trienol, and ergosta-7,22,24(28)-trienol, respectively. The former three strains were characterized as defective in delta 5-desaturation, delta 8--delta 7 isomerization, and C-24 transmethylation. Strain N3H was found to be defective in delta 5-desaturation as well as in delta 24(28)-reduction. However, the defect of N26 and N3H was suggested to be leaky, since small amounts of ergosterol and ergosta-7,22-dienol were found in these mutants, respectively. In N15, an accumulation (2% in total sterols) of the compound likely to be hydroxylated sterol was found. By aerobic adaptation of these strains, the accumulation of these strains, the accumulations of ergosta-7,22-dienol (22 mg/g dry cells), ergosta-7,22,24(28)-trienol (24 mg), ergosta-8,24(28)-dienol (18 mg), and cholesta-8,24-dienol (22 mg) reached a maximum in N3, N3H, N15, and N26 after 20, 20, 30, and 30 h, respectively. These strains appear to be useful for making 14C-labeled and non-labeled preparations of the above sterols.     

The Arabidopsis dwf7/ste1 mutant is defective in the delta7 sterol C-5 desaturation step leading to brassinosteroid biosynthesis.

Lesions in brassinosteroid (BR) biosynthetic genes result in characteristic dwarf phenotypes in plants. Understanding the regulation of BR biosynthesis demands continued isolation and characterization of mutants corresponding to the genes involved in BR biosynthesis. Here, we present analysis of a novel BR biosynthetic locus, dwarf7 (dwf7). Feeding studies with BR biosynthetic intermediates and analysis of endogenous levels of BR and sterol biosynthetic intermediates indicate that the defective step in dwf7-1 resides before the production of 24-methylenecholesterol in the sterol biosynthetic pathway. Furthermore, results from feeding studies with 13C-labeled mevalonic acid and compactin show that the defective step is specifically the Delta7 sterol C-5 desaturation, suggesting that dwf7 is an allele of the previously cloned STEROL1 (STE1) gene. Sequencing of the STE1 locus in two dwf7 mutants revealed premature stop codons in the first (dwf7-2) and the third (dwf7-1) exons. Thus, the reduction of BRs in dwf7 is due to a shortage of substrate sterols and is the direct cause of the dwarf phenotype in dwf7.     

Control of rRNA synthesis in Escherichia coli at increased rrn gene dosage. Role of guanosine tetraphosphate and ribosome feedback.

The effects of extra, plasmid-borne rRNA genes on the synthesis rate of rRNA in Escherichia coli were examined by measuring the fraction of total RNA synthesis that is rRNA and tRNA (rs/rt), the cytoplasmic concentration of guanosine tetraphosphate (ppGpp), and the absolute rates of RNA and protein synthesis. Experiments were carried out in different growth media and with two different strains of E. coli, B/r and K-12. The results indicated: 1) increased rrn gene dosage from either intact or defective rrn genes reduced bacterial growth rates and ribosome activity (protein synthesis rate/average ribosome), and increased rs/rt. 2) Extra intact, but not extra defective, plasmid-borne rrn genes caused the level of ppGpp to be increased in comparison to the pBR322-carrying control strain. 3) As a function of ppGpp, rs/rt was increased with either intact or defective rrn genes. 4) The rRNA synthesis rate/rrn gene was reduced in the presence of extra rrn genes; this reduction in gene activity was greater with intact than with defective rrn genes. An analysis of these results showed that they are consistent with the ppGpp hypothesis of rRNA control but not with a feedback effector role of translating ribosomes.     

The effect of oxygen deficiency on the stability of the yeast Saccharomyces cerevisiae to the polyenic antibiotic nystatin

Oxygen deficiency was shown to affect the resistance of different Saccharomyces cerevisiae strains to nystatin, a polyenic antibiotic. This resistance decreased upon oxygen deficiency in the wild-type strains alpha'1 and 7A-P192 as well as in the mutant strains NYS 2, NYS 3 and NYS 4, but remained unchanged in the mutants NYS-1 and NYS 5. The qualitative composition of sterols was studied in the strains with a modified ergosterol synthesis, which were grown under the conditions of oxygen deficiency. Such conditions exerted a considerable effect on the accumulation of intermediate products in ergosterol biosynthesis.     

The genetic basis of fluconazole resistance development in Candida albicans

Infections by the opportunistic fungal pathogen Candida albicans are widely treated with the antifungal agent fluconazole that inhibits the biosynthesis of ergosterol, the major sterol in the fungal plasma membrane. The emergence of fluconazole-resistant C. albicans strains is a significant problem after long-term treatment of recurrent oropharyngeal candidiasis (OPC) in acquired immunodeficiency syndrome (AIDS) patients. Resistance can be caused by alterations in sterol biosynthesis, by mutations in the drug target enzyme, sterol 14alpha-demethylase (14DM), which lower its affinity for fluconazole, by increased expression of the ERG11 gene encoding 14DM, or by overexpression of genes coding for membrane transport proteins of the ABC transporter (CDR1/CDR2) or the major facilitator (MDR1) superfamilies. Different mechanisms are frequently combined to result in a stepwise development of fluconazole resistance over time. The MDR1 gene is not or barely transcribed during growth in vitro in fluconazole-susceptible C. albicans strains, but overexpressed in many fluconazole-resistant clinical isolates, resulting in reduced intracellular fluconazole accumulation. The activation of the gene in resistant isolates is caused by mutations in as yet unknown trans-regulatory factors, and the resulting constitutive high level of MDR1 expression causes resistance to other toxic compounds in addition to fluconazole. Disruption of both alleles of the MDR1 gene in resistant C. albicans isolates abolishes their resistance to these drugs, providing genetic evidence that MDR1 mediates multidrug resistance in C. albicans.     

The pathophysiology and mechanisms of NP-C disease

The molecular isolation of NPC1 and NPC2, the genes defective in patients with Niemann-Pick disease type C (NP-C), has heralded in an exponential increase in our understanding of this syndrome and thus of human intracellular sterol transport. Despite this, neither the mechanisms of action nor the substrates for these putative transporters have been defined. In this overview, we describe our perspectives on the current awareness of the genetic determination and cellular biology of this syndrome, with emphasis on the underlying events that lead to neurodegeneration and the manner in which they might eventually be treated.     

Autophagy genes mediate the effect of calcineurin on life span in C. elegans

Calcineurin (CaN) is a serine/threonine phosphatase, activated by Ca²⁺/calmodulin (Ca²⁺/CaM). CaN is known to regulate various cellular responses in different organisms. A recent study showed an extended life span in the calcineurin mutants of C. elegans. In this study, we report that calcineurin defective strains exhibit enhanced autophagy. In addition, we found two essential autophagy genes (bec-1 and atg-7) are required for the life-span extension in calcineurin null mutants [cnb-1(jh103)]. Thus, for the first time we suggest that autophagy genes are required for the life-span regulation in calcineurin defective C. elegans strains.     

Aerobic isolation of an ERG24 null mutant of Saccharomyces cerevisiae.

The ERG24 gene, encoding the C-14 sterol reductase, has been reported to be essential to the aerobic growth of Saccharomyces cerevisiae. We report here, however, that strains with null mutations in the ERG24 gene can grow on defined synthetic media in aerobic conditions. These sterol mutants produce ignosterol (ergosta-8,14-dienol) as the principal sterol, with no traces of ergosterol. In addition, we mapped the ERG24 gene to chromosome XIV between the MET2 and SEC2 genes. Our results indicate that ignosterol can be a suitable sterol for aerobic growth of S. cerevisiae on synthetic media and that inactivation of ERG24 is only conditionally lethal.     

Overlapping functions of the yeast oxysterol-binding protein homologues

The Saccharomyces cerevisiae genome encodes seven homologues of the mammalian oxysterol-binding protein (OSBP), a protein implicated in lipid trafficking and sterol homeostasis. To determine the functions of the yeast OSBP gene family (OSH1-OSH7), we used a combination of genetics, genomics, and sterol lipid analysis to characterize OSH deletion mutants. All 127 combinations and permutations of OSH deletion alleles were constructed. Individual OSH genes were not essential for yeast viability, but the elimination of the entire gene family was lethal. Thus, the family members shared an essential function. In addition, the in vivo depletion of all Osh proteins disrupted sterol homeostasis. Like mutants that affect ergosterol production, the viable combinations of OSH deletion alleles exhibited specific sterol-related defects. Although none of the single OSH deletion mutants was defective for growth, gene expression profiles revealed that each mutant had a characteristic molecular phenotype. Therefore, each gene performed distinct nonessential functions and contributed to a common essential function. Our findings indicated that OSH genes performed a multitude of nonessential roles defined by specific subsets of the genes and that most shared at least one essential role potentially linked to changes in sterol lipid levels.