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M Bard

Publications and source records attributed to M Bard.

At least 37 records · Page 2Linked to original sources
PubMed2000

Cloning and sequencing of the Candida albicans C-4 sterol methyl oxidase gene (ERG25) and expression of an ERG25 conditional lethal mutation in Saccharomyces cerevisiae.

The ERG25 gene encoding the Candida albicans C-4 sterol methyl oxidase was cloned and sequenced by complementing a Saccharomyces cerevisiae erg25 mutant with a C. albicans genomic library. The Erg25p is comprised of 308 amino acids and shows 65 and 38% homology to the enzymes from S. cerevisiae and Homo sapiens, respectively. The protein contains three histidine clusters common to nonheme iron-binding enzymes and an endoplasmic reticulum retrieval signal as do the proteins from S. cerevisiae and humans. A temperature-sensitive (ts) conditional lethal mutation of the C. albicans ERG25 was isolated and expressed in S. cerevisiae. Sequence analysis of the ts mutant indicated an amino acid substitution within the region of the protein encompassed by the histidine clusters involved in iron binding. Results indicate that plasmid-borne conditional lethal mutants of target genes have potential use in the rescue of Candida mutations in genes that are essential for viability.

Amino Acid Sequence↗
PubMed2000

Identification of a novel family of nonclassic yeast phosphatidylinositol transfer proteins whose function modulates phospholipase D activity and Sec14p-independent cell growth.

Yeast phosphatidylinositol transfer protein (Sec14p) is essential for Golgi function and cell viability. We now report a characterization of five yeast SFH (Sec Fourteen Homologue) proteins that share 24-65% primary sequence identity with Sec14p. We show that Sfh1p, which shares 64% primary sequence identity with Sec14p, is nonfunctional as a Sec14p in vivo or in vitro. Yet, SFH proteins sharing low primary sequence similarity with Sec14p (i.e., Sfh2p, Sfh3p, Sfh4p, and Sfh5p) represent novel phosphatidylinositol transfer proteins (PITPs) that exhibit phosphatidylinositol- but not phosphatidylcholine-transfer activity in vitro. Moreover, increased expression of Sfh2p, Sfh4p, or Sfh5p rescues sec14-associated growth and secretory defects in a phospholipase D (PLD)-sensitive manner. Several independent lines of evidence further demonstrate that SFH PITPs are collectively required for efficient activation of PLD in vegetative cells. These include a collective requirement for SFH proteins in Sec14p-independent cell growth and in optimal activation of PLD in Sec14p-deficient cells. Consistent with these findings, Sfh2p colocalizes with PLD in endosomal compartments. The data indicate that SFH gene products cooperate with "bypass-Sec14p" mutations and PLD in a complex interaction through which yeast can adapt to loss of the essential function of Sec14p. These findings expand the physiological repertoire of PITP function in yeast and provide the first in vivo demonstration of a role for specific PITPs in stimulating activation of PLD.

Base Sequence↗
PubMed1999

Characterization of the Saccharomyces cerevisiae ERG27 gene encoding the 3-keto reductase involved in C-4 sterol demethylation.

The last unidentified gene encoding an enzyme involved in ergosterol biosynthesis in Saccharomyces cerevisiae has been cloned. This gene, designated ERG27, encodes the 3-keto sterol reductase, which, in concert with the C-4 sterol methyloxidase (ERG25) and the C-3 sterol dehydrogenase (ERG26), catalyzes the sequential removal of the two methyl groups at the sterol C-4 position. We developed a strategy to isolate a mutant deficient in converting 3-keto to 3-hydroxy-sterols. An ergosterol auxotroph unable to synthesize sterol or grow without sterol supplementation was mutagenized. Colonies were then selected that were nystatin-resistant in the presence of 3-ketoergostadiene and cholesterol. A new ergosterol auxotroph unable to grow on 3-ketosterols without the addition of cholesterol was isolated. The gene (YLR100w) was identified by complementation. Segregants containing the YLR100w disruption failed to grow on various types of 3-keto sterol substrates. Surprisingly, when erg27 was grown on cholesterol- or ergosterol-supplemented media, the endogenous compounds that accumulated were noncyclic sterol intermediates (squalene, squalene epoxide, and squalene dioxide), and there was little or no accumulation of lanosterol or 3-ketosterols. Feeding experiments in which erg27 strains were supplemented with lanosterol (an upstream intermediate of the C-4 demethylation process) and cholesterol (an end-product sterol) demonstrated accumulation of four types of 3-keto sterols identified by GC/MS and chromatographic properties: 4-methyl-zymosterone, zymosterone, 4-methyl-fecosterone, and ergosta-7,24 (28)-dien-3-one. In addition, a fifth intermediate was isolated and identified by (1)H NMR as a 4-methyl-24, 25-epoxy-cholesta-7-en-3-one. Implications of these results are discussed.

Amino Acid Sequence↗
PubMed1999

Transcriptional regulation of the squalene synthase gene (ERG9) in the yeast Saccharomyces cerevisiae.

The ergosterol biosynthetic pathway is a specific branch of the mevalonate pathway. Since the cells requirement for sterols is greater than for isoprenoids, sterol biosynthesis must be regulated independently of isoprenoid biosynthesis. In this study we explored the transcriptional regulation of squalene synthase (ERG9) in Saccharomyces cerevisiae, the first enzyme dedicated to the synthesis of sterols. A mutant search was performed to identify genes that were involved in the regulation of the expression of an ERG9-lacZ promoter fusion. Mutants with phenotypes consistent with known sterol biosynthetic mutations (ERG3, ERG7, ERG24) increased expression of ERG9. In addition, treatment of wild-type cells with the sterol inhibitors zaragozic acid and ketoconazole, which target squalene synthase and the C-14 sterol demethylase respectively, also caused an increase in ERG9 expression. The data also demonstrate that heme mutants increased ERG9 expression while anaerobic conditions decreased expression. Additionally, the heme activator protein transcription factors HAP1 and HAP2/3/4, the yeast activator protein transcription factor yAP-1, and the phospholipid transcription factor complex INO2/4 regulate ERG9 expression. ERG9 expression is decreased in hap1, hap2/3/4, and yap-1 mutants while ino2/4 mutants showed an increase in ERG9 expression. This study demonstrates that ERG9 transcription is regulated by several diverse factors, consistent with the idea that as the first step dedicated to the synthesis of sterols, squalene synthase gene expression and ultimately sterol biosynthesis is highly regulated.

Base Sequence↗
PubMed1999

Effects of load and tone on the mechanics of isolated human bronchial smooth muscle.

Isotonic and isometric properties of nine human bronchial smooth muscles were studied under various loading and tone conditions. Freshly dissected bronchial strips were electrically stimulated successively at baseline, after precontraction with 10(-7) M methacholine (MCh), and after relaxation with 10(-5) M albuterol (Alb). Resting tension, i.e., preload determining optimal initial length (Lo) at baseline, was held constant. Compared with baseline, MCh decreased muscle length to 93 +/- 1% Lo (P < 0.001) before any electrical stimulation, whereas Alb increased it to 111 +/- 3% Lo (P < 0.01). MCh significantly decreased maximum unloaded shortening velocity (0.045 +/- 0.007 vs. 0.059 +/- 0.007 Lo/s), maximal extent of muscle shortening (8.4 +/- 1.2 vs. 13.9 +/- 2.4% Lo), and peak isometric tension (6.1 +/- 0.8 vs. 7.2 +/- 1.0 mN/mm2). Alb restored all these contractile indexes to baseline values. These findings suggest that MCh reversibly increased the number of active actomyosin cross bridges under resting conditions, limiting further muscle shortening and active tension development. After the electrically induced contraction, muscles showed a transient phase of decrease in tension below preload. This decrease in tension was unaffected by afterload levels but was significantly increased by MCh and reduced by Alb. These findings suggest that the cross bridges activated before, but not during, the electrically elicited contraction may modulate the phase of decrease in tension below preload, reflecting the active part of resting tension.

Albuterol↗
PubMed1998

Characterization of the Saccharomyces cerevisiae ERG26 gene encoding the C-3 sterol dehydrogenase (C-4 decarboxylase) involved in sterol biosynthesis.

All but two genes involved in the ergosterol biosynthetic pathway in Saccharomyces cerevisiae have been cloned, and their corresponding mutants have been described. The remaining genes encode the C-3 sterol dehydrogenase (C-4 decarboxylase) and the 3-keto sterol reductase and in concert with the C-4 sterol methyloxidase (ERG25) catalyze the sequential removal of the two methyl groups at the sterol C-4 position. The protein sequence of the Nocardia sp NAD(P)-dependent cholesterol dehydrogenase responsible for the conversion of cholesterol to its 3-keto derivative shows 30% similarity to a 329-aa Saccharomyces ORF, YGL001c, suggesting a possible role of YGL001c in sterol decarboxylation. The disruption of the YGL001c ORF was made in a diploid strain, and the segregants were plated onto sterol supplemented media under anaerobic growth conditions. Segregants containing the YGL001c disruption were not viable after transfer to fresh, sterol-supplemented media. However, one segregant was able to grow, and genetic analysis indicated that it contained a hem3 mutation. The YGL001c (ERG26) disruption also was viable in a hem 1Delta strain grown in the presence of ergosterol. Introduction of the erg26 mutation into an erg1 (squalene epoxidase) strain also was viable in ergosterol-supplemented media. We demonstrated that erg26 mutants grown on various sterol and heme-supplemented media accumulate nonesterified carboxylic acid sterols such as 4beta, 14alpha-dimethyl-4alpha-carboxy-cholesta-8,24-dien-3be ta-ol and 4beta-methyl-4alpha-carboxy-cholesta-8,24-dien-3beta-o l, the predicted substrates for the C-3 sterol dehydrogenase. Accumulation of these sterol molecules in a heme-competent erg26 strain results in an accumulation of toxic-oxygenated sterol intermediates that prevent growth, even in the presence of exogenously added sterol.

Amino Acid Sequence↗
PubMed1998

Biochemistry, cell biology and molecular biology of lipids of Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is a powerful experimental system to study biochemical, cell biological and molecular biological aspects of lipid synthesis. Most but not all genes encoding enzymes involved in fatty acid, phospholipid, sterol or sphingolipid biosynthesis of this unicellular eukaryote have been cloned, and many gene products have been functionally characterized. Less information is available about genes and gene products governing the transport of lipids between organelles and within membranes, turnover and degradation of complex lipids, regulation of lipid biosynthesis, and linkage of lipid metabolism to other cellular processes. Here we summarize current knowledge about lipid biosynthetic pathways in S. cerevisiae and describe the characteristic features of the gene products involved. We focus on recent discoveries in these fields and address questions on the regulation of lipid synthesis, subcellular localization of lipid biosynthetic steps, cross-talk between organelles during lipid synthesis and subcellular distribution of lipids. Finally, we discuss distinct functions of certain key lipids and their possible roles in cellular processes.

Biological Transport↗
PubMed1998

Sequencing, disruption, and characterization of the Candida albicans sterol methyltransferase (ERG6) gene: drug susceptibility studies in erg6 mutants.

The rise in the frequency of fungal infections and the increased resistance noted to the widely employed azole antifungals make the development of new antifungals imperative for human health. The sterol biosynthetic pathway has been exploited for the development of several antifungal agents (allylamines, morpholines, azoles), but additional potential sites for antifungal agent development are yet to be fully investigated. The sterol methyltransferase gene (ERG6) catalyzes a biosynthetic step not found in humans and has been shown to result in several compromised phenotypes, most notably markedly increased permeability, when disrupted in Saccharomyces cerevisiae. The Candida albicans ERG6 gene was isolated by complementation of a S. cerevisiae erg6 mutant by using a C. albicans genomic library. Sequencing of the Candida ERG6 gene revealed high homology with the Saccharomyces version of ERG6. The first copy of the Candida ERG6 gene was disrupted by transforming with the URA3 blaster system, and the second copy was disrupted by both URA3 blaster transformation and mitotic recombination. The resulting erg6 strains were shown to be hypersusceptible to a number of sterol synthesis and metabolic inhibitors, including terbinafine, tridemorph, fenpropiomorph, fluphenazine, cycloheximide, cerulenin, and brefeldin A. No increase in susceptibility to azoles was noted. Inhibitors of the ERG6 gene product would make the cell increasingly susceptible to antifungal agents as well as to new agents which normally would be excluded and would allow for clinical treatment at lower dosages. In addition, the availability of ERG6 would allow for its use as a screen for new antifungals targeted specifically to the sterol methyltransferase.

Amino Acid Sequence↗
PubMed1998

Effects of initial length on intrinsic tone in guinea pig tracheal smooth muscle.

In the guinea pig, tracheal smooth muscle (TSM) exhibits intrinsic tone (IT). The active nature of IT suggests that it could be influenced by muscle length and load. In the guinea pig, IT is entirely suppressed by the cyclooxygenase inhibitor indomethacin. IT could be measured as the difference between resting tone before and after indomethacin addition. We examined, in electrically stimulated TSM strips (n = 9), the influence of initial muscle length (Li) on IT, the relationship between IT and the maximum extent of relaxation (DeltaF1), and the influence of indomethacin on active isometric force. When Li decreased from 100 to 75% of optimal Li, there was a significant decrease in IT (from 12.0 +/- 0.2 to 5.3 +/- 0.1 mN; P < 0.001). Over the range of Li studied, DeltaF1 underestimated the amount of IT, but there was a close linear relationship between DeltaF1 and IT (r = 0.9). Compared with the basal state, indomethacin increased active isometric force (from 9.5 +/- 1.0 to 19.7 +/- 2.0 mN at optimal Li; P < 0.001) and induced its length dependency. In guinea pig TSM, Li was an important determinant of IT.

Animals↗
PubMed1998

Hyperfiltration in African-American patients with type 2 diabetes. Cross-sectional and longitudinal data.

OBJECTIVE: Hyperfiltration may play a role in the development of diabetic nephropathy. African-American patients with diabetes have more than a fourfold increase in end-stage renal disease. The purpose of this study is to evaluate the impact of hyperfiltration on renal function in African-American patients with type 2 diabetes. RESEARCH DESIGN AND METHODS: Renal function of 194 African-American patients with diagnosed type 2 diabetes from 1 month to 36 years was assessed by studies of isotopic glomerular filtration rate (GFR), serum creatinine, creatinine clearance, and 24-h urinary albumin excretion rates. Thirty-four patients with a duration of diagnosed type 2 diabetes from 1 month to 10 years were found to have hyperfiltration (GFR > or = 140 ml.min-1.1.73 m-2). Fifteen of these patients received longitudinal follow-up of renal function for as long as 15 years after the initial study. RESULTS: Hyperfiltration is present in 15 (36%) of 42 patients whose duration of diagnosed type 2 diabetes is < 1 year, and it persists for up to 10 years in 14-20% of patients with diagnosed type 2 diabetes. Patients with hyperfiltration are younger than their counterparts without hyperfiltration when matched for duration of diagnosed diabetes. When followed over time, those patients with hyperfiltration were not more likely to develop impaired renal function as measured by GFR or creatinine clearance. CONCLUSIONS: Hyperfiltration does not identify patients at risk for deterioration in renal function.

Adult↗
PubMed1998

Accelerated obstructive pulmonary disease in HIV infected patients with bronchiectasis.

Human immunodeficiency virus (HIV) infection has been associated with a wide spectrum of pulmonary disease. We report three HIV-seropositive patients with rapidly worsening airway obstruction associated with bronchiectasis. All subjects (age range 33-39 yrs) were cigarette smokers. Two had previously used intravenous drugs. The CD4 lymphocyte count ranged 40-250 cells x mm(-3). All individuals had complained of increasing dyspnoea for 3-6 months. Within 1 yr, they all developed severe airway obstruction with a decrease in both forced expiratory volume in one second (FEV1) and ratio of FEV1 to forced vital capacity (FEV1/FVC) to less than 60% of predicted value, and a decrease in mean forced expiratory flow at 25-75% of the forced vital capacity (FEF25-75) to less than 35% of predicted value. Computed tomography of the chest disclosed bilateral dilated and thickened bronchi. No classical causes of genetic or acquired bronchiectasis were identified in our patients. Recurrent bacterial bronchitis occurred in the follow-up period of the three patients. In conclusion, unusually rapid airway obstruction associated with bronchiectasis should be added to the wide spectrum of respiratory complications of human immunodeficiency virus infection.

Adult↗
PubMed1997

A yeast sterol auxotroph (erg25) is rescued by addition of azole antifungals and reduced levels of heme.

Genetic disruption of the Saccharomyces cerevisiae C-4 sterol methyl oxidase ERG25 gene leads to sterol auxotrophy. We have characterized a suppression system that requires two mutations to restore viability to this disrupted strain. One suppressor mutation is erg11, which is blocked in 14alpha-demethylation of lanosterol and is itself an auxotroph. The second suppressor mutation required is either slu1 or slu2 (suppressor of lanosterol utilization). These mutations are leaky versions of HEM2 and HEM4, respectively; addition of exogenous hemin reverses the suppressing effects of slu1 and slu2. Suppression of erg25 by erg11 slu1 (or erg11 slu2) results in a slow-growing strain in which lanosterol, the first sterol in the pathway, accumulates. This result indicates that endogenously synthesized lanosterol can substitute for ergosterol and support growth. In the triple mutants, all but 1 (ERG6) of the 13 subsequent reactions of the ergosterol pathway are inactive. Azole antibiotics (clotrimazole, ketoconazole, and itraconazole) widely used to combat fungal infections are known to do so by inhibiting the ERG11 gene product, the 14alpha-demethylase. In this investigation, we demonstrate that treatment of the sterol auxotrophs erg25 slu1 or erg25 slu2 with azole antibiotics paradoxically restores viability to these strains in the absence of sterol supplementation via the suppression system we have described.

Antifungal Agents↗
PubMed1997

A guide to finding and evaluating best practices health care information on the Internet: the truth is out there?

BACKGROUND: The advent of virtually free Internet access has opened large vistas of health care information to those willing to invest a small amount of time and energy learning how to perform searches using browser software. Health care providers, organizations, and professional associations, among many others, publish "best practices" information for both administrative and clinical audiences, making these recommendations among the fastest-growing types of health care information appearing on the World Wide Web. The problem is how to find best practices among the wealth of resources on the Internet and then how to separate the proverbial wheat from the chaff. WHO IS SEEKING BEST PRACTICES ON THE INTERNET? Best practice describes a process or technique whose employment results in improved patient and/or organizational outcomes. Health care providers, managed care organizations, administrators, payers, and policy analysts are all interested in improving the quality of health care and are likely to be customers of best practices informational resources. HOW TO EVALUATE THE QUALITY OF BEST PRACTICES INFORMATION? Once the information is available on the Internet, the problem for the searcher shifts from one of quantity to quality. The best practices information seeker should stop and ask a number of questions about the quality of information, its sources, and the methods used to obtain it. CONCLUSION: The "truth" may be out there some-where in cyberspace, but locating best practices information and evaluating its quality require new skills and patience and time to practice and develop them to the point of efficiency.

Authorship↗