Showing papers on "Sterol published in 1996"

Vaccines containing a saponin and a sterol

Abstract: The invention relates to a vaccine composition comprising an antigen, an immunologically active saponin fraction and a sterol.

Sterol Esterification in Yeast: A Two-Gene Process

Abstract: Unesterified sterol modulates the function of eukaryotic membranes. In human cells, sterol is esterified to a storage form by acyl-coenzyme A (CoA): cholesterol acyl transferase (ACAT). Here, two genes are identified, ARE1 and ARE2, that encode ACAT-related enzymes in yeast. The yeast enzymes are 49 percent identical to each other and exhibit 23 percent identity to human ACAT. Deletion of ARE2 reduced sterol ester levels to approximately 25 percent of normal levels, whereas disruption of ARE1 did not affect sterol ester biosynthesis. Deletion of both genes resulted in a viable cell with undetectable esterified sterol. Measurements of [14C]acetate incorporation into saponified lipids indicated down-regulation of sterol biosynthesis in the are1 are2 mutant cells. With the use of a consensus sequence to the yeast and human genes, an additional number of the ACAT gene family was identified in humans.

Sterol regulation of acetyl coenzyme A carboxylase: a mechanism for coordinate control of cellular lipid

Abstract: Transcription from the housekeeping promoter for the acetyl coenzyme A carboxylase (ACC) gene, which encodes the rate-controlling enzyme of fatty acid biosynthesis, is shown to be regulated by cellular sterol levels through novel binding sites for the sterol-sensitive sterol regulatory element binding protein (SREBP)-1 transcription factor. The position of the SREBP sites relative to those for the ubiquitous auxiliary transcription factor Sp1 is reminiscent of that previously described for the sterol-regulated low density lipoprotein receptor promoter. The experiments provide molecular evidence that the metabolism of fatty acids and cholesterol, two different classes of essential cellular lipids, are coordinately regulated by cellular lipid levels.

Cyclodextrin-mediated removal of sterols from monolayers: effects of sterol structure and phospholipids on desorption rate.

Abstract: In this study, we have examined a number of parameters which affect the rate of sterol desorption from a model membrane surface (a monolayer at the air/water interface) to cyclodextrins (CD) in the aqueous subphase. The desorption experiments were carried out at a constant lateral surface pressure with a zero-order trough, which allowed for a determination of desorption rates which were unaffected by monolayer substrate concentration. At a surface pressure of 20 mN/m (30 degrees C), 0.9 mM beta-CD caused a desorption of about 13 pmol of cholesterol per minute and square centimeter of monolayer area. The desorption of cholesterol proceeded linearly as a time function and was sensitive to the concentration of beta-CD in the subphase. The rate of cholesterol desorption increased as the monolayer surface pressure increased (3->35 mN/m) but decreased slightly with increasing temperature (15->30 degrees C). The rate of sterol desorption appeared to be influenced by the relative polarity of the sterols. Oxidized sterols desorbed significantly faster than cholesterol (e.g., 4-cholesten-3-one desorbed 8.4-fold faster than cholesterol), whereas less polar sterols desorbed at slower rates [e.g., 20(R)-isoheptyl-5-pregnen-3 beta-ol, a cholesterol analogue with a ten-carbon branched side chain, desorbed at 1/10 of the rate of cholesterol]. Cholesterol desorption from a monolayer membrane containing both cholesterol and a phospholipid was much slower than from a pure cholesterol monolayer. When the effect of dipalmitoylphosphatidylcholine and N-palmitoylsphingomyelin on cholesterol desorption rate was compared, it was found that cholesterol desorption was much more retarded from sphingomyelin monolayers as compared to that from phosphatidylcholine monolayers. Taken together, the results of this study show that the beta-CD-enhanced desorption of cholesterol (and other sterols) from monolayer membranes is influenced by the polarity of the desorbing molecules, as well as by lipid/lipid interactions in the membranes. Since beta-CD has no surface activity of its own, it appears to be a useful, nonintrusive catalyzer of cholesterol desorption and is expected to become a valuable probe in membrane and cell research.

beta-Sitosterol inhibits HT-29 human colon cancer cell growth and alters membrane lipids.

Abstract: The purpose of the present study was to examine the effect of beta-sitosterol, the main dietary phytosterol on the growth of HT-29 cells, a human colon cancer cell line. In addition, the incorporation of this phytosterol into cellular membranes and how this might influence the lipid composition of the membranes were investigated. Tumor cells were grown in DMEM containing 10% FBS and supplemented with sterols (cholesterol or beta-sitosterol) at final concentrations up to 16 microM. The sterols were supplied to the media in the form of sterol cyclodextrin complexes. The cyclodextrin used was 2-hydroxypropyl-beta-cyclodextrin. The sterol to cyclodextrin molar ratio was maintained at 1:300. The study indicated that 8 and 16 microM beta-sitosterol were effective at cel growth inhibition as compared to cholesterol or to the control (no sterol supplementation). After supplementation with 16 microM beta-sitosterol for 9 days, cell growth was only one-third that of cells supplemented with equimolar concentration of cholesterol. No effect was observed on total membrane phospholipid concentration. At 16 microM beta-sitosterol supplementation, membrane cholesterol was reduced by 26%. Cholesterol supplementation resulted in a significant increase in the cholesterol/phospholipid ratio compared to either beta-sitosterol supplemented cells or controls. There was a 50% reduction in membrane sphingomyelin (SM) of cells grown in 16 microM beta-sitosterol. Additional changes were observed in the fatty acid composition of minor phospholipids of beta-sitosterol supplemented cells, such as SM, phosphatidylserine (PS), and phosphatidylinositol (PI). Only in the case of PI, was there an effect of these fatty acid changes on the unsaturation index, beta-sitosterol incorporation resulted in an increase in the U.I. It is possible that the observed growth inhibition by beta-sitosterol may be mediated through the influence of signal transduction pathways that involve membrane phospholipids.

Cloning and characterization of ERG25, the Saccharomyces cerevisiae gene encoding C-4 sterol methyl oxidase

Abstract: We have cloned the Saccharomyces cerevisiae C-4 sterol methyl oxidase ERG25 gene. The sterol methyl oxidase performs the first of three enzymic steps required to remove the two C-4 methyl groups leading to cholesterol (animal), ergosterol (fungal), and stigmasterol (plant) biosynthesis. An ergosterol auxotroph, erg25, which fails to demethylate and concomitantly accumulates 4,4-dimethylzy-mosterol, was isolated after mutagenesis. A complementing clone consisting of a 1.35-kb Dra I fragment encoded a 309-amino acid polypeptide (calculated molecular mass, 36.48 kDa). The amino acid sequence shows a C-terminal endoplasmic reticulum retrieval signal KKXX and three histidine-rich clusters found in eukaryotic membrane desaturases and in a bacterial alkane hydroxylase and xylene monooxygenase. The sterol profile of an ERG25 disruptant was consistent with the erg25 allele obtained by mutagenesis.

Importance of a Novel Oxidative Mechanism for Elimination of Intracellular Cholesterol in Humans

Abstract: We have recently demonstrated that cultured human alveolar macrophages efficiently convert cholesterol into excretable 27-oxygenated products. We show here that increasing the intracellular concentration of cholesterol by a factor of 10 leads to about a twofold increase in the excretion of 27-oxygenated products from cultured macrophages. Inhibition of the sterol 27-hydroxylase caused a significant intracellular accumulation of cholesterol. A direct comparison was made between flux of cholesterol and 27-oxygenated products from macrophages preloaded with [4-14C]cholesterol. Under the specific conditions employed with fetal calf serum in the culture medium, the flux of 27-oxygenated products was about 10% of that of cholesterol. Since the sterol 27-hydroxylase, which converts cholesterol to 27-oxygenated products, is present in many cell types, we suggest that 27-oxygenation is a general mechanism for removal of intracellular cholesterol. To evaluate this hypothesis, we measured the net uptake by the human liver of circulating 27-oxygenated products, which was found to be about 20 mg/24 h. This uptake corresponds to approximately 4% of the bile acid production, assuming quantitative conversion into bile acids. It is concluded that the 27-hydroxylase pathway is of significance for elimination of extrahepatic cholesterol.

Origin of Cholesterol in Myelin

Abstract: We review some of the older literature concerning metabolic turnover of cholesterol in the nervous system. The overall picture is that incorporation of radioactive precursors into brain cholesterol is roughly proportional to the rate of myelination and that, once incorporated, radioactive cholesterol is relatively stable metabolically. We outline a strategy for demonstrating the source (local synthesis or uptake from circulation) of cholesterol in brain. The experimental design involves determining the rate of accumulation of cholesterol; this is calculated as the increasing amounts of sterol in brain at successive time intervals during development. The rate of appearance of newly synthesized cholesterol is determined from incorporation of radioactivity from 3H20 (injected i.p. several hours prior to sacrifice) into cholesterol. The radioactivity associated with the sterol fractions and the specific activity of body water determined from the serum can be used to calculate the absolute amount of sterol newly synthesized during the time when 3H20 was present. The results obtained demonstrated that all of the bulk cholesterol accumulating in brain can be accounted for by newly synthesized cholesterol. None of the radioactive cholesterol came from the circulation, since cholesterol feeding suppressed cholesterol biosynthesis in the liver and specific radioactivity of circulating cholesterol was negligible. Thus, almost all cholesterol accumulating in brain during development is locally synthesized.

Transcriptional regulation by ergosterol in the yeast saccharomyces cerevisiae

Abstract: Sterol biosynthesis in the yeast Saccharomyces cerevisiae is an energy-expensive, aerobic process, requiring heme and molecular oxygen. Heme, also synthesized exclusively during aerobic growth, not only acts as an enzymatic cofactor but also is directly and indirectly responsible for the transcriptional control of several yeast genes. Because of their biosynthetic similarities, we hypothesized that ergosterol, like heme, may have a regulatory function. Sterols are known to play a structural role in membrane integrity, but regulatory roles have not been characterized. To test possible regulatory roles of sterol, the promoter for the ERG3 gene, encoding the sterol C-5 desaturase, was fused to the bacterial lacZ reporter gene. This construct was placed in strains making aberrant sterols, and the effect of altered sterol composition on gene expression was monitored by beta-galactosidase activity. The absence of ergosterol resulted in a 35-fold increase in the expression of ERG3 as measured by beta-galactosidase activity. The level of ERG3 mRNA was increased as much as ninefold in erg mutant strains or wild-type strains inhibited in ergosterol biosynthesis by antifungal agents. The observed regulatory effects of ergosterol on ERG3 are specific for ergosterol, as several ergosterol derivatives failed to elicit the same controlling effect. These results demonstrate for the first time that ergosterol exerts a regulatory effect on gene transcription in S. cerevisiae.

Cholesterol-induced interfacial area condensations of galactosylceramides and sphingomyelins with identical acyl chains

Abstract: Interest in the in-plane interactions that occur between cholesterol and simple sphingolipids has been stimulated by recent discoveries in virology and cell biology. Semliki Forest virus requires cholesterol in target membranes for binding, but the fusion event is dependent upon simple sphingolipids such as sphingomyelin, ceramide, or galactosylceramide being present in target membranes (Nieva et al., 1994; Wilschut et al., 1995). Other envelope viruses, such as HIV,1 are enriched in sphingomyelin and possess high cholesterol:phospholipid ratios compared to host cell plasma membranes (Aloia et al., 1993). With regard to the sorting of integral membrane proteins in the Golgi apparatus, the cholesterol–sphingolipid concentration is hypothesized to play a critical role in formation of membrane microdomains that differ in thickness from other regions. During sorting, Golgi proteins, but not plasma membrane proteins, may be excluded from the thicker, cholesterol-rich microdomains due to dimensional and compositional differences in their transmembrane helices (Bretscher & Munro, 1993). The trafficking, sorting, and localization of proteins bearing covalently attached glycosylphosphatidylinositol (GPI) lipid anchors also has been linked to specialized membrane regions enriched in cholesterol, glycosylceramides, and sphingomyelin [ for recent review, see Parton and Simons (1995)]. Determining and verifying the molecular details of these interesting cell biological processes requires that the nature of the in-plane interactions between cholesterol and simple sphingolipids be clearly defined. An effective way to investigate the in-plane interactions between cholesterol and other lipids is by Langmuir film balance approaches [e.g. Phillips (1972)]. An important advantage of studying lipid–lipid interactions using the monolayer approach is that the range of molecular areas known to occur in membrane systems can be investigated systematically while avoiding the mesomorphic changes that often occur in bulk hydrated dispersions (e.g. bilayer vesicles) as lipid composition is varied. Also, the in-plane lipid–lipid interactions can be isolated from the influences of changing transbilayer compositional distributions that may occur in bilayer systems when cholesterol mole fractions are varied [e.g. Sankaram and Thompson (1991) and Harris et al. (1995)]. For these reasons, monolayer investigations of sphingolipid–cholesterol interactions have proven useful (Lund-Katz et al., 1988; Johnston & Chapman, 1988; Gronberg et al., 1991; Slotte et al., 1993; Ali et al., 1994; Smaby et al., 1994; Bittman et al., 1994). In these earlier studies, interactions between cholesterol and different sphingolipids were evaluated by directly measuring the average cross-sectional molecular areas at various mixing ratios and surface pressures. The extent to which the average molecular area in a mixed composition film decreased below that predicted by summing the known molecular areas of the pure lipids (apportioned by mole fraction) provided a measure of the area condensation induced by cholesterol. This classic monolayer approach originally was used to show the marked condensing effect that cholesterol has on fluid-phase phosphoglycerides [e.g. Phillips, (1972) and references therein]. As a result, the extent of lipid monolayer condensation induced by cholesterol sometimes has been used as an indicator of affinity or interaction strength that cholesterol has for various phosphoglycerides or sphingolipids [e.g. de Kruyff et al., (1973), Lund-Katz et al. (1988), Gronberg et al. (1991), and Slotte et al. (1993)]. Using the area condensation approach, Johnston and Chapman (1988) and Slotte et al. (1993) investigated the mixing behavior of cholesterol with galactosylceramides (GalCer) containing their naturally occurring, mixed complement of acyl chains. Slotte et al. (1993) concluded that GalCer does not interact with cholesterol because no area condensation was observed. Subsequently, we showed that significant molecular area condensation does occur when cholesterol is mixed with GalCer if the acyl composition imparts fluid-phase behavior to this sphingolipid prior to mixing with cholesterol (Ali et al., 1994). Even when the acyl structure of GalCer was modified so as to maximize area condensation upon mixing with cholesterol, larger effects were observed with egg or bovine brain sphingomyelin (SM) than with GalCer when these sphingolipids were in comparable phase states prior to mixing with cholesterol (Ali et al., 1994). Recently, we noted that the acyl chain heterogeneity of egg and bovine brain SM significantly broadened the surface pressure range of their phase transitions compared to those of their predominant molecular species (e.g. egg SM = 86% 16:0 SM; bovine brain SM = 63% 18:0 SM) (Smaby et al., 1996). An examination of earlier studies, in which the cholesterol-induced condensations of SMs and other lipids had been compared, revealed that phase transitional broadening was probably an unrecognized contributor that enhanced the area condensations of SM. Here, we have synthesized different GalCer and SM species containing identical acyl chains (e.g. oleate, nervonate, and linoleate) as well as the predominant acyl residues in egg SM (palmitate) and in bovine SM (stearate). We have investigated the mixing behavior of these sphingolipids and cholesterol with two aims in mind: (1) to assess the contribution of phase transitional broadening to the cholesterol-induced condensations of naturally occurring SMs and (2) to carefully compare not only the changes in SM and GalCer cross-sectional area induced by cholesterol but also the effect that the sterol has on the molecular interfacial-packing density of GalCer relative to that of SM.

The immunosuppressant SR 31747 blocks cell proliferation by inhibiting a steroid isomerase in Saccharomyces cerevisiae.

Abstract: SR 31747 is a novel immunosuppressant agent that arrests cell proliferation in the yeast Saccharomyces cerevisiae, SR 31747-treated cells accumulate the same aberrant sterols as those found in a mutant impaired in delta 8- delta 7-sterol isomerase. Sterol isomerase activity is also inhibited by SR 31747 in in vitro assays. Overexpression of the sterol isomerase-encoding gene, ERG2, confers enhanced SR resistance. Cells growing anaerobically on ergosterol-containing medium are not sensitive to SR. Disruption of the sterol isomerase-encoding gene is lethal in cells growing in the absence of exogenous ergosterol, except in SR-resistant mutants lacking either the SUR4 or the FEN1 gene product. The results suggest that sterol isomerase is the target of SR 31747 and that both the SUR4 and FEN1 gene products are required to mediate the proliferation arrest induced by ergosterol depletion.

Centripetal cholesterol flux from extrahepatic organs to the liver is independent of the concentration of high density lipoprotein-cholesterol in plasma

Abstract: High density lipoproteins (HDLs) play a role in two processes that include the amelioration of atheroma formation and the centripetal flow of cholesterol from the extrahepatic organs to the liver. This study tests the hypothesis that the flow of sterol from the peripheral organs to the liver is dependent upon circulating HDL concentrations. Transgenic C57BL/6 mice were used that expressed variable amounts of simian cholesteryl ester-transfer protein (CETP). The rate of centripetal cholesterol flux was quantitated as the sum of the rates of cholesterol synthesis and low density lipoprotein-cholesterol uptake in the extrahepatic tissues. Steady-state concentrations of cholesterol carried in HDL (HDL-C) varied from 59 to 15 mg/dl and those of apolipoprotein AI from 138 to 65 mg/dl between the control mice (CETPc) and those maximally expressing the transfer protein (CETP+). There was no difference in the size of the extrahepatic cholesterol pools in the CETPc and CETP+ animals. Similarly, the rates of cholesterol synthesis (83 and 80 mg/day per kg, respectively) and cholesterol carried in low density lipoprotein uptake (4 and 3 mg/day per kg, respectively) were virtually identical in the two groups. Thus, under circumstances where the steady-state concentration of HDL-C varied 4-fold, the centripetal flux of cholesterol from the peripheral organs to the liver was essentially constant at approximately 87 mg/day per kg. These studies demonstrate that neither the concentration of HDL-C or apolipoprotein AI nor the level of CETP activity dictates the magnitude of centripetal cholesterol flux from the extrahepatic organs to the liver, at least in the mouse.

Antiproliferative Effects of △24(25) Sterol Methyl Transferase Inhibitors on Trypanosoma (Schizotrypanum) cruzi: In vitro and in vivo Studies

Abstract: We have studied the antiproliferative effects of two sterol analogs previously reported as potent inhibitors of Δ24(25) sterol methyl transferase (E.C. 2.1.1.43) of yeasts and fungi on epim