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At least 19 recordsLinked to original sources

PubMed2005

Cellular sterol ester synthesis in plants is performed by an enzyme (phospholipid:sterol acyltransferase) different from the yeast and mammalian acyl-CoA:sterol acyltransferases.

A gene encoding a sterol ester-synthesizing enzyme was identified in Arabidopsis. The cDNA of the Arabidopsis gene At1g04010 (AtPSAT) was overexpressed in Arabidopsis behind the cauliflower mosaic virus 35S promoter. Microsomal membranes from the leaves of overexpresser lines catalyzed the transacylation of acyl groups from phosphatidylethanolamine to sterols. This activity correlated with the expression level of the AtPSAT gene, thus demonstrating that this gene encodes a phospholipid:sterol acyltransferase (PSAT). Properties of the AtPSAT were examined in microsomal fractions from the tissues of an overexpresser. The enzyme did not utilize neutral lipids, had the highest activity with phosphatidylethanolamine, had a 5-fold preference for the sn-2 position, and utilized both saturated and unsaturated fatty acids. Various sterols and sterol intermediates, including triterpenic precursors, were acylated by the PSAT, whereas other triterpenes were not. Sterol selectivity studies showed that the enzyme is activated by end product sterols and that sterol intermediates are preferentially acylated by the activated enzyme. This indicates that PSAT both regulates the pool of free sterols as well as limits the amount of free sterol intermediates in the membranes. Two T-DNA insertion mutants in the AtPSAT gene, with strongly reduced (but still measurable) levels of sterol esters in their tissues, had no detectable PSAT activity in the microsomal fractions, suggesting that Arabidopsis possess other enzyme(s) capable of acylating sterols. The AtPSAT is the only intracellular enzyme found so far that catalyzes an acyl-CoA-independent sterol ester formation. Thus, PSAT has a similar physiological function in plant cells as the unrelated acyl-CoA:sterol acyltransferase has in animal cells.

Acyltransferases↗

PubMed2001

Dietary plant sterols alter the serum plant sterol concentration but not the cholesterol precursor sterol concentrations in young children (the STRIP Study). Special Turku Coronary Risk Factor Intervention Project.

Plant sterol supplementation reduces serum cholesterol concentration but may increase serum plant sterol concentrations, especially in children. We determined whether natural dietary plant sterols derived mainly from vegetable oil or margarine in early childhood affect serum concentrations of plant sterols (campesterol and sitosterol) and cholesterol precursor sterols (Delta-8 cholestenol, desmosterol, and lathosterol), reflecting endogenous cholesterol synthesis. We measured the serum sterol concentrations using gas liquid chromatography in 20 healthy 13-mo-old intervention children in a randomized, prospective study designed to decrease exposure of the children to known environmental atherosclerosis risk factors and in 20 control children. The diet of the intervention children was rich in plant sterols due to replacement of milk fat with vegetable fat, whereas the diet of the control children contained only small amounts of plant sterols. The intervention children consumed twice as much plant sterols as the control children (P < 0.001). Their serum concentrations of campesterol and sitosterol were 75% and 44% higher, respectively, than those in the control children (P < 0.001 for both), but serum cholesterol precursor sterol concentrations did not differ between the two groups. We conclude that doubling dietary plant sterol intake almost doubles serum plant sterol concentrations in 13-mo-old children, but has no effect on endogenous cholesterol synthesis. Relative intestinal absorption of natural plant sterols from the diet in early childhood is similar to that in adults.

Arteriosclerosis↗

PubMed1988

Inhibitors of sterol synthesis. Chemical syntheses, properties and effects of 4,4-dimethyl-15-oxygenated sterols on sterol synthesis and on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in cultured mammalian cells.

The chemical syntheses of a number of 4,4-dimethyl substituted 15-oxygenated sterols have been pursued to permit evaluation of their activity in the inhibition of the biosynthesis of cholesterol and other biological effects. Described herein are the first chemical syntheses of 4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-en-3 beta-ol-15-one, 3 beta,15 alpha-diacetoxy-4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-ene, 3 beta-acetoxy-4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-en-15 beta-ol, 4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta,15 alpha-diol, 4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta,15 beta-diol, 4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-en-15 alpha-ol-3-one, 3 beta-benzoyloxy-4,4-dimethyl-5 alpha-cholest-8(14)-ene-7 alpha,15 alpha-diol, 7 alpha,15 alpha-diacetoxy-3 beta-benzoyloxy-4,4-dimethyl-5 alpha-cholest-8(14)-ene, 4,4-dimethyl-5 alpha-cholest-8(14)-en-3 beta-ol-15-one and 3 beta,7 alpha,15 alpha-tri-o-bromobenzoyloxy-5 alpha-cholest-8(14)-ene. Also prepared for use in the biological experiments were 4,4-dimethyl-5 alpha-cholest-7-ene-3 beta,15 alpha-diol, 4,4-dimethyl-5 alpha-cholest-8-ene-3 beta,15 alpha-diol and 4,4-dimethyl-5 alpha-cholest-8(14)-ene-3 beta,7 alpha,15 alpha-triol. The effects of twelve 4,4-dimethyl substituted 15-oxygenated sterols and of four 4,4-dimethyl substituted 32-oxygenated sterols on sterol synthesis and on the level of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity were evaluated in mouse L cells. With the exception of 4,4-dimethyl-5 alpha-cholest-8(14)-ene-3 beta,7 alpha,15 alpha-triol, all of the 4,4-dimethyl substituted 15-oxygenated sterols caused a 50% inhibition of sterol synthesis at less than 10(-6) M and six of the 4,4-dimethyl substituted 15-oxygenated sterols caused a 50% inhibition of sterol synthesis at less than 10(-7) M. 4,4-Dimethyl-14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta,15 alpha-diol caused a 50% decrease in sterol synthesis at 10(-8) M. The potencies of the 4,4-dimethyl substituted 15-oxygenated and C-32-oxygenated sterols with respect to inhibition of sterol synthesis and suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity have been compared with those of the corresponding sterols lacking the 4,4-dimethyl substitution.

Animals↗

PubMed2003

Sterol effects and sites of sterol accumulation in Caenorhabditis elegans: developmental requirement for 4alpha-methyl sterols.

Caenorhabditis elegans requires sterol, usually supplied as cholesterol, but this is enzymatically modified, and different sterols can substitute. Sterol deprivation decreased brood size and adult growth in the first generation, and completely, reversibly, arrested growth as larvae in the second. After one generation of sterol deprivation, 10 ng/ml cholesterol allowed delayed laying of a few eggs, but full growth required 300 ng/ml. C. elegans synthesizes two unusual 4alpha-methyl sterols (4MSs), but each 4MS supported only limited growth as the sole sterol. However, addition of only 10 ng of cholesterol to 1,000 ng of 4MS restored full growth and egg-laying, suggesting that both a 4MS and an unmethylated sterol are required for development. Filipin stained sterols in only a few specific cells: the excretory gland cell, two amphid socket cells, two phasmid socket cells and, in males, spicule socket cells. Sterols were also present in the pharynx and in the intestine of feeding animals in a proximal-to-distal gradient. This non-random sterol distribution, the low concentration requirements, and the effects of 4MSs argues that sterols are unlikely to be used for bulk structural modification of cell membranes, but may be required as hormone precursors and/or developmental effectors.

Animals↗

PubMed1988

Inhibition of sterol synthesis by delta 5-sterols in a sterol auxotroph of yeast defective in oxidosqualene cyclase and cytochrome P-450.

Synthesis of ergosterol is demonstrated in the GL7 mutant of Saccharomyces cerevisiae. This sterol auxotroph has been thought to lack the ability to synthesize sterols due both to the absence of 2,3-oxidosqualene cyclase and to a heme deficiency eliminating cytochrome P-450 which is required in demethylation at C-14. However, when the medium sterol was 5 alpha-cholestan-3 beta-ol, 5 alpha-cholest-8(14)-en-3 beta-ol, or 24 beta-methyl-5 alpha-cholest-8(14)-en-3 beta-ol, sterol synthesis was found to proceed yielding 1-3 fg/cell of ergosterol (24 beta-methylcholesta-5,7,22E-trien-3 beta-ol). Ergosterol was identified by mass spectroscopy, gas and high performance liquid chromatography, ultraviolet spectroscopy, and radioactive labeling from [3H]acetate. Except for some cholest-5-en-3 beta-ol (cholesterol) which was derived from the 5 alpha-cholestan-3 beta-ol, the stanol and the two 8(14)-stenols were not significantly metabolized confirming the absence of an isomerase for migration of the double bond from C-8(14) to C-7. Drastic reduction of ergosterol synthesis to not more than 0.06 fg/cell was observed when the medium sterol either had a double bond at C-5, as in the case of cholesterol, or could be metabolized to a sterol with such a bond. Thus, both 5 alpha-cholest-8(9)-en-3 beta-ol and 5 alpha-cholest-7-en-3 beta-ol (lathosterol) were converted to cholesta-5,7-dien-3 beta-ol (7-dehydrocholesterol), and the presence of the latter dienol depressed the level of ergosterol. The most attractive of the possible explanations for our observations is the assumption of two genetic compartments for synthesis of sterols, one of which has and one of which has not been affected by the two mutations. The ability, despite the mutations, to synthesize small amounts of ergosterol which could act to regulate the cell cycle may also explain why this mutant can grow aerobically with cholesterol (acting in the bulk membrane role) as the sole exogenous sterol.

Cholesterol↗

PubMedDate not supplied

Determination of plant sterol oxidation products in plant sterol enriched spreads, fat blends, and plant sterol concentrates.

Plant sterols (PS) are very stable molecules but may undergo oxidation due to the presence of a double bond in the ring structure. In order to assess whether this occurs during heating and storage, an analytical procedure was developed for the determination of concentration levels and identity of PS oxidation products in functional food ingredients and products. The method is based on cold saponification, solvent extraction of unsaponifiables, isolation of sterol oxidation products by means of liquid chromatography, and final analysis by gas chromatography (GC) with flame ionization detection. Identification of the key PS oxidation products was performed by means of GC-mass spectrometry (GC-MS). Isotope dilution MS was used to verify the absence of the formation of potential artifacts by the method. The method described is applicable to spreads (containing 20-65% water), oils, sterol esters, pure sterols, and fat extracts from food. The between-day reproducibility of the total content of sterol oxidation products in control samples sample was 8%, and of individual sterol oxidation products, 6-15%. The recovery of sterol oxidation products was 91%. The limit of detection was 0.1 mg/kg.

Gas Chromatography-Mass Spectrometry↗

PubMed1978

Variations in the total sterols, free sterols and sterol esters concentration during embryogenesis in Dysdercus similis (Freeman).

In the present study fluctuations in the total sterols, free sterols and sterols esters concentration has been recorded day to day during embryogenesis. Oscillations in the total sterol correlated with the biosynthesis and/or utilization of cholesterol occur in the eggs while free sterols are used in the maintenance of cell membranes. The role of sterol ester is not known but their increase may be correlated with the accretion of oleic and linoleic fatty acids.

Animals↗

PubMed1995

Sterol absorption and sterol balance in phytosterolemia evaluated by deuterium-labeled sterols: effect of sitostanol treatment.

Absorption of dietary cholesterol, campesterol, and sitosterol, cholesterol balance, and fecal excretion of plant sterols were determined in three unrelated patients with phytosterolemia and three healthy volunteers during constant intake of cholesterol and plant sterols using accurate gas-liquid chromatography-mass spectrometry techniques. Each subject received a mixture of [26,26,26,27,27,27-2H6]cholesterol, [6,7,7-2H3]sitostanol, and [6,7,7-2H3]campesterol together with two non-absorbable markers, [5,6,22,23-2H4]sitostanol and chromic oxide. Feces were collected from days 5 to 7 and absorption of different sterols was calculated from the intestinal disappearance of the different sterols relative to [5,6,22,23-2H4]sitostanol and chromic oxide. The results obtained by the two markers were not different and the absorption of cholesterol averaged 53 +/- 4% for the patients (mean +/- SD) and 43 +/- 3% for the volunteers. Campesterol absorption averaged 24 +/- 4% in patients and 16 +/- 3% in healthy volunteers, whereas sitosterol absorption averaged 16 +/- 1% and 5 +/- 1%, respectively. Cholesterol synthesis expressed by body weight varied considerably in the two groups but appeared to be about 5 times lower in patients than in controls. Administration of a high dose of sitostanol (0.5 g t.i.d.) to two patients was followed by a reduction in cholesterol absorption by 24% and 44%, an increase in fecal output of cholesterol and steroids derived from cholesterol and plant steroids, and a marked reduction of serum cholesterol, campesterol, and sitosterol. Under the conditions used, inhibition of cholesterol absorption by sitostanol was not followed by a significant rise in cholesterol synthesis. The time of observation was, however, too short to allow final conclusion on this. The results show that the absolute difference in absorption rate of different sterols between the patients and healthy volunteers was about the same. As a consequence, increasing hydrophobicity causes a relative decrease of absorption rates. Thus, patients with phytosterolemia seem to have a generally increased absorption of sterols rather than a loss of a specific discriminatory mechanism, and oral administration of sitostanol seems to be an interesting new approach for treatment of phytosterolemia.

Absorption↗

PubMed2001

Differential effects of sterol regulatory binding proteins 1 and 2 on sterol 12 alpha-hydroxylase. SREBP-2 suppresses the sterol 12 alpha-hydroxylase promoter.

The most important pathway for the catabolism and excretion of cholesterol in mammals is the formation of bile acids. Improper regulation of this pathway has implications for atherosclerosis, cholesterol gallstone formation, and some lipid storage diseases. Sterol 12 alpha-hydroxylase (12 alpha-hydroxylase) is required for cholic acid biosynthesis. The alpha(1)-fetoprotein transcription factor FTF is crucial for the expression and the bile acid-mediated down-regulation of 12 alpha-hydroxylase. Cholesterol, on the other hand, down-regulates expression of the 12 alpha-hydroxylase gene. In this study, we show that the two sterol regulatory binding proteins (SREBPs) have opposite effects on the 12 alpha-hydroxylase promoter. SREBP-1 activated the 12 alpha-hydroxylase promoter, as it does with many other cholesterol-regulated genes. In contrast, SREBP-2 suppressed 12 alpha-hydroxylase promoter activity. SREBP-1 mediates the cholesterol-down-regulation of 12 alpha-hydroxylase promoter by binding to two inverted sterol regulatory elements found approximately 300 nucleotides from the transcriptional initiation site. SREBP-2 mediated suppression of 12 alpha-hydroxylase without binding to its promoter. Data are presented suggesting that SREBP-2 suppresses the 12 alpha-hydroxylase promoter by interacting with FTF. This is the first report of a promoter responding oppositely to two members of the SREBP family of transcription factors. These studies provide a novel function and mode of action of a SREBP protein.

Animals↗

PubMed1975

Sterol balance studies in the rat. Effects of dietary cholesterol and beta-sitosterol on sterol balance and rate-limiting enzymes of sterol metabolism.

Sterol balance measurements using isotopic and chromatographic techniques were carried out in rats fed diets containing beta-sitosterol (0.8%) and cholesterol (1.2%). The activities of the rate-limiting enzymes of cholesterol synthesis (beta-hydroxy-beta-methylglutaryl-CoA reductase, EC 1.1.1.34) and bile acid synthesis (cholesterol 7 alpha-hydroxylase) were determined in the same animals. Cholesterol feeding increased cholesterol absorption from 1.2 to 70 mg/day. The increased absorption was compensated for by inhibition of hepatic cholesterol synthesis, enhanced conversion of cholesterol to bile acids (from 13.7 to 27.3 mg/day) and a slight increase in the excretion of endogenous neutral steroids (from 7.7 to 11.2 mg/day). Despite the adaptation there was accumulation of cholesterol in the liver (from 2.2 to 9.2 mg/g). Beta-Sitosterol feeding inhibited cholesterol absorption (calculated absorption was zero). In these rats there was enhanced cholesterol synthesis (from 20.0 to 28.8 mg/day, but no change in the rates of bile acid formation. Measurements of the activities of the rate-limiting enzymes showed fair correlation with cholesterol-bile acid balance. In cholesterol fed animals, beta-hydroxy-beta-methylglutaryl-CoA reductase was inhibited 80% and cholesterol 7 alpha-hydroxylase was enhanced 61%. In beta-sitosterol-fed animals, the reductase was increased 2-fold and cholesterol 7 alpha-hydroxylase was not significantly different from controls.

Animals↗

PubMed2005

Effect of high plant sterol-enriched diet and cholesterol absorption inhibitor, SCH 58235, on plant sterol absorption and plasma concentrations in hypercholesterolemic wild-type Kyoto rats.

BACKGROUND AND AIMS: Plant sterols are widely distributed in human diet but are poorly absorbed so that their plasma levels are very low. However, when fed in large amounts, they lower plasma cholesterol levels by interfering with cholesterol absorption. We have studied the effect of 4 weeks of feeding a chow diet supplemented with 1% plant sterols [brassicasterol (6.3%), campesterol (28.5%), stigmasterol (15.6%) and sitosterol (49.6%)], with or without SCH 58235 (a derivative of ezetimibe), 30 mg/kg per day, known to suppress intestinal cholesterol absorption, on plasma, tissue, biliary, and fecal sterols in Wistar and wild-type Kyoto (WKY) rats, and their metabolism by intestinal bacteria. METHODS: After 2 weeks of feeding control or experimental diet, rats were given [3alpha-(3)H]sitosterol intravenously and [4-(14)C]sitosterol by mouth, and blood was collected after 1, 2, 3, and 5 days after labeling to determine sitosterol absorption. Feces were collected during the last 3 days and freeze dried. At the end of feeding, bile fistulas were created in 3 rats of each strain and bile was collected for 1 hour. All rats were then sacrificed and plasma and liver were collected for sterol measurements and activities of hepatic HMG-CoA reductase, cholesterol 7alpha-hydroxylase, and cholesterol 27-hydroxylase. RESULTS: Wild-type Kyoto rats were hypercholesterolemic compared to Wistar rats and had increased plant sterols in the plasma. Plasma cholesterol tended to be lower in WKY rats after feeding with plant sterol-enriched diet whereas plant sterol levels rose to approximately 31% of plasma sterols in WKY and 14% in Wistar rats. However, brassicasterol and stigmasterol, with a double bond at C-22, constituted less than 3.5% of total plasma plant sterols. After feeding, biliary plant sterols increased 2.25-fold in Wistar and 1.5-fold in WKY rats, suggesting less hepatic clearance in WKY rats. SCH 58235 feeding significantly increased plasma as well as biliary cholesterol levels in both the untreated and plant sterol-fed WKY rats, and the plasma plant sterols showed a tendency to increase but did not reach significant level. Intestinal bacteria in both rat strains metabolized all plant sterols to mainly the 5beta-H-stanols. However, the C-22 double bond was stable to bacterial degradation. Intestinal absorption of sitosterol and cholesterol was increased 1.5- and 1.3-fold, respectively, in the WKY rats as compared to the Wistar rats, and plant sterol feeding lowered absorption of these sterols in both strains. Absorption of both these sterols was also lowered in SCH 58235-treated rats in both strains and was further lowered when SCH 58235 and plant sterols were simultaneously fed. The activity of the rate-limiting enzyme, HMG-CoA reductase, was increased 1.57-fold in Wistar rats and 1.27-fold in WKY rats that were fed plant sterols as compared to untreated rats. CONCLUSIONS: (1) Plant sterol absorption was increased whereas hepatic elimination of all sterols was diminished in WKY rats accounting for elevated cholesterol and plant sterol levels. (2) The 1% plant sterol-enriched diet tended to lower plasma cholesterol levels whereas SCH 58235 feeding significantly increased plasma cholesterol levels in the WKY rats. (3) Intestinal absorption of sterols with C-22 double bond is diminished and the side-chain double bond is resistant to intestinal bacteria.

Animals↗

PubMed1994

Effect of sterol side-chain structure on sterol-phosphatidylcholine interactions in monolayers and small unilamellar vesicles.

In this study we have characterized the monolayer behavior of analogues of cholesterol having different side-chain structures and their interaction with phosphatidylcholines in mixed monolayers and small unilamellar vesicles (SUVs). Two series of side-chain analogues of cholesterol were synthesized, one with an unbranched side chain (the n-series, from 3 to 7 carbons in length), and the other with a single methyl-branched side chain (the iso-series, from 5 to 10 carbons in length). The length and conformation of the sterol side chain markedly influenced both the mean molecular area of the pure sterols and their monolayer stability (i.e., collapse pressure). Shorter side chains gave smaller mean molecular areas and decreased monolayer stability. The sterols from the n-series also had smaller mean molecular areas than the corresponding sterols in the iso-series. In mixed 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/sterol monolayers (equimolar ratio; at 22 degrees C), all of the sterols tested decreased the monolayer stability as judged by the lower collapse pressure with sterol than without sterol. A similar trend was observed in mixed monolayers containing 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), except that sterols from the iso-series with a chain length of 8 or 10 carbon atoms actually stabilized the monolayer compared with the sterol-free SOPC monolayer. The ability of the sterols to condense the molecular packing of DPPC was similar with all sterols (3-5% condensation at 10 mN/m), irrespective of the length or structure of the side chain. 5-Androsten-3 beta-ol, however, which lacks the side chain, did not at all condense the monolayer packing of DPPC. With SOPC mixed monolayers, all side chain containing sterols caused a 18-20% condensation (at 10 mN/m) of monolayer packing. The condensing effect of 5-androsten-3 beta-ol on SOPC packing was again much smaller (about 10%) compared with that of the side-chain sterols. The rate of sterol oxidation by cholesterol oxidase (at 37 degrees C) in DPPC-containing SUVs increased as a function of increasing the side-chain length (iso-series). With sterols from the n-series, the same trend was seen, except that the n-C7 analogue was oxidized much slower than the n-C4, n-C5, and n-C6 analogues. With SOPC SUVs, a similar side-chain dependent oxidation pattern was observed. Our results support and extend previous knowledge about the importance of the sterol side chain in determining sterol-sterol and sterol-phospholipid interactions, both in mono- and bilayers.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1996

Fibroblast membrane sterol kinetic domains: modulation by sterol carrier protein-2 and liver fatty acid binding protein.

The mechanism(s) of intracellular sterol trafficking among subcellular organelle membranes is not well understood. Relative contributions of vesicular, sterol carrier protein, and membrane sterol domain pathways are not resolved. A sterol kinetic assay was used to resolve multiple sterol domains in microsome (MICRO), mitochondria (MITO), and plasma (PM) membrane: exchangeable, 20-40% of total; non-exchangeable, 60-80% of total. Spontaneous sterol transfer between dissimilar donor and acceptor membranes was vectorial and depended both on acceptor and donor membrane properties. For example, sterol transfer from PM to MICRO or to MITO, or from MICRO to MITO was 3- to 5-fold slower as compared to sterol movement in the opposite direction. Sterol carrier protein-2 (SCP-2) stimulated sterol transfer in most donor/acceptor membrane combinations by decreasing exchange half-time but not domain size. SCP-2 enhanced sterol transfer selectively: PM-MICRO (12-fold); MITO-MITO, MICRO-MICRO, MICRO-PM (3-fold); PM-PM (1.4-fold); PM-MITO, MICRO-MITO (no effect). Thus, SCP-2-mediated sterol movement was vectorial and not necessarily down a membrane sterol concentration gradient. In contrast, liver fatty acid binding protein (L-FABP) revealed a modest (2-fold) stimulatory effect on sterol transfer only between PM-MITO and MICRO-MICRO. In conclusion, in vitro studies of sterol transfer among isolated subcellular membranes provided kinetic evidence for sterol domains in microsomes and mitochondria as well as plasma membranes. Furthermore, both spontaneous and protein-mediated sterol transfer appeared vectorial and selective in nature.

Animals↗

PubMed1995

Modification of the sterol composition of Trypanosoma (Schizotrypanum) cruzi epimastigotes by delta 24(25)-sterol methyl transferase inhibitors and their combinations with ketoconazole.

We report a detailed analysis of the sterol composition of Trypanosoma cruzi epimastigotes grown in the absence or presence of two sterol analogs previously reported as inhibitors of delta 24(25) sterol methyltransferase (24(25)-SMT,E.C.2.1.1.43) in yeast and fungi, a cholestanol analog with a 6-membered aza ring as side chain (22,26-azasterol) and 24-(R,S),25-epiminolanosterol, as well as combinations of these compounds with the C14 demethylase inhibitor ketoconazole. Both sterol analogs produced a dose-dependent reduction in the incorporation of radioactivity from [methyl-14C]methionine with IC50 values of 640 nM and 70 nM for 22,26-azasterol and 24,25-(R,S)-epiminolanosterol, respectively, indicating a specific inhibition of 24(25)-SMT. Correspondingly, it was found that the sterols present in control cells (ergosterol, 24-ethylcholesta-5,7,22-trien-3 beta-ol and precursors) were almost completely replaced by zymosterol (cholesta-8,24-dien-3 beta-ol) or a mixture of zymosterol, cholesta-7,24-dien-3 beta-ol and cholesta-5,7,24-trien-3 beta-ol when the parasites were exposed to the minimal growth inhibitory concentrations of 22,26-azasterol and 24-(R,S),25-epiminolanosterol, respectively. At sub-optimal concentrations of the inhibitors a complete disappearance of the 24-ethyl sterols was observed and a concomitant increase in the proportion of 24-methyl sterols, particularly delta 24(24') sterols. This showed that in T. cruzi the second methenylation step (catalyzed by delta 24(24') sterol methyl transferase) was significantly more sensitive to these inhibitors than the first and that the sterol analogs were also powerful inhibitors of the delta 24(24') sterol reductase. In growth-arrested epimastigotes resulting from their treatment with low (1-3 microM) concentrations of either sterol analog combined with sub optimal (100-300 nM) levels of ketoconazole the main sterol was lanosterol with no evidence 24-methylenedihydrolanosterol, the main sterol found in cells treated with growth inhibitory concentrations of the azole alone. Taken together, these results indicated that 24-alkyl sterols are essential growth factors for T. cruzi and that the preferred substrate of the delta 24(25) sterol methyl transferase in this organism is zymosterol.

Animals↗

PubMed2002

Alternative pathways of sterol synthesis in yeast. Use of C(27) sterol tracers to study aberrant double-bond migrations and evaluate their relative importance.

Yeast produce traces of aberrant sterols by minor alternative pathways, which can become significant when normal metabolism is blocked by inhibitors or mutations. We studied sterols generated in the absence of the delta(8)-delta(7) isomerase (Erg2p) or delta(5) desaturase (Erg3p) by incubating three mutant strains of Saccharomyces cerevisiae with 5 alpha-cholest-8-en-3beta-ol, 8-dehydrocholesterol (delta(5,8) sterol), or isodehydrocholesterol (delta(6,8) sterol), together with the corresponding 3 alpha-3H isotopomer. Nine different incubations gave altogether 16 sterol metabolites, including seven delta(22E) sterols formed by action of the yeast C-22 desaturase (Erg5p). These products were separated by silver-ion high performance liquid chromatography (Ag(+)-HPLC) and identified by gas chromatography-mass spectrometry, nuclear magnetic resonance spectroscopy, and radio-Ag(+)-HPLC. When delta(8)-delta(7) isomerization was blocked, exogenous delta(8) sterol underwent desaturation to delta(5,8), delta(6,8), and delta(8,14) sterols. Formation of delta(5,8) sterol was strongly favored over delta(6,8) sterol, but both pathways are essentially dormant under normal conditions of sterol synthesis. The delta(5,8) sterol was metabolically almost inert except for delta(22) desaturation, whereas the delta(6,8) sterol was readily converted to delta(5,7), delta(5,7,9(11)), and delta(7,9(11)) sterols. The combined results indicate aberrant metabolic pathways similar to those in mammalian systems. However, delta(5,7) sterol undergoes only slight isomerization or desaturation in yeast, an observation that accounts for the lower levels of delta(5,8) and delta(5,7,9(11)) sterols in wild-type yeast compared to Smith-Lemli-Opitz individuals.

Cholestadienols↗

PubMed2005

Role of sterol superlattice in free radical-induced sterol oxidation in lipid membranes.

We developed a new fluorescence assay for sterol oxidation and used it to study the relationship between free radical-induced sterol oxidation and membrane sterol lateral organization. This assay used dehydroergosterol (DHE) as both a membrane probe and a membrane component. Sterol oxidation was induced by a free radical generator, AAPH (2,2'-azobis(2-amidinopropane)dihydrochloride). Using this new assay, we found that, in unilamellar vesicles composed of DHE and 1-palmitoyl-2-oleoyl-l-alpha-phosphatidylcholine (POPC), the initial rate of DHE oxidation induced by AAPH changed with membrane sterol content in an alternating manner, exhibiting a local maximum at 20.3, 22.2, 25.0, 32.3, and 40.0 mol % DHE. These mole fractions correspond to the critical sterol mole fractions C(r) predicted for maximal sterol superlattice formation. In three-component bilayers composed of POPC, cholesterol, and DHE (fixed at 1 and 5 mol %), the initial rate of AAPH-induced DHE oxidation exhibited a biphasic change whenever the total sterol mole fraction, irrespective of the DHE content, was near C(r), indicating that the correlation between sterol oxidation and sterol superlattice formation revealed in this study is not an artifact due to the use of the fluorescent cholesterol analogue DHE. The alternating variation of AAPH-induced sterol oxidation with sterol content also appeared in multicomponent unilamellar vesicles containing bovine brain sphingomyelins (bbSPM), POPC, and DHE. The present work and our previous study on cholesterol oxidase-induced sterol oxidation [Wang et al. (2004) Biochemistry 43, 2159-2166] suggest that sterol oxidation in general, either by reactive oxygen species or by enzymes, may be regulated by the extent of sterol superlattice in the membrane and thus regulated by the membrane sterol content in a fine-tuning manner.

Amidines↗

PubMed1995

An Arabidopsis mutant deficient in sterol biosynthesis: heterologous complementation by ERG 3 encoding a delta 7-sterol-C-5-desaturase from yeast.

The mutant STE 1 was isolated by screening an ethylmethane sulfonate (EMS)-mutagenized population of Arabidopsis thaliana which consisted of 22,000 M2 plants divided into 1100 pools of 20 plants by gas chromatography of sterols extracted from small leaf samples. STE 1 was characterized by the accumulation of three delta 7-sterols concomitantly with the decrease of the three corresponding delta 5-sterols which are the end products of the sterol pathway in wild-type leaves. The structure of these delta 7-sterols was determined after two steps of purification on HPLC, by gas chromatography coupled with mass spectrometry (GC-MS) and proton nuclear magnetic resonance spectrometry (1H-NMR). The accumulation of delta 7-sterols suggested that the mutant is deficient in the activity of the delta 7-sterol-C-5-desaturase. Genetic analysis showed that the accumulation of delta 7-sterols was due to a single recessive nuclear mutation. The mutant line STE 1 was backcrossed four times to the wild-type. The resulting STE 1 plants had wild-type morphology and set seeds normally, suggesting that the delta 7-sterols in STE 1 are good surrogates of physiologically active delta 5-sterols to sustain normal development. STE 1 roots were transformed with the Saccharomyces cerevisiae ERG 3 gene encoding the delta 7-sterol-C-5-desaturase under the control of the CaMV 35S promoter. Seven transgenic STE 1 root-derived calli showed an increase in delta 5-sterols and a concomitant decrease in delta 7-sterols in comparison with STE 1 untransformed root-derived calli. Northern blot analysis using the ERG 3 probe showed a strong expression of ERG 3 in three of the seven transgenic calli. These results suggest that the accumulation of delta 7-sterols in the STE 1 mutant is due to a deficiency of the delta 7-sterol-C-5-desaturation step in the plant sterol biosynthesis pathway.

Arabidopsis↗

PubMed1983

The effect of AY-9944 on yeast sterol and sterol ester metabolism.

The effects of the hypocholesterolemic drug AY-9944 (trans-1,4-bis(2-chlorobenzylaminoethyl)cyclohexane dihydrochloride) at two concentrations (10(-4) M and 5 X 10(-4) M) on the synthesis of sterols and sterol esters by Saccharomyces cerevisiae were investigated. Although growth was not markedly affected by the drug, there was a decrease in the free sterol to sterol ester ratio with increased drug concentration. A concomitant increase in the saturated fatty acids esterified to sterol relative to the unsaturated fatty acids was also noted in response to increased drug concentration. Ergosterol accounted for 94.7% of the free sterol in the control culture and for 87.8% of the 5 X 10(-4) M drug-treated culture, respectively. However, in the sterol ester fraction, the ergosterol content decreased from a value of 45.1% in the control culture to 2.4% in the 5 X 10(-4) M AY-9944 treated culture. The sterol ester fraction simultaneously showed increased levels of the delta 8 sterol, fecosterol, in response to increased drug concentration from a 7.4% control value to 57.4% in the 5 X 10(-4) M drug-treated culture. The accumulation of the delta 8 sterol suggests that the site of action of the drug is probably at the delta 8 to delta 7 isomerase step in the biosynthesis of ergosterol. The fact that ergosterol is retained as the major free sterol suggests a biological advantage to the retention of this particular sterol. In addition, the near normal growth in the presence of the drug, in spite of the occurrence of an altered sterol ester profile, indicates that the composition of the sterol ester fraction is not as critical as the free sterol fraction.

Cholesterol↗