Comparative responses of the yeast mutant strain GL7 to lanosterol,cycloartenol, and cyclolaudenol

Under anaerobic growth conditions the isomeric 4,4′,14-trimethylcholestane derivatives lanosterol and, more efficiently, cycloartenol satisfy the sterol requirement of the yeast sterol auxotroph $\text{Saccharomyces cerevisiae}$ strain GL7. Aerobic mutant growth is supported only by cycloartenol and not by lanosterol, suggesting different structural requirements for aerobic and anaerobic cells. It is proposed that the non-planar conformation imposed by the 9,19-cyclopropane ring of cycloartenol moderates the adverse membrane effects of the nuclear methyl groups at C-4 and C-14. Under both aerobic and anaerobic conditions cyclolaudenol, a C-24-methyl derivative of cycloartenol, is a significantly more effective sterol source for strain GL7 than cycloartenol. This result is in keeping with the predominance of C-24-methyl sterols (ergosterol) in wild-type yeast.     

Effects of cycloartenol and lanosterol on artificial and natural membranes

Cycloartenol, a 9,19-cyclopropane sterol which is isomeric with lanosterol, showed an ability intermediate between lanosterol and cholesterol to increase the microviscosity of lecithin vesicles, to serve as a growth factor for the sterol auxotroph $\text{Mycoplasma capricolum}$, and to increase the microviscosity of $\text{M. capricolum}$ membranes. The corresponding membrane effects of cyclolaudenol which contains a methyl group added to C-24 of the isooctenyl side chain of cycloartenol are more like those shown by lanosterol. We propose that the enhanced effectiveness of cycloartenol over lanosterol is due to a more favorable spatial disposition of the angular 14α-methyl group on the α-face of the molecule promoting more effective van der Waals contacts between the phospholipid fatty acyl chains and the sterol α-face. Side chain alkylation appears to perturb such contacts, reducing the effectiveness of cyclolaudenol for competent membrane function in $\text{M. capricolum}$.     

Phytosterols and Other Lipids as Survival Factors for Tetrahymena at 0–5 C

SYNOPSIS. Tetrahymena pyriformis syngen 1, mating type II, (optimal growth temperature ∼ 37 C) ordinarily dies out in 5-14 days at 0-5 C. Dying cells were lumpy, suggesting membrane damage. By supplying crude soy lecithin, survival at 0-5 C was prolonged (after growth in peptone-yeast-dextrin) to at least 22 weeks. Crude soy sterols or sitosterol or stigmasterol, and antioxidant, e.g., Ionox 330 or ascorbylpalmitate, permitted survival of cells in suspension or in growth media for at least 16-22 weeks. These sterols are known to protect against triparanol toxicity, which suggested that triparanol, which blocks cholesterol synthesis in higher animals, might enhance cold-induced injury. Triparanol was more toxic at 0–5 than at 28 C for cell suspensions and cells in growth medium; this toxicity was annulled by crude soy lecithin or β-sitosterol, the only phytosterol tested. The synthetic medium intended as a control on the crude media became toxic at 0–5 C. Protection against cold damage is discussed as a means of elucidating the role of sterols—especially phytosterols—and other lipids in maintaining the integrity of the ciliate cell membrane.     

The effect of triparanol on the composition of free and esterified sterols of Saccharomyces cerevisiae.

Triparanol altered the sterol composition of Saccharomyces cerevisiae and promoted an increase in the steryl ester and total sterol per organism. The accumulation of Δ⁸ sterols, both free and esterified, in the presence of triparanol indicates that a major effect of the compound in yeast is the inhibition of the Δ^8→7 isomerase. Isolation of ergosta-5,8(9),22-trien-3 β-ol, hitherto detected only in ergosterol-deficient yeast mutants, further supports the concept that all of the other metabolic alterations required for the conversion of lanosterol to ergosterol can occur without the necessity of Δ^8→7 isomerization.     

Effect of ethanol on the sterols of the fission yeast Schizosaccharomyces pombe

Abstract Ergosterol, lanosterol and two further unidentified sterols were detected and quantified in Schizosaccharomyces pombe cell extracts. In cells grown under anaerobic conditions, the levels of these sterols were dramatically reduced with a concomitant increase of their squaline precursor as compared with cells growing under aerobic conditions. Presence of ethanol resulted in a decrease in the sterol content under aerobic conditions. On the contrary, under anaerobic conditions presence of ethanol resulted in a three-fold increase of total sterols. Lanosterol was the main constituent of this elevation. It is suggested that lanosterol in parallel with unsaturated fatty acids is responsible for maintaining membrane integrity of S. pombe cells growing in the presence of ethanol.     

Lipid Composition of the Marine Ciliates Pleuronema sp. and Fabrea salina: Shifts in Response to Changes in Diet1

ABSTRACT. The composition and incorporation of lipids in two marine ciliates, Pleuronema sp. and Fabrea salina, was examined following growth on either an algal or bacterial diet. When allowed to feed on a natural bacterial community, Pleuronema sp. synthesized the triterpenoid alcohol gammaceran-3β-ol (tetrahymanol) and two hopanoids (hopan-3β-ol and one uncharacterized hopanoid). When fed the marine alga Isochrysis galbana, F. salina contained the major algal sterol 24-methylcholesta-5, 22-dien-3β-ol and several long chain ketones specific to the alga. In both ciliates, fatty acids composition showed a general correspondence to that of the diet. Using a series of antibiotic treatments to alter the bacterial prey community, and thus fatty acid composition of the ciliate's diet, promoted changes in the fatty acid composition of Pleuronema sp. to resemble that of the bacterial prey. The addition of a mixture of algal sterols to a bacterized culture of another scuticociliate, Parauronema acutum, inhibited tetrahymanol synthesis and resulted in the incorporation of sterols into the ciliate.     

Microbial Eukaryotes that Lack Sterols

It is widely held that sterols are key cyclic triterpenoid lipids in eukaryotic cell membranes and are synthesized through oxygen‐dependent multienzyme pathways. However, there are known exceptions―ciliated protozoans, such as Tetrahymena, along with diverse low‐oxygen‐adapted eukaryotes produce, instead of sterols, the cyclic triterpenoid lipid tetrahymanol that does not require molecular oxygen for its biosynthesis. Here, we report that a number of anaerobic microbial eukaryotes (protists) utilize neither sterols nor tetrahymanol in their membranes. The lack of detectable sterol‐like compounds in their membranes may provide an opportunity to reconsider the physiological function of sterols and sterol‐like lipids in eukaryotes.     

The effect of cholesterol on ubiquinone and tetrahymanol biosynthesis in Tetrahymena pyriformis.

The biosynthesis of ubiquinone-8 from radioactive mevalonate by cultures of Tetrahymena pyriformis is demonstrated. Under normal conditions the incorporation of this radioactive precursor into ubiquinone and the triterpenoid alcohol tetrahymanol reflects the amounts of these two compounds in the cell. Growth of T. pyriformis in the presence of cholesterol results in a complete inhibition of incorporation of radioactive mevalonate into tetrahymanol while there is a corresponding increase of radioactive incorporation into ubiquinone. This increased incorporation of mevalonic acid into ubiquinone must reflect a reduced level of mevalonic acid in the cell under these conditions and is not due to increased ubiquinone biosynthesis, indicating tight regulation of the pathway prior to mevalonate formation.     

Lipid modification during cytodifferentiation of Tetrahymena vorax. Whole cell phospholipids and triacylglycerols of microstomal and macrostomal phenotypes

Microstomal cells of the ciliate Tetrahymena vorax V2S can be induced to undergo cytodifferentiation to form an alternate phenotype known as the macrostomal cell; however, sublines of T. vorax exist that respond differently to methods that induce macrostomal cell formation. The phospholipid- and triacylglycerol-bound fatty acid compositions of microstomal and macrostomal cells of a high-transforming subline (designated 3-C) were determined and compared to similar data from cells of a low-transforming subline (designated Ala). Differences in fatty acid composition were found between the two phenotypes as well as between the different sublines. Some change in the distribution of radioactive acetate and lauric acid into phospholipid classes of the different subline was observed, and evidence was also obtained that indicated changes in the relative amounts of the sterol-like pentacyclic triterpenoid tetrahymanol. A limited analysis of the lipid composition of stomatin revealed the presence of small amounts of tetrahymanol, phospholipid and free fatty acid. Stomatin is the naturally produced material obtained from T. pyriformis that triggers differentiation in T. vorax. The existence of a low-transforming subline provides a powerful experimental tool for elucidating the underlying biochemical and molecular mechanisms that control cytodifferentiation in T. vorax and possibly in other eukaryotic cells.     

Microsomal synthesis of cholesterol from squalene, Lanosterol, and desmosterol. Evidence for the presence of two noncatalytic activator proteins in the 105,000 g supernatant fraction from brain, heart, and kidney

The biosynthesis of C₂₇ sterols (used as a generic term for 3 β-hydroxysterols containing 27 carbon atoms) from squalene and lanosterol, of cholesterol from desmosterol, and of lanosterol from squalene by microsomal fractions from adult rat heart, kidney, and brain was investigated. These conversions required the presence of 105,000g supernatant fraction. Heat treatment of the supernatant fractions resulted in a significant loss of their capacity to stimulate the conversion of squalene to sterols, but the capacity to stimulate conversion of lanosterol to C₂₇ sterols and desmosterol to cholesterol was unaffected. The stimulatory activity (for the conversion of all three substrates) of both the heated and unheated supernatant fractions was lost on treatment with trypsin. Thus the soluble fraction appears to contribute at least two essential protein components for the overall conversion of squalene to cholesterol; one a heat labile protein, which functions in the squalene to lanosterol sequence, and the other a heat-stable protein, which is operative in the pathway between lanosterol and cholesterol. Hepatic supernatant factors required for cholesterol synthesis by liver microsomal enzymes function with heart, kidney, and brain microsomal enzymes in stimulating sterol synthesis from squalene and sterol precursors. Moreover, heart, kidney, and brain supernatant fractions prepared in 100 mm phosphate buffer stimulated cholesterol synthesis from squalene and other sterol precursors by liver microsomes. The supernatant fractions of the extrahepatic tissues prepared in 20 mm phosphate buffer lacked the ability to stimulate the biosynthesis of lanosterol from squalene by liver microsomes but were able to stimulate the conversion of lanosterol to C₂₇ sterols or conversion of desmosterol to cholesterol. These findings indicate that the heat-stable protein factor present in the supernatant fractions from extrahepatic tissues is perhaps identical to that in liver, but that the heat-labile factor in extrahepatic tissues, which catalyzes the cyclization of squalene to lanosterol, differs in some respect from that in liver.     

Inhibition by the fungicide fenpropimorph of cholesterol biosynthesis in 3T3 fibroblasts.

Fenpropimorph (N-[3-(p-t-butylphenyl)-2-methylpropyl]-cis-2,6-dimethylmorpholine), a morpholine fungicide known to be an inhibitor of sterol biosynthesis in fungi and in higher plants, was demonstrated to be an efficient inhibitor of cholesterol biosynthesis in cultured Swiss 3T3 fibroblasts. Treatment of the mammalian cells with fenpropimorph resulted in a dose-dependent inhibition of [14C]acetate incorporation into the C27 sterols [IC50 (concentration causing half-maximal inhibition) = 0.5 microM], which was accompanied by an accumulation of polar sterols and a decrease in cellular hydroxymethylglutaryl-CoA reductase activity. Exposure of the cells to the drug affected cell growth. Analysis of the sterols in the growth-arrested and in the pulse-labelled cells indicate that fenpropimorph has, in the sterol-biosynthetic pathway, target enzymes in mammalian cells different from those in the other phyla. Whereas in plants and fungi fenpropimorph mainly affects sterol isomerases and reductases, in the fibroblasts its main target seems to be the demethylation of lanosterol.     

Supplementation with sterols improves food quality of a ciliate for Daphnia magna

Experimental results provide evidence that trophic interactions between ciliates and Daphnia are constrained by the comparatively low food quality of ciliates. The dietary sterol content is a crucial factor in determining food quality for Daphnia. Ciliates, however, presumably do not synthesize sterols de novo. We hypothesized that ciliates are nutritionally inadequate because of their lack of sterols and tested this hypothesis in growth experiments with Daphnia magna and the ciliate Colpidium campylum. The lipid content of the ciliate was altered by allowing them to feed on fluorescently labeled albumin beads supplemented with different sterols. Ciliates that preyed upon a sterol-free diet (bacteria) did not contain any sterols, and growth of D. magna on these ciliates was poor. Supplementation of the ciliates' food source with different sterols led to the incorporation of the supplemented sterols into the ciliates' cells and to enhanced somatic growth of D. magna. Sterol limitation was thereby identified as the major constraint of ciliate food quality for Daphnia. Furthermore, by supplementation of sterols unsuitable for supporting Daphnia growth, we provide evidence that ciliates as intermediary grazers biochemically upgrade unsuitable dietary sterols to sterols appropriate to meet the physiological demands of Daphnia.     

Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in human hepatoma cell line Hep G2. Effects of inhibitors of cholesterol synthesis on enzyme activity.

Incubating Hep G2 cells for 18 h with triparanol, buthiobate and low concentrations (less than 0.5 microM) of U18666A, inhibitors of desmosterol delta 24-reductase, of lanosterol 14 alpha-demethylase and of squalene-2,3-epoxide cyclase (EC 5.4.99.7) respectively, resulted in a decrease of the HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase activity. However, U18666A at concentrations higher than 3 microM increased the HMG-CoA reductase activity in a concentration-dependent manner. None of these inhibitors influenced directly the reductase activity in Hep G2 cell homogenates. Analysis by t.l.c. of 14C-labelled non-saponifiable lipids formed from either [14C]acetate or [14C]mevalonate during the cell incubations confirmed the sites of action of the drugs used. Beside the 14C-labelled substrates of the blocked enzymes and 14C-labelled cholesterol, another non-saponifiable lipid fraction was observed, which behaves as polar sterols on t.l.c. This was the case with triparanol and at those concentrations of U18666A that decreased the reductase activity, suggesting that polar sterols may play a role in suppressing the reductase activity. In the presence of 30 microM-U18666A (sterol formation blocked) the increase produced by simultaneously added compactin could be prevented by addition of mevalonate. This indicates the existence of a non-sterol mevalonate-derived effector in addition to a sterol-dependent regulation. LDL (low-density lipoprotein), which was shown to be able to decrease the compactin-induced increase in reductase activity, could not prevent the U18666A-induced increase. On the contrary, LDL enhanced the U18666A effect, showing that the LDL regulation is not merely the result of introducing cholesterol to the cells.     

The influence of hopanoids on growth of Mycoplasma mycoides

Hopane glycolipid, isolated from Bacillus acidocaldarius, was studied in its ability to support growth from Mycoplasma mycoides var. capri as substitute for sterols. All attempts to culture Mycoplasma on hopane glycolipid failed. When added together with cholesterol, the effect of HGL was a reduction in growth rate.The hopanoid diplopterol moderately supported growth of Mycoplasma. This effect was strikingly enhanced in diplopterol adapted cultures. Application of diplopterol via dipalmitoyl-phosphatidylcholine liposomes resulted in an improved growth rate. These results suggested that diplopterol can fullfill sterol function in Mycoplasma membranes.Abbreviations HGL Hopane glycolipid 1-(O--N-acylglucosaminyl)-2,3,4-tetrahydroxypentane-29-hopane - diplopterol 22-hydroxy-hopane - DPPC dipalmitoyl-phosphatidylcholine - PC phosphatidylcholine     

The presence of acyl-CoA: cholesterol acyltransferase in Tetrahymena pyriformis W

1. The esterification of cholesterol was studied in Tetrahymena pyriformis an organism which does not synthesize sterols nor are sterols required for growth. 2. Microsomes catalyzed the esterification of cholesterol in the presence of oleoyl-CoA but not oleic acid or lecithin. 3. The enzyme has a similar sterol substrate specificity to that of mammalian acyl-CoA: cholesterol acyltransferase (ACAT) and was inhibited by the specific ACAT inhibitor 58-035. 4. The enzyme is constitutive since activity was observed in cells grown in sterol-free medium when cholesterol was added to the in vitro assay.     

Characterization of the structure of a 4-methyl-delta 8,24-cholestadien-3 beta-ol isolated from rat skin

A new sterol has been isolated from the skin of rats treated with triparanol. Its chromatographic behavior on silicic acid-Celite columns and in gas-liquid chromatographic systems indicated it to be a 4-methyl-Delta(8,24)-cholestadien-3beta-ol. The specific rotation, the delayed color reaction with Liebermann-Burchard reagent, and the nuclear magnetic resonance (NMR) data support the Delta(8(9))-unsaturation. Previous workers have shown that triparanol treatment results in an accumulation of Delta(24)-unsaturated sterols in animal tissues. Consonant with this observation, the infrared, NMR, and mass spectrometric data confirm the presence of a C-24(25) unsaturated side chain in this sterol.     

Sterol biosynthesis via cycloartenol and other biochemical features related to photosynthetic phyla in the amoeba Naegleria lovaniensis and Naegleria gruberi

The sterols and sterol precursors of two amoebae of the genus Naegleria, Naegleria lovaniensis and Naegleria gruberi were investigated. Cycloartenol, the sterol precursor in photosynthetic organisms, is present in both amoebae. In N. lovaniesis, it is accompanied by lanosterol and parkeol, as well as by the 24,25-dihydro derivatives of these triterpenes. One of the most striking features of these amoebae is the accumulation of 4 alpha-methylsterols which are present in similar amounts as those of 4,4-desmethylsterols (3-5 mg/g, dry weight). 4 alpha-Methylergosta-7,22-dienol was identified as a new compound. Ergosterol was the major 4,4-desmethylsterol, accompanied by small amounts of C27 and other C28 sterols. Treatment of N. lovaniensis with fenpropimorph modified the sterol pattern of this amoeba and inhibited its growth. This fungicide, known to inhibit steps of sterol biosynthesis in fungi and plants, induced the disappearance of 4 alpha-methyl-delta 7-sterols and the appearance of the unusual delta 6,8,22-ergostatrienol as in A. polyphaga. These results might be explained by a partial inhibition of the delta 8----delta 7 isomerase, the small amounts of delta 7-sterols formed being converted into ergosterol which is still present in fenpropimorph-exposed cells. De novo sterol biosynthesis in N. lovaniensis was shown by incorporation of [1-14C]acetate into sterols and sterol precursors, especially cycloartenol. Lanosterol and parkeol were not significantly labelled. Furthermore, [3-3H]squalene epoxide was efficiently cyclized by a cell-free system of this amoeba into cycloartenol, and again no significant radioactivity was detected in lanosterol and parkeol. This shows that cycloartenol, the sterol precursor in plants and algae, is also the sterol precursor in Naegleria species, and that these amoebae, like A. polyphaga, are related by some biosynthetic pathways to photosynthetic phyla. Lanosterol, the sterol precursor in non-photosynthetic phyla (animal and fungi) and parkeol are more likely dead-ends of this biosynthetic pathway. The peculiar phylogenetic position of these protozoa was further emphasized by the action of indole acetic acid and other auxine-like compounds on their growth. Indeed amoebic growth was enhanced in the presence of these higher plant growth hormones. The differences in the sterol composition of the protozoa we have hitherto examined is related to their sensitivity toward polyene macrolide antibiotics.(ABSTRACT TRUNCATED AT 400 WORDS)     

The Effect of Specific Sterols on Cell Size and Fatty Acid Composition of Tetrahymena pyriformis W

The size and fatty acid composition of Tetrahymena pyriformis W cells were influenced by the provision of a nutritional supplement of ergosterol, cholesterol, or tetrahymanol, but not of 20-isocholesterol. Ergosterol and cholesterol addition led to a reduction in cellular volume, an increase in glycerophospholipid saturated fatty acid content, and an increase in palmitoleic acid and its metabolic products when compared to unsupplemented controls. Tetrahymanol supplementation resulted in an increase in cellular volume, a decrease in saturated fatty acid content, and a reduction in palmitoleic acid and derivatives. 20-Isocholesterol was accumulated by the cells; however, this compound had no effect on any of the parameters followed in this investigation and had only a small depressant effect on tetrahymanol biosynthesis. Ergosterol and cholesterol had the same impact on the ciliates, even though the ergosterol-supplemented cells contained approximately three times as much free sterol as did cholesterol-grown cells. The amount of the free cholesterol and metabolic products in supplemented cultures was similar to the amount of tetrahymanol present in control cultures. This observation suggests that the cells recognize qualitative differences among the various polycyclic alcohols rather than responding to the amount of sterol present. Increased cellular levels of tetrahymanol led to a response unlike that of the true sterols, which again suggests that the high degree of specificity depends on the structure of the added polycyclic alcohol. The changes in fatty acid composition may be required to maintain proper interaction of the polar lipids and the polycyclic alcohols to give an appropriate degree of membrane fluidity.