The utilization of sterols and other lipids by insect cells grown in vitro

Cells derived from Antheraea eucalypti were grown in vitro in a medium containing triglycerides, diglycerides, free fatty acids, and sterols as the main ‘neutral’ lipids. The sterol content of the medium was derived chiefly from the haemolymph component. The ‘neutral’ lipids in the cells were triglycerides, free fatty acids, and sterols. During growth over 6 days there was a quantitative balance between cholesterol and β-sitosterol gained by the cells and those sterols removed from the medium when allowance was made for losses from sterile medium. Cells metabolized more triglycerides and free fatty acids than they incorporated.     

Free and bound sterols in seedlings of Cucumis sativus

Free sterols, sterol esters, sterol monoglucosides and sterol acylmonoglucosides have been obtained from 10 days old seedlings of Cucumis sativus. Free sterols and sterol esters consist mainly of Δ⁷ di- and triunsaturated sterols, whereas Δ⁷ mono-unsaturated and Δ⁵ mono- and diunsaturated sterols predominate in the glucosides and acylglucosides. Both acetates and derivatives of higher fatty acids, mainly linoleic and linolenic acids, have been found in the sterol esters. Sterol acylglucosides contain mostly saturated fatty acids, palmitic and stearic acids being the main components.     

Influence of lignin and oxygen on the growth and the lipid formation of the fungus Lentinus tigrinus

During cultivation of the filamentous fungus Lentinus tigrinus on a medium containing lignin, a high oxygen content stimulated the growth of the fungus and contributed to the yield of lipids. A high content of phosphatidic acid and a reduction in the level of phosphatidylethanolamine and phosphatidylserine were first detected in the composition of phospholipids. Changes in the composition of neutral lipids, such as variation in the ratio of esterified and free sterols, have occurred; thus, the amount of sterol esters reduced simultaneously with a decrease in the content of free fatty acids. Based on the obtained results, the possible role of phosphatidic acid as a second messenger in the process of the consumption of lignin by the fungus Lentinus tigrinus is discussed.     

Les sterols de l'Ascomycete Leptosphaeria typhae

A mixture of C₂₈ and C₂₉ sterols have been isolated from Leptosphaeria typhae grown in vitro on “oat water” and characterized by GLC and MS. Mono-, di- and tri-unsaturated sterols are present in the extracts of fungi cultivated both in the dark and in the light but the sterol composition is different. The influence of “oat water” on sterol structure has been determined by comparison with the sterols of the same fungus grown on synthetic medium in the dark.     

Sterol distribution in intracellular organelles isolated from tobacco leaves

All membrane-containing fractions isolated from tobacco leaves contained free sterols, sterol glycosides, and sterol esters. The three sterol forms increased, on a dry weight basis, with a decrease in particle size. The supernatant fraction contained only trace amounts of sterol. The major sterols in all cellular fractions, in the order of decreasing amounts, were: stigmasterol, β-sitosterol, campesterol, and cholesterol. The 500g pellet contained the largest percentage of free sterol, while the 46,000g pellet contained the largest percentage of esterified sterol. The individual sterol composition of the free sterol and sterol glycoside fraction was very similar; however, the composition of the sterol ester fraction varied widely among intracellular fraction. The intracellular distribution pattern of cholesterol-¹⁴C added to the isolation medium provided evidence that the intracellular sterol distribution pattern is not an artifact. These results support the suggestion that sterols in plant cells may have a physiological function associated with membranes.     

Sterols and fatty acids from three species of mangrove

The hydrolysis of steryl esters on thin-layer chromatographic plates by porcine pancreatic lipase is described. The sterols and fatty acids produced were separated on the same plate, recovered, and analysed by gas-liquid chromatography for their compositions. Synthetic cholesteryl esters containing various saturated and unsaturated fatty acids and synthetic steryl oleates with various sterols were lipolysed along with steryl esters of Acanthus ilicifolius, Bruguiera gymnorhiza and Rhizophora mucronata mangrove leaves. The major sterol was sitosterol which was accompanied by cholesterol, campesterol, stigmasterol and 28-isofucosterol. In addition, stigmast-7-en-3β-ol was present in R. mucronata leaves. The component fatty acids found in all three species were 16:0, 18:0, 18:1, 18:2 and 18:3. The relative proportions of the sterols and fatty acids were significantly different from the chemotaxonomic standpoint. The results obtained by carrying out plate lipolysis for 45 min at 40° compared well with those produced by conventional chemical hydrolysis.     

Effectiveness of the fatty acid and sterol composition of seeds for the chemotaxonomy of Coffea subgenus Coffea

The chemotaxonomic relationships between Coffea (subgenus Coffea) species have been poorly studied to date and the compounds tested so far - chlorogenic acids, diterpenoids and purine alkaloids - did not enable the establishment of phylogenetic relationships analogous to those revealed by chloroplast and nuclear DNA studies. In the present study, the relationships between African Coffea species were assessed on the basis of their seed lipid composition. Fatty acids and sterols were determined in 59 genotypes belonging to 17 distinct Coffea species/origins. Principal Component Analysis of fatty acid and sterol data enabled easy identification of the few species for which one or several compounds could serve as a quantitative signature. Hierarchical Clustering classified the Coffea species in seven groups with both fatty acids and sterols. However, while groupings based on seed fatty acid composition showed remarkable ecological and geographical coherence, no phylogeographic explanation was found for the clusters retrieved from sterol data. When compared with previous phylogenetic studies, the groups deduced from seed fatty acid composition were remarkably congruent with the clades inferred from nuclear and plastid DNA sequences.     

Sterols,steryl esters and fatty acids in some Vicia faba seeds

Free and esterified sterol levels in seeds of five cultivars of Vicia faba were determined. Sitosterol was the most abundant free sterol, followed by stigmasterol and campesterol. Cholesterol could not be detected. Esters were generally present in greater quantities than the free form of the sterols. The fatty acid content of the plants is also reported.     

Oxygen Consumption by Anaerobic Saccharomyces cerevisiae under Enological Conditions: Effect on Fermentation Kinetics

The anaerobic growth of the yeast Saccharomyces cerevisiae normally requires the addition of molecular oxygen, which is used to synthesize sterols and unsaturated fatty acids (UFAs). A single oxygen pulse can stimulate enological fermentation, but the biochemical pathways involved in this phenomenon remain to be elucidated. We showed that the addition of oxygen (0.3 to 1.5 mg/g [dry mass] of yeast) to a lipid-depleted medium mainly resulted in the synthesis of the sterols and UFAs required for cell growth. However, the addition of oxygen during the stationary phase in a medium containing excess ergosterol and oleic acid increased the specific fermentation rate, increased cell viability, and shortened the fermentation period. Neither the respiratory chain nor de novo protein synthesis was required for these medium- and long-term effects. As de novo lipid synthesis may be involved in ethanol tolerance, we studied the effect of oxygen addition on sterol and UFA auxotrophs (erg1 and ole1 mutants, respectively). Both mutants exhibited normal anaerobic fermentation kinetics. However, only the ole1 mutant strain responded to the oxygen pulse during the stationary phase, suggesting that de novo sterol synthesis is required for the oxygen-induced increase of the specific fermentation rate. In conclusion, the sterol pathway appears to contribute significantly to the oxygen consumption capacities of cells under anaerobic conditions. Nevertheless, we demonstrated the existence of alternative oxygen consumption pathways that are neither linked to the respiratory chain nor linked to heme, sterol, or UFA synthesis. These pathways dissipate the oxygen added during the stationary phase, without affecting the fermentation kinetics.     

Reversal of cerulenin-induced inhibition of phospholipids and sterol synthesis by exogenous fatty acids/sterols in Epidermophyton floccosum

Cerulenin, a specific inhibitor of fatty acids and sterol biosynthesis inhibited the growth of Epidermophyton floccosum, which was reversed when growth medium was supplemented with palmitic acid and sterols. Unsaturated fatty acids partially restored the growth. Cerulenin inhibited both phospholipid and sterol biosynthesis (60-70%) at the minimum inhibitory concentration (0.5 microgram/ml) as demonstrated by [32P]orthophosphoric acid and [14C]acetate incorporation into the respective lipids. Cerulenin-induced inhibition of phospholipid and sterol synthesis was dose dependent up to 0.5 microgram/ml. Exogenously supplied fatty acids and sterols restored the biosynthesis of phospholipids in cerulenin-treated cultures, while that of sterols was enhanced. The biosynthesis of both saturated and unsaturated fatty acids was inhibited by cerulenin.     

Lipids of the antarctic sea ice diatom Nitzschia cylindrus

The sterol and neutral, glyco- and phospholipid fatty acid profiles of the sea ice diatom Nitzschia cylindrus, isolated from McMurdo Sound, Antarctica, are reported. Two sterols were detected, trans-22-dehydrocholesterol (66% of total sterols) and cholesterol (34%); no sterols containing alkyl groups at the C₂₄ position were present. The major fatty acids in N. cylindrus, 16:1Δ9c, 14:0, 16:0, 20:5Δ5,8,11,14,17 and 20:4Δ5,8,11,14, were typical of previous reports of diatom fatty acids. A number of long-chain monounsaturated fatty acids were also detected, with higher relative proportions present in the phospholipid fraction. GC-MS analysis of the dimethyldisulphide adducts of these monounsaturated components showed that 24: 1Δ13c, 24:1Δ15c, 26:1Δ15c and 26:1Δ17c were the major components. The distribution of these fatty acids suggests that chain elongation of monounsaturated fatty acids was occurring in N. cylindrus. The proposed chain lengthening occurring for N. cylindrus represents, to our knowledge, the first report of possible chain lengthening of monounsaturated fatty acids in microscopic algae. These features, the presence of long-chain monounsaturated fatty acids and the sterol profile, may allow the input of this alga into benthic marine sediments or food webs to be monitored.     

An investigation of the mechanisms for sterol synthesis and dietary sterol bioconversion in the heterotrophic protists Oxyrrhis marina and Gyrodinium dominans

The ability of the marine heterotrophic protists Oxyrrhis marina and Gyrodinium dominans to synthesize sterols de novo and modify dietary sterols was investigated using ¹³C-labeled substrates. De novo sterol synthesis of O. marina was determined by incorporation of ¹³C acetate into the culture medium. For G. dominans which has low tolerance of acetate, a protozoan prey Perkinsus marinus that cannot synthesize sterols, was cultured with ¹³C acetate then fed to G. dominans. Both heterotrophs utilized dietary ¹³C to synthesize fatty acids de novo, but not sterols. The ability of O. marina and G. dominans to alkylate, saturate, and desaturate dietary sterols was tested using P. marinus incorporated with ¹³C-labeled cholesterol as prey. O. marina did not modify the dietary ¹³C-cholesterol, but G. dominans produced 5 labeled sterols (brassicasterol, C28:1, and unknown C28, C29 and C30 sterols) indicating that G. dominans has the ability to desaturate and alkylate dietary cholesterol. The ability of O. marina and G. dominans to dealkylate dietary sterols was tested by feeding them gelatin acacia microspheres (GAMs) containing ¹³C-labeled brassicasterol. Neither heterotroph dealkylated brassicasterol to make cholesterol, but G. dominans alkylated and saturated brassicasterol to make 2 sterols (C29:1 and C30:0). The lack of dealkylation of brassicasterol by both protist species suggests problems with the substrate and/or delivery system since previous studies suggest that dealkylation of brassicasterol occurs when either species is fed algae containing this sterol.     

The effect of altered membrane sterol composition on the temperature dependence of yeast mitochondrial ATPase

The sterol content of cells of $\text{Saccharomyces cerevisiae}$ was manipulated by growing the organism anaerobically in a medium containing excess supplements of unsaturated fatty acids and a range of supplements of ergosterol. Anaerobic mitochondrial precursor structures were isolated whose membrane lipids contain the same fatty acid composition but whose sterol content varies from 7 to 105 mg/g mitochondrial protein. Arrhenius plots of the mitochondrial ATPase activity of the different preparations show a discontinuity with Arrhenius activation energies of about +40 and +80 KJ/mole, respectively, above and below the transition temperature. However, the temperature of the transition is markedly dependent on sterol composition, and increases by up to 17° as the sterol content of the mitochondria is progressively decreased. These results support the concepts that membrane lipid composition influences the activity of membrane-bound enzymes, and that sterols promote the gel to liquid phase transition in biological membranes.     

Effect of altered sterol composition on the salt tolerance of Zygosaccharomyces rouxii

To obtain mutants containing altered sterol composition and sterol contents, nystatin-resistant mutants were isolated in Zygosaccharomyces rouxii. Two of nine mutants isolated were resistant toward 20 μg of nystatin per ml, while the other seven showed resistance toward 50 μg per ml. However, the seven mutants could not grow at 35°C. TN5, a mutant of the first group, showed the same sterol composition as the wild type strain, with ergosterol and zymosterol as major sterols, whereas it contained free sterols about 70% of those of the wild type. TN1 and TN3, representative mutants of the second group, had altered sterol compositions, containing three major sterols, zymosterol, ergosta-5,7,24-trienol, and an unidentified sterol. TN1 and TN3 could not grow in YPD medium containing more than 8% NaCl, whereas TN5 grew in the same medium containing 15% NaCl after a longer lag phase than the wild type strain. TN1 and TN3, in particular TN3, when incubated in YPD medium containing 15% NaCl, leaked significant amounts of glycerol. Protoplasts of these mutants were more labile than those of the wild-type cells. These facts suggest that the amount and kind of ergosterol in the cell membrane might be concerned with the salt tolerance of Z. rouxii.     

Annual Variation in the Effect of Red Light on Sterol Biosynthesis in Digitalis purpurea L

The effect of varying sequences of red and far red light on sterol biosynthesis in etiolated seedlings of Digitalis purpurea L. was examined. Red light caused a marked increase in the amounts of free and glycosidic sterols and a small decrease in esterified sterols during the first 4 hours after illumination. Far red light elicited the same response but to a lesser degree. Exposure to red followed by far red light or the reverse caused little or no increase in the amounts of free and glycosidic sterols. The magnitude of the increase in the amounts of sterols varied, depending upon the season in which the experiments were performed. The largest increments were obtained during the summer and fall, whereas the smallest were observed during the winter and spring. Correlation of these data with previous observations of an annual cycle in the sterol content of Digitalis seedlings showed that the maximum stimulation in sterol biosynthesis occurs when the endogenous level of sterols is minimal.

Sterol monoglycosides, acylmonoglycosides, and an unidentified sterol conjugate from the lipid extracts were quantitated. Changes in conjugated sterol content were related to the particular light conditions of each experiment. The results are discussed in terms of physiological cycles and the possible influence of hormones upon the control of sterol biosynthesis in Digitalis.

     

Lipids and sterols of Pinus sylvestris L. sapwood and heartwood

Summary The lipid and sterol content and composition of three lipid fractions (free fatty acids/ sterols, triacylglycerols and sterol/triterpenoid esters) extracted from three stem discs of Pinus sylvestris were assessed to investigate metabolic changes related to heartwood formation. The wood was separated into (1) cambial zone, (2) outer sapwood, (3) inner sapwood, (4) transition zone, (5) outer heartwood and 6) inner heart-wood. The fractions were separated by thin-layer chromatography (TLC) and analysed by gas-liquid chromatography (GLC). The amount of fatty acids of sapwood triacylglycerols was about 1.5% (dry wt.) but a large reduction occurred in the transition zone. In contrast, noticeable amounts of free fatty acids were present only in the heart-wood. The most important fatty acids in the sapwood fractions were 16:0, 18:0, 18:1, 18:2 (the dominant fatty acid in all fractions), 18:3 and 20:3. Together 18:1 and 18:2 formed about 70% of the total triacylglycerol fatty acids. Of the sterol/ triterpenoid esters, 18:2 and 18:3 were predominant. The fatty acid composition of all fractions changed in the transition zone. The sterols found were sitosterol, stigmastanol, campesterol and campestanol. The amount of sterol esters increased towards the heartwood, and the amount of free sterols was lowest in the inner sapwood. Sitosterol was the dominant sterol in both free sterols and sterol esters.     

Lipid biomarkers in Symbiodinium dinoflagellates: new indicators of thermal stress

Lipid content and fatty acid profiles of corals and their dinoflagellate endosymbionts are known to vary in response to high-temperature stress. To better understand the heat-stress response in these symbionts, we investigated cultures of Symbiodinium goreauii type C1 and Symbiodinium sp. clade subtype D1 grown under a range of temperatures and durations. The predominant lipids produced by Symbiodinium are palmitic (C16) and stearic (C18) saturated fatty acids and their unsaturated analogs, the polyunsaturated fatty acid docosahexaenoic acid (C22:6, n-3; DHA), and a variety of sterols. Prolonged exposure to high temperature causes the relative amount of unsaturated acids within the C18 fatty acids in Symbiodinium tissue to decrease. Thermal stress also causes a decrease in abundance of fatty acids relative to sterols, as well as the more specific ratio of DHA to an algal 4-methyl sterol. These shifts in fatty acid unsaturation and fatty acid-to-sterol ratios are common to both types C1 and D1, but the apparent thermal threshold of lipid changes is lower for type C1. This work indicates that ratios among free fatty acids and sterols in Symbiodinium can be used as sensitive indicators of thermal stress. If the Symbiodinium lipid stress response is unchanged in hospite, the algal heat-stress biomarkers we have identified could be measured to detect thermal stress within the coral holobiont. These results provide new insights into the potential role of lipids in the overall Symbiodinium thermal stress response.     

Fatty Acid and Sterol Composition of Mucor genevensis in Relation to Dimorphism and Anaerobic Growth

Fatty acid and sterol content and composition were determined for the dimorphic mold, Mucor genevensis, grown under a variety of experimental conditions. Fatty acids account for 6 to 9% of the dry weight of aerobically grown mycelium, and 70 to 80% of these are unsaturated. The organism contains γ-linolenic acid which is characteristic for Phycomycetes, and in sporangiospores this compound represents 40% of the total fatty acids. Of the sterols found in mycelium, 80% is ergosterol, and stigmasterol was positively identified as one of the minor components. In anaerobically grown yeastlike cells, sterol content is less than 10% of the level found in aerobically grown cells, and fatty acids amount to less than 2% of the dry weight. These fatty acids are predominantly short chain and less than 10% are unsaturated. Yeastlike cells obtained under aerobic conditions by growth in the presence of phenethyl alcohol have fatty acid and sterol compositions characteristic of aerobically grown mycelium. It is concluded that the dimorphology of the organism is not directly related to lipid composition.