The effects of temperature acclimation on membrane sterols and phospholipids of Neurospora crassa

Experiments were conducted to examine the effects of temperature acclimation on sterol and phospholipid biosynthesis in Neurospora crassa. Cultures grown at high (37 degrees C) and low (15 degrees C) temperatures show significant differences in free and total sterol content, sterol/phospholipid ratios and distribution of major phospholipid species in total lipids and two functionally distinct membrane fractions. The ratio of free sterols to phospholipids in total cellular lipids from 15 degrees C cultures was found to be about one-half that found at 37 degrees C, whereas sterol/phospholipid ratios of mitochondrial and microsomal membranes were found to be higher at the low growth temperature. Total sterol and phospholipid biosynthetic rates showed parallel reductions in cultures acclimating to a shift from 37 to 15 degrees C growth conditions. Distribution of [14C]acetate label into free sterols was significantly lower under these conditions, however; indicating an increase in the conversion rate of sterols to sterol esters at the lower temperature. Mitochondrial and microsomal membrane fractions showed distinct phospholipid distributions which also differed from total lipid distributions at the two growth temperatures. In each case there was a consistent decrease in phosphatidylcholine and a corresponding increase in phosphatidylethanolamine as growth temperatures were lowered.     

Environmentally-induced changes in the neutral lipids and intracellular vesicles of Saccharomyces cerevisiae and Kluyveromyces fragilis

Saccharomyces cerevisiae, grown aerobically or anaerobically under conditions which induce a requirement for a sterol and an unsaturated fatty acid, synthesized approximately the same amounts of neutral lipid and intracellular low-density vesicles, although the neutral lipids in aerobically-grown cells contained more esterified sterol and less triacylglycerol than those in anaerobically-grown cells. Kluyveromyces fragilis synthesized much less neutral lipid and a smaller quantity of low-density vesicles than S. cerevisiae whether grown at 30°C (generation time 1.1 h) or 20°C (generation time 2.1 h). Both yeasts synthesized highly saturated triacylglycerols, relatively unsaturated phospholipids, and esterified sterols with an intermediate degree of unsaturation irrespective of the conditions under which they were grown. Free sterols in the yeasts were rich in ergosterol and 22(24)-dehydroergosterol, while the esterified sterol fractions were richer in zymosterol.     

Reversal of dietary-induced hyperlipidemia in Biomphalaria glabrata (Gastropoda).

1. To determine if dietary induced hyperlipidemia in snails could be reversed, thin-layer chromatography studies were done on lipids in the digestive gland-gonad (DGG) complex of Biomphalaria glabrata fed either hen's egg yolk for 2 weeks (Group A), yolk for 1 week followed by leaf lettuce for 1 week (Group B), or lettuce for 2 weeks (Group C). 2. The major lipid fractions in Group A were triacylglycerols, phosphatidylethanolamine, and phosphatidylcholine, along with lesser amounts of sterol esters, methyl esters, free fatty acids, free sterols and unidentified phospholipids. 3. Groups B and C had about one-half the amount of triacylglycerols than Group A and did not show sterol esters or methyl esters. 4. The triacylglycerol increase of the DGG resulting from the hen's egg diet could be reversed by returning the snails to the lettuce diet.     

Schistosoma haematobium: neutral lipid composition and release by adults maintained in vitro

Thin-layer chromatographic analyses showed that the major neutral lipid fractions of whole-worm extracts of male and female adult Schistosoma haematobium were free sterols, triacylglycerols and sterol esters. Worm-free incubates of adult worm-pairs contained free sterols only. The major fractions of worm-free incubates from separated worms were free fatty acids and free sterols; traces of triacylglycerols and sterol esters were also detected. Females incubated in a group of ten released more free fatty acids than ten incubated singly. Males incubated singly released more free sterols than a similar number incubated in a group. Females released more free sterols than males.     

Sterols and frost hardening of winter rape

Winter rape ( Brassica napus L., var. olifera cv. Górczanski) seedlings were exposed to hardening conditions and the content and composition of free sterols as well as the ratio of free sterols to total phospholipids were determined. There was a reduction in free sterol content in the leaves at the most advanced stage of hardening. The ratio of free sterol to total phospholipids was significantly reduced by hardening due to a decrease in the level of the former and an increase in that of the latter compounds. There was a negative correlation between this ratio and the temperature at which half of the seedlings died. Thus, adaptation of membranes to temperature takes place also at the level of sterol-phospholipid interactions. Exposing seedlings already hardened to freezing temperatures caused injury higher than 50%, and brought about a drastic increase in the level of free sterols and an elevation in the ratio of free sterols to phospholipids. The results are discussed in terms of a possible role of the molecular architecture of membranes in surviving at subzero temperatures.     

Lipid composition of plasma membranes isolated from sunflower seedlings grown under water-stress

Plasma membranes were isolated by aqueous two-phase-partitioning from sunflower ( Helianthus annuus cv. Isabel) seedlings grown both under field irrigation and dryland conditions. Water-stressed plants showed a decrease in the leaf water potential and in the osmotic potential at full turgor, with the turgor pressure remaining at positive values. Dryland conditions also induced a reduction in the bulk modulus of elasticity. Plasma membranes of irrigated plants were characterized by high contents of phospholipids (68% of total lipids), free sterols (15. 7%) and glycolipids (9. 1%), mainly glycosphingolipids and steryl glycosides. Diacylglycerols, triacylglycerols and free fatty acids were also present. The major phospholipids were phosphatidylcholine and phosphatidylethanolamine with smaller amounts of phosphatidylinositol and phosphatidylglycerol. Following water stress, the plasma membranes showed a reduction of about 24 and 31% in total lipids and phospholipids, respectively. Also the amounts of glycolipids and diacylglycerols decreased significantly upon water stress. There was no change in free fatty acids, however, and triacylglycerols and free sterols increased. As a consequence, the free sterol to phospholipid molar ratio increased from 0. 4 to 0. 7 under water deficit conditions. The ratio of phosphatidylcholine to phosphatidylethanolamine increased from 1. 1 (control plants) to 1. 6 (water-stressed plants), while phosphatidic acid rose to 4% of total phospholipids. Dehydration did not result in any substantial change in the unsaturation level of the individual lipid classes, however. The results show that dryland conditions resulted in a marked alteration in the lipid composition of the sunflower leaf plasma membrane     

Metabolic interconversion of free sterols and steryl esters in Saccharomyces cerevisiae.

The interconversion of free and esterified sterols was followed radioisotopically with [U-14C]acetate and [methyl-14C]methionine. In pulse-chase experiments, radioactivity first appeared mainly in unesterified sterols in exponential-phase cells. Within one generation time, the label equilibrated between the free and esterified sterol pools and subsequently accumulated in steryl esters in stationary-phase cells. When the sterol pools were prelabeled by growing cells aerobically to the stationary phase and the cells were diluted into unlabeled medium, the prelabeled steryl esters returned to the free sterol form under several conditions. (i) During aerobic growth, the prelabeled sterols decreased from 80% to 45% esters in the early exponential phase and then returned to 80% esters as the culture reached the stationary phase. (ii) Under anaerobic conditions, the percentage of prelabeled steryl esters declined continuously. When growth stopped, only 15% of the sterols remained esterified. (iii) In the presence of an inhibitor of sterol biosynthesis, which causes accumulation of a precursor to ergosterol, prelabeled sterols decreased to 40% steryl esters while the precursor was found preferentially in the esterified form. These results indicate that the bulk of the free sterol and steryl ester pools are freely interconvertible, with the steryl esters serving as a supply of free sterols. Furthermore, there is an active cellular control over what types of sterol are found in the free and esterified sterol pools.     

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.     

Modulation of yeast plasma membrane composition of a yeast sterol auxotroph as a function of exogenous sterol

Plasma membranes isolated from a yeast sterol auxotroph (RD5-R) grown on 1, 5, and 15 micrograms ml-1 exogenous concentrations of sterol showed no discontinuity in plots of steady-state fluorescence anisotropy. Liposomes constructed from phospholipid and sterol extracted from RD5-R grown on different sterols indicated that exogenously supplied sterol modulated cellular phospholipids such that lipid-phase transitions were avoided. Liposomes derived from sterol and phospholipid extracted from the same culture exhibited no lipid-phase transitions. However, when phospholipid extracted from a culture grown on a specific sterol was mixed with sterol extracted from a heterologous culture grown on a different sterol to form liposomes, discontinuities were detected in the anisotropy measurements of the liposomes produced. Quantitative analyses revealed that the exogenously supplied sterol coordinately regulated specific phospholipid species, fatty acid composition, and sterol to phospholipid ratios in yeast auxotrophs.     

Fragility of plasma membranes in Saccharomyces cerevisiae enriched with different sterols.

Saccharomyces cerevisiae NCYC 366, grown under strictly anaerobic conditions to induce requirements for an unsaturated fatty acid (supplied by Tween 80) and a sterol, contained free sterol fractions enriched to the extent of 67 to 93% with the exogenously supplied sterol (campesterol, cholesterol, 7-dehydrocholesterol, 22, 23-dihydrobrassicasterol, beta-sitosterol, or stigmasterol). Cells enriched in any one of the sterols did not differ in volume, growth rate, contents of free sterol, esters and phospholipids, or phospholipid composition. Cholesterol-enriched cells contained about 2% more lipid than cells enriched in any of the other sterols, which was largely accounted for by increased contents of triacylglycerols and, to a lesser extent, esterified sterols. Phospholipids were enriched to the extent of about 52 to 63% with C18:1 residues. Cells enriched in ergosterol or stigmasterol were slightly less susceptible to the action of a wall-digesting basidiomycete glucanase than cells enriched with any one of the other sterols. The capacity of the plasma membrane to resist stretching, as indicated by the stability and volume of spheroplasts suspended in hypotonic solutions of buffered sorbitol (particularly in the range 0.9 to 0.7 M), was greater with spheroplasts enriched in sterols with an unsaturated side chain at C17 (ergosterol or stigmasterol) than with any of the other sterols. Plasma membranes were obtained from spheroplasts enriched in cholesterol or stigmasterol and had free sterol fractions containing 70 and 71%, respectively, of the sterol supplied exogenously to the cells. The sterol-phospholipid molar ratios in these membranes were, respectively, 1:7 and 1:8.     

Cholesterol biosynthesis by the cornea. Comparison of rates of sterol synthesis with accumulation during early development

The origin of the cholesterol needed by the cornea for growth and cell turnover was addressed by comparing absolute rates of sterol synthesis with rates of sterol accumulation during early development of the rabbit. Linearity of incorporation of 3H2O and [14C]mevalonate into digitonin-precipitable sterols with time of incubation in vitro and a lack of accumulation of 14C in intermediates of sterol biosynthesis indicated that tritiated water can validly be used to measure rates of sterol synthesis by the cornea. The rate of sterol synthesis per unit weight of rabbit cornea was constant between 14 and 60 days of age at an average 1.03 nmol of 3H of 3H2O incorporated/mg dry cornea per 8 h. Essentially all of the synthesized cholesterol and most of the cholesterol mass was present in corneal epithelium. The cumulative sterol synthesized over the 46-day period studied exceeded the observed rate of cholesterol accumulation by sixfold. Cholesterol synthesized in excess of the growth requirement was likely used to support turnover of the epithelium which was estimated at 9 days. Removal of cholesterol from the cornea by excretion into tear fluid and clearance by high density lipoproteins are also considered.     

Lipids and lipolytic enzymes in the trunkwood ofRobinia pseudoacacia L. during heartwood formation

The radial distribution of membrane and storage lipids was determined in the trunkwood ofRobinia pseudoacacia L. The trees were felled in November at the time of heartwood formation and fluctuations in the amount and composition of phospholipids, free sterols, steryl esters, diand triacylglycerols, and free fatty acids were investigated across the sapwood-heartwood boundary. The individual compounds were identified and quantified by thin layer chromatography, enzymatic and colorimetric assays, and by capillary gas chromatography. Phospholipids show a significant decrease towards the boundary area, and in the heartwood only trace amounts can be detected. The same pattern is observed for free sterols in the sapwood; in the heartwood, however, they reach maximum values with increasing depth of the trunk. Steryl esters exhibit a complementary behaviour by accumulating at the periphery of the heartwood. No concentration changes are found in the total amounts of diacylglycerols and free fatty acids. In contrast, the triacylglycerol concentration declines steadily across the trunk. With regard to qualitative composition, free fatty acids and the fatty acid moieties of the esterified constituents vary in their chain length from 14 to 24 carbon atoms and have up to three double bonds. A radial gradient in the ratio saturated/unsaturated fatty acids can be observed: except for the phospholipid fraction the relative amounts of unsaturated fatty acids increase in centripetal direction. Seven phospholipids were identified: phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, diphosphatidylglycerol, and phosphatidic acid, which constitutes the major proportion. In the sterol group, sitosterol is the most abundant component. The composition of the esterified sterols remains constant across the trunk cross-section, whereas the relative frequencies of individual free sterols change markedly.     

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.     

Sterol composition of yeast organelle membranes and subcellular distribution of enzymes involved in sterol metabolism.

Organelles of the yeast Saccharomyces cerevisiae were isolated and analyzed for sterol composition and the activity of three enzymes involved in sterol metabolism. The plasma membrane and secretory vesicles, the fractions with the highest sterol contents, contain ergosterol as the major sterol. In other subcellular membranes, which exhibit lower sterol contents, intermediates of the sterol biosynthetic pathway were found at higher percentages. Lipid particles contain, in addition to ergosterol, large amounts of zymosterol, fecosterol, and episterol. These sterols are present esterified with long-chain fatty acids in this subcellular compartment, which also harbors practically all of the triacylglycerols present in the cell but very little phospholipids and proteins. Sterol delta 24-methyltransferase, an enzyme that catalyzes one of the late steps in sterol biosynthesis, was localized almost exclusively in lipid particles. Steryl ester formation is a microsomal process, whereas steryl ester hydrolysis occurs in the plasma membrane and in secretory vesicles. The fact that synthesis, storage, and hydrolysis of steryl esters occur in different subcellular compartments gives rise to the view that ergosteryl esters of lipid particles might serve as intermediates for the supply of ergosterol from internal membranes to the plasma membrane.     

Light-mediated regulation of phospholipid synthesis in Rhodopseudomonas sphaeroides.

The relationship between the culture levels of guanosine-5'-diphosphate-3'-diphosphate (ppGpp) and the rates of synthesis and accumulation of cellular phospholipids was examined in cultures of Rhodopseudomonas sphaeroides that had been subjected to immediate decreases in incident light intensity. After a high-to-low light transition of high-light-adapted cells, an immediate inhibition of total cellular phospholipid production occurred coincident with a rapid accumulation of culture ppGpp. The inhibition of phospholipid accumulation occurred at the level of phospholipid synthesis rather than turnover, and both the extent of ppGpp accumulation and the degree of inhibition of phospholipid synthesis were directly dependent upon the magnitude of the light transition. Maximum inhibition (greater than 90%) of the rate of cellular phospholipid synthesis occurred after transitions from 5,350 to 268 1x and lower, including transitions to the dark, with comparable inhibition being exerted upon the rates of synthesis of individual species of phospholipids. Reinitiation of culture phospholipid accumulation in cultures shifted from 5,350 to 1,070 1x and lower occurred 65 to 70 min subsequent to the downshift in light intensity, apparently irrespective of the culture level of ppGpp.     

Essential fatty acid uptake and esterification in primary culture of rat hepatocytes

Primary cultures of adult rat hepatocytes were used to compare the uptake and esterification of essential polyunsaturated fatty acids (18:2, 20:3 and 20:4 of the n-6 series) with those of palmitic and oleic acids. The uptake of unesterified fatty acids was linearly related to the free fatty acid/albumin molar ratio for 14 h and did not depend on the unbound free fatty acid level. Whatever the initial free fatty acid/albumin molar ratio, it dropped to 0.5 +/- 0.1 mM after 14 h, thus showing that hepatocytes have a high capacity for clearing free fatty acids from the medium at high free fatty acid/albumin molar ratios. The free fatty acid uptake become saturable when the free fatty acid and albumin concentrations were raised and the free fatty acid/albumin ratio remained constant. This strongly suggests that albumin-hepatocyte interaction mediates free fatty acid uptake. This uptake was identical whatever the fatty acid tested and did not depend on the relative amounts of fatty acids when they were added simultaneously. Triacylglycerol accumulation and synthesis, monitored by labelled fatty acids, were related to the free fatty acid/albumin molar ratio and exhibited no specificity for the series of fatty acids tested. Triacylglycerols were enriched in all the fatty acids tested by up to 60%, and fatty acid incorporation into diacylglycerols and triacylglycerols reflected the free fatty acid composition of the medium. By contrast, neither the level nor the synthesis of phospholipids varied with free fatty acid/albumin, but the rate of phospholipid turnover depended on the fatty acids tested. Accumulation of these acids was smaller in phospholipids than in triacylglycerols. When linoleic and arachidonic acids were added together, phospholipids (especially phosphatidylethanolamine and phosphatidylinositol) were more enriched in arachidonic acid than triacylglycerols. This might be due to the specificity for fatty acid of the enzymes involved in phospholipid metabolism.     

The study on the interaction between phytosterols and phospholipids in model membranes

Sterols are one of the major components of cellular membranes. Although in mammalian membranes cholesterol is a predominant sterol, in the human organism plant sterols (phytosterols) can also be found. Phytosterols, especially if present in concentrations higher than normal (phytosterolemia), may strongly affect membrane properties. In this work, we studied phytosterol-phospholipid interactions in mixed Langmuir monolayers serving as model membranes. Investigated were two phytosterols, beta-sitosterol and stigmasterol and a variety of phospholipids, both phosphatidylethanolamines and phosphatidylcholines. The phospholipids had different polar heads, different length and saturation of their hydrocarbon chains. The interactions between molecules in mixed sterol/phospholipid films were characterized with the mean area per molecule (A(12)) and the excess free energy of mixing (DeltaG(Exc)). The effect of the sterols on the molecular organization of the phospholipid monolayers was analyzed based on the compression modulus values. It was found that the incorporation of the phytosterols into the phospholipid monolayers increased their condensation. The plant sterols revealed higher affinity towards phosphatidylcholines as compared to phosphatidylethanolamines. The phytosterols interacted more strongly with phospholipids possessing longer and saturated chains. Moreover, both the length and the saturation of the phosphatidylcholines influenced the stoichiometry of the most stable complexes. Our results, compared with those presented previously for cholesterol/phospholipid monolayers, allowed us to draw a conclusion that the structure of sterol (cholesterol, beta-sitosterol, stigmasterol) does not affect the stoichiometry of the most stable complexes formed with particular phospholipids, but influences their stability. Namely, the strongest interactions were found for cholesterol/phospholipids mixtures, while the weakest for mixed systems containing stigmasterol.     

The prostacyclin analogue iloprost and prostaglandin E1 suppress sterol synthesis in freshly isolated human mononuclear leukocytes

The effects of the stable prostacyclin analogue iloprost, prostaglandin E1 and prostaglandin F2 alpha on sterol synthesis were investigated in freshly isolated human mononuclear leukocytes. Incubation of cells for 6 h in a medium containing lipid-depleted serum led to a 3-fold rise in the rate of sterol synthesis from [14C]acetate or tritiated water. Iloprost and prostaglandin E1 added in increasing concentrations at zero time resulted in an inhibition of the synthesis of sterols, the suppression being 50 and 55% at a concentration of 1 mumol/1, respectively. Both prostaglandins yielded a sigmoidal log dose-effect curve. In contrast, prostaglandin F2 alpha had no influence on sterol synthesis up to a concentration of 1 mumol/1. The action of the prostacyclin analogue and prostaglandin E1 on the relative rate of sterol synthesis was not immediate, since the prostaglandins had no effect when given at 6 h to the incubation medium, and the incorporation of [14C]acetate into sterols was measured thereafter. The results suggest that prostacyclin and prostaglandin E1 affect cholesterol synthesis and therefore may play a role in the regulation of cellular cholesterol homeostasis and in the development of atherosclerosis.     

Regulation of sterol biosynthesis inSaccharomyces cerevisiae

Sterol synthesis inSaccharomyces cerevisiae was primarily controlled by the growth rate. At low specific growth rates the intermediates of ergosterol biosynthesis prevailed in cells. At the same time, the total sterol content reached about 6% of dry matter whereas the content of ergosterol was only 2–2.5%, which seems to be the maximum value forS. cerevisiae. After esterification with fatty acids these sterol intermediates are stored in lipid globules together with reserve triacylglycerols. The sporulatingS. cerevisiae cells contained 3.5% sterols and 1.5% ergosterol of dry matter.     

The biosynthesis of the free sterols and sterol esters of Neurospora crassa.

The composition of the free and esterified sterols in Neurospora crassa was examined as a function of incubation time in starvation medium containing [2-14C]mevalonic acid. The 14C incorporation was monitored in nuclear methylated and 4,14-desmethyl sterol fractions. After 7 h incubation, sterol esterification had increased from an initial 5% in the log phase culture to 48% of the total sterol pool, with a concomitant decrease in free sterols. The relationship of the free and esterified sterol components in ergosterol biosynthesis is discussed.