Binding of sterols affects membrane functioning and sphingolipid composition in wheat roots

The present work was devoted to the exploration of the role of sterols in the functioning of membranes in root cells. Membrane characteristics and composition of the membrane lipids in the roots of wheat (Triticum aestivum L.) seedlings treated with exogenous cholesterol and antibiotic nystatin, which specifically binds with endogenous sterols, were analyzed. Cholesterol caused a fall of membrane potential, acidification of the incubation medium, decrease in potassium leakage of roots, and increase in the level of exogenous superoxide radical. Similarly to cholesterol, the application of nystatin also induced the depolarization of the plasma membrane, but in contrast with cholesterol it was accompanied by alkalinization of the incubation medium and decrease in the level of exogenous superoxide radical. Analysis of membrane lipids showed that following nystatin treatment the total sterol content in roots did not change, while the level of complex sphingolipids represented mainly by glycoceramides became higher. Using mass spectrometry with electrospray ionization (+ESI-MS) for the analysis of the glycoceramide composition, we showed that nystatin induced changes in the ratios of molecular species of glycoceramides. It was suggested that the modification of the sterol component of plasma membrane could influence membrane functioning by changing the sphingolipid composition of lipid rafts.     

The replacement of cholesterol by phytosterols and the increase of total sterol content in model erythrocyte membranes

The activity of phytosterols on human organism includes the ability of these compounds to incorporate into membranes. In the consequence the plant sterols are able to increase total sterol concentration in membrane or/and to replace cholesterol molecules. The aim of this work was to compare the influence of both these effects on the properties of model erythrocyte membranes. Moreover, the interactions between the plant sterols (β-sitosterol and stigmasterol) and saturated–monounsaturated phosphatidylcholine were investigated and the condensing and ordering potency of these phytocompounds on membrane phospholipids were thoroughly analyzed. It was found that the addition of the plant sterols into model membrane modifies the condensation, ordering and interactions in the system. Moreover, the replacement of mammalian sterol by phytosterol more strongly influences the model system than even a 10% increase of total sterol concentration induced by the incorporation of the plant sterol, at constant content of cholesterol. The investigated plant sterols at their lower concentration in the mixed system are of similar effect on its properties. At higher content stigmasterol was found to modify the properties of model membrane more strongly than β-sitosterol.     

Influence of sterol structure on yeast plasma membrane properties

Fluorescence anisotropy measurements indicated that physical changes occured in the lipids of plasma membranes of yeast sterol mutants but not in the plasma membrane of an ergosterol wild-type. Parallel experiments with model membrane liposomes verified that the physical changes in lipids observed in the sterol mutants are dependent on the sterol present and not the phospholipid composition. In addition, the physical changes in lipids observed in liposomes derived from wild-type phospholipids were eliminated by addition of ergosterol but persisted in the presence of cholesterol, cholestanol, ergostanol, or sterols from the sterol mutants. No physical changes in lipids were observed, however, in plasma membranes from a sterol auxotroph, even when the auxotroph was grown on cholesterol or cholestanol. The lack of physical changes in lipids in the sterol auxotroph may reflect the ability of the auxotroph to modify its phospholipid composition with respect to its sterol composition. These results indicate that high specificity ‘sparking’ sterol is not required for the regulation of overall bulk lipid properties of the plasma membrane.     

Brachymeria lasus and Pachycrepoideus vindemiae: Sterol requirements during larval growth of two hymenopterous insect parasites reared in vitro on chemically defined media

Brachymeria lasus and Pachycrepoideus vindemiae failed to develop in vitro on sterol-free artificial media, and dietary acetate and squalene failed to maintain and/or support growth. The sterols, cholesterol, cholestanol, β-sitosterol, 7-dehydrocholesterol, and cholesterol linoleate were all utilized and maintained larvae of both species. Larval survival and development rate were greatest with cholesterol followed by cholestanol, β-sitosterol and 7-dehydrocholesterol. Although cholesterol linoleate maintained larvae little growth occurred and mortality was high. Cholestanol followed by β-sitosterol and 7-dehydrocholesterol displayed partial cholesterol sparing activity. Cholesterol linoleate had little effect on larval growth when fed with suboptimal levels of cholesterol or cholestanol. Both species contained 5 to 10% of the total body lipids as free sterol with traces of sterol ester. The major free sterol appears to be cholesterol.     

Biogas digestate as a sustainable phytosterol source for biotechnological cascade valorization

Every year, several million tonnes of anaerobic digestate are produced worldwide as a by-product of the biogas industry, most of which is applied as agricultural fertilizer. However, in the context of a circular bioeconomy, more sustainable uses of residual digestate biomass would be desirable. This study investigates the fate of the sterol lipids β-sitosterol and cholesterol from the feedstocks to the final digestates of three agricultural and one biowaste biogas plants to assess if sterols are degraded during anaerobic digestion or if they remain in the digestate, which could provide a novel opportunity for digestate cascade valorization. Gas chromatographic analyses showed that feedstock sterols were not degraded during anaerobic digestion, resulting in their accumulation in the digestates to up to 0.15% of the dry weight. The highest concentrations of around 1440 mg β-sitosterol and 185 mg cholesterol per kg dry weight were found in liquid digestate fractions, suggesting partial sterol solubilization. Methanogenic batch cultures spiked with β-sitosterol, cholesterol, testosterone and β-oestradiol confirmed that steroids persist during anaerobic digestion. Mycobacterium neoaurum was able to transform digestate sterols quantitatively into androstadienedione, a platform chemical for steroid hormones, without prior sterol extraction or purification. These results suggest that digestate from agricultural and municipal biowaste is an untapped resource for natural sterols for biotechnological applications, providing a new strategy for digestate cascade valorization beyond land application.     

Studies on β-sitosterol and ceramide-induced alterations in the properties of cholesterol/sphingomyelin/ganglioside monolayers

Phytosterol—β-sitosterol promotes apoptosis in various cancer cells and inhibits their growth. Supplementation of cancer cells with this compound causes modifications in membrane composition, namely, substitution of cholesterol (Chol), decrease of sphingomyelin (SM) content and increase of ceramide (Cer) level. The aim of this work was to investigate the influence of partial replacement of cholesterol by plant sterol, substitution of sphingomyelin by ceramide and both these factors simultaneously on the properties of the monolayers composed of major lipids identified in breast cancer membranes, namely Chol/SM/GM3 mixtures. Brewster Angle Microcopy experiments and the analysis of the isotherms recorded during films compression and resulting parameters evidenced that β-sitosterol weakens the interactions between molecules, decreases films stability and condensation. The influence of ceramide on sterol/SM/GM3 films was reflected in strong modifications of their texture, however, the morphology of monolayer was determined by the structure of sterol present in the system. It was also found, that simultaneous replacement of 50 mol% of Chol and SM by phytosterol and Cer, respectively, induces lipids segregation, which is manifested in large diversity of phases observed in BAM images. To facilitate the analysis of the data collected for multicomponent monolayers, the properties of selected sterol/GM3, sterol/Cer, SM/GM3, Cer/GM3 binary films were also investigated. The obtained results evidenced that the studied herein modifications in the composition of Chol/SM/GM3 monolayer, reflecting compositional alterations induced by phytosterol in cancer membranes, strongly affect the organization of model system, therefore they should be considered in the studies on anticancer mechanism of β-sitosterol.     

Changes in Sterol Composition during Greening of Etiolated Barley Shoots

The following sterols were identified in barley shoots: stigmasterol, β-sitosterol, campesterol, and cholesterol. The total sterol content of green and etiolated tissue was 2.84 and 3.20 milligrams per gram dry weight, respectively. The free sterols accounted for most of the difference in total sterol content. The sterol ester, sterol glycoside, and acylated sterol glycoside contents of green and etiolated barley shoots were essentially the same. Etiolated tissue had twice as much total β-sitosterol as stigmasterol, while green tissue had equal amounts of these two sterols. The campesterol and cholesterol content was the same in green and etiolated tissue. This same sterol composition pattern held true for the free, glycosidic, and acylated glycosidic sterols; however, the sterol ester fraction had a completely different composition pattern. The esterified stigmasterol content was quite low in green and etiolated tissue, and campesterol was the second largest esterfied sterol component in etiolated tissue. Etiolated barley seedlings exposed to light had a shift in the ratio of free stigmasterol to β-sitosterol in favor of stigmasterol; however, no correlation was observed between chlorophyll synthesis and shift in sterol composition.     

Sterols in Germinating Seeds and Developing seedlings of Longleaf Pine, Pinus palustris

Sterols in germinating embryos and young seedlings of longleaf pine (Pinus palustris Mill.) were identified and quantities determined for different periods after germination. Sterol analyses were performed by gas-liquid chromatography (GLC) and verified by combination of GLC-mass spectrometry. Campesterol and β-sitosterol were two major sterols which accounted for most of the sterol composition while stigmasterol was present in very small amounts. No cholesterol was revealed by GLC-mass spectrometry although there was a minor peak appearing on the sterol gas-liquid chromatograms with a retention time close to that of authentic cholesterol. By fractionation, three different forms of sterols were obtained: steryl esters, steryl glycosides, and free sterols. The sterols were mainly found in the esterified fraction, while steryl glycosides and free sterols only made up a small portion of the total sterol value. The total sterol content in general increased during seedling development, and this increase reflected mainly a change in steryl esters. The low levels of both free and glycosidic sterols remained nearly unchanged throughout the experimental germination period.     

Sterol and fatty acid composition of polyene macrolide antibiotic resistant Torulopsis glabrata.

Successive reculturing of Torulopsis glabrata on media containing increasing concentration of the polyene macrolide antibiotics nystalin or lucensomycin resulted in the segregation of cultures resistant to these antibiotics. Isolates resistant to lucensomycin showed good resistance to nystatin, and vice versa. Analysis of the sterols and fatty acids of sensitive and polyene resistant T. glabrata revealed that compositional changes occurred in both classes of lipids upon acquistion of resistance. The sterol composition of nystatin and lucensomycin resistant cultures possessed reduced amounts of, or no ergosterol (the major sterol of the sensitive parent culture), and increased amounts of sterols which were biogenetically more primitive than ergosterol. Resistant cultures in which ergosterol was absent possessed a fatty acid composition that did not differ significantly from the parent sensitive culture grown under identical conditions. Resistant cultures containing significantly reduced amounts of ergosterol were found to possess altered fatty acid compositions. Generally it was observed that these latter cultures possessed fatty acids containing shorter and more saturated chains. These results are considered to indicate that alteration in both lipid and sterol composition is involved in determination of culture resistance to polyene macrolides.     

Lipid acyl chain-dependent effects of sterols in Acholeplasma laidlawii membranes.

Acholeplasma laidlawii was grown with different fatty acids for membrane lipid synthesis (saturated straight- and branched-chain acids and mono- and di-unsaturated acids). The ability of 12 different sterols to affect cell growth, lipid head group composition, the order parameter of the acyl chains, and the phase equilibria of in vivo lipid mixtures was studied. The following two effects were observed with respect to cell growth: with a given acyl chain composition of the membrane lipids, growth was stimulated, unaffected, reduced, or completely inhibited (lysis), depending on the sterol structure; and the effect of a certain sterol depended on the acyl chain composition (most striking for epicoprostanol, cholest-4-en-3-one, and cholest-5-en-3-one, which stimulated growth with saturated acyl chains but caused lysis with unsaturated chains). The three lytic sterols were the only sterols that caused a marked decrease in the ratio between the major lipids monoglucosyldiglyceride and diglucosyldiglyceride and hence a decrease in bilayer stability when the membranes were enriched in saturated (palmitoyl) chains. With these chains correlations were found for several sterols between the glucolipid ratio and the order parameter of the acyl chains, as well as the lamellar-reversed hexagonal phase transition, in model systems. A shaft experiment revealed a marked decrease in the ratio of monoglucosyldiglyceride to diglucosyldiglyceride with the lytic sterols in unsaturated (oleoyl) membranes. The two cholestenes induced nonlamellar phases in in vivo mixtures of oleoyl A. laidlawii lipids. The order parameters of the oleoyl chains were almost unaffected by the sterols. Generally, the observed effects cannot be explained by an influence of the sterols on the gel-to-liquid crystalline phase transition.     

Where sterols are required for endocytosis

Sterols are essential membrane components of eukaryotic cells. Interacting closely with sphingolipids, they provide the membrane surrounding required for membrane sorting and trafficking processes. Altering the amount and/or structure of free sterols leads to defects in endocytic pathways in mammalian cells and yeast. Plasma membrane structures functioning in the internalization step in mammalian cells, caveolae and clathrin-coated pits, are affected by cholesterol depletion. Accumulation of improper plasma membrane sterols prevents hyperphosphorylation of a plasma membrane receptor in yeast. Once internalized, sterols still interact with sphingolipids and are recycled to the plasma membrane to keep an intracellular sterol gradient with the highest amount of free sterols at the cell periphery. Interestingly, cells from patients suffering from sphingolipid storage diseases show high intracellular amounts of free cholesterol. We propose that the balanced interaction of sterols and sphingolipids is responsible for protein recruitment to specialized membrane domains and their functionality in the endocytic pathway.     

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.     

Energy metabolism in wheat root cells under modification of plasma membrane permeability by antibiotic nystatin

This paper reports changes in ion transport and energy metabolism of plant cells during short- and long-term expositions, resp., to antibiotic nystatin, which is known to specifically bind with plasma membrane sterols to form channels. The excised roots of 5 days old wheat seedlings were used as a model system in this research. It has been shown that treatment of excised roots with nystatin leads to activation of energy metabolism expressed as an increase of respiration and heat production by root cells. Furthermore, in the presence of nystatin increased pH of incubation medium, plasma membrane depolarization and a significant loss of potassium ions were observed. Nystatin-induced stimulation of respiration was prevented by malonate, an inhibitor of succinate dehydrogenase, electron acceptor dichlorophenolindophenol, and AgNO3, an inhibitor of H(+)-ATPase. Based on the data obtained it can be suggested that nystatin-induced stimulation of respiration is related to electron transport activation via mitochondrial respiratory chain, and is connected with activation of plasmalemma proton pump. Moreover, nystatin-induced increase of oxygen consumption was prevented by cerulenin, an inhibitor of fatty acid and sterol synthesis. This indicates that additional sterols and phospholipids may be synthesized in root cells to "heal" nystatin-caused damage of plasma membrane. A supposed chain of events of cell response to nystatin action may by as following: formation of nystatin channels-influx of protons--depolarization of plasmalemma-efflux of potassium ions-disturbance of ion homeostasis--activation of H(+)-ATPase work-increase in energy "requests" for H(+)-ATPase function--increase in the rate of oxygen consumption and heat production. The increased energy production under the action of nystatin, may provide the work of proton pump and synthesis of sterols and phospholipids, which are necessary for membrane regeneration.     

Plasma Membrane Lipid Alterations Associated with Cold Acclimation of Winter Rye Seedlings (Secale cereale L. cv Puma)

Highly enriched plasma membrane fractions were isolated from leaves of nonacclimated (NA) and acclimated (ACC) rye (Secale cereale L. cv Puma) seedlings. Collectively, free sterols, steryl glucosides, and acylated steryl glucosides constituted >50 mole% of the total lipid in both NA and ACC plasma membrane fractions. Glucocerebrosides containing hydroxy fatty acids constituted the major glycolipid class of the plasma membrane, accounting for 16 mole% of the total lipid. Phospholipids, primarily phosphatidylcholine and phosphatidylethanolamine with lesser amounts of phosphatidylglycerol, phosphatidic acid, phosphatidylserine, and phosphatidylinositol, comprised only 32 mole% of the total lipid in NA samples. Following cold acclimation, free sterols increased from 33 to 44 mole%, while steryl glucosides and acylated steryl glucosides decreased from 15 to 6 mole% and 4 to 1 mole%, respectively. Sterol analyses of these lipid classes demonstrated that free β-sitosterol increased from 21 to 32 mole% (accounting for the increase in free sterols as a class) at the expense of sterol derivatives containing β-sitosterol. Glucocerebrosides decreased from 16 to 7 mole% of the total lipid following cold acclimation. In addition, the relative proportions of associated hydroxy fatty acids, including 22:0 (h), 24:0 (h), 22:1 (h), and 24:1 (h), were altered. The phospholipid content of the plasma membrane fraction increased to 42 mole% of the total lipid following cold acclimation. Although the relative proportions of the individual phospholipids did not change appreciably after cold acclimation, there were substantial differences in the molecular species. Di-unsaturated molecular species (18:2/18:2, 18:2/18:3, 18:3/18:3) of phosphatidylcholine and phosphatidylethanolamine increased following acclimation. These results demonstrate that cold acclimation results in substantial changes in the lipid composition of the plasma membrane.     

Dietary Sterol Preference in the Silkworm,Bombyx mori

Since insects are unable to biosynthesize sterols de novo, sterols must be obtained from dietary sources. Although it has been reported that β-sitosterol is crucial for larval growth in the silkworm, Bombyx mori, little has been investigated concerning the dietary selection of sterols by Bombyx larvae. Here, we demonstrate that Bombyx larvae have the following sterol preference: β-sitosterol >> ergosterol > cholesterol = stigmasterol. Interestingly, Bombyx larvae preferred ergosterol, an inhibitory sterol on larval growth, indicating that sterol selection following first contact of the diet with the mouthpart might be different from the sterol recognition mechanism present in sterol metabolism.     

Externalization of phosphatidylserine from inner to outer layer may alter the effect of plant sterols on human erythrocyte membrane - The Langmuir monolayer studies

One of the factors, which can strongly modify the cell membrane composition, is disordering in membrane asymmetry, resulting from redistribution of lipids from inner to outer layer. Such a disturbance may affect the behavior of various biologically active compounds incorporating into membranes. In this contribution, the relationship between the amounts of phosphatidylserine (PS) in the model outer layer of human erythrocyte (RBC) membrane and the effect induced by a plant sterol (β-sitosterol) was verified. The experiments were performed on multicomponent Langmuir films imitating red blood cell (RBC) membrane, differing in the contents of PS (0%; 5% and 10%) into which the plant sterol was incorporated in various concentrations. The analysis of experimental results (surface pressure-area isotherms complemented with Brewster Angle Microscopy (BAM) proved that the presence of phosphatidylserine molecules, depending on their contents in the mixed monolayer mimicking RBC membrane, changes its properties and exerts influence on the effect of plant sterol on the model system. The addition of phytosterol into the monolayer that lacks or contains only 5% of PS was found to be of rather weak effect on the properties of the system. However, in the case of the model membrane containing the increased amount (10%) of PS, the incorporation of plant sterol strongly affects the interactions between molecules and caused thermodynamic destabilization of the monolayer imitating RBC membrane. These results allow one to suggest that externalization of phosphatidyserine to the outer membrane leaflet may differentiate the effect of plant sterols on cell membranes of various origins.     

Filipin/sterol complexes in fertilized and unfertilized sea urchin egg membranes

Sea urchin (Arbacia punctulata) eggs and zygotes were treated with filipin in an effort to examine changes in membrane sterols at fertilization. The plasma membrane of treated unfertilized eggs possessed numerous filipin/sterol complexes, while fewer complexes were associated with membranes delimiting cortical granules, demonstrating that the plasmalemma is relatively rich in β-hydroxysterols in comparison to cortical granule membrane. Following fusion with the plasmalemma, membrane formerly delimiting cortical granules underwent a dramatic alteration in sterol composition, as indicated by a rapid increase in the number of filipin/sterol complexes. In contrast, portions of the zygote plasma membrane, derived from the plasmalemma of the unfertilized egg, displayed little or no change in filipin/sterol composition. Other than regions of the plasma membrane engaged in endocytosis, the plasmalemma of the zygote possessed a homogeneous distribution of filipin/sterol complexes and appeared similar to that of the unfertilized egg. These results demonstrate that following its fusion with the egg plasmalemma, membranes, formerly delimiting cortical granules, undergo a dramatic alteration in sterol composition. Changes in the localization of filipin/sterol complexes are discussed in reference to alterations in egg plasmalemmal function at fertilization.     

Studies on the Sterol of Bombyx mori L

In whole stages of Bombyx silkworm it was shown with gas-liquid chromatographic systems that silkworm-sterols consist of three sterols such as cholesterol, β-sitosterol, and campesterol, at least, and the sterols in the 5th instar larva contain afore-mentioned three sterols and an additional unknown sterol. Moreover, the sterol composition of the silkworm was studied in various stages.     

Role of ergosterol in growth inhibition of Saccharomyces cerevisiae by syringomycin E

The antifungal activity of the lipodepsipeptide syringomycin E from Pseudomonas syringae pv. syringae is modulated by sterols. To study the requirement of the predominant fungal sterol, ergosterol, in syringomycin E action, the sterol composition of Saccharomyces cerevisiae sterol auxotroph strain FY-14 was modified and sensitivity to syringomycin E examined. Cells containing solely ergosterol, cholesterol, β-sitosterol or stigmasterol were sensitive to syringomycin E with the latter two being the most sensitive. Cells containing growth-promoting cholesterol were the most sensitive and those with growth-promoting ergosterol the least sensitive. It is concluded that sensitivity to syringomycin E is modulated by growth-promoting sterols and does not necessarily require ergosterol.     

Sterols as dietary markers for Drosophila melanogaster

During cold acclimation fruit flies switch their feeding from yeast to plant food, however there are no robust molecular markers to monitor this in the wild. Drosophila melanogaster is a sterol auxotroph and relies on dietary sterols to produce lipid membranes, lipoproteins and molting hormones. We employed shotgun lipidomics to quantify eight major food sterols in total lipid extracts of heads and genital tracts of adult male and female flies. We found that their sterol composition is dynamic and reflective of fly diet in an organ-specific manner. Season-dependent changes observed in the organs of wild-living flies suggested that the molar ratio between yeast (ergosterol, zymosterol) and plant (sitosterol, stigmasterol) sterols is a quantifiable, generic and unequivocal marker of their feeding behavior suitable for ecological and environmental population-based studies. The enrichment of phytosterols over yeast sterols in wild-living flies at low temperatures is consistent with switching from yeast to plant diet and corroborates the concomitantly increased unsaturation of their membrane lipids.