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.     

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.     

Relationship Between the Sterol Content of Yeast Cells and Their Fermentation Activity in Grape Must

In grape must of high sugar concentration, yeast growth, the viability rate of “resting” yeast cells, and fermentation activity were stimulated under certain conditions of aeration and temperature. This stimulation might be interpreted as being a result of the yeast cell sterol content. The addition of certain sterols to the fermenting medium was able to increase this sterol content. According to aeration conditions of the medium, which determined the sterol content of yeasts, the sterols added in the medium acted as (i) growth factors, (ii) fermentation inhibitors, and (iii) survival factors for the yeast.     

Sterol and Squalene Content of a Docosahexaenoic-Acid-Producing Thraustochytrid: Influence of Culture Age, Temperature, and Dissolved Oxygen

Thraustochytrid strain ACEM 6063, rich in omega-3 polyunsaturated fatty acids, was cultured at 15°C and 20°C in high (>40%) and low (<5%) dissolved oxygen (DO), and at 25°C in low-DO media. Samples were taken 4, 2, and 0 days before each culture reached peak biomass (T₋₄, T₋₂, and T_p, respectively). Twenty sterols, 13 of which were identified, were detected. Predominant were cholest-5-en-3β-ol, 24-ethylcholesta-5,22E-dien-3β-ol, 24-methylcholesta-5,22E-dien-3β-ol, and 2 coeluting sterols, one of which was 24-ethylcholesta-5,7,22-trien-3β-ol. These 4 sterols comprised 50% to 90% of total sterols. Cultures grown at high DO had simpler sterol profiles than those grown at low DO. Only the 4 sterols mentioned above were present at more than 3% of total sterols in high-DO cultures. In low-DO cultures, up to 6 additional sterols were present at more than 3% of total sterols. Culture age, temperature, and DO influenced squalene and sterol content. Total sterols (as a proportion of total lipids) decreased with increasing culture age. If organisms such as ACEM 6063 are to be used for commercial production of lipid products for human consumption, both their sterol content and factors influencing sterol production need to be characterized thoroughly. Received January 8, 2001; accepted March 6, 2001.     

Effects of lysine deprivation and serum stimulation on the synthesis of non-histone chromosomal proteins by confluent chick fibroblasts

Cellular sterol content and sterol metabolism were studied in diploid human kidney cells in early passages in culture. Cholesterol, the main cellular sterol, was present at levels similar to those reported for other cultured mammalian kidneys. Cholesterol biosynthesis was characterized by a slow conversion of sterol precursors with accumulation of lanosterol and 27 carbon-atom sterols. In the absence of exogenous cholesterol, kidney cells grew slowly and intracellular cholesterol decreased; however, sterol formation from labelled acetate was stimulated. These results suggest that the cholesterol concentration in the culture medium influences the rate of sterol formation by the kidney cell. Furthermore, cholesterol appears to be essential to cultured human kidney and de novo synthesis by the cells in culture is not adequate to meet their requirements for growth.     

Effect of sterol side-chain structure on the feed-back control of sterol biosynthesis in yeast

We measured the incorporation of radiolabeled methionine and acetate into the sterol component of G204, a Saccharomyces cerevisiae mutant strain which is partially heme competent. By comparing the amount of label incorporated into the sterol pool of a control culture, to which no exogenous sterol was added, with a culture which had various sterols added to the growth medium, we were able to determine the specific structural features of ergosterol which facilitate its ability to restrict the sterol biosynthetic pathway. These experiments demonstrate that sterols which contain both a C22 unsaturation and a C24 methyl group are capable of reducing sterol biosynthesis by approx. 50%, regardless of B-ring structure. We examined the regulatory properties of various oxysterols; 24,25-epoxylanosterol reduced endogenous biosynthesis by 49%, whereas all cholesterol derivatives tested, including 25-hydroxycholesterol, had little effect. A new procedure for the synthesis of ergosterol peroxides is also described.     

Sterol biosynthesis de nova via cycloartenol by the soil amoeba Acanthamoeba polyphaga.

The soil amoeba Acanthamoeba polyphaga is capable of synthesizing its sterols de novo from acetate. The major sterols are ergosterol and poriferasta-5,7,22-trienol. Furthermore C28 and C29 sterols of still unknown structure with an aromatic B-ring are also synthesized by the amoeba. The first cyclic sterol precursor is cycloartenol, which is the sterol precursor in all photosynthetic phyla. No trace of lanosterol, which is the sterol precursor in animals and fungi, could be detected. These results show that at least some of the biochemical processes of Acanthamoeba polyphaga might be phylogenetically related to those of unicellular algae. Addition of exogenous sterols to the culture medium does not influence the sterol biosynthesis and the sterol composition of the cells.     

Sterols and sterylglycosides of oats (Avena sativa). Distribution in the leaf tissue and medium-induced glycosylation of sterols during protoplast isolation

Sterols, sterylglycosides (SG), acylated sterylglycosides (ASG) and steroidal saponins of primary leaves of oat ( Avena sativa L. cv. Flämingskrone) were analyzed by thin-layer chromatography, gas-liquid chromatography and high-performance liquid chromatography. Intact leaves, epidermis preparations, epidermis-stripped leaves, isolated protoplasts and chloroplasts were compared. The mesophyll contained 79% of the total leaf sterols, 80% of the SG and 78% of the ASG, but only 33–67% of the saponins. Free sterols, SG and ASG were mainly localized within the mesophyll, whereas steroidal saponins were localized in the epidermis to a significantly higher extent. The sterol parts consisted mainly of sitosterol, stigmasterol. cholesterol. Δ⁵-avenasterol, Δ⁷-avenasterol, campesterol and Δ⁷-cholestenol, and were quantitatively different in different sterol groups. A higher percentage of sitosterol at the expense of stigmasterol was typical for SG and ASG as compared to free sterols. Only minor differences in the sterol composition were found in a given sterol group when isolated from different tissues. Isolated protoplasts contained only 5–9% of the sterols present in mesophyll cells, indicating that the major part of the free sterols was lost during isolation. Exposure of radioactively labelled leaf segments to either buffer or digestion medium induced rapid transformation of sterols to SG and ASG as shown by the shift of radioactivity from free sterols to the glyeosides. This suggests that two sterol pools exist in the cell: one in the plasmalemma, which is accessible to medium-induced transformation, and a second non-accessible pool in the interior membranes (e.g. chloroplasts) of the cell.     

Effects of thiamine and pyridoxine on the content and composition of sterols in Saccharomyces carlsbergensis 4228

The level of sterols in $\text{S. carlsbergensis}$ 4228 cells grown aerobically on a synthetic medium fortified with thiamine was significantly low compared with that in the control cells. The levels of free and esterified sterols in the thiamine-cells were 60% and 10% of the corresponding sterol levels in the control cells, respectively. Analysis by gas-liquid chromatography of non-saponifiable lipids extracted from the cells revealed that the amounts of squalene, lanosterol and two unidentified sterols were higher than those in the control cells and that ergosterol and zymosterol, major sterols in the control cells, were not present. These effects of thiamine on the content and composition of sterols were abolished by the addition of pyridoxine to the medium.     

Studies on the sterols and sterol esters of the intracellular organelles of maize shoots

1. The composition of the esterified and unesterified sterols of the nuclear, chloroplastidic, mitochondrial and microsomal fractions of 21-day-old maize shoots was examined. 2. The microsomal and mitochondrial fractions contain the bulk of the sterols of the tissue. 3. Only 1% of the sterol isolated from all the organelles is esterified. 4. The nuclear fraction has the greatest proportion of esterified sterol and the microsomal fraction the least. 5. 4-Demethyl sterols constitute the bulk of both esterified and unesterified sterols in all organelle fractions. 6. Cholesterol is the major esterified 4-demethyl sterol of the nuclear and chloroplastidic fractions, but only the nuclear fraction has an appreciable proportion of unesterified cholesterol. 7. Sterol esters of linolenic acid are more abundant in the mitochondrial and microsomal fractions than in the other two fractions.     

The effect of sterols and haemin on the growth of the rumen ciliate Ophryoscolex caudatus and some other Entodiniomorphid protozoa

Seven isolates of Ophryoscolex caudatus have been cultured anaerobically in vitro (at a population density of 56/ml) for an average of 18 months each in the presence of bacteria on a reduced buffered salts medium containing prepared fresh rumen fluid with the daily addition of ground wheat and dried grass and with twice weekly dilution of the culture with an equal volume of fresh medium. The ground wheat and dried grass could be replaced by ground wheat coated with β-sitosterol, stigmasterol, ergosterol or α-spinasterol and with β-sitosterol the population density increased to 110/ml. Haemin further increased the population density obtained in the presence of sterol by 9–160%. The population density of cultures of Epidinium ecaudatum caudatum was also increased by sterols and haemin, that of Polyplastron multivesiculatum by sterols only, and some sterols and haemin, under certain conditions, increased that of Entodinium caudatum.     

STEROLS OF 14 SPECIES OF MARINE DIATOMS (BACILLARIOPHYTA)1

The sterol compositions of 14 species of marine diatoms were determined by gas chromatography and gas chromatography-mass spectrometry. A variety of sterol profiles were found. The sterols 24-methylcholesta-5,22E-dien-3β-ol, cholest-5-en-3β-ol, and 24-methylcholesta-5,24(28)-dien-3β-ol, previously described as the most common sterols found in diatoms, were major sterols in only a few of the species. In light of this and other recent data, it is clear that these three sterols are not typical constituents of many diatom species. Most of the centric species examined had 24-methylcholesta-5,24(28)-dien-3β-ol and 24-methylcholest-5-en-3β-ol as two of their major sterols. The exception was Rhizosolenia setigera, which possessed cholesta-5,24-dien-3β-ol as its single major sterol. In contrast to the centric species, the pennate diatoms examined did not have any particular sterols common to most species. Minor levels ofΔ⁷-sterols, rarely found in large amounts in diatoms, were found in four species. C₂₉sterols were found in many species; seven contained 24-ethylcholest-5-en-3β-ol and three contained 24-ethylcholesta-5,22E-dien-3β-ol, reinforcing previous suggestions that C₂₉ sterols are not restricted to higher plants and macroalgae. 24-Ethylcholesta-5,22E-dien-3β-ol may prove to be useful for taxonomy of the genus Amphora and the order Thalassiophysales. A major sterol of Fragilaria pinnata was the uncommon algal sterol 23,24-dimethylcholesta-5,22E-dien-3β-ol. Cholesta-5,24-dien-3β-ol was the only sterol found in the culture of Nitzschia closterium. This differed from previous reports of 24-methylcholesta-5,22E-dien-3β-ol as the single major sterol in N. closterium. Two C₂₈ sterols possessing an unusual side chain were found in Thalassi-onema nitzschioides, a C_28:2 sterol (16%) and a C_28:1 sterol in lower abundance (2.5%), which may be 23-methylcholesta-5,22E-dien-3β-ol and 23-methyl-5α-cholest-22E-en-3β-ol, respectively. The species Cylindrotheca fusiformis, T. nitzschioides, and Skeletonema sp. may be useful as direct sources of cholesterol in mariculture feeds due to their moderate to high content of this sterol.     

Die quantitative verteilung der sterine und sterinderivate in organen von Solanum dulcamara

The following sterols were found in the roots, stems, leaves, unripe and ripe fruits of Solanum dulcamara: cholesterol, sitosterol, stigmasterol, campesterol, brassicasterol, isofucosterol and 24-methylenecholesterol. The most abundant components are cholesterol, sitosterol and stigmasterol (77–84%). In all parts of the plant the sterols are present in the free form and as esters, glycosides and acylated glycosides. The total sterol content and the content of combined forms were determined photometrically. In the leaves 58% of the sterols were found in the form of glycoside (26%), acylated glycoside (29%) and ester (2%). In the roots only 25% of the sterol were found in combined form. In the other organs the ratio of free and combined sterols was intermediate. In all cases, the ester fraction was the least.     

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.     

Towards a commercially potential process: Enzymatic recovery of phytosterols from plant oil deodoriser distillates mixture

In order to examine the industrial potential to indirectly isolate phytosterols from deodoriser distillates (DODs), enzymatic transesterification of an industrial rapeseed and soybean oil DOD mixture with bioethanol was investigated using commercial lipases and a few newly immobilised preparations of lipases. The lipases from different sources and differing preparation forms were evaluated, in terms of thermostability, enzyme efficiency, and toleration of ethanol. Lipozyme 435 and Lipozyme NS-40044 TLL were found to be most effective biocatalysts in catalysing ethanolysis of glycerides and steryl esters from DODs. The optimum conditions are 10% enzyme load (wt% of DODs), ethanol/DODs of 3.0:1.0 (mol/mol), water content 0.125% (based on the weight of total mixture), and reaction at 30 °C for 5 h. The results demonstrated that >95% sterols can be recovered as free form (>85% sterol esters were liberated as free sterols within 4 h). With this process, the system was simplified as fatty acid ethyl esters and free sterol as major components, where free sterols can be recovered via solvent extraction or molecular distillation. Furthermore, a reuse study of enzyme in consecutive batch reactions demonstrated an excellent operation stability and reusability of Lipozyme 435 and Lipozyme NS-40044 TLL with the developed process. This work indicated that the industrially refined waste DODs can be directly subjected to an enzymatic process for high efficacy recovery of phytosterol without any pre-process, driven by robust lipase preparations.     

Sterol content and efficiency of ion uptake by roots of maize genotypes

Data of compartmental analysis of sulphate were compared with the sterol content of roots of differently yielding maize genotypes. In conditions of steady state nutrient supply, sterol content was significantly correlated only with sulphate efflux (co). This increased at increasing concentration of sterols in the roots. Influx to cytoplasm (oc) was evaluated after sulphate deprivation leading to an induced rate of sulphate uptake. This was negatively correlated with sterol content, which was lower in the high than in the low yielding genotypes. When the highest yield genotype was grown at different sulphate concentrations, influx, efflux, root content of sulphate and sterols were positively correlated with the concentration of sulphate in the nutrient medium. Sterol content in roots appears to be controlled by both the genetic settlement and the nutritional status in maize. Low sterol content is connected with a high efficiency of sulphate utilization.     

Composition of sterols from arthrospores and mycelia of the fungus Mucor hiemalis

Sterol composition of the arthrospores and mycelium of the fungus Mucor hiemalis 1156 was studied by the method of chromatography-mass spectrometry. Along with ergosterol, the major sterol of the culture studied, ten minor sterol were identified, which were either precursors or products of ergosterol degradation. The content of individual sterols differed substantially in arthrospores and mycelium, which represent different stages of ontogenetic development of the fungus. In arthrospores, the content of ergosterol was lower than in mycelium (55.9 and 78%, respectively). Among the precursors of ergosterol, methylated sterols predominated in arthrospores (24.1% versus 11.6% in mycelium). Eburicol and 4,4-dimethylfecosterol were the major methylated sterols of arthrospores (10.6 and 8.1%, respectively). In addition, two uncommon and extremely rare sterols, 1-dihydro-dehydroneoergosterol and dehydroneoergosterol, were identified (for the first time in M. hiemalis). These substances, containing a complex system of conjugated double bonds in their A and B rings, are the products of ergosterol degradation. The data on sterol composition are discussed in terms of their morphogenetic implication.     

The effects of altered membrane sterol composition on oxidative phosphorylation in a haem mutant of Saccharomyces cerevisiae

1. The sterol, unsaturated fatty acid and cytochrome contents of cells of a delta-aminolaevulinate synthase mutant of Saccharomyces cerevisiae are manipulated by growing the organism in media containing defined supplements of delta-aminolaevulinate and other porphyrin intermediates. 2. If unsaturated fatty acids are added to the growth medium as Tween 80, sterol content and respiratory cytochromes alone are manipulated. 3. In the presence of delta-aminolaevulinate (10-50mg/1) cells exhibit moderate to high respiratory activity, but growth yields are low, indicating a loss of oxidative phosphorylation. This is associated with the depletion of membrane lipids, either unsaturated fatty acids and sterols together or sterols alone. 4. Sterol depletion leads to the loss of coupled mitochondrial oxidative phosphorylation in vitro. 5. The lesion in oxidative phosphorylation is associated with an increase in the passive permeability of sterol-depleted mitochondria to protons. 6. Arrhenius plots of mitochondrial permeability to protons indicate that the activation energy for proton entry increases as the sterol content of the membranes decreases. 7. Studies on a cytoplasmic petite mutant isolated from strain ole-3, which lacks a functional membrane-bound protein-translocating adenosine triphosphatase, indicate that proton permeability of the petite mitochondria varies as a function of sterol composition in the same way as that of ole-3 grande mitochondria. This indicates that sterols alone are probably directly responsible for the increased proton entry, owing to a reorganization of the lipid in the membrane. 8. Supplemented ole-3 cells with a normal lipid composition and normal or higher than normal respiratory activities have a growth efficiency only 65% of that of the wild-type, indicating that a further lesion in energy metabolism may be present.     

Sterol Composition of the Arthrospores and Mycelium of the Fungus Mucor hiemalis

Sterol composition of the arthrospores and mycelium of the fungus Mucor hiemalis 1156 was studied by the method of chromatography–mass spectrometry. Along with ergosterol, the major sterol of the culture studied, ten minor sterols were identified, which were either precursors or products of ergosterol degradation. The content of individual sterols differed substantially in arthrospores and mycelium, which represent different stages of ontogenetic development of the fungus. In arthrospores, the content of ergosterol was lower than in mycelium (55.9 and 78.0%, respectively). Among the precursors of ergosterol, methylated sterols predominated in arthrospores (24.1% versus 11.6% in mycelium). Eburicol and 4,4-dimethylfecosterol were the major methylated sterols of arthrospores (10.6 and 8.1%, respectively). In addition, two uncommon and extremely rare sterols, 1-dihydro-dehydroneoergosterol and dehydroneoergosterol, were identified (for the first time in M. hiemalis). These substances, containing a complex system of conjugated double bonds in their A and B rings, are the products of ergosterol degradation. The data on sterol composition are discussed in terms of their morphogenetic implication.     

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.