Principal Sterol Research Articles

Sterols of eustigmatophytes

The oyster cannot synthesize sterols from smaller molecules but must obtain them from its diet, which consists of detritus and small organisms, i.e., mostly single-celled algae. Algae differ widely in their effectiveness as oyster food. Small (< 5 microns) algae which are abundant in sterols and polyunsaturated fatty acids appear to be most effective. Recent studies have shown the occurrence of cholesterol in strains of the unicellular algae Tetraselmis, Chaetoceros and Skeletonema, sometimes in large quantities. In the study reported here, six isolates of a recently constructed algal class, the Eustigmatophyceae, have been examined for sterols and fatty acids by gas chromatography and gas chromatography/mass spectrometry. All strains were shown to contain cholesterol as the principal sterol. Two isolates contained large amounts of total sterol (400-1000 fg/cell), and one (Sticho 0-18) also contained large amounts of eicosapentaenoic acid (20:5n-3). These biochemical characteristics are desirable in a potential food source for oysters.

Biosynthesis and transformation of tremorgenic indolediterpenoids by Penicillium paxilli and Acremonium lolii

Two novel metabolites of Penicillium paxilli were isolated and the structures determined, by mass spectrometry and 1H NMR spectroscopy, as 7α-hydroxy-13-desoxy paxilline and 10β-hydroxy-13-desoxy paxilline. Chromatographically similar compounds were also recognized in fermentations of Acremonium lolii producing paxilline. Another indole-diterpenoid, 7α-hydroxy paxilline, which was apparently not tremorgenic, was characterized from P. paxilli, and recognized chromatographically in A. lolii in which it was shown by radiolabelling experiments to arise from transformation of the tremorgenic mycotoxin paxilline. The significance of the new compounds in biosynthetic interrelationships between the various indole-diterpenoid metabolises of Acremonium, Aspergillus, Claviceps and Penicillium is discussed. Other notable aromatic amino acid-derived metabolises of A. lolii were tryptophol and tyrosol that were excreted from mycelia, the principal sterol of which was 5α-ergosta-7,22 E-dien-3β-ol.

Sterols and alkenones of Isochrysis

Sterols were identified and quantified in six marine microalgal strains identified as belonging to the prymnesiophyte genus Isochrysis, several of these strains are in wide use in the commerical mariculture industry. One strain contained only cholesterol, and another contained a complex mixture of Δ 5-sterols and dihydroxysterols. Two strains contained brassicasterol and two others contained epibrassicasterol as the principal sterol. The strains containing brassicasterol or epibrassicasterol also contained long chain alkenones characteristic of some members of the Prymnesiophyceae; whereas, those without brassicasterol or epibrassicasterol contained no long chain alkenones. These qualitative biochemical differences appear to have taxonomic significance and may be important in the value of these algae as live feeds for rearing marine invertebrates.

Sterols and phytyl esters of Arabidopsis thaliana under normal and chilling temperatures

Sitosterol was the principal sterol in Arabidopsis thaliana with campesterol being second most abundant. Small amounts of cholesterol, stigmasterol, campestanol, brassicasterol, stigmastanol, fucosterol and isofucosterol were identified by GC-MS. Free sterol made up 88% of the total sterol, but both esters and glycosides were detected. No significant changes in sterol composition were noted in Arabidopsis thaliana or in mutant PM-11 in response to chilling temperatures. Phytyl esters accumulated in mutant PM-11 after two or more days of chilling temperature, but did not accumulate in the wild type under chilling conditions. The fatty acids in these phytyl esters are atypical of the total fatty acids of Arabidopsis and are dominated by short chain, saturated fatty acids and hexadecatrienoic acid (16:3). The accumulation of these phytyl esters would be consistent with chlorophyll degradation, inhibition of late stages in fatty acid biosynthesis and degradation of the chloroplast.

Identification of two novel dihydroxysterols fromPavlova

AbstractPavlova gyrans andP. lutheri were cultured, and the dihydroxysterols were isolated from the free sterol and the polar sterol fractions. Four dihydroxysterols were detected in amounts greater than 1% of total sterol by gas chromatography and were analyzed by gas chromatography/mass spectrometry. The two principal sterols were isolated by chromatography on alumina followed by hydrophobic Sephadex column chromatography. The two sterols appeared to differ by having either a methyl or an ethyl group at C−24; they were termed “methylpavlovol” and “ethylpavlovol.” Analysis by 400 MHz nuclear magnetic resonance showed that methylpavlovol is 4α,24β‐dimethylcholestan‐3β,4β‐diol and ethylpavlovol is 4α‐methyl,24β‐ethylcholestan‐3β,4β‐diol. The 4α‐methyl,4β‐hydroxy configuration has not been observed previously in a natural sterol. Dihydroxysterols make up approximately one‐third of the total sterols in thesePavlova species. Neither the biosynthetic origin of these dihydroxysterols nor their role in the biochemistry ofPavlova is known.

Sterols of Tetraselmis (Prasinophyceae)

1. 1. Sterols were identified from 11 isolates of Tetraselmis, a unicellular Prasinophyte alga used frequently as food in mariculture. 2. 2. The principal sterol in eight isolates was either 24-methylenecholesterol or 24-methylcholesterol; the latter was determined to be campesterol in all cases. 3. 3. Campesterol is the first 24α sterol to be reported in the Prasinophyceae. 4. 4. In the remaining three isolates, cholesterol was the principal sterol with smaller amounts of 24-methylenecholesterol and campesterol present; in two of these strains total sterol approached 3% of dry weight. 5. 5. This is the first report of cholesterol as the principal sterol of a Prasinophyte; the C28 sterols found in Tetraselmis are the dominant sterols in most Prasinophyceae studied to date.

Sterols ofChaetoceros andSkeletonema

AbstractDietary sterol is required by the oyster for growth, and sterol is believed to be obtained primarily from dietary phytoplankton. Seven isolates ofChaetoceros and one ofSkeletonema, which are of potential use as oyster food, were analyzed for sterol composition using gas chromatography, high‐performance liquid chromatography and gas chromatography/mass spectrometry.Skeletonema and five isolates ofChaetoceros contained cholesterol as their major sterol. Two other isolates ofChaetoceros also contained cholesterol, but 24‐methylenecholesterol was the principal sterol. Cholesterol has rarely been reported as the major sterol from phytoplankton. In view of the widespread occurrence ofSkeletonema andChaetoceros in the marine environment, these algae could be an important source of the oyster's cholesterol.

Brefeldin A reversibly blocks early but not late protein transport steps in the yeast secretory pathway.

We have found that brefeldin A (BFA) inhibited the growth of an ise1 mutant of Saccharomyces cerevisiae. Genetic complementation and mapping studies demonstrated that ise1 was allelic to erg6, a gene required for the biosynthesis of the principal membrane sterol of yeast, ergosterol. Treatment of ise1 cells with BFA resulted in an immediate block in protein transport through the secretory pathway. Vacuolar carboxypeptidase Y (CPY) and the secreted pheromone alpha-factor accumulated as both the core glycosylated (ER) and alpha 1,6 mannosylated (early Golgi) forms in drug-treated cells. The modification of alpha-factor with alpha 1,6 mannose in BFA-treated cells did not appear to result from retrograde transport of the alpha 1,6 mannosyl-transferase into the ER. We found that transport of CPY from medial and late Golgi compartments to the vacuole was unaffected by BFA, nor was secretion of alpha 1,3 mannosylated alpha-factor or invertase blocked by BFA. The effects of BFA on the secretory pathway were also reversible after brief exposure (< 40 min) to the drug. We suggest that the primary effect of BFA in S. cerevisiae is restricted to the ER and the alpha 1,6 mannosyltransferase compartment of the Golgi complex.

The sterol composition of powdery mildews

The sterol composition of conidia of powdery mildews of apple ( Podosphaera leucotricha), barley ( Erysiphe graminis f.sp. hordei), and cucurbits ( Sphaerotheca fuliginea) was very similar, and simple compared with that of other plant pathogenic fungi. Ergosta-5,24(24 1)-dien-3β-ol was the principal sterol in all three species, accompanied by much smaller amounts of 5α-ergosta-7,24(24 1)-dien-3β-ol in apple and barley mildews, and cholesterol in the cucurbit mildew. All the ergostane derivatives found share the structural feature of a 24(24 1)-double bond, suggesting that in powdery mildews sterol side-chain biosynthesis does not proceed beyond the initial introduction of C-24 1.

Sterols of Cajanus cajan and three other leguminosae seeds

The occurrence of 24-epiclerosterol, three Δ 8(14)-sterols [24α- and 24β-methyl-5α-cholest-8(14)-en-3β-ols and 24α-ethyl-5α-cholest-8(14)-en-3β-ol] and four 14α-methyl-Δ 9(11)-sterols [14α,24α- and 14α,24β-dimethyl-5α-cholest-9(11)-en-3β-ols, 14α-methyl-24α-ethyl-5α-cholest-9(11)-en-3β-ol and 14α-methyl-24β-ethyl-5a-cholesta-9(11),25-dien-3β-ol] has been demonstrated in the seeds of four Leguminosae species, Cajanus cajan, Cicer arietinum, Pisum sativum and Vigna mungo. These compounds were minor sterol constituents. In addition, 24α-ethyl-5α-cholest-9(11)-en-3β-ol and 24-methylenepollinastanol were isolated and identified from V. mungo seeds. Sitosterol was the principal sterol in all of the four Leguminosae seeds investigated.

24β-methyl-5α-cholest-9(11)-en-3β-ol, two 24β-alkyl-Δ 5,7,9(11)-sterols and other 24β-alkylsterol from Chlorella vulgaris

Two 24β-alkyl-Δ 5,7-sterols, ergosterol (51% of the sterol mixture) and 7-dehydroporiferasterol (30%), were the principal sterols of Chlorella vulgaris. In addition two 24β-methyl-Δ 9(11)-sterols, 24β-methyl-5α-cholest-9(11)-en-3β-ol and 14α,24β-dimethyl-5α-cholest-9(11)-en-3β-ol, and two 24β-alkyl-Δ 5,7,9(11)-sterols, 9(11)-dehydroergosterol and 24β-ethylcholesta-5,7,9(11),22 E-tetraen-3β-ol, were isolated and identified from among the other Δ 5- and Δ 7-sterols. This is the first report of the natural occurrence of 24β-methyl-5α-cholest-9(11)-en-3β-ol as well as the first report of the detection of 24β-ethylcholesta-5,7,9(11),22 E-tetraen-3β-ol in an alga.

Unusual tetraene sterols in some phytoplankton

AbstractSterols were analyzed from four phytoplankton strains which are under investigation as possible sources of food for oysters in culture. One strain ofPyramimonas contained only 24‐methylenecholesterol as a major sterol component.Pyramimonas grossii, Chlorella autotrophica andDunaliella tertiolecta each contained a complex mixture of C28 and C29 sterols with Δ7, Δ5,7 and Δ5,7,9(11) nuclear double bond systems. Sterols were found both with and without the C‐22 side chain double bond. Ergosterol and 7‐dehydroporiferasterol were the principal sterols in each of the latter three species, which also contained the rare tetraene sterols, 24‐methylcholesta‐5,7,9(11),22‐tetraen‐3β‐ol and 24‐ethylcholesta‐5,7,9(11),22‐tetraen‐3β‐ol.

STEROLS OF CEPHALEUROS (TRENTEPOHLIACEAE), A PARASITIC GREEN ALGA1

ABSTRACT24‐Ethylcholesterol, 24‐ethylcholesta‐5,7,22‐trienol, dihydrolanosterol, 24‐ethylcholesta‐7,22‐dienol, and 4‐methyl‐24‐ethylcholesta‐7,22‐dienol were identified in cultured Cephaleuros in small quantities. Cholesterol made up 19% of the total sterol. The principal sterol, making up 65% of the total sterol, was 4,24‐dimethylcholest‐7‐enol, a new algal sterol. This is the first report of this sterol as the principal sterol of any living organism.

Free and combined sterols ofPavlova gyrans

AbstractPavlova gyrans, a unicellular alga of interest as food for oysters, was cultured axenically and examined for sterol composition. Desmethyl monohydroxy sterols, which are frequently seen in algae, made up 40% of the total sterols and were observed primarily in the free sterol fraction. The principal sterols of this group were 5‐ergostenol, poriferasterol, and clionasterol, as well as some poriferast‐22‐enol and poriferastanol. Several “methyl” sterols with unusual structures made up 27% of the total sterols. The principal “methyl sterols” were 4α‐methyl ergostanol, 4α‐methyl poriferastanol, and 4α‐methyl poriferast‐22‐enol. Methyl sterols were found primarily in the ester fraction. Also observed was a new class of dihydroxysterols composing 33% of the total sterols. These sterols are structurally related to the methyl and desmethyl sterols ofPavlova but contain an extra nuclear hydroxyl which can be acetylated when present on a desmethyl sterol, but which is nonreactive with acetic anhydride in 4α‐methyl sterols. None of these sterols were observed in ester form but are concentrated in the acid‐hydrolyzable, bound fraction. The unique nature of these sterols suggests potential taxonomic utility.

Sterols of some diatoms

Abstract Sterols were identified from seven species of axenically cultured diatoms which may be used for oyster food. Five species contained 24-methylenecholesterol as the major sterol. Stigmasterol was the principal sterol in Amphora coffaeformis while 24-ethylcholesterol was the major sterol in Navicula pelliculosa . Each of the four species of the order Centrales had 24-methylcholesterol, which was all 22-dihydrobrassicasterol (24β-methyl epimer). The 24-methylcholesterol from Nitzschia brevirostris (order Pennales) was all campesterol (24α-methyl epimer). From this and previously published works on diatom sterols, it appears that sterols from the order Centrales are 24β-oriented and those from the order Pennales are 24α-oriented.

Inhibitors of ergosterol biosynthesis and growth of the trypanosomatid protozoan Crithidia fasciculata.

Six nitrogen-, sulfur- and cyclopropane-containing derivatives of cholestanol were examined as inhibitors of growth and sterol biosynthesis in the trypanosomatid protozoan Crithidia fasciculata. The concentrations of inhibitors in the culture medium required for 50% inhibition of growth were 0.32 microM for 24-thia-5 alpha,20 xi-cholestan-3 beta-ol (2), 0.009 microM for 24-methyl-24-aza-5 alpha,20 xi-cholestan-3 beta-ol (3), 0.95 microM for (20,21),(24,-25)-bis-(methylene)-5 alpha,20 xi-cholestan-3 beta-ol (4), 0.13 microM for 22-aza-5 alpha,20 xi-cholestan-3 beta-ol (5), and 0.3 microM for 23-azacholestan-3-ol (7). 23-Thia-5 alpha-cholestan-3 beta-ol (6) had no effect on protozoan growth at concentrations as high as 20 microM. Ergosterol was the major sterol observed in untreated C. fasciculata, but significant amounts of ergost-7-en-3 beta-ol, ergosta-7,24(28)-dien-3 beta-ol, ergosta-5,7,22,24(28)-tetraen-e beta-ol, cholesta-8,24-dien-3 beta-ol, and, in an unusual finding, 14 alpha-methyl-cholesta-8,24-dien-3 beta-ol were also present. When C. fasciculata was cultured in the presence of compounds 2 and 3, ergosterol synthesis was suppressed, and the principal sterol observed was cholesta-5,7,24-trien-3 beta-ol, a sterol which is not observed in untreated cultures. The presence of this trienol strongly suggests that 2 and 3 specifically inhibit the S-adenosylmethionine:sterol C-24 methyltransferase but do not interfere with the normal enzymatic processing of the sterol nucleus. When C. fasciculata was cultured in the presence of compounds 5 and 7, the levels of ergosterol and ergost-7-en-3 beta-ol were suppressed, but the amounts of the presumed immediate precursors of these sterols, ergosta-5,7,22,24(28)-tetraen-3 beta-ol and ergosta-7,24-(28)-dien-3 beta-ol, respectively, were correspondingly increased. These findings suggest that 5 and 7 specifically inhibit the reduction of the delta 24(28) side chain double bond. When C. fasciculata was cultured in the presence of compound 4, ergosterol synthesis was suppressed, but the sterol distribution in these cells was complex and not easily interpreted. Compound 6 had no significant effect on sterol synthesis in C. fasciculata.

Sterol composition in five families of the order Caryophyllales

The sterols of 31 species in the order Caryophyllales were examined including representatives from the families Achatocarpaceae (4 species), Gyrostemonaceae (6 species), Aizoaceae (11 species), Nyctaginaceae (5 species), and Didiereaceae (5 species). In Aizoaceae, Drosenthemum hispidium had Δ 0 sterols as its principal sterols, while all other species of Aizoaceae and all species of Achatocarpaceae, Gyrostemonaceae, Nyctaginaceae, and Didiereaceae contained Δ 5-sterols as principal sterols. None of the species examined in the above families contained Δ 7-sterols as principal sterols. This is in marked contrast to a recent report on five other families of the order Caryophyllales where the principal sterols in 38 of 57 species examined were Δ 7-sterols.

Characterization of acylmono-, mono-, di-, tri- and tetraglycosylsterol and saponin in Adzuki bean (Vigna angularis) seeds.

Five sterylglycosides (acylmono-, mono-, di tri- and tetraglycosylsterol) and a saponin were isolated from Adzuki beans and characterized. In the glycosylsterols, the principal component sterols were sitosterol and stigmasterol; the major sugar component was glucose. The glucose units were shown to be linked by β1,6-bonds. The three oligoglycosylsterols were shown to be gentiobiosylsterol, gentiotriosylsterol and gentiotetraosylsterol; the latter two are novel sterylglycosides. The saponin was identified as glucopyranosyl-(β1→2)-glucopyranurosyl-(β1→3')-soyasapogenol B (Azukisaponin I) which had previously been found in Adzuki beans.

The yeast gene ERG6 is required for normal membrane function but is not essential for biosynthesis of the cell-cycle-sparking sterol.

In Saccharomyces cerevisiae, methylation of the principal membrane sterol at C-24 produces the C-28 methyl group specific to ergosterol and represents one of the few structural differences between ergosterol and cholesterol. C-28 in S. cerevisiae has been suggested to be essential for the sparking function (W. J. Pinto and W. R. Nes, J. Biol. Chem. 258:4472-4476, 1983), a cell cycle event that may be required to enter G1 (C. Dahl, H.-P. Biemann, and J. Dahl, Proc. Natl. Acad. Sci. USA 84:4012-4016, 1987). The sterol biosynthetic pathway in S. cerevisiae was genetically altered to assess the functional role of the C-28 methyl group of ergosterol. ERG6, the putative structural gene for S-adenosylmethionine: delta 24-methyltransferase, which catalyzes C-24 methylation, was cloned, and haploid strains containing erg6 null alleles (erg6 delta 1 and erg6 delta ::LEU2) were generated. Although erg6 delta cells are unable to methylate ergosterol precursors at C-24, they exhibit normal vegatative growth, suggesting that C-28 sterols are not essential in S. cerevisiae. However, erg6 delta cells exhibit pleiotropic phenotypes that include defective conjugation, hypersensitivity to cycloheximide, resistance to nystatin, a severely diminished capacity for genetic transformation, and defective tryptophan uptake. These phenotypes reflect the role of ergosterol as a regulator of membrane permeability and fluidity. Genetic mapping experiments revealed that ERG6 is located on chromosome XIII, tightly linked to sec59.

The yeast gene ERG6 is required for normal membrane function but is not essential for biosynthesis of the cell-cycle-sparking sterol.

In Saccharomyces cerevisiae, methylation of the principal membrane sterol at C-24 produces the C-28 methyl group specific to ergosterol and represents one of the few structural differences between ergosterol and cholesterol. C-28 in S. cerevisiae has been suggested to be essential for the sparking function (W. J. Pinto and W. R. Nes, J. Biol. Chem. 258:4472-4476, 1983), a cell cycle event that may be required to enter G1 (C. Dahl, H.-P. Biemann, and J. Dahl, Proc. Natl. Acad. Sci. USA 84:4012-4016, 1987). The sterol biosynthetic pathway in S. cerevisiae was genetically altered to assess the functional role of the C-28 methyl group of ergosterol. ERG6, the putative structural gene for S-adenosylmethionine: delta 24-methyltransferase, which catalyzes C-24 methylation, was cloned, and haploid strains containing erg6 null alleles (erg6 delta 1 and erg6 delta ::LEU2) were generated. Although erg6 delta cells are unable to methylate ergosterol precursors at C-24, they exhibit normal vegatative growth, suggesting that C-28 sterols are not essential in S. cerevisiae. However, erg6 delta cells exhibit pleiotropic phenotypes that include defective conjugation, hypersensitivity to cycloheximide, resistance to nystatin, a severely diminished capacity for genetic transformation, and defective tryptophan uptake. These phenotypes reflect the role of ergosterol as a regulator of membrane permeability and fluidity. Genetic mapping experiments revealed that ERG6 is located on chromosome XIII, tightly linked to sec59.