Incorporation of methionine-[methyl-2H3] and mevalonic acid-[2-14C,(4R)-4-3H1] into Phycomyces blakesleeanus sterols

Ergosterol, episterol, 4α-methyl-5α-ergosta-8,24(28)-dien-3β-ol and 24-methylene-24,25-dihydrolanosterol, isolated from Phycomyces blakesleeanus grown in the presence of methionine-[methyl-²H₃], each contained two deuterium atoms; lanosterol, however, was unlabelled. The ¹⁴C:³H atomic ratio of the following sterols isolated from P. blakesleeanus grown in the presence of mevalonic acid-[2-¹⁴C,(4R)-4-³H₁], was: ergosterol, 5:3; episterol, 5:4; ergosta-5,7,24(28)-trien-3β-ol, 5:3; 4α-methyl-5α-ergosta-8,24(28)-dien-3β-ol, 5:4; 24-methylene-24,25-dihydrolanosterol, 6:5; lanosterol, 6:5. The significance of these results in terms of ergosterol biosynthesis is discussed.     

Effect of ay-9944 on sterol biosynthesis in Chlorella sorokiniana

When Chlorella sorokiniana was grown in the presence of 4 ppm AY-9944 total sterol production was unaltered in comparison to control cultures. However, inhibition of sterol biosynthesis was shown by the accumulation of a number of sterols which were considered to be intermediates in sterol biosynthesis. The sterols which were found in treated cultures were identified as cyclolaudenol, 4α,14α-dimethyl-9β,19-cyclo-5α-ergost-25-en-3β-ol, 4α,14α-dimethyl -5α-ergosta-8,25-dien-3β-ol, 14α-methyl-9β,19-cyclo-5α-ergost-25-en-3β-ol, 24-methylpollinastanol, 14α-methyl-5α-ergost-8-en-3β-ol, 5α-ergost -8(14)-enol, 5α-ergost-8-enol, 5α-ergosta-8(14),22-dienol, 5α-ergosta-8,22-dienol, 5α-ergosta-8,14-dienol, and 5α-ergosta-7,22-dienol, in addition to the normally occurring sterols which are ergosterol, 5α-ergost-7-enol, and ergosta-5,7-dienol.The occurrence of these sterols in the treated culture indicates that AY-9944 is an effective inhibitor of the Δ⁸ → Δ⁷ isomerase and Δ¹⁴-reductase, and also inhibits introduction of the Δ²²-double bond. The occurrence of 14α-dimethyl-5α-ergosta-8,25-dien-3β-ol and 14α-methyl-9β,19-cyclo-5α-ergost -25-en-3β-ol is reported for the first time in living organisms. The presence of 25-methylene sterols suggests that they, and not 24-methylene derivatives, are intermediates in the biosynthesis of sterols in C. sorokiniana.     

Sterols of the lichen Pseudevernia furfuracea

A mixture of C₂₇, C₂₈ and C₂₉ sterols was isolated from the lichen Pseudevernia furfuracea and characterized by means of GLC and MS. Mono-, di- and tri-unsaturated sterols were identified as well as a small amount of fully saturated sterols (stanols). Only a part of the total sterols present in the lichen tissue was easily extractable with organic solvents. Another portion was only obtained after saponification of the lichen residue remaining after extraction with organic solvents. The composition of these two fractions difrered considerably, the former contained predominantly 5a,8a-epidioxy-5a-ergosta-6,22-dien-3β-ol (ergosterol peroxide) and 24-ethylcholesta-5,22-dien-3β-ol while in the latter 24-ethylcholesta-5,22-dien- 3β-ol and C₂₈ triene sterols were the main components.     

Biosynthesis of Δ5.23 sterols in etiolated coleoptiles from Zea mays

In addition to the previously found ergosta-5, E-23-dien-3β-ol and 5α-ergosta-7, E-23-dien-3β-ol, the following Δ23 sterols have been identified in etiolated maize coleoptiles: cyclosadol, 4α, 14α-dimethyl-5α-ergosta-8, E-23-dien-3β-ol, 4α, 14α-dimethyl-9β, 19-cyclo-5α-ergosta-8, E-23-dien-3β-ol and 4α-methyl-5α-ergosta-7, E-23-dien-3β-ol. The incubation of maize coleoptile microsomes in the presence of cycloartenol and of [¹⁴C-methyl]S-adenosyl methionine gave a mixture of labelled 24-methylene cycloartanol and cyclosadol. No trace of cyclolaudenol could be detected in these conditions. It is suggested that Δ²³ sterols are products of the C-24 methyltransferase reaction and they probably do not arise from a Δ²⁴ → Δ²³ isomerization occurring at a later stage of the biosynthesis. The Δ¹³-sterols may play an intermediary role in the biosynthesis of 24-methyl sterols in this plant material.     

Two new sterols from Chlorella ellipsoidea

Eight sterols were observed in Chlorella ellipsoidea and the four major components were identified as ergosterol, 5α-ergost-7-en-3β-ol, 22-trans-ergosta-5,8(9),22-trien-3β-ol and ergosta-5,8(9)-dien-3β-ol. This is the first report of the latter two sterols from green plants.     

Sterol esters of Phycomyces blakesleeanus

The sterol esters of Phycomyces blakesleeanus are based on ergosterol, episterol, ergosta-7-en-3β-ol, ergosta-7,22-dien-3β-ol, cholesterol, lan     

Dehydrodinosterol,dinosterone and related sterols of a non-photosynthetic dinoflagellate, Crypthecodinium cohnii

The heterotrophic dinoflagellate Crypthecodinium cohnii contained the 4α-methyl sterols, dinosterol, dehydrodinosterol (4α,23,24-trimethylcholesta-5,22-dien-3β-ol) and the tentatively identified 4α,24-dimethyl-cholestan-3β-ol and 4α,24-dimethylcholest-5-en-3β-ol. The major 4-demethyl sterol was cholesta-5,7-dien-3β-ol which was accompanied by a smaller amount of cholesterol and traces of several other C₂₇,C₂₈ and C₂₉ sterols. In addition, a 3-oxo-steroid fraction was isolated and the major component identified as dinosterone (4α,23,24-trimethylcholest-22-en-3-one). The possible biosynthetic relationships of these compounds are discussed.     

Isolation of 4α-methyl-5α-Ergosta-8, 24 (28)-dien-3β-ol from ester fractions of nutritionally deprived neurospora crassa

A method of isolating pure fractions of 4α-methyl-5α-ergosta-8, 24 (28)-dien-3β-ol for sterol intermediate studies is described. Starvation cultures of $\text{Neurospora crassa}$ readily incorporate exogenous mevalonic acid into the sterol ester fraction. Isolation involves a simple solvent extraction and two chromatograms. Only the ester fraction yielded the required purity. Radioactive 4α-methyl-5α-ergosta-8, 24 (28)-dien-3β-ol is readily produced from DL-[2-¹⁴C] mevalonic acid.     

Analysis of sterols in selected bloom-forming algae in China

Sterols, a group of stable lipid compounds, are often used as biomarkers in marine biogeochemical studies to indicate sources of organic matter. In this study, sterols in 13 species of major bloom-forming algae in China, which belong to Dinophyceae, Bacillariophyceae, Ulvophyceae, and Pelagophyceae, were analyzed with gas chromatography-mass spectrometry (GC–MS) to test their feasibility in representing different types of harmful algal blooms (HABs). It was found that (24Z)-stigmasta-5,24-dien-3β-ol (28-isofucosterol) was a major sterol component in green-tide forming macroalga Ulva prolifera. In bloom-forming dinoflagellates Alexandrium spp., Prorocentrum micans and Scrippsiella trochoidea, (22E)-4α,23-dimethyl-5α-ergost-22-en-3β-ol (dinosterol) was detected in addition to cholest-5-en-3β-ol (cholesterol), (22E)-ergosta-5,22-dien-3β-ol, (22E)-stigmasta-5,22-dien-3β-ol and other minor sterol components. In brown-tide forming pelagophyte Aureococcus anophagefferens, (24E)-24-propylcholesta-5,24-dien-3β-ol ((24E)-24-propylidenecholesterol) and (24Z)-24-propylcholesta-5,24-dien-3β-ol ((24Z)-24-propylidenecholesterol) were detected together with cholesterol, (22E)-stigmasta-5,22-dien-3β-ol, stigmast-5-en-3β-ol and campest-5-en-3β-ol. Among the selected bloom-forming diatoms, Chaetoceros sp. and Pseudo-nitzschia spp. only produced cholesterol, while Cylindrotheca closterium produced solely (22E)-ergosta-5,22-dien-3β-ol. Sterol content in four bloom-forming algal species correlates well with their biomass or abundance. It's proposed that 28-isofucosterol could serve as a promising biomarker for green algae in green-tide studies. Dinosterol and (24Z)-24-propylidenecholesterol can be used as potential biomarkers to represent bloom-forming dinoflagellates and pelagophytes, while (22E)-ergosta-5,22-dien-3β-ol is not a good indicator for diatoms.     

Detection of the ergosterol and episterol isomers lichesterol and fecosterol in nystatin-resistant mutants of Neurospora crassa

Wild-type Neurospora crassa is completely inhibited by 5 ppm nystatin. Ultraviolet-induced mutants have been isolated that grow in the presence of 60 ppm of the antibiotic. Gas-liquid chromatographic, mass spectroscopic, and nuclear magnetic resonance analyses showed the wild-type sterols to be ergosterol (ergosta-5,7,22-trien-3β-ol) and episterol (ergosta-7,24(28)-dien-3β-ol) in a 3:1 ratio. The mutants contained lichesterol (ergosta-5,8,22-trien-3β-ol) and fecosterol (ergosta-8,24(28)-dien-3β-ol) in a 2:1 ratio, differing from the wild type only in the position of the B-ring unsaturation. A deficiency of an ergosta-8,24 (28)-dien-3β-ol:ergosta-7,24(28)-dien-3β-ol isomerase is indicated.     

Sterols of Testudodinium testudo (formerly Amphidinium testudo): Production of the Δ8(14) sterol gymnodinosterol and chemotaxonomic relationship to the Kareniaceae

Testudodinium testudo is a peridinin-containing dinoflagellate recently renamed from Amphidinium testudo. While T. testudo has been shown via phylogenetic analysis of small subunit ribosomal RNA genes to reside in a clade separate from the genus Amphidinium, it does possess morphological features similar to Amphidinium sensu stricto. Previous studies of Amphidinium carterae and Amphidinium corpulentum have found the sterols to be enriched in Δ⁸⁽¹⁴⁾ sterols, such as 4α-methyl-5α-ergosta-8(14),24(28)-dien-3β-ol (amphisterol), uncommon to most other dinoflagellate taxa and thus considered possible biomarkers for the genus Amphidinium. Here, we provide an examination of the sterols of T. testudo and show they are dominated not by amphisterol, but rather by a different Δ⁸⁽¹⁴⁾ sterol, (24R)-4α-methyl-5α-ergosta-8(14),22-dien-3β-ol (gymnodinosterol), previously thought to be a major sterol only within the Kareniaceae genera Karenia, Karlodinium, and Takayama. Also found to be present at low levels were 4α-methyl-5α-ergosta-8,14,22-trien-3β-ol, a sterol previously observed in Karenia brevis to be an intermediate in the production of gymnodinosterol, and cholesterol, a sterol common to many other dinoflagellates. The presence of gymnodinosterol in T. testudo is the first report of this sterol as the sole major sterol in a dinoflagellate outside of the Kareniaceae. The implication of this chemotaxonomic relationship to the Kareniaceae is discussed.     

Effect of AY-9944 on sterol biosynthesis in suspension cultures of bramble cells

Bramble suspension cultures normally contain Δ⁵ sterols (sitosterol, campesterol, and isofucosterol). When the cells were grown in a medium supplemented with AY-9944, their content of Δ⁵ sterols was greatly decreased and Δ⁸ sterols accumulated. Six Δ⁸ sterols, including three new compounds, (24R)-24-ethyl-5α-cholest-8-en-3β-ol, stigmasta-8,Z-24(28)-dien-3β-ol, and 4α-methyl-stigmasta-8,Z-24(28)-dien-3β-ol, were identified. AY-9944 probably inhibited the Δ⁸→Δ⁷ isomerase. A stable cell line growing permanently in an AY-supplemented medium was obtained.     

Sterols of Amphidinium species in the Operculatum Clade: Predominance of cholesterol instead of Δ8(14) sterols previously considered Amphidinium-specific biomarkers

The dinoflagellates Amphidinium carterae and Amphidinium corpulentum have been previously characterized as having Δ⁸⁽¹⁴⁾-nuclear unsaturated 4α-methyl-5α-cholest-8(14)-en-3β-ol (C_28:1) and 4α-methyl-5α-ergosta-8(14),24(28)-dien-3β-ol (amphisterol; C_29:2) as predominant sterols, where they comprise approximately 80% of the total sterol composition. These two sterols have hence been considered as possible major sterol biomarkers for the genus. Here, we have examined the sterols of four recently identified species of Amphidinium (Amphidinium fijiense, Amphidinium magnum, Amphidinium theodori, and Amphidinium tomasii) that are closely related to Amphidinium operculatum as part of what is termed the Operculatum Clade to show that each species has its sterol composition dominated by the common dinoflagellate sterol cholesterol (cholest-5-en-3β-ol; C_27:1), which is found in many other dinoflagellate genera, rather than Δ⁸⁽¹⁴⁾ sterols. While the Δ⁸⁽¹⁴⁾ sterols 4α-methyl-5α-cholest-8(14)-en-3β-ol and 4α,23,24-trimethyl-5α-cholest-8(14),22E-dien-3β-ol (C_30:2) were present as minor sterols along with another common dinoflagellate sterol, 4α,23,24-trimethyl-5α-cholest-22E-en-3β-ol (dinosterol; C_30:1), in some of these four species, amphisterol was not conclusively observed. From a chemotaxonomic perspective, while this does reinforce the genus Amphidinium's ability to produce Δ⁸⁽¹⁴⁾ sterols, albeit here as minor sterols, these results demonstrate that caution should be used when considering Δ⁸⁽¹⁴⁾ sterols, especially amphisterol, as Amphidinium-specific biomarkers within these species where cholesterol is the predominant sterol.     

Effect of fenarimol on sterol biosynthesis in suspension cultures of bramble cells

Bramble suspension cultures normally contain Δ⁵-sterols (sitosterol, campesterol and isofucosterol). When the cells were grown in a medium supplemented with fenarimol, 14α-methyl sterols accumulated. Eight 14α-methyl sterols, including two new compounds, 4α,14α-dimethyl-stigmasta-8,Z-24(28)-dien-3β-ol and 14α-methyl-stigmasta-8,Z-24(28)-dien-3β-ol, were identified. Fenarimol probably inhibited the 14α-methyl demethylation. Cell lines growing permanently in 2 fenarimol-supplemented medium were obtained.     

14α-methyl-5α-ergosta-9(11)24(28)-dien-3β-ol a sterol from Gynostemma pentaphyllum

A new sterol isolated from the aerial parts ofGynostemma pentaphyllum has been shown to be 14α-methyl-5α-ergosta-9(11),24(28)-dien-3β-ol.     

Novel dinoflagellate 4α-methylated sterols from four caribbean gorgonians

Fourteen 4α-methyl sterols have been isolated from the gorgonians Briareum asbestinum, Gorgonia mariae, Muriceopsis flavida and Pseudoplexaura wagenaari, including the following five new sterols: 4α-methyl-24-methylene-5α-cholestan-3β-ol, (24R)-4α, 24-dimethyl-5α-cholesta-7,22-dien-3,β-ol, 4α,24S(or 23ξ)-dimethyl-5α-cholest-7-en-3β-ol, (22E, 24R)-4α,23,24-trimethyl-5α-cholesta-7,22-dien-3β-ol and (24R)-4α,24-dimethyl-5α-cholesta-8(14),22-dien-3β-ol. There is strong evidence that these 4α-methyl sterols are synthesized by the algal (dinoflagellate) symbionts (zooxanthellae) of the gorgonians. It is suggested that analysis of 4Δ-methyl sterol mixtures isolated from a zooxanthellae-bearing invertebrate, collected in several different geographic locations, might give information on the specificity of the symbiotic association between a given animal species and a particular strain of zooxanthellae.     

Sterols with unusual nuclear unsaturation from three cultured marine dinoflagellates

Several new 4α-methyl sterols with unusual unsaturation in the Δ⁸⁽¹⁴⁾-or Δ¹⁴-positions, 4α,24S-dimethyl-5α-cholest-8 (14)-en-3β-ol, 4α-methyl-24ξ-ethyl-5α-cholest-8(14)-en-3β-ol, 4α-methyl-24(Z)-ethylidene-5α-cholest-8(14)- en-3β-ol, 4α,23 (or 22),24ξ-trimethyl-5α-cholesta-8(14),22-dien-3β-ol, 4α,24S(or 23ξ)-dimethyl-5α-cholest-14-en-3β-ol and 14-dehydrodinosterol, have been isolated from extracts of the cultured marine dinoflagellates Amphidinium carterae, A. corpulentum and Glenodinium sp. 4α-Methyl-24ξ-ethyl-5α-cholestan-3β-ol was isolated from the steryl ester fraction of Glenodinium sp. The structures of these new sterols are based upon extensive 360 MHz ¹H NMR and MS analyses.     

Sterols from Candida lipolytica yeast grown on n-alkanes

The sterols of Candida lipolytica grown on n-alkanes were isolated by reverse phase HPLC and found to be mainly ergosterol, with small quantities of ergost-7-en-3β-ol, ergosta-7,22-dien-3β-ol, ergosta-7,24(28)-dien-3β-ol and ergosta-5,7,9(11),22-tetraen-3β-ol.     

Inhibition of Sterol Biosynthesis in Chlorella sorokiniana by Triparanol

When Chlorella sorokiniana was cultured in the presence of 1 mg/1 triparanol succinate, there was a 42% reduction in total sterol concentration. Algal biomass was reduced by approximately the same amount. In addition to the cycloartenol, cyclolaudenol, 24-methyl-pollinastanol, ergosta-5, 7-dien-3β-ol, and ergosterol that occur in control culture, pollinastanol, 14α-methyl-5α-ergost-8-en-3β-ol, 5α-ergosta-8, 14, 22-trien-3β-ol, 5α-ergosta-8(14), 22-dien-3β-ol, 5α-ergosta-8(9), 22-dien-3β-ol, 5α-ergosta-8, 14-dien-3β-ol, 5α-ergost-8(9)-3n-3β-ol, 5α-ergost-8(14)-en-3β-ol, 5α-ergosta-7, 22-dien-3β-ol, and 5α-ergost-7-en-3β-ol were isolated and identified from triparanol succinate-treated cells. A biosynthetic pathway for sterol biosynthesis in this organism is postulated based on all the sterols that were isolated and identified in triparanol-treated cultures of C. sorokiniana. Cyclolaudenol appears to be the product of the first alkylation at C-24 in this organism rather than the more common 24-methylene cycloartanol. Since 24-methylene sterols are needed for the second alkylation reaction, this would explain the absence of C-29 sterols in C. sorokiniana. Four of the sterols identified in C. sorokiniana are reported for the first time in a living organism. They are: 24-methyl pollinastanol, 5α-ergosta-8, 14, 22-trien-3β-ol, 5α-ergosta-8(14), 22-dien-3β-ol and 5α-ergost-8(14)-en-3β-ol.     

Sterols of the unicellular algae Nematochrysopsis roscoffensis and Chrysotila lamellosa: isolation of (24E)-24-n-propylidenecholesterol and 24-n-propylcholesterol

Two rare C₃₀-sterols, (24E)-24-n-propylidenecholest-5-en-3β-ol and 24-n-propylcholest-5-en-3β-ol, and (24S)-24-ethylcholesta-5,22-dien- 3β-ol (stigmasterol) are the major sterols of Nematochrysopsis roscoffensis, a Chrysophyte of the Sarcinochrysidales order. This unique sterol composition is different from the sterol contents of other Chrysophytes and justifies the peculiar position of the Sarcinochrysidales, which are by some characteristics morphologically and biologically related to the Phaeophyceae. The presence of (24S)-24-methylcholesta-5,22-dien-3β-ol (24-epibrassicasterol) as a major sterol in Chrysotila lamellosa is in accordance with the few previous results obtained from other Prymnesiophyceae, although the presence of the other major sterol, (24R)-24-ethylcholesta-5,22-dien-3β-ol (poriferasterol) has never been reported in these algae.