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.     

Inhibition of ergosterol biosynthesis in saccharomyces cerevisiae and Ustilago maydis by tridemorph,fenpropimorph and fenpropidin

The structurally related fungicides, tridemorph, fenpropimorph and fenpropidin have been shown to inhibit the sterol Δ¹⁴-reductase and Δ⁸→Δ⁷-isomerase during ergosterol biosynthesis in Saccharomyces cerevisiae and Ustilago maydis. However, although the three fungicides are able to inhibit both enzymes, tridemorph inhibits the Δ⁸-Δ⁷-isomerase better than the Δ¹⁴-reductase whilst the reverse is true for fenpropidin and to a lesser extent for fenpropimorph.     

Ergosterol biosynthesis in Mucor pusillus

Ergosterol, 22-dihydroergosterol, obtusifoliol and 24-methylene-24,25-dihydrolanosterol, isolated from Mucor pusillus grown in the presence of methionine-[methyl-²H₃], each contained two deuterium atoms; lanosterol, however, was unlabelled. Ergosterol and 22-dihydroergosterol, isolated from M. pusillus grown in the presence of mevalonic acid-[2-¹⁴C, (4R)-4-³H₁] had ¹⁴C:³H atomic ratios of 5:3. The significance of these results in terms of sterol biosynthesis in this organism in general and alkylation at C-24 in particular is discussed.     

Biosynthesis of sitosterol in barley seedlings

A method is described for the chemical synthesis of stigmasta-5,24-dien-3β-ol-[26-¹⁴C] and (24S)-24-ethylcholesta-5,25-dien-3β-ol-[26-¹⁴C] (clerosterol). 28-Isofucosterol-[7-³H₂] fed to developing barley seedlings (Hordeum vulgare) was incorporated into sitosterol and stigmasterol confirming the utilisation of a 24-ethylidene sterol intermediate in 24α-ethyl sterol production in this plant. Also, the use of mevalonic acid-[2-¹⁴C(4R)-4-³H₁] verified the loss of the C-25 hydrogen of 28-isofucosterol during its conversion into sitosterol and stigmasterol in agreement with the previously postulated isomerisation of the 24-ethylidene sterol to a Δ²⁴⁽²⁵⁾-sterol prior to reduction. However, feeding stigmasta-5,24-dien-3β-ol [26-¹⁴C] to barley seedlings gave very low incorporation into sitosterol. Attempts to trap radioactivity from mevalonic-[2-¹⁴C(4R)-4-³H₁] in stigmasta-5,24-dien-3β-ol when this unlabelled sterol was administered to barley seedlings gave only a very small incorporation although both 28-isofucosterol and sitosterol were labelled.     

The mechanism of alkylation at C-24 during clionasterol biosynthesis in Monodus subterraneus

Clionasterol isolated from Monodus subterraneus grown in the presence of methionine-[methyl-²H₃] contained four ²H atoms showing the participation of a 24-ethylidene sterol intermediate in its biosynthesis. Clionasterol isolated from M. subterraneus grown in the presence of mevalonic acid-[2-¹⁴C,(4R)-4-³H₁ had a ¹⁴C:₃H atomic ratio of 5:3 indicating that the 24-ethylidene sterol intermediate is reduced directly to clionasterol and not isomerized to a Δ²⁴-sterol which is then reduced.     

Investigations on the Δ23-, Δ24(28)- and Δ25-Sterols of zea mays

The sterols of Zea mays shoots were isolated and characterized by TLC, HPLC, GC/MS and ¹H NMR techniques. In all, 22 4-demethyl sterols were identified and they included trace amounts of the Δ²³-, Δ²⁴- and Δ²⁵-sterols, 24-methylcholesta-5,E-23-dien-3β-ol, 24-methylcholesta-5,Z-23-dien-3β-ol, 24-methylcholesta-5,25-dien-3β-ol, 24-ethylcholesta-5,25-dien-3β-ol and 24-ethylcholesta-5,24-dien-3β-ol. In the 4,4-dimethyl sterol fraction, cycloartenol and 24-methylenecycloartanol were the major sterol components but small amounts of the Δ²³-compound, cyclosadol, and the Δ²⁵-compound, cyclolaudenol, were recognized. These various Δ²³- and Δ²⁵-sterols may have some importance in alternative biosynthetic routes to the major sterols, particularly the 24β-methylcholest-5-en-3β-ol component of the C₂₈-sterols. Radioactivity from both [2-¹⁴C]MVA and [methyl-¹⁴C]methionine was incorporated by Z. mays shoots into the sterol mixture. Although 24-methylene and 24-ethylidene sterols were relatively highly labelled, the various Δ²³- and Δ²⁵-sterols contained much lower levels of radioactivity, which is possibly indicative of their participation in alternative sterol biosynthetic routes. (24R)-24-Ethylcholest-5-en-3β-ol (sitosterol) had a significantly higher specific activity than the 24-methylcholest-5-en-3β-ol indicating that the former is synthesized at a faster rate.     

Studies on the C-24 configurations of Δ7-sterols in theseeds of Cucurbita maxima

Uncertainties surrounding the structures of the Δ⁷-sterols in the seeds of Cucurbita maxima have been resolved. Seven components were found by TLC, GLC, HPLC, mass spectrometry and ¹H NMR. They were 24β-ethyl-5α-cholesta-7,22,25(27)-trien-3β-ol, 24β-ethyl-5α-cholesta-7,25(27)-dien-3gb-ol, avenasterol, spinasterol, 24-dihydrospinasterol, 24ζ-methyllathosterol and 25(27)-dehydrofungisterol. The ¹H NMR spectra indicated that the sterols with an ethyl substituent at C-24 occurred in the absence of their C-24 epimers. This seems to be the first instance of the detection of 25(27)-dehydrofungisterol in a higher plant.     

Small nuclear RNA and VA RNA in nuclear ribonucleoprotein fibrils from adenovirus-2 infected HeLa cells

The small RNA of hnRNP¹ were studied in HeLa cells infected with adenovirus-2. At 15 h post-infection, when 50–60 % of the hnRNA was of viral origin, all the small nuclear RNA of hnRNP from non-infected cells were present in hnRNP from infected cells. The small, virus-encoded VA RNA could not be detected by staining like the snRNA but only after labeling. It represented less than 1 % of the small nuclear RNA in hnRNP. The low level of VA RNA in hnRNP as compared to that of the small nuclear RNA does not favor the hypothesis of a similar function for these 2 classes of small RNA.     

Codisterol and other Δ5-sterols in the seeds of Cucurbita maxima

A substantial amount (ca 18%) of the sterol found in the seeds of Cucurbita maxima had a Δ-bond and consisted of seven components. They were identified as 25(27)-dehydroporiferasterol, clerosterol, isofucosterol, stigmasterol, sitosterol, campesterol and codisterol. The C-24 configuration of each of the sterols was unequivocally established by a ¹H NMR spectral comparison with authentic standards. This is the first time codisterol has been found in a higher plant and also the first time the structures and configurations of the Δ⁵-sterols from a Cucurbitaceae species have been clearly characterized.     

The stereochemistry of biosynthesis of decaprenoxanthin in a cell-free system

Intact cells of Flavobacterium dehydrogenans grown on glucose or acetate did not incorporate mevalonic acid-[¹⁴C]. After treatment with lysozyme the protoplasts were lysed by sonication in a dilute medium containing mevalonic acid-[¹⁴C] and the cell-free system produced incorporated label into uncyclized C₄₀, monocyclic C₄₅ and bicyclic C₅₀ carotenoids of which decaprenoxanthin was the most abundant.With mevalonate-[2-¹⁴C,4R-4-³H₁] the ¹⁴C:³H ratios of the carotenoids showed that the hydrogen atoms at C-2 and C-6 of the ring and that at C-3 of the 1-hydroxy, 2-methyl but-2-ene-4-yl residues of decaprenoxanthin were derived from the 4-pro-R hydrogen atom of mevalonic acid.Mevalonate-[2-¹⁴C,2R-2-³H₁] and mevalonate-[2-¹⁴C,2S-2-³H₁] gave ratios which showed that the C-4 hydrogen atoms of decaprenoxanthin were derived from the 2-pro-S hydrogen atom of mevalonic acid.     

Sterols of Candida tropicalis grown on N-alkanes

Six sterols isolated from the yeast Candida tropicalis were identified as ergosterol (major component), (22E)-ergosta-5,7,9(11),22-tetraen-3β     

The conversion of 5α-lanost-24-ene-3β,9α-diol and parkeol into poriferasterol by the alga Ochromon as malhamensis

The 4,4-dimethylsterols 4α-lanost-24-ene-3β,9α-diol-[2-³H₂] and parkeol-[2-³H₂] were synthesized from lanosterol and subsequently incubated with cultures of Ochromonas malhamensis. 5α-Lanost-24-ene-3β,9α-diol was converted into poriferasterol with three times the efficiency of parkeol. Clionasterol was also found to be labelled from both parkeol and 5α-lanost-24-ene-3β,9α-diol. No significant incorporation of radioactivity into sterols was obtained after feeding 5α-lanost-24-ene-3β,9α-diol to higher plants, the chlorophyte alga Trebouxia, yeast or a cell free homogenate of rat liver.     

Role of endogenous and exogenous cholesterol in liver as the precursor for bile acids in rats

There is considerable evidence suggesting that compartmentalized functional pools of cholesterol in the liver contribute differently to the formation of bile acids as the precursor. The present paper deals with the incorporation of [1-¹⁴C]acetate and of [1,2-³H]cholesterol carried on lipoproteins (LDL and HDL) into biliary bile acids in perfused rat livers and bile-fistula rats. The results showed that endogenous cholesterol synthesized newly from [1-¹⁴C]acetate in the liver was incorporated into both cholic acid and chenodeoxycholic acid in a similar way, while exogenous lipoprotein-[1,2-³H]cholesterol delivered to hepatocytes from hepatic circulation was incorporated into chenodeoxycholic acid at a higher rate.     

Cyclization of squalene-2,3-epoxide to 10α-cucurbita-5,24-dien-3β-ol by microsomes from Cucurbita maxima seedlings

Labelled 10α-cucurbita-5,24-dien-3β-ol was obtained from [3-³H]squalene-2,3-epoxide incubated with microsomes of Cucurbita maxima seedlings. By contrast the lanostane triterpenoids [2-³H]cycloartenol, [2-³H]parkeol and their corresponding derivatives [2,12-³H]11-ketocycloartenol and [2-³H]24,25-dihydro-90α,11a-epoxyparkeol, incubated under the same conditions, gave no rearranged products with a cucurbitane skeleton. These results suggest that biosynthesis of cucurbitane triterpenoids from squalene-2,3-epoxide occurs through direct cyclization without the intermediacy of lanostane-type triterpenoids such as cycloartenol or parkeol. Labelled cycloartenol, α-amyrin, β-amyrin and 24-methylenecycloartanol were also obtained from [3-³H]squalene-2,3-epoxide in the same enzymatic system yielding labelled 10α-cucurbitadienol.     

The sterol composition of mushrooms

Sterols were isolated from ten mushrooms, Hygrocybe punicea, Lampteromyces japonicus, Leucopaxillus giganteus, Lentinus edodes, Flammulina velutipes, Amanita caesarea, Coprinus atramentarius, Russula foetens, R. nigricans and R. senecis. The compositions of the sterol fractions were determined by GLC, combined GC/MS, and ¹H NMR. Ergosterol was present in all the mushrooms. Other sterols found were 5α-cholest-7-en-3β-ol and ergosta-5,7-dien-3β-ol. Ergosta-5,8,22-trien-3β-ol was isolated from F. velutipes.     

The identification of 24-methylene-24,25-dihydrolanosterol and other possible ergosterol precursors in Phycomyces blakesleeanus and Agaricus campestris

The following sterols have been isolated from the fungi, Phycomyces blakesleeanus and Agaricus campestris: ergosterol, lanosterol, 24-methylene-24,25-dihydrolanosterol and episterol. 4,4-Dimethyl-5α-ergosta-8.24(28)-dien-3β-ol and 4α-methyl-5α-ergosta-8,24(28)-dien-3β-ol have been tentatively identified. Evidence for the incorporation of label from l-methionine-[methyl-¹⁴C] into some of these sterols in P. blakesleeanus has been obtained. The significance of these sterols in ergosterol biosynthesis is discussed.     

Overexpression of a sterol C-24(28) reductase increases ergosterol production in Saccharomyces cerevisiae

Three plasmids, pHX4, pHXA4 and pHXC4, containing sterol C-24(28) reductase gene (ERG4) under the control of ERG4, ADH1 or CUP1 promoters, respectively, and the copper resistance gene as the selection marker were constructed, and they were then introduced into Saccharomyces cerevisiae. Ergosterol production in recombinant strains was enhanced. Under the optimal culture condition, ergosterol content in recombinant strains YEH56(pHX4), YEH56(pHXA4) and YEH56(pHXC4) was 1.2, 1.4 and 1.5-fold (47 mg g^–1) of that in the original strain.     

Biosynthesis of the tigloyl esters in Datura: The role of 2-methylbutyric acid

Datura innoxia plants were wick fed with (±)-2-methylbutyric acid-[1-¹⁴C] and harvested after 7 days. The root alkaloids 3α,6β-ditigloyloxytropane and 3α,6β-ditigloyloxytropan-7β-ol were isolated and degraded. In each case the radioactivity was located in the ester carbonyl group indicating that this acid is an intermediate in the biosynthesis of tiglic acid from l-isoleucine. On the other hand, (±)-2-hydroxy-2-methylbutyric acid-[1-¹⁴C], which was fed to hydroponic cultures of Datura innoxia alongside isoleucine[U-¹⁴C] positive control plants, is not an intermediate.     

24α-Methyl-5α-cholest-7-en-3β-ol from seed oil of Helianthus annuus

24-Methyl-5α-cholest-7-en-3β-ol (24-methyllathosterol) isolated from the seed oil of Helianthus annuus was shown to have 24α-configuration by ¹H NMR spectroscopy. The stereochemistry at C-24 of some other 24-alkylsterols isolated from this plant material also was determined.     

The stereochemistry of hydroxylation of the carotenoid lutein in Calendula officinalis

Excised, opening inflorescences of Calendula officinalis incorporated (3RS, 5R)- and (3RS, 5S)-[2-¹⁴C,5-³H₁]mevalonates into the carotenoid fraction. The ¹⁴C:³H ratios of lutein isolated from these tissues showed the hydrogen atom at C-3 of the β-ring is derived from the 5-pro-S position of mevalonate, while that at C-3 of the ε-ring is derived from the 5-pro-R position of mevalonate. Oxidation of lutein to monoketolutein showed that both hydrogen atoms at the C-15,15′ central double bond are derived from the 5-pro-R position of mevalonate.