Mechanism of alkylation at C-24 during ergosterol biosynthesis in Phycomyces blakesleeanus

Ergosterol isolated from Phycomyces blakesleeanus grown in the presence of methionine-[methyl-²H₃] contained two ²H atoms showing that one ²H atom is lost during transmethylation. Ergosterol isolated from P. blakesleeanus grown in the presence of mevalonic acid-[2-¹⁴C,(4R)-4-³H₁] had a ¹⁴C:³H atomic ratio of 5:3. Chemical degradation of 2,3-dimethylbutanal obtained by ozonolysis of the doubly-labelled ergosterol showed that the ³H atom originally at C-24 of lanosterol is transferred to C-25 of ergosterol during transmethylation. The mechanism of formation of the ergosterol side chain in P. blakesleeanus is presented.     

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.     

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.     

Four new and other 4α-methylsterols in the seeds of Solanaceae

Four new 4α-methylsterols in the seeds of Solanaceae were identified as 31-norlanost-9(11)-enol, 24-methyl-31-norlanost-9(11)-enol, 4α,24-dimethylcholesta-7,24-dienol and 4α-methyl-24-ethylcholesta-7,24-dienol. The other 4α-methylsterols identified in the seeds were 31-norcycloartanol, 31-norcycloartenol, cycloeucalenol, 31-norlanost-8-enol, 31-norlanosterol, obtusifoliol, 4α,14α,24-trimethylcholesta-8,24-dienol, 4α-methylcholest-8-enol, lophenol, 24-methyllophenol, 24-ethyllophenol, gramisterol and citrostadienol. The distribution of these seventeen 4α-methyl- sterols in the seeds of eight species of the Solanaceae was determined.     

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.     

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.     

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.     

hnRNP proteins interact with nascent Adenovirus RNA

By using Adenovirus 2 infected HeLa cells labeled during very brief pulses of (³H)Uridine, we have shown that nascent chains of heterogenous nuclear RNA (hnRNA) were already associated with proteins to form ribonucleoprotein particles (hnRNP). It was also shown that the small Ad2 specific VA RNA was not associated with these hnRNP.     

Carbon-carbon bond cleavage of fucosterol-24, 28-oxide by cell-free extracts of silkworm Bombyx mori.

The 1500 X g supernatant of the silkworm $\text{Bombyx mori}$ gut homogenate catalyzed the conversion of 24, 28-epoxystigmast-5-en-3β-ol(III) to cholesta-5, 24-dien-3β-ol(IV) which is a key step of stigmast-5-en-3β-ol(I) dealkylation in the insects. A structural analog 24, 28-imino-stigmast-5-en-3β-ol(VI) was a potent inhibitor of this conversion.     

Five 3β, 6α-dihydroxysterols in organ-pipe cactus

The sterol diol fraction from the lipids of organ-pipe cactus, Stenocereus thurberi, was separated into five compounds: macdougallin, peniocerol, cyclostenol, stenocereol and thurberol. The last three compounds have not been described before. All compounds were characterized by physical and spectroscopic properties.     

Configuration at C-24 of sterols from the liverwort Palavicinnia lyellii

The 4-desmethylsterol fraction of the liverwort Palavicinnia lyellii is composed of 36% 24β-methylcholest-5-en-3β-ol (dihydrobrassicasterol), 16% 24α-methylcholest-5-en-3β-ol (campesterol), 33% 24α-ethylcholest-5-en-3β-ol (sitosterol) and 15% 24ξ-ethylcholesta-5,22-dien-3β-ol.     

Heterogeneous nuclear ribonucleoprotein A3 is the liver nuclear protein binding to age related increase element RNA of the factor IX gene

Background

In the ASE/AIE-mediated genetic mechanism for age-related gene regulation, a recently identified age-related homeostasis mechanism, two genetic elements, ASE (age-related stability element) and AIE (age-related increase element as a stem-loop forming RNA), play critical roles in producing specific age-related expression patterns of genes.

Principal Finding

We successfully identified heterogeneous nuclear ribonucleoprotein A3 (hnRNP A3) as a major mouse liver nuclear protein binding to the AIE-derived RNAs of human factor IX (hFIX) as well as mouse factor IX (mFIX) genes. HnRNP A3 bound to the AIE RNA was not phosphorylated at its Ser³⁵⁹, while hnRNP A3 in the mouse liver nuclear extracts was a mixture of phosphorylated and unphosphorylated Ser³⁵⁹. HepG2 cells engineered to express recombinant hFIX transduced with adenoviral vectors harboring an effective siRNA against hnRNP A3 resulted in a substantial reduction in hFIX expression only in the cells carrying a hFIX expression vector with AIE, but not in the cells carrying a hFIX expression vector without AIE. The nuclear hnRNP A3 protein level in the mouse liver gradually increased with age, while its mRNA level stayed age-stable.

Conclusions

We identified hnRNP A3 as a major liver nuclear protein binding to FIX-AIE RNA. This protein plays a critical role in age-related gene expression, likely through an as yet unidentified epigenetic mechanism. The present study assigned a novel functional role to hnRNP A3 in age-related regulation of gene expression, opening up a new avenue for studying age-related homeostasis and underlying molecular mechanisms.     

Comparison of proteins bound to heterogeneous nuclear RNA and messenger RNA in HeLa cells.

Ribonucleoprotein particles containing either heterogeneous nuclear RNA or polyribosomal messenger RNA were isolated from growing HeLa cells in order to compare their respective protein components. The major obstacle to analysing the proteins bound to HeLa cell mRNA proved to be the cosedimentation of a large fraction of the mRNP² particles with ribosomal subunits following puromycin or EDTA disassembly of polyribosomes. This was circumvented by oligo(dT)-cellulose chromatography, in which essentially all of the ribosomal subunits passed through the column without retention, while approximately 80% of the pulse-labeled, poly(A)-containing mRNP became bound and could be eluted with formamide. Polyacrylamide gel electrophoresis of the non-bound fraction (ribosomal subunits) revealed polypeptides between 15,000 and 55,000 molecular weight, with no detectable components greater than 55,000. The oligo-(dT)-bound mRNP contained a much simpler protein complement, consisting of three major components having molecular weights of 120,000, 76,000 and 52,000.In the case of the nuclear ribonucleoprotein particles that contain heterogeneous nuclear RNA, oligo(dT)-cellulose chromatography revealed two classes of particles. The first contained 10 to 20% of the hnRNA, did not bind to oligo(dT)-cellulose in 0.25 m-NaCl, 10 mm-sodium phosphate buffer, pH 7.0 (4 °C), and contained primarily a single polypeptide component having an estimated molecular weight of 40,000 (“informofers”). A second population of hnRNP particles comprised approximately 80% of the hnRNA, displayed strong binding to oligo(dT)-cellulose at 0.25 m-NaCl, and contained a very complex population of proteins, having molecular weights between 40,000 and 180,000, the same as unfractionated hnRNP. The results indicate that, at the resolution of gel electrophoresis and at the sensitivity of Coomassie blue dye, the proteins bound to HeLa cell hnRNA are qualitatively distinct from those bound to polyribosomal mRNA and, in addition, that the hnRNP proteins are the more complex of the two. These results are discussed in relation to the possible nucleotide sequence elements in hnRNA and mRNA to which these specific proteins are bound.     

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.     

Direct measurement of androgen receptors in cultured sertoli cells

Cytoplasmic and nuclear forms of macromolecules with the properties of androgen receptors have been demonstrated by direct labeling techniques in cultured Sertoli cells. The cytoplasmic form was excluded from Sephadex G-200 and could be distinguished from androgen binding protein (ABP) on the basis of size, heat stability, relative electrophoretic mobility, and binding complex dissociation rate. When cultured Sertoli cells were incubated with ³H-testosterone, a time- and temperature-dependent accumulation of label into the nuclear fraction was observed, 46% of which crystallized as authentic testosterone. Specific binding was saturable with an apparent association constant of 0.4nM^?1. Approximately 30% of the nuclear bound hormone was extracted within 1 h by 0.4M KCl and 34% of this was associated with macromolecular species as measured by gel filtration. Unlabeled androgens and to some degree progestogens competed with ³H-testosterone for binding sites. These data constitute direct evidence that Sertoli cells contain androgen receptors.     

Synthesis and anti-glioma activity of 25(R)-spirostan-3β,5α,6β,19-tetrol

Malignant gliomas are common and aggressive brain tumours in adults. The rapid proliferation and diffuse brain migration are the main obstacles to successful treatment. Here, we show 25(R)-spirostan-3β,5α,6β,19-tetrol, a polyhydroxy steroid, is capable of suppressing proliferation and migration of C6 malignant glioma cells in a concentration-dependent manner. The compound 25(R)-spirostan-3β,5α,6β,19-tetrol was synthesised by seven steps starting from diosgenin in 8.55% overall yield. The structures of the synthetic compounds were characterised by infrared (IR), ¹H nuclear magnetic resonance (NMR), ¹³C NMR spectra and EA.     

24β-Ethyl-31-norlanosta-8,25(27)-dien-3β-ol and 24β-ethyl-25(27)-dehydrolophenol in seeds of three cucurbitaceae species

The structures of two 4α-methylsterols is isolated from Cucumis sativus(Cucurbitaceae) seeds were determined based mainly on their ¹³CNMR spectra as 24β-ethyl-31-norlanosta-8,25(27)-dien-3β-ol and 24β-ethyl-25(27)- dehydrolophenol, respectively, of which the former is a new sterol from natural sources. These two 4α-methylsterols were identified in the seeds of two other Cucurbitaceae species, Lagenaria leucantha var. Gourda and Citrullus battich. The probable biogenetic significance of the two 4α-methylsterols is discussed. Other 4α-methylsterols identified in the seeds of the three Cucurbitaceae species were obtusifoliol, cycloeucalenol and gramisterol.