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Showing papers on "Lanosterol published in 1993"


Journal ArticleDOI
TL;DR: The cloning and characterization of CAS1, an Arabidopsis thaliana gene encoding cycloartenol synthase, is reported, which should be generally applicable to genes responsible for secondary metabolite biosynthesis.
Abstract: Whereas vertebrates and fungi synthesize sterols from epoxysqualene through the intermediate lanosterol, plants cyclize epoxysqualene to cycloartenol as the initial sterol. We report the cloning and characterization of CAS1, an Arabidopsis thaliana gene encoding cycloartenol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, cycloartenol forming), EC 5.4.99.8]. A yeast mutant lacking lanosterol synthase [(S)-2,3-epoxysqualene mutase (cyclizing, lanosterol forming), EC 5.4.99.7] was transformed with an A. thaliana cDNA yeast expression library, and colonies were assayed for epoxysqualene mutase activity by thin-layer chromatography. One out of approximately 10,000 transformants produced a homogenate that cyclized 2,3-epoxysqualene to the plant sterol cycloartenol. This activity was shown to be plasmid dependent. The plasmid insert contains a 2277-bp open reading frame capable of encoding an 86-kDa protein with significant homology to lanosterol synthase from Candida albicans and squalene-hopene cyclase (EC 5.4.99.-) from Bacillus acidocalcarius. The method used to clone this gene should be generally applicable to genes responsible for secondary metabolite biosynthesis.

222 citations


Journal ArticleDOI
01 Jul 1993-Lipids
TL;DR: In contrast, nicotinic acid and particularly adenosine caused moderate inhibition of HMG-CoA reductase activity and of cholesterol biosynthesis suggesting that these compounds participate, at least in part, in the early inhibition of sterol synthesis by garlic extracts.
Abstract: Exposure of primary rat hepatocytes and human HepG2 cells to water-soluble garlic extracts resulted in the concentration-dependent inhibition of cholesterol biosynthesis at several different enzymatic steps. At low concentrations, sterol biosynthesis from [14C]acetate was decreased in rat hepatocytes by 23% with an IC50 (half-maximal inhibition) value of 90μg/mL and in HepG2 cells by 28% with an IC50 value of 35 μg/mL. This inhibition was exerted at the level of hydroxymethylglutaryl-COA reductase (MHG-CoA reductase) as indicated by direct enzymatic measurements and the absence of inhibition if [14C]mevalonate was used as a precursor. At high concentrations (above 0.5 mg/mL), inhibition of cholesterol biosynthesis was not only seen at an early step where it increased considerably with dose, but also at later steps resulting in the accumulation of the precursors lanosterol and 7-dehydrocholesterol. No desmosterol was formed which, however, was a major precursor accumulating in the presence of triparanol. Thus, the accumulation of sterol precursors seem to be of less therapeutic significance during consumption of garlic, because it requires concentrations one or two orders of magnitude above those affecting HMG-CoA reductase. Alliin, the main sulfur-containing compound of garlic, was without effect itself. If converted to allicin, it resulted in similar changes of the sterol pattern. This suggested that the latter compound might contribute to the inhibition at the late steps. In contrast, nicotinic acid and particularly adenosine caused moderate inhibition of HMG-CoA reductase activity and of cholesterol biosynthesis suggesting that these compounds participate, at least in part, in the early inhibition of sterol synthesis by garlic extracts.

136 citations


Journal ArticleDOI
01 Nov 1993-Lipids
TL;DR: Results show that these double disruption strains are viable and that spontaneously arising suppressors of theERG11 disruption areerg3 mutants, compared to similar mutants ofCandida albicans that are viable in the absence of theerg3 lesion.
Abstract: The identification of the precise structural features of yeast sterol molecules required for the essential “sparking” function has been a controversial area of research. Recent cloning and gene disruption studies inSaccharomyces cerevisiae have shown that C-24 methylation (ERG6), C-5 desaturation (ERG3) and Δ8-Δ7 isomerization (ERG2) are not required, while C-14 demethylation (ERG11) and C-14 reduction (ERG24) are each required for aerobic viability. Earlier observations had indicated that C-14 demethylase deficient strains could be restored to aerobic growth by suppressor mutations that caused a deficiency in C-5 desaturase. These strains were reported to synthesize some ergosterol, indicating that they contained leaky mutations in bothERG11 andERG3, thereby making it imposssible to determine whether the removal of the C-14 methyl group was required for aerobic viability. The availability of theERG11 andERG3 genes has been used in this study to construct strains that contain null mutants in bothERG11 andERG3. Results show that these double disruption strains are viable and that spontaneously arising suppressors of theERG11 disruption areerg3 mutants. Theerg11 mutants ofS. cerevisiae are compared to similar mutants ofCandida albicans that are viable in the absence of theerg3 lesion.

112 citations


Journal ArticleDOI
TL;DR: Cultures of Saccharomyces cerevisiae strain GL7 auxotrophic for sterol were incubated with a series of sterols and sterol-like molecules (tetracyclic and pentacyClic triterpenoids) in order to determine the structural requirements of sterol for bulk membrane function.

96 citations


Journal ArticleDOI
TL;DR: The complete inhibition of ergosterol synthesis and the accumulation of the 4,4,14-trimethylsterol and of the 3-ketosteroid together with the absence of sterols, such as 14 alpha-methylfecosterol and lanosterol, are at the origin of the high activity of itraconazole against C. neoformans.
Abstract: As in other pathogenic fungi, the major sterol synthesized by Cryptococcus neoformans var. neoformans is ergosterol. This yeast also shares with most pathogenic fungi a susceptibility of its cytochrome P-450-dependent ergosterol synthesis to nanomolar concentrations of itraconazole. Fifty percent inhibition of ergosterol synthesis was reached after 16 h of growth in the presence of 6.0 +/- 4.7 nM itraconazole, and complete inhibition was reached at approximately 100 nM itraconazole. This inhibition coincided with the accumulation of mainly eburicol and the 3-ketosteroid obtusifolione. The radioactivity incorporated from [14C]acetate in both compounds represents 64.2% +/- 12.9% of the radioactivity incorporated into the sterols plus squalene extracted from cells incubated in the presence of 10 nM itraconazole. The accumulation of obtusifolione as well as eburicol indicates that itraconazole inhibits not only the 14 alpha-demethylase but also (directly or indirectly) the NADPH-dependent 3-ketosteroid reductase, i.e., the enzyme catalyzing the last step in the demethylation at C-4. This latter inhibition obviates the synthesis of 4,4-demethylated 14 alpha-methylsterols that may function at least partly as surrogates of ergosterol. Eburicol and obtusifolione are unable to support cell growth, and the 3-ketosteroid has been shown to disturb membranes. The complete inhibition of ergosterol synthesis and the accumulation of the 4,4,14-trimethylsterol and of the 3-ketosteroid together with the absence of sterols, such as 14 alpha-methylfecosterol and lanosterol, which can partly fulfill some functions of ergosterol, are at the origin of the high activity of itraconazole against C. neoformans. Fifty percent inhibition of growth achieved after 16 h of incubation in the presence of 3.2 +/- 2.6 nM itraconazole.

85 citations


Journal ArticleDOI
TL;DR: The premise that oxylanosterols regulate HMG-CoA reductase expression through a post-transcriptional process which may be distinct from other previously described sterol regulatory mechanisms is supported.

44 citations


Journal ArticleDOI
01 Nov 1993-Steroids
TL;DR: Radiolabeling experiments indicated that there are two pathways of sterol biosynthesis in E. fraudratix and these proceeds through squalene to lanosterol which is further metabolized to produce the triterpene saponins and its xyloside.

44 citations


Journal ArticleDOI
TL;DR: Three 32-methylated analogs of the intermediates generated during the removal of the 14 alpha-methyl group by P-450DM, compounds 17a, 17b, and 18, have been prepared and their biochemical activities assessed and all three compounds were found to be direct inhibitors of P- 450DM.
Abstract: Lanosterol 14 alpha-methyl demethylase (P-450DM) is the cytochrome P-450 monooxygenase which oxidatively removes the 14 alpha-methyl group of lanosterol. This demethylation is considered to be a rate-limiting step in the conversion of lanosterol to cholesterol. The intermediates in this transformation are known to bind very tightly to P-450DM and have been implicated in the regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) activity, the rate-limiting enzyme in overall cholesterol biosynthesis. Three 32-methylated analogs of the intermediates generated during the removal of the 14 alpha-methyl group by P-450DM, compounds 17a, 17b, and 18, have been prepared and their biochemical activities assessed. All three compounds were found to be direct inhibitors of P-450DM. These compounds were also shown to suppress HMGR activity by reducing the level of enzyme protein.

25 citations


Journal ArticleDOI
TL;DR: 19-Azasqualene 2,3-epoxide was active on SO cyclase from rat and pig liver with an IC 50 of 1.5 μM in pig, while in SO cyclases of yeast ( S cerevisiae and C albicans ) microsomes it was 20–30-fold less active.

24 citations


Book ChapterDOI
TL;DR: The 14α-demethylation is an essential reaction of steroid biosynthesis by eukaryotes, because most of the functional steroids have no 14-methyl group.
Abstract: Lanosterol 14α-demethylase (cytochrome P45014DM) is a housekeeping enzyme occurring widely in eukaryotes. It is a cytochrome P450 mono-oxygenase catalyzing the conversion of lanosterol or 24,25-dihydrolanosterol (DHL) to the 14-demethylated and 14,15-desaturated derivatives by removing the 14α-methyl group (C32) as formic acid (Fig. 1) (Watkinson and Akhtar 1969; Alexander et al. 1972; Gibbons and Mitropoulos 1973; Mitropoulos et al. 1976; Aoyama and Yoshida 1978; Aoyama et al. 1984; Trzaskos et al. 1986). The 14α-demethylation is an essential reaction of steroid biosynthesis by eukaryotes, because most of the functional steroids have no 14-methyl group.

22 citations


Journal ArticleDOI
01 Nov 1993-Plasmid
TL;DR: Two plasmids were obtained which allow triazole and imidazole resistance in Saccharomyces cerevisiae and can be used on a dominant selection marker to transform wild-type yeasts and to confer imidrazole and triazoles resistance in industrial fermentation.

Journal ArticleDOI
Yuko Aoki1, F Yoshihara1, M Kondoh1, Y Nakamura1, Noboru Nakayama1, Mikio Arisawa1 
TL;DR: Ro 09-1470 preferentially inhibited the yeast P-450(14DM), showing a 50% inhibitory concentration (IC50) of 0.47 to approximately 1.1 microM, which is much lower than the IC50s for rat hepatic P- 450s catalyzing cholesterol biosynthesis, p-nitroanisol O-demethylation, aniline hydroxylation, and aminopyrine N-dem methylation.
Abstract: Ro 09-1470 is a new antifungal agent that belongs to a series of compounds characterized by a tetrahydropyran skeleton with glycine and alkenyl side chains and that inhibits P-450 lanosterol C-14 demethylase (P-450(14DM)) of fungi (Y. Aoki, T. Yamazaki, M. Kondoh, Y. Sudoh, N. Nakayama, Y. Sekine, H. Shimada, and M. Arisawa, J. Antibiot. 45:160-170, 1992; S. Matsukuma, T. Ohtsuka, H. Kotaki, H. Sawairi, T. Sano, K. Watanabe, N. Nakayama, Y. Itezono, M. Fujiu, N. Shimma, K. Yokose, and T. Okuda, J. Antibiot. 45:151-159, 1992). We have studied the compound9s mode of interaction with fungal P-450(14DM) and its selectivity for the fungal versus mammalian P-450 enzymes. Ro 09-1470 bound to the Saccharomyces cerevisiae P-450(14DM) by coordinating to the heme with one-to-one stoichiometry. Unlike the azole compounds, it interacted with both ferric and ferrous heme. It was active also against the P-450(14DM) of Candida albicans. Ro 09-1470 preferentially inhibited the yeast P-450(14DM), showing a 50% inhibitory concentration (IC50) of 0.47 to approximately 1.1 microM, which is much lower than the IC50s for rat hepatic P-450s catalyzing cholesterol biosynthesis (IC50 = 341 microM), p-nitroanisol O-demethylation (> 1,000 microM), aniline hydroxylation (> 1,000 microM), and aminopyrine N-demethylation (920 microM). The degree of selectivity for yeast P-450 was higher than that of ketoconazole.

01 Jan 1993
TL;DR: This result shows that ignosterol is able to efficiently replace ergosterol as bulk membrane component and that the fen1-1 mutation eliminates the specific ergostol requirement in yeast.
Abstract: Screening for resistance to fenpropimorph was under- taken in order to isolate yeast mutants affected in the regulation of the ergosterol pathway. Among the mutants isolated, one bearing the recessive fen1-1 mutation was characterized by a 1.~fold increase in the ergosterol level and a general resistance to sterol biosynthesis inhibitors. The fenl-I mutation was linked to MATlocus on chromo some III. The measurement of enzyme activities involved in the ergosterol pathway revealed that isopenteuyl di- phosphate (IPP) isomerase activity was specifically in- creased 1.5~fold as compared to the wild type strain. However, overexpression of IPP isomerase in the wild type strain was not by itself sufficient to lead to sterol increase or resistance to sterol biosynthesis inhibitors, showing that IPP isomerase is not a limiting step in the pathway. The fen1-1 mutation permits viability in aerc~ biosis of yeast disrupted for sterol-14 reductase in absence of exogenous ergosterol supplementation, whereas the co~ responding strain bearing the wild type FEN1 allele grows only in anaerobiosis. This result shows that ignosterol is able to efficiently replace ergosterol as bulk membrane component and that the fen1-1 mutation eliminates the specific ergosterol requirement in yeast. Lipids 28, 907-912 (1993). The sterol biosynthetic pathway is the target of power- ful fungicides. Among them, the most commonly used against pathogenic fungi are azoles and morpholine derivatives. Azoles, such as ketoconazole, flusilazol or LAB 170250 F, inhibit the C-14 lanosterol demethylase step (1-3) through the binding of the fungicide's hetero- cyclic nitrogen atom to the protohaem iron of cytochrome P450 lanosterol demethylase (4). Morpholines block two later steps downstream in the ergosterol pathway, /114 sterol reductase and As-h 7 sterol isomerase (5). These compounds, which are likely to be protonated at physio- logical pH, share some structural and electronic similari- ties with the sterol carbocationic high energy intermedi- ates involved in the A

Journal ArticleDOI
TL;DR: New lanosterol and 27-norlanosterol oligosaccharides were found in the bulbs of Chionodoxa gigantea by extensive 2D NMR analysis as well as by comparison with spectral data of known compounds.

Journal ArticleDOI
TL;DR: 2,3:18,19-dioxidosqualene was found to be the best inhibitor within the compounds assayed (IC 50 =0.11μM), although other dioxides also exhibited a potent inhibitory activity.
Abstract: The preparation and characterization of dioxidosqualenes 4-10 is reported. Treatment of the appropriate epoxysqualene 1, 2, or 3 with NBS followed by chromatographic purification afforded the corresponding epoxybromohydrins 11-14 as diastereomeric mixtures, with the exception of compound 11, which could be separated into the respective racemates 11a and 11b. Further dehydrobromination with NaH in THF led to the respective dioxidosqualenes 4-8 in good conversion yields. Dioxides 9 and 10 were isolated from the crude reaction mixture of the treatment of epoxide 2 with dimethyldioxirane. Characterization of compounds 4-10 was carried out by combining 1 H and 13 C NMR spectral means with positive GC-MS-CI analysis. The GC-MS-CI analysis inclued the identification of the carbonyl compounds resulting from the cleavage of dioxido derivatives with periodic acid. Finally, data on the activity of dioxidosqualenes as oxidosqualene-lanosterol cyclase (OSLC) inhibitors in rat liver microsomes are also presented. In this respect, 2,3:18,19-dioxidosqualene was found to be the best inhibitor within the compounds assayed (IC 50 =0.11μM), although other dioxides also exhibited a potent inhibitory activity. The fact that these compounds could be potentially generated in an organism constitutes a remarkable difference relative to other OSLC inhibitors described to date

Journal ArticleDOI
TL;DR: An isozyme of cytochrome P-450 catalyzing lanosterol 14α-demethylation was purified from human liver using column chromatography, including immunoaffinity chromatography and the apparent Km value for 24,25-dihydrolanosterol was found to be 27 μM.

Journal ArticleDOI
TL;DR: It is suggested that lanosterol in parallel with unsaturated fatty acids is responsible for maintaining membrane integrity of S. pombe cells growing in the presence of ethanol.
Abstract: Ergosterol, lanosterol and two further unidentified sterols were detected and quantified in Schizosaccharomyces pombe cell extracts. In cells grown under anaerobic conditions, the levels of these sterols were dramatically reduced with a concomitant increase of their squaline precursor as compared with cells growing under aerobic conditions. Presence of ethanol resulted in a decrease in the sterol content under aerobic conditions. On the contrary, under anaerobic conditions presence of ethanol resulted in a three-fold increase of total sterols. Lanosterol was the main constituent of this elevation. It is suggested that lanosterol in parallel with unsaturated fatty acids is responsible for maintaining membrane integrity of S. pombe cells growing in the presence of ethanol.

Journal ArticleDOI
01 Feb 1993-Steroids
TL;DR: The inhibitory effect of SSF-109 on cholesterol synthesis in isolated hepatocytes was studied using a radio-high-performance liquid chromatography system, and the results were compared with those of other inhibitors, triparanol and AMO-1618.


01 Jan 1993
TL;DR: Ergosterol, lanosterol and two further unidentified sterols were detected and quantified in Schizosaccharomyces pombe cell extracts in this paper, and the levels of these sterols are dramatically reduced with a concomitant increase of their squalene precursor as compared with cells growing under aerobic conditions.
Abstract: Ergosterol, lanosterol and two further unidentified sterols were detected and quantified in Schizosaccharomyces pombe cell extracts. In cells grown under anaerobic conditions, the levels of these sterols were dramatically reduced with a concomitant increase of their squalene precursor as compared with cells growing under aerobic conditions. Presence of ethanol resulted in a decrease in the sterol content under aerobic conditions. On the contrary, under anaerobic conditions presence of ethanol resulted in a three-fold increase of total sterols. Lanosterol was the main constituent of this elevation. It is suggested that lanosterol in parallel with unsaturated fatty acids is responsible for maintaining membrane integrity of S. pombe cells growing in the presence of ethanol.

Journal ArticleDOI
TL;DR: In this article, a method for the introduction of 23-keto and 26-carboxyl groups (Synthesis of a genoderic acid analog) into lanosterol side chain by use of hydroboration and allylboration as key reactions is described.


01 Jan 1993
TL;DR: The results demonstrate that the effects of water-soluble extracts of garlic powder on cholesterol biosynthesis in rat hepatocytes are due to distinct compounds present in garlic which may contribute to the hypocholesterolemic effect of garlic observed in rats and man.
Abstract: Water-soluble extracts of garlic powder inhibit cholesterol-biosynthesis at early and late steps of the pathway. In this study we have tried to identify garlic compounds associated with these different inhibitory actions wh ich are predominant at low and high concentrations, respectively. Alliin, neither alone nor after incubation with alliinase, was able to inhibit sterol synthesis from 14C-acetate in cultured rat hepatocytes at the early step. Likewise, allicin caused no reduction, while ajoen diminished sterol synthesis by maximally 16 %. Other compounds containing allyl- and/or sulphur-groups (including y­ glutamyl-peptides) did not inhibit, whereas nicotinamide and particularly adenosine caused some inhibition. From all these compounds only allicin and ajoen at high concentrations interacted with the conversion of lanosterol to cholesterol. 80th substances led to the accumulation of lanosterol as weil as some 7-dehydrocholesterol at the expense of cholesterol. Desmosterol was not accumulating. These results demonstrate that the effects of water-soluble extracts of garlic powder on cholesterol biosynthesis in rat hepatocytes are due to distinct compounds present in garlic which may contribute to the hypocholesterolemic effect of garlic observed in rats and man.