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Showing papers on "Lanosterol published in 1989"


Journal ArticleDOI
TL;DR: It is suggested that growth can occur through utilisation of 14-methyl fecosterol, produced by a combination of azole inhibition and defective sterol C5-6 desaturation.

187 citations


Journal ArticleDOI
TL;DR: Oxygenated lanosterol derivatives, which were isolated from Ganoderma lucidum or their derivatives obtained by chemical conversion, were tested for their effect on cholesterol biosynthesis from 24,25-dihydrolanosterol by rat hepatic subcellular 10,000 x g supernatant fraction.
Abstract: Oxygenated lanosterol derivatives, which were isolated from Ganoderma lucidum (Polyporaceae) or their derivatives obtained by chemical conversion, were tested for their effect on cholesterol biosynthesis from 24,25-dihydrolanosterol by rat hepatic subcellular 10,000 x g supernatant fraction. The sterol (VI, 40 microM) with 7-oxo and 15 alpha-hydroxy groups potently inhibited the synthesis of cholesterol from [24,25-3H]-24,25-dihydrolanosterol (18 microM).

121 citations



Journal ArticleDOI
TL;DR: The azole antifungals ketoconazole and itraconazole possess in vitro antileishmanial activity against Leishmania mexicana mexicanana amastigotes in macrophages and all [14C]acetate was incorporated in ergosterol, suggesting an inhibition in cholesterol synthesis in the host cells.

66 citations


Journal ArticleDOI
TL;DR: Strains of Saccharomyces cerevisiae deleted in the NADPH-cytochrome P450 reductase gene by transplacement are 200-fold more sensitive to ketoconazole, an inhibitor of the cytochromeP450 lanosterol 14 alpha-demethylase.

64 citations


Journal ArticleDOI
TL;DR: Observations on the metabolism of 32-hydroxy-24,25-dihydrolanosterol indicate that the 14 alpha-demethylation of lanosterol catalyzed by cytochrome P-45014DM proceeds with three step monooxygenations via the 32-Hydroxy and 32-oxo intermediates, and the cy tochrome mediates this sequential reaction without releasing the intermediates.

54 citations


Journal ArticleDOI
TL;DR: Results demonstrate the double-bond requirement for both components of the dem methylation sequence and suggest that the olefinic electrons at delta 7 or delta 8 but not delta 6 may participate directly during demethylation.

51 citations


Journal ArticleDOI
TL;DR: The inhibition of HMG-CoA reductase activity and cholesterol synthesis by vitamin D3 and 25-hydroxy vitamin D 3 was also observed in other cell culture lines such as human skin fibroblasts (GM-43), transformed human liver cells (Hep G2), and mouse peritoneal macrophages (J-774).

47 citations


Journal ArticleDOI
TL;DR: The synthesis of these intermediary sterols, not found normally in H4 cells, is particularly pronounced in cultures kept in lipid-depleted media that contain the inhibitors and proceeds by the use of endogenous substrates at the expense of cholesterol.

46 citations


Journal ArticleDOI
TL;DR: The purification of cytochrome P-450-dependent 14 alpha-sterol demethylase (P-450DM) from the important opportunistic fungal pathogen, Candida albicans, is described and it is judged to be highly purified on the basis of Coomassie Brilliant Blue staining of protein.
Abstract: The purification of cytochrome P-450-dependent 14 alpha-sterol demethylase (P-450DM) from the important opportunistic fungal pathogen, Candida albicans, is described. Optimal purification (875-fold) was achieved by extracting the cytochrome from microsomes with sodium cholate followed by hydroxyapatite, octyl-Sepharose and CM-Sepharose chromatographies, giving a cytochrome preparation of 17.5 nmol/mg of protein. By the use of SDS/polyacrylamide-gel electrophoresis the cytochrome was judged to be highly purified on the basis of Coomassie Brilliant Blue staining of protein. The Mr of P-450DM was estimated to be 51,000. The absorption spectrum of oxidized P-450DM was characteristic of a low-spin cytochrome, and its reduced CO complex had a Soret absorption peak at 447 nm. When reconstituted in a model membrane system of dilauroylphosphatidylcholine with NADPH and O2, P-450DM catalysed the complete 14 alpha-demethylation of lanosterol, which was inhibited by CO. The cytochrome appeared to have a high degree of substrate specificity; it was unable to oxidize a number of xenobiotic compounds in the reconstituted assay.

38 citations


Journal ArticleDOI
TL;DR: The hydrogen bond formation between the 3-hydroxy group of lanosterol and the specific amino acid residue in the substrate site is assumed to be essential for orienting the substrate in the substrates site of the cytochrome.

Journal ArticleDOI
TL;DR: Results revealed that the 8-double bond of lanosterol plays an important role in the enzyme-substrate interaction of cytochrome P-450(14DM) and the sterol-cytochrome complexes and kinetics indicated that 6-lanostene-3 beta,32-diol and lanostane-3 alpha-demethylase could not interact with the substrate site of the cy tochrome.

Journal ArticleDOI
TL;DR: A novel assay for cytochrome P-450-dependent 14 alpha-sterol demethylase of the important opportunistic fungal pathogen, Candida albicans, is described, and there was a reciprocal relationship between the amount of radioactivity incorporated into desmethylated sterols and that lost from lanosterol.
Abstract: A novel assay for cytochrome P-450-dependent 14 alpha-sterol demethylase of the important opportunistic fungal pathogen, Candida albicans, is described. The enzyme was assayed in microsomal preparations (microsomes) by measuring the incorporation of [14C]lanosterol into (4,14)-desmethylated sterols. The efficacy of different cell-breakage methods was compared; desmethylated-sterol biosynthesis was maximal when cells were broken with a Braun disintegrator. The solubilization of [14C]lanosterol with detergent in the assay system was essential for enzyme activity, which was enhanced considerably when microsomes were gassed with O2. Under these conditions, there was a reciprocal relationship between the amount of radioactivity incorporated into desmethylated sterols and that lost from lanosterol. The major radiolabelled desmethylated sterol was ergosterol. The enzyme had an apparent Km of 52.73 +/- 2.80 microM and an apparent Vmax of 0.84 +/- 0.14 nmol/min per mg of protein (n = 3). Enzyme activity was decreased greatly when microsomes were treated with CO or the triazole antifungal ICI 153066.

Journal ArticleDOI
TL;DR: The results demonstrate for the first time the direct reduction in situ of an aziridine ring to a tertiary amine and the stereocontrolled deamination of a C-25 aminosteroid to produce a delta 24, rather than a delta 25(27), steroid.

Journal ArticleDOI
J M Trzaskos1, M J Henry1
TL;DR: The results indicate that selectivity of azole antimycotics is not due to inherent sensitivity differences between fungal and mammalian 14 alpha-demethylase; rather, in crude enzyme systems, a protective effect is afforded by other susceptible isozymes present in mammalian systems.
Abstract: Flusilazole inhibits the 14 alpha-demethylation of [24,25-3H-2]dihydrolanosterol by yeast and rat liver cell-free preparations. The 50% inhibitory concentration of demethylation shows that the yeast system is 100 times more sensitive than the rat system. Purified rat liver P-45014DM is about 10 times more sensitive to flusilazole than crude rat liver microsomes. Binding constants in microsomes indicate that yeast preparations (Kd, 21 nM) bind flusilazole with a higher affinity than rat liver (Kd, 496 nM). Purified rat liver P-45014DM (Kd, 45 nM) binds flusilazole with an affinity similar to that of the yeast enzyme. The P-450-dependent cholesterol 7 alpha-hydroxylase in rat liver microsomes is more sensitive to flusilazole than P-45014DM. The results indicate that selectivity of azole antimycotics is not due to inherent sensitivity differences between fungal and mammalian 14 alpha-demethylase; rather, in crude enzyme systems, a protective effect is afforded by other susceptible isozymes present in mammalian systems.

Journal ArticleDOI
TL;DR: In this paper, the sterol precursors of photosynthetic and nonphotosynthetic organisms respectively were transfromed into the 4,4,14-demethyl sterols in most but not all, of a variety of marine and freshwater sponges.

Journal ArticleDOI
TL;DR: The involvement of oxygenated cholesterol precursors in the regulation of HMG-CoA reductase activity was studied by examining the effect of ketoconazole on the metabolism of mevalonic acid, lanost-8-ene-3 beta,32-diol and the lanosterol metabolites in liver subcellular fractions and hepatocyte cultures.
Abstract: The involvement of oxygenated cholesterol precursors in the regulation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase activity was studied by examining the effect of ketoconazole on the metabolism of mevalonic acid, lanosterol and the lanosterol metabolites, lanost-8-ene-3 beta,32-diol,3 beta-hydroxylanost-8-en-32-al and 4,4-dimethylcholesta-8,14-dien-3 beta-ol, in liver subcellular fractions and hepatocyte cultures. Inhibition of cholesterol synthesis from mevalonate by ketoconazole at concentrations up to 30 microM was due exclusively to a suppression of cytochrome P-450LDM (LDM = lanosterol demethylase) activity, resulting in a decreased rate of lanosterol 14 alpha-demethylation. No enzyme after the 14 alpha-demethylase step was affected. When [14C]mevalonate was the cholesterol precursor, inhibition of cytochrome P450LDM was accompanied by the accumulation of several labelled oxygenated sterols, quantitatively the most important of which was the C-32 aldehyde derivative of lanosterol. There was no accumulation of the 24,25-oxide derivative of lanosterol, nor of the C-32 alcohol. Under these conditions the activity of HMG-CoA reductase declined. The C-32 aldehyde accumulated to a far greater extent when lanost-8-ene-3 beta,32-diol rather than mevalonate was used as the cholesterol precursor in the presence of ketoconazole. With both precursors, this accumulation was reversed at higher concentrations of ketoconazole in liver subcellular fractions. A similar reversal was not observed in hepatocyte cultures.

Journal ArticleDOI
TL;DR: It can be concluded that the nonsaponifiable metabolite essential for cell growth is not newly synthesized cholesterol, as inhibitors affected cell division only when they were added to the culture medium before the decline of cholesterol synthesis stimulation.

Journal ArticleDOI
TL;DR: Several 14α-aminomethyl-substituted lanosterol derivatives have been synthesized involving a complete Δ7,8to Δ8,9isomerization; these compounds are inhibitors of fungal ergosterol biosynthesis and are active against intact Candida and dermatophyte strains.
Abstract: Several 14-α-aminomethyl-substituted lanosterol derivatives have been synthesized involving a complete Δ7,8to Δ8,9isomerization; these compounds are inhibitors of fungal ergosterol biosynthesis and are active against intact Candida and dermatophyte strains.

Book ChapterDOI
01 Jan 1989
TL;DR: The presence of lanosterol and the results of labelling studies with [2–14C]mevalonic acid indicate that the major promastigote C28-sterols are produced by the routes shown in Fig. 1.
Abstract: The major sterol identified [1] in the promastigotes of several Leishmania species is ergosta-5,7,24(28)-trien-3β-ol (11, Fig 1) and this is often accompanied by variable amounts of ergosta-5,7,22E-trien-3β-ol (ergosterol, 12). The presence of lanosterol (1) and a range of other trace sterols (2–10) in Leishmania [1,2], together with the results of labelling studies with [2–14C]mevalonic acid [2–4], indicate that the major promastigote C28-sterols are produced by the routes shown in Fig. 1. Similar metabolic routes are operative in many fungi which elaborate ergosterol (12) as the major sterol. When Leishmania promastigotes are cultured on a medium containing serum the cells contain considerable amounts of cholest-5-en-3β-ol (cholesterol) [1, 2]. This cholesterol is derived from the serum lipoproteins and it does not appear to be metabolised by the promastigotes to C28 sterols such as 11 or 12 [2].

Journal ArticleDOI
TL;DR: In this paper, the epoxide function located in the central part of the tetracyclic steroidal skeleton allows the evaluation of epoxidation-induced shifts (EIS) for several carbons.
Abstract: The 13C chemical shifts of triterpenoidal olefins and epoxides related to lanosterol are described and discussed. The epoxide function located in the central part of the tetracyclic steroidal skeleton allows the evaluation of epoxidation-induced shifts (EIS) for several carbons. The most characteristic and valuable for stereochemical assignments are the EIS values for γ-carbons C-1, C-5, C-12, C-19 and C-28 in 8α,9α-epoxides, and C-1 and C-19 in 9α,11α-epoxides. The C-α effect (EIS −55 to −79 ppm) is found to be susceptible to minor conformational distortions of the molecular framework. Characteristic signals are discussed in terms of their predictive value for each group of analysed compounds.

Journal ArticleDOI
TL;DR: A rapid and sensitive kinetic assay of lanosterol 14α-demethylation has been developed and analyzed and the percentage of turnover monitored by the novel tritium release assay was comparable to that observed by conventional GC methods.

Journal ArticleDOI
TL;DR: Comparison of each gas chromatography-mass spectrum and RtR value with those of the metabolite of DHL, indicated that the metabolites could be inferred to be 27-nor-4,4-dimethyl-5 alpha-cholesta-8,14-dien-3 beta-ol, 26,27-dinor- 4, 4- dimethyl- 5 alpha- cholesta -8, 14- dien- 3 Beta-ol.
Abstract: 27-Nor-24, 25-dihydrolanosterol (27-nor-DHL), 26, 27-dinor-24, 25-dihydrolanosterol (26, 27-dinor-DHL), and 25, 26, 27-trinor-24, 25-dihydrolanosterol (25, 26, 27-trinor-DHL), analogs of 24, 25-dihydrolanosterol (DHL) which have no C-27 carbon, C-26, 27 carbons and C-25, 26, 27 carbons, were converted to the corresponding 14-demethylated products using a reconstituted monooxygenase system from rat liver microsomes which contained cytochrome P-45014DM catalyzing lanosterol 14-demethylation and reduced nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P-450 reductase in the presence of NADPH and molecular oxygen. Each metabolite showed a relative retention time (RtR)of 0.72 with respect to each substrate in high-performance liquid chromatography (HPLC) on a reversed-phase column. Comparison of each gas chromatography-mass spectrum and RtR value with those of the metabolite of DHL, 4, 4-dimethyl-5α-cholesta-8, 14-dien-3β-ol, indicated that the metabolites could be inferred to be 27-nor-4, 4-dimethyl-5α-cholesta-8, 14-dien-3β-ol, 26, 27-dinor-4, 4-dimethyl-5α-cholesta-8, 14-dien-3β-ol, and 25, 26, 27-trinor-4, 4-dimethyl-5α-cholesta-8, 14-dien-3β-ol. However, 24, 25, 26, 27-tetranor- and 23, 24, 25, 26, 27-pentanor analogs of DHL and 20-iso-24, 25-dihydrolanosterol were not metabolized by the reconstituted enzyme system.

Book ChapterDOI
01 Jan 1989
TL;DR: Several membrane lipid classes of Leishmania promastigotes and amastigote are characteristic of plant cell membranes and are attractive targets for chemotherapeutic drugs, since some reactions are unique to plants and others are more sensitive to drugs than are comparahle reactinns in vertehrate svstems.
Abstract: Several membrane lipid classes of Leishmania promastigotes and amastigotes are characteristic of plant cell membranes Among them are sterols, ω3 and cyclopropane fatty acyl groups of phosphoglycerides and inositolphosphosphingolipids1 The enzymatic reactions in the biosynthesis of those lipids are attractive targets for chemotherapeutic drugs, since some reactions are unique to plants and others are more sensitive to drugs than are comparahle reactinns in vertehrate svstems

Journal ArticleDOI
01 Mar 1989-Steroids
TL;DR: The inability to detect 2 in nature coupled with the findings that 1 but not 2 is metabolized to cholesterol by H4 cells is interpreted to imply that the biosynthetic inclusion of the delta 8(9)-bond during the cyclization process of squalene-oxide to a tetracyclic product is an evolutionary adaptation selected for.

Journal ArticleDOI
01 Mar 1989-Steroids
TL;DR: Labeled 4,4-dimethyl-5 alpha -cholesta-8,24-dien-3 beta-ol (4,4,dimethylzymosterol) was prepared by incubating labeled mevalonate with rat liver extracts in the presence of arsenite and lanosterol.

Journal ArticleDOI
01 Mar 1989-Steroids
TL;DR: In the subcellular fractions, dihydrolanosterol, the 3 beta,32-diol and the 32-aldehyde were each metabolized to more polar sterols, in addition to cholesterol, and ketoconazole also inhibited the formation of these polar substances.


Journal ArticleDOI
TL;DR: The metabolism of delta 8-CHO was inhibited by 7-oxo-24,25-dihydrolanosterol (6, 7-Oxo-DHL), which is a potent inhibitor of cholesterol biosynthesis from lanosterol or DHL, however, the metabolism of Delta 8- CHO was less inhibited by7-oxi-D HL than that of DHL.
Abstract: Metabolism of 32-oxo-24, 25-dihydrolanosterols (3β-hydroxylanost-8-en-32-al (4, Δ8-CHO) and 3β-hydroxylanost-7-en-32-al (5, Δ7-CHO)) was studied in a reconstituted system consisting of rat liver partially purified cytochrome P-450, which catalyzes lanosterol 14-demethylation (P-45014DM), and reduced nicotineamide adenine dinucleotide phosphate (NADPH)-cytochrome P-450 reductase. The reconstituted system converted Δ8-CHO (4) to 4, 4-dimethyl-5α-cholesta-8, 14-dien-3β-ol (2, 8, 14-Diene), which corresponds to the 14-deformylated product. Δ7-CHO (5), the isomer of Δ8-CHO (4), was not converted to the corresponding 14-deformylated product. The apparent Km value of cytochrome P-45014DM for Δ8-CHO (4) was about 1/20 of that for 24, 25-dihydrolanosterol (1, DHL). The metabolism of Δ8-CHO (4) was inhibited by 7-oxo-24, 25-dihydrolanosterol (6, 7-oxo-DHL), which is a potent inhibitor of cholesterol biosynthesis from lanosterol or DHL (1). However, the metabolism of Δ8-CHO (4) was less inhibited by 7-oxo-DHL (6) than that of DHL (1).