Composition of Pneumocystis carinii neutral lipids and identification of coenzyme Q10 as the major ubiquinone homolog.

The lipids of purified preparations of Pneumocystis carinii carinii freshly isolated from infected rats were analyzed and compared with those of whole lungs from normal and methylprednisolone-immunosuppressed uninfected rats. In this study, the neutral lipid fraction was examined in detail; the relative concentrations of individual classes making up this fraction were quantified. Of particular interest was the nature of the organism's ubiquinone (coenzyme Q, CoQ) fraction because atovaquone, a hydroxynaphtho-quinone (566C80) analog of ubiquinone, is efficacious in the treatment of P.

Progesterone stimulation of HMG-CoA reductase activity in cultured cells.

In a previous study we showed that progesterone (PG) stimulated HMG-CoA reductase (HMGR) activity in rat intestinal epithelial cells (IEC-6) incubated in the presence or absence of low-density lipoprotein (LDL) [1,2]. In the present study we examined further the mechanism of this stimulation. We observed that the stimulation of HMGR activity by PG was completely prevented by cycloheximide.

Occurrence of specific sterols in Pneumocystis carinii.

We have examined the sterol composition and biosynthesis of rat Pneumocystis carinii. We found a number of lipid components among the nonsaponifiable fraction that appear unique to P. carinii.

Sphingomyelinase treatment of low density lipoprotein and cultured cells results in enhanced processing of LDL which can be modulated by sphingomyelin.

The addition of neutral sphingomyelinase from S. aureus to the medium of rat intestinal epithelial cell cultures (IEC-6) containing added human low density lipoprotein (LDL) resulted in two- to fivefold increases in LDL uptake and degradation. This overall effect was shown to be the combined result of sphingomyelinase activity on the composition of the LDL particle and a separate action directly on the cells when native LDL was incubated with sphingomyelinase from S.

Plasma membrane sphingomyelin and the regulation of HMG-CoA reductase activity and cholesterol biosynthesis in cell cultures.

We have examined the mechanism of the inhibition of cholesterol synthesis in cells treated with exogenous sphingomyelinase. Treatment of rat intestinal epithelial cells (IEC-6), human skin fibroblasts (GM-43), and human hepatoma (HepG2) cells in culture with sphingomyelinase resulted in a concentration- and time-dependent inhibition of the activity of HMG-CoA reductase, a key regulatory enzyme in cholesterol biosynthesis. The following observations were obtained with IEC-6 cells.

Differential regulation of low density lipoprotein suppression of HMG-CoA reductase activity in cultured cells by inhibitors of cholesterol biosynthesis.

Treatment of rat intestinal epithelial cells (IEC-6 cells) with lanosterol 14 alpha-demethylase inhibitors, ketoconazole and miconazole, had similar effects on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity and cholesterol biosynthesis but the drugs differed in their ability to prevent the low density lipoprotein (LDL) suppression of reductase activity. Miconazole, at concentrations that inhibited the metabolism of lanosterol and epoxylanosterol to the same degree as ketoconazole, did not prevent low density lipoprotein action on reductase activity, whereas ketoconazole totally abolished the low density lipoprotein action on reductase activity. Both drugs caused: 1) a biphasic response in reductase activity such that at low concentrations (less than 2 microM) reductase activity was inhibited and at high concentrations (greater than 5 microM) the activity returned to control or higher than control levels; 2) an inhibition of metabolism of lanosterol to cholesterol, and 24(S), 25-epoxylanosterol to 24(S), 25-epoxycholesterol.

Effect of vitamin D3 derivatives on cholesterol synthesis and HMG-CoA reductase activity in cultured cells.

Treatment of logarithmically growing rat intestinal epithelial cells (IEC-6) in culture with vitamin D3 (cholecalciferol), 25-hydroxy vitamin D3 (25-hydroxy cholecalciferol), 1,25-dihydroxy vitamin D3 (1,25-dihydroxycholecalciferol), and 24,25 dihydroxy vitamin D3 (24(R),25-dihydroxycholecalciferol), caused an inhibition of the cholesterol biosynthetic pathway at two separate sites. At concentrations greater than 2 micrograms/ml, the hydroxylated forms of vitamin D3 caused an accumulation of methyl sterols indicating an inhibition of lanosterol demethylation. Vitamin D3, however, had little effect on lanosterol demethylation.

Identification of regulatory sites in the biosynthesis of ubiquinone in the perfused rat heart.

The biosynthesis of ubiquinone was studied in an isolated perfused beating heart preparation from adult male rats to determine rate-limiting steps in the biosynthetic pathway. The isolated heart could incorporate p-hydroxy[U-14C]benzoate into ubiquinones (ubiquinone-9 and -10) and two other lipids which were identified as 3-nonaprenyl 4-hydroxybenzoate and 3-decaprenyl 4-hydroxybenzoate. No other lipids could be detected.

Regulation of sterol biosynthesis and of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in cultured cells by progesterone.

Treatment of rat intestinal epithelial cells in culture (IEC-6) with progesterone (10 micrograms/ml) caused a strong inhibition of cholesterol biosynthesis as indicated by a decreased incorporation of radiolabel from [3H]acetate. This inhibition was accompanied by an accumulation of radioactivity in an intermediate which coeluted with authentic desmosterol upon high performance liquid chromatography (HPLC). In addition, treatment of cells with progesterone caused lesser accumulation of radiolabel in products with retention times (RT) of 7.

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and cholesterol biosynthesis by oxylanosterols.

Treatment of rat intestinal epithelial cell cultures with the oxidosqualene cyclase inhibitor, 3 beta-[2-(diethylamino)-ethoxy]androst-5-en-17-one (U18666A), resulted in an accumulation of squalene 2,3:22,23-dioxide (SDO). When U18666A was withdrawn and the cells were treated with the sterol 14 alpha-demethylase inhibitor, ketoconazole, SDO was metabolized to a product identified as 24(S),25-epoxylanosterol. To test the biological effects and cellular metabolism of this compound, we prepared 24(RS),25-epoxylanosterol by chemical synthesis.

Modulation of regulatory oxysterol formation and low density lipoprotein suppression of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity by ketoconazole. A role for cytochrome P-450 in the regulation of HMG-CoA reductase in rat intestinal epithelial cells.

The effects of ketoconazole, a lanosterol demethylase and cytochrome P450 inhibitor, on the regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.

Detergent-solubilization, purification, and characterization of membrane-bound 3-hydroxy-3-methylglutaryl-coenzyme A reductase from radish seedlings.

3-Hydroxy-3-methylglutaryl-CoA reductase (NADPH) was solubilized with polyoxyethylene ether (Brij) W-1 from a heavy-membrane fraction, sedimented at 16000 X g from a cell-free homogenate of four-day-old, dark-grown radish seedlings (Raphanus sativus L.). Approximately 350-fold purification of the solubilized enzyme activity was achieved by (NH4)2SO4 precipitation followed by column chromatography on DEAE-Sephadex A-50, blue-dextran-agarose and HMG-CoA-hexane-agarose.

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase by oxysterol by-products of cholesterol biosynthesis. Possible mediators of low density lipoprotein action.

Regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.

Stimulation of rat liver 4-hydroxybenzoate: polyprenyl transferase activity by a cytosolic protein factor; evidence for a polyprenyl pyrophosphate transport protein.

Rat liver postmicrosomal supernatant contains a factor which stimulates the 4-hydroxybenzoate:polyprenyl transferase activity of whole mitochondria and inner mitochondrial membrane fragments. The factor involved appears to be a heat stable, nondializable protein sensitive to tryptic hydrolysis and has been partially purified. Since this protein binds nonaprenyl pyrophosphate and stimulates its transport into mitochondria but shows no similar effect with 4-hydroxybenzoate, it is suggested that this protein also acts as an all trans polyprenyl pyrophosphate carrier protein.

Effects of 3 beta-[2-(diethylamino)ethoxy]androst-5-en-17-one on the synthesis of cholesterol and ubiquinone in rat intestinal epithelial cell cultures.

The relationship between cholesterol and ubiquinone synthesis in rat intestinal epithelial cell cultures was examined by using 3 beta-[2-(diethylamino)ethoxy]androst-5-en-17-one hydrochloride (U18666A). Addition of U18666A to cells caused a greater than 90% inhibition of incorporation of [3H]acetate into cholesterol and an apparent large increase in the incorporation of [3H]acetate and [3H]mevalonate into ubiquinone. However, the incorporation of 4-hydroxy[U-14C]benzoate, a ring precursor of ubiquinone, was unchanged.

Short term reversible modulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in isolated epithelial cells from rat ileum. Regulation of phosphorylation dephosphorylation by bicarbonate.

In ileal epithelial cells isolated through incubation of gut loops in phosphate-buffered saline containing EDTA, 3-hydroxy-3-methylglutaryl coenzyme A reductase was found to exist prdominantly in an active (dephosphorylated) form. However, in cells obtained by scraping the intestine, the enzyme was mostly inactive (phosphorylated) and could be activated several-fold on incubation with a crude phosphatase preparation. Reductase activity in microsomes from ileal epithelial cells could be inactivated extensively by incubation with Mg .