scholarly journals The effect of carbon monoxide on the nature of the accumulated 4,4-dimethyl sterol precursors of cholesterol during its biosynthesis from [2-14C]mevalonic acid in vitro

1973 ◽  
Vol 132 (3) ◽  
pp. 439-448 ◽  
Author(s):  
Geoffrey F. Gibbons ◽  
Konstantinos A. Mitropoulos
Keyword(s):  
Gas Phase ◽  
Cholesterol Biosynthesis ◽  
Quantitative Difference ◽  
Specific Radioactivity ◽  
Mevalonic Acid ◽  
Sterol Fraction ◽  
Side Chain ◽  
Gas Phases ◽  
Physicochemical Methods

Cholesterol biosynthesis was studied in rat liver subcellular fractions incubated with dl-[2-14C]mevalonic acid under gas phases consisting of either N2+O2 (90:10) or CO+O2 (90:10). CO inhibits cholesterol biosynthesis from [2-14C]mevalonic acid and results in a large accumulation of radioactive 4,4-dimethyl sterols. Separation of the components of the 4,4-dimethyl sterol fraction showed that lanosterol and dihydrolanosterol are the major components that accumulate during cholesterol biosynthesis in an atmosphere containing CO, whereas 14-demethyl-lanosterol and 14-demethyldihydrolanosterol are the major components of the much less intensely radioactive 4,4-dimethyl sterol fraction isolated from incubations with N2+O2 as the gas phase. The identities of lanosterol, dihydrolanosterol and 14-demethyldihydrolanosterol were confirmed by both radiochemical and physicochemical methods, including g.l.c. and combined g.l.c.–mass spectrometry. CO therefore results in a qualitative as well as a quantitative difference in the 4,4-dimethyl sterol fraction which arises during cholesterol biosynthesis from mevalonic acid. The specific radioactivity of the [14C]lanosterol biosynthesized in the presence of CO was lower than that of its companion, [14C]dihydrolanosterol. The relative amounts of 4,4-dimethyl-Δ24-sterols and 4,4-dimethyl-24,25-dihydrosterols present in each type of incubation suggest that enzymic reduction of the sterol side chain occurs predominantly at a stage after that of lanosterol.

scholarly journals The compartmentation of non-esterified and esterified cholesterol in the superovulated rat ovary

1971 ◽  
Vol 123 (2) ◽  
pp. 143-152 ◽  
Author(s):  
A. P. F. Flint ◽  
D. T. Armstrong
Keyword(s):  
Microsomal Fraction ◽  
Specific Radioactivity ◽  
Subcellular Fractions ◽  
Side Chain ◽  
Electron Microscopic ◽  
Side Chain Cleavage ◽  
Chain Cleavage ◽  
Cleavage Enzyme ◽  
Rat Ovary

1. The specific radioactivities of non-esterified and esterified cholesterol, progesterone and 20α-hydroxypregn-4-en-3-one were determined in slices of superovulated rat ovary after incubation with [1-14C]acetate in vitro for various times. The specific radioactivities of progesterone and 20α-hydroxypregn-4-en-3-one were equal, and (during the fourth hour of incubation) exceeded those of the non-esterified cholesterol and the esterified cholesterol by factors of 2.8 and 7.6 respectively. 2. After separation of homogenates of superovulated rat ovary slices previously incubated with [14C]acetate into subcellular fractions by differential centrifugation, the specific radioactivities of non-esterified cholesterol in the cytosol, mitochondria, lipid-containing storage granules and microsomal fraction were 1220, 1510, 1420 and 4020d.p.m./μmol respectively; the corresponding values for the specific radioactivity of the esterified cholesterol were 600, 700, 730 and 760d.p.m./μmol. The specific radioactivities of progesterone and 20α-hydroxypregn-4-en-3-one were equal in all fractions; the corresponding mean specific radioactivity of progesterone+20α-hydroxypregn-4-en-3-one was 6150d.p.m./μmol. 3. By using glutamate dehydrogenase and cytochrome (a+a3) as mitochondrial markers, the presence of cholesterol side-chain cleavage enzyme was demonstrated in microsomal fraction free of mitochondrial contamination. 4. The specific radioactivities of ovarian non-esterified and esterified cholesterol, progesterone and 20α-hydroxypregn-4-en-3-one were determined up to 8h after the intravenous injection of [4-14C]cholesterol into superovulated rats. At all times the specific radioactivities of progesterone and 20α-hydroxypregn-4-en-3-one were equal to the specific radioactivity of non-esterified cholesterol and exceeded, by up to 3.3-fold, that of the esterified cholesterol. 5. It is concluded that non-esterified cholesterol formed from [14C]acetate in the endoplasmic reticulum equilibrates slowly with non-esterified cholesterol in other subcellular fractions, and is preferentially converted into steroids. Such a mechanism presupposes the operation of a microsomal cholesterol side-chain cleavage enzyme using non-esterified cholesterol as its substrate. Unrelated evidence is presented in support of the existence of such an enzyme. The results are discussed in the light of other biochemical and electron-microscopic findings relating to the compartmentation of cholesterol in steroidogenic tissues.

scholarly journals Inhibition of Cholesterol Biosynthesis in Ovine Ovarian Follicles in vitro by Human Chorionic Gonadotrophin

1978 ◽  
Vol 31 (4) ◽  
pp. 405 ◽  
Author(s):  
TJ Douglas ◽  
RP Hamilton ◽  
RF Seamark
Keyword(s):  
Cholesterol Biosynthesis ◽  
Mevalonic Acid ◽  
Ovarian Follicles ◽  
Chorionic Gonadotrophin ◽  
Human Chorionic Gonadotrophin

Cholesterol biosynthesis from DL-[2-14C]mevalonic acid ([l4C]MVA) was demonstrated in ovine ovarian foIlicles and isolated thecal tissues and granulosaI ceIls incubated in vitro. Thecal tissues more readily synthesized cholesterol than did granulosal ceIls when incubated separately, but in the intact follicle the newly synthesized cholesterol distributed evenly between the two tissue layers, indicating that the theca could act as a supplementary source of cholesterol for the granulosal ceIls.

Simvastatin-induced endothelial cell detachment and microparticle release are prenylation dependent

2008 ◽  
Vol 100 (09) ◽  
pp. 489-497 ◽  
Author(s):  
Michaela Diamant ◽  
Maarten E. Tushuizen ◽  
Mohammed N. Abid-Hussein ◽  
Chi M. Hau ◽  
Anita N. Böing ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Disease Risk ◽  
Umbilical Vein ◽  
Cholesterol Biosynthesis ◽  
Cardiovascular Disease Risk ◽  
Mevalonic Acid ◽  
Caspase 8 ◽  
Cell Detachment ◽  
Induced Apoptosis

SummaryStatins reduce cardiovascular disease risk and affect endothelial function by cholesterol-dependent and independent mechanisms. Recently, circulating (detached) endothelial cells and endothelial microparticles (EMP) have been associated with endothelial functioning in vitro and in vivo.We investigated whether simvastatin affects endothelial detachment and release of EMP. Human umbilical vein endothelial cells (HUVECs) were incubated with clinically relevant concentrations of simvastatin (1.0 and 5.0 µM), with or without mevalonic acid (100 µM) or gera-nylgeranylpyrophosphate (GGPP; 20 µM) for 24 hours, and analyzed by flowcytometry andWe stern blot. Simvastatin at 1.0 and 5.0 µM increased cell detachment from 12.5 ± 4.1% to 26.0 ± 7.6% (p=0.013) and 28.9 ± 2.2% (p=0.002) as well as EMP release (p=0.098 and p=0.041, respectively).The majority of detached cells was apoptotic, although the fraction of detached cells that showed signs of apoptosis (>70%) was unaffected by simvastatin. Detached cells and EMP contained caspase 3 and caspase 8,but not caspase 9. Restoring either cholesterol biosynthesis and prenylation (mevalonate) or prenylation alone (GGPP) reversed all simvastatin-induced effects on cell detachment and EMP release. Adherent cells showed no signs of simvastatin-induced apoptosis. Simvastatin promotes detachment and EMP release by inhibiting prenylation, presumably via a caspase 8-dependent mechanism. We hypothesize that by facilitating detachment and EMP release, statins improve the overall condition of the remaining vascular endothelium.

scholarly journals Prenylation of mammalian Ras protein in Xenopus oocytes.

1990 ◽  
Vol 10 (11) ◽  
pp. 5945-5949 ◽  
Author(s):  
R Kim ◽  
J Rine ◽  
S H Kim
Keyword(s):  
Biological Activity ◽  
Posttranslational Modification ◽  
Xenopus Oocytes ◽  
Cholesterol Biosynthesis ◽  
Mevalonic Acid ◽  
Xenopus Laevis Oocytes ◽  
Ras Protein ◽  
C Terminus

Ras protein requires an intermediate of the cholesterol biosynthetic pathway for posttranslational modification and membrane anchorage. This step is necessary for biological activity. Maturation of Xenopus laevis oocytes induced by an oncogenic human Ras protein can be inhibited by lovastatin or compactin, inhibitors of the synthesis of mevalonate, an intermediate of cholesterol biosynthesis. This inhibition can be overcome by mevalonic acid or farnesyl diphosphate, a cholesterol biosynthetic intermediate downstream of mevalonate, but not by squalene, an intermediate after farnesyl pyrophosphate in the pathway. This study supports the idea that in Xenopus oocytes, the Ras protein is modified by a farnesyl moiety or its derivative. Furthermore, an octapeptide with the sequence similar to the C-terminus of the c-H-ras protein inhibits the biological activity of Ras proteins in vivo, suggesting that it competes for the enzyme or enzymes responsible for transferring the isoprenoid moiety (prenylation) in the oocytes. This inhibition of Ras prenylation by the peptide was also observed in vitro, using both Saccharomyces cerevisiae and Xenopus oocyte extracts. These observations show that Xenopus oocytes provide a convenient in vivo system for studies of inhibitors of the posttranslational modification of the Ras protein, especially for inhibitors such as peptides that do not penetrate cell membranes.

scholarly journals The preparation and purification of isolated rat corpus-luteum cells and their use in studying the relationship between cholesterol biosynthesis and the lutropin-stimulated formation of progesterone

1980 ◽  
Vol 192 (2) ◽  
pp. 559-567 ◽  
Author(s):  
B C McNamara ◽  
C E G Cranna ◽  
R Booth ◽  
D A Stansfield
Keyword(s):  
Cyclic Amp ◽  
Corpus Luteum ◽  
Cholesterol Biosynthesis ◽  
Gradient Centrifugation ◽  
Side Chain ◽  
Luteal Cells ◽  
Side Chain Cleavage ◽  
Chain Cleavage ◽  
In Cells

Isolated luteal cells, prepared from superovulated rat ovaries by digestion with collagenase, were subjected to density-gradient centrifugation on Percoll to give a more highly purified preparation of luteal cells than has been reported previously. The cells formed progesterone when incubated in vitro; lutropin stimulated this steroidogenesis. Progesterone formation was linear for at least 2 h; a minimal lutropin concentration of 1.0 ng/ml was needed for stimulation and concentrations of 3.0 and 100 ng/ml gave half-maximal and maximal responses respectively. The cells were unresponsive towards hormones other than lutropin. Exposure to lutropin raised the cellular cyclic AMP concentration, and dibutyryl cyclic AMP, but not dibutyryl cyclic GMP, was as effective in stimulating steroidogenesis as was lutropin. Aminoglutethimide, an inhibitor of cholesterol side-chain cleavage, completely blocked progesterone formation by the cells, showing cholesterol side-chain cleavage to be an obligatory step in steroidogenesis by these cells. Neither the activity of 3-hydroxy-3-methylglutaryl-CoA reductase nor the incorporation of radioactively labelled acetate or mevalonate into cholesterol by cells incubated in vitro were detectable unless the rats had been treated previously with 4-aminopyrazolo[3,4-d]pyrimidine. In cells from rats so treated, compactin was found to block almost completely the incorporation of radioactively labelled acetate, but not of mevalonate, into cholesterol, indicating that this inhibitor acts in corpus luteum in the same way as it does in other tissues. In cells from rats not treated with 4-aminopyrazolo[3,4-d]pyrimidine compactin had no effect on progesterone formation in vitro, showing cholesterol biosynthesis to be unnecessary for the rapid steroidogenic response by luteal cells to lutropin.

scholarly journals Cholesterol and mevalonic acid are independent requirements for the in vitro proliferation of human bone marrow granulocyte progenitor cells: studies using ML-236B

Blood ◽  
1983 ◽  
Vol 61 (4) ◽  
pp. 667-671
Author(s):  
PC Hoffman ◽  
CM Richman ◽  
RA Larson ◽  
S Yachnin
Keyword(s):  
Bone Marrow ◽  
Dna Synthesis ◽  
Colony Formation ◽  
Mononuclear Cells ◽  
Cholesterol Biosynthesis ◽  
Mevalonic Acid ◽  
Human Bone ◽  
Hmg Coa Reductase ◽  
Coa Reductase

ML-236B is a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, the key regulatory enzyme in the sequence that catalyzes the conversion of acetate to mevalonic acid in cholesterol biosynthesis. This compound caused marked inhibition of human bone marrow granulocyte progenitor cell (CFU-C) proliferation, the 50% inhibitory concentration (IHD50) being 2.0 X 10(6)M. Inhibition of colony formation was reversed by mevalonic acid but not by cholesterol. ML-236B also inhibited DNA synthesis and acetate incorporation into cholesterol in marrow mononuclear cells (IHD50 = 5.6 x 10(6)M and 3.2 x 10(7)M, respectively). No inhibition of mevalonate incorporation into cholesterol was observed. These results differ from those observed with 25-hydroxycholesterol, another inhibitor of HMG CoA reductase. The latter compound also inhibited CFU-C proliferation and cholesterol biosynthesis from acetate; inhibition of colony formation was reversed by cholesterol but not by mevalonic acid. In addition, 25- hydroxycholesterol inhibited cholesterol synthesis from mevalonic acid precursor. We conclude that: (1) ML-236B is a potent inhibitor of CFU-C proliferation, DNA synthesis, and cholesterol biosynthesis from acetate precursor in marrow mononuclear cells; (2) the effects of ML-236B are completely reversed by mevalonic acid but not by cholesterol, suggesting that mevalonic acid per se or one or more of its nonsterol products are critical for cell growth; (3) the inhibitory effects of 25- hydroxycholesterol on CFU-C proliferation and cholesterol biosynthesis are not solely a result of its inhibition of HMG CoA reductase, but are due in part to inhibition of enzymatic steps distal to mevalonic acid in the sterol synthetic pathway; and (4) mevalonic acid and cholesterol are independent requirements for CFU-C proliferation and differentiation in vitro.

scholarly journals Cholesterol and mevalonic acid are independent requirements for the in vitro proliferation of human bone marrow granulocyte progenitor cells: studies using ML-236B

Blood ◽  
1983 ◽  
Vol 61 (4) ◽  
pp. 667-671 ◽  
Author(s):  
PC Hoffman ◽  
CM Richman ◽  
RA Larson ◽  
S Yachnin
Keyword(s):  
Bone Marrow ◽  
Dna Synthesis ◽  
Colony Formation ◽  
Mononuclear Cells ◽  
Cholesterol Biosynthesis ◽  
Mevalonic Acid ◽  
Human Bone ◽  
Hmg Coa Reductase ◽  
Coa Reductase

Abstract ML-236B is a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, the key regulatory enzyme in the sequence that catalyzes the conversion of acetate to mevalonic acid in cholesterol biosynthesis. This compound caused marked inhibition of human bone marrow granulocyte progenitor cell (CFU-C) proliferation, the 50% inhibitory concentration (IHD50) being 2.0 X 10(6)M. Inhibition of colony formation was reversed by mevalonic acid but not by cholesterol. ML-236B also inhibited DNA synthesis and acetate incorporation into cholesterol in marrow mononuclear cells (IHD50 = 5.6 x 10(6)M and 3.2 x 10(7)M, respectively). No inhibition of mevalonate incorporation into cholesterol was observed. These results differ from those observed with 25-hydroxycholesterol, another inhibitor of HMG CoA reductase. The latter compound also inhibited CFU-C proliferation and cholesterol biosynthesis from acetate; inhibition of colony formation was reversed by cholesterol but not by mevalonic acid. In addition, 25- hydroxycholesterol inhibited cholesterol synthesis from mevalonic acid precursor. We conclude that: (1) ML-236B is a potent inhibitor of CFU-C proliferation, DNA synthesis, and cholesterol biosynthesis from acetate precursor in marrow mononuclear cells; (2) the effects of ML-236B are completely reversed by mevalonic acid but not by cholesterol, suggesting that mevalonic acid per se or one or more of its nonsterol products are critical for cell growth; (3) the inhibitory effects of 25- hydroxycholesterol on CFU-C proliferation and cholesterol biosynthesis are not solely a result of its inhibition of HMG CoA reductase, but are due in part to inhibition of enzymatic steps distal to mevalonic acid in the sterol synthetic pathway; and (4) mevalonic acid and cholesterol are independent requirements for CFU-C proliferation and differentiation in vitro.

Effect of inhibition of sterol delta 14-reductase on accumulation of meiosis-activating sterol and meiotic resumption in cumulus-enclosed mouse oocytes in vitro

Reproduction ◽  
2000 ◽  
pp. 171-179 ◽  
Author(s):  
L Leonardsen ◽  
M Stromstedt ◽  
D Jacobsen ◽  
KS Kristensen ◽  
M Baltsen ◽  
...  
Keyword(s):  
Cholesterol Biosynthesis ◽  
Cumulus Cell ◽  
Mevalonic Acid ◽  
Cumulus Cells ◽  
Control Culture ◽  
Meiotic Maturation ◽  
Human Follicular Fluid ◽  
Steroid Synthesis ◽  
Mouse Oocytes

Two sterols of the cholesterol biosynthetic pathway induce resumption of meiosis in mouse oocytes in vitro. The sterols, termed meiosis-activating sterols (MAS), have been isolated from human follicular fluid (FF-MAS, 4,4-dimethyl-5 alpha-cholest-8,14,24-triene-3 beta-ol) and from bull testicular tissue (T-MAS, 4,4-dimethyl-5 alpha-cholest-8,24-diene-3 beta-ol). FF-MAS is the first intermediate in the cholesterol biosynthesis from lanosterol and is converted to T-MAS by sterol delta 14-reductase. An inhibitor of delta 7-reductase and delta 14 reductase, AY9944-A-7, causes cells with a constitutive cholesterol biosynthesis to accumulate FF-MAS and possibly other intermediates between lanosterol and cholesterol. The aim of the present study was to evaluate whether AY9944-A-7 added to cultures of cumulus-oocyte complexes (COC) from mice resulted in accumulation of MAS and meiotic maturation. AY9944-A-7 stimulated dose dependently (5-25 mumol l-1) COC to resume meiosis when cultured for 22 h in alpha minimal essential medium (alpha-MEM) containing 4 mmol hypoxanthine l-1, a natural inhibitor of meiotic maturation. In contrast, naked oocytes were not induced to resume meiosis by AY9944-A-7. When cumulus cells were separated from their oocytes and co-cultured, AY9944-A-7 did not affect resumption of meiosis, indicating that intact oocyte-cumulus cell connections are important for AY9944-A-7 to exert its effect on meiosis. Cultures of COC with 10 mumol AY9944-A-7 l-1 in the presence of [3H]mevalonic acid, a natural precursor for steroid synthesis, resulted in accumulation of labelled FF-MAS, which had an 11-fold greater amount of radioactivity incorporated per COC compared with the control culture without AY9944-A-7. In contrast, incorporation of radioactivity into the cholesterol fraction was reduced 30-fold in extracts from the same oocytes. The present findings demonstrate for the first time that COC can synthesize cholesterol from mevalonate and accumulate FF-MAS in the presence of AY9944-A-7. Furthermore, AY9944-A-7 stimulated meiotic maturation dose dependently, indicating that FF-MAS, and possibly other sterol intermediates of the cholesterol synthesis pathway, play a central role in stimulating mouse oocytes to resume meiosis. The results also indicate that oocytes may not synthesize steroids from mevalonate.

Prenylation of mammalian Ras protein in Xenopus oocytes

1990 ◽  
Vol 10 (11) ◽  
pp. 5945-5949
Author(s):  
R Kim ◽  
J Rine ◽  
S H Kim
Keyword(s):  
Biological Activity ◽  
Posttranslational Modification ◽  
Xenopus Oocytes ◽  
Cholesterol Biosynthesis ◽  
Mevalonic Acid ◽  
Xenopus Laevis Oocytes ◽  
Ras Protein ◽  
C Terminus

Ras protein requires an intermediate of the cholesterol biosynthetic pathway for posttranslational modification and membrane anchorage. This step is necessary for biological activity. Maturation of Xenopus laevis oocytes induced by an oncogenic human Ras protein can be inhibited by lovastatin or compactin, inhibitors of the synthesis of mevalonate, an intermediate of cholesterol biosynthesis. This inhibition can be overcome by mevalonic acid or farnesyl diphosphate, a cholesterol biosynthetic intermediate downstream of mevalonate, but not by squalene, an intermediate after farnesyl pyrophosphate in the pathway. This study supports the idea that in Xenopus oocytes, the Ras protein is modified by a farnesyl moiety or its derivative. Furthermore, an octapeptide with the sequence similar to the C-terminus of the c-H-ras protein inhibits the biological activity of Ras proteins in vivo, suggesting that it competes for the enzyme or enzymes responsible for transferring the isoprenoid moiety (prenylation) in the oocytes. This inhibition of Ras prenylation by the peptide was also observed in vitro, using both Saccharomyces cerevisiae and Xenopus oocyte extracts. These observations show that Xenopus oocytes provide a convenient in vivo system for studies of inhibitors of the posttranslational modification of the Ras protein, especially for inhibitors such as peptides that do not penetrate cell membranes.

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