Cholesterol Biosynthesis and Its Regulation in Dissociated Cell Cultures of Fetal Rat Brain: Developmental Changes and the Role of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase

Primary cultures of cells dissociated from fetal rat brain were utilized to define the developmental changes in cholesterol biosynthesis and the role of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in the regulation of these changes. Cerebral hemispheres of fetal rats of 15-16 days of gestation were dissociated mechanically into single cells and grown in the surface-adhering system. Cholesterol biosynthesis, studied as the rate of incorporation of [14C]acetate into digitonin-precipitable sterols, was shown to exhibit two distinct increases in synthetic rates, a prominent increase after 6 days in culture and a smaller one after 14 days in culture. Parallel measurements of HMG-CoA reductase activity also demonstrated two discrete increases in enzymatic activity, and the quantitative and temporal aspects of these increases were virtually identical to those for cholesterol synthesis. These data indicate that cholesterol biosynthesis undergoes prominent alterations with maturation and suggest that these alterations are mediated by changes in HMG-CoA reductase activity. The timing of the initial prominent peak in both cholesterol biosynthesis and HMG-CoA reductase activity at 6 days was found to be the same as the timing of the peak in DNA synthesis, determined as the rate of incorporation of [3H]thymidine into DNA. The second, smaller peak in reductase activity and sterol biosynthesis at 14 days occurred at the time of the most rapid rise in activity of the oligodendroglial enzyme, 2':3'-cyclic nucleotide 3'-phosphohydrolase (CNP). These latter observations suggest an intimate relationship of the sterol biosynthetic pathway with cellular proliferation and with oligodendroglial differentiation in developing mammalian brain.     

Relation of Cholesterol to Oligodendroglial Differentiation in C-6 Glial Cells

Abstract: The relation of cellular cholesterol content to a biochemical expression of oligodendroglial differentiation was studied in cultured C-6 glial cells. Induction of the oligodendroglial marker enzyme 2′: 3′-cyclic nucleotide 3′-phosphohydrolase (CNP) was determined after alteration of the sterol content of cellular membranes by exposure to compactin, a specific inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol synthesis. The sterol content and as a consequence, the sterol/phospholipid molar ratio of C-6 glial cells were decreased by treating the cells, in 10% lipoprotein-poor serum, with various concentrations of compactin for 24 h. The degrees of sterol depletion thus produced were maintained for 48 h after removal of the compactin if the cells were maintained in serum-free medium, the culture conditions necessary for induction of CNP in untreated cells. Forty-eight hours after removal of serum, no induction of CNP occurred in cells previously treated with 0.5 μg/ml of compactin, whereas untreated cells exhibited a three- to fourfold increase in CNP activity. Intermediate degrees of sterol depletion resulted in intermediate degrees of inhibition of the CNP induction. Moreover, the morphological expressions of glial differentiation observed in the untreated cells did not occur in the sterol-depleted cells. That the effect of compactin on the induction of CNP relates to depletion of sterol was indicated by the finding that when low-density lipoprotein was added to the compactin-treated cells, the induction of CNP, the morphological expressions of differentiation and the sterol/phospholipid molar ratios were preserved. The degree of sterol depletion that totally prevented the induction of CNP had no effect on (Na⁺+ K⁺)-activated ATPase activity, total protein synthesis and cell viability. The data define a critical role for sterol in oligodendroglial differentiation in this model system.     

Relation of Cellular Phospholipid Composition to Oligodendroglial Differentiation in C-6 Glial Cells

The relation of the polar head group composition of cellular phospholipids to a biochemical expression of oligodendroglial differentiation was studied in cultured C-6 glial cells. Induction of the oligodendroglial enzyme, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP), was determined after alteration of the polar head group composition of phospholipids by exposure of the cells to choline analogues, especially N,N'-dimethylethanolamine. To accomplish the phospholipid alteration, cells were grown in the presence of the analogue in medium free of exogenous lipid, i.e., first for 24 h in 10% delipidated serum and then for 48 h in serum-free medium. The 48-h exposure to serum-free medium resulted in untreated C-6 cells in a several fold increase in CNP activity, but in cells treated with 2.5 mM N,N'-dimethylethanolamine, total inhibition of this induction was observed. A graded, concentration-dependent inhibitory effect of the analogue on the induction of CNP was defined. The effect of the analogue was relatively specific, e.g., the activity of another plasma membrane enzyme of C-6 cells, (Na+ + K+)-activated ATPase, was not affected. Morever, there was no evidence of a toxic effect of the analogue; thus, total protein synthesis and cell growth were not altered, and the induction of CNP in serum-free medium recurred after removal of the analogue. N,N'-Dimethylethanolamine was shown to be incorporated into cellular phospholipids, primarily at the expense of phosphatidylcholine. The data define an important role for the polar head group composition of membrane phospholipids in oligodendroglial differentiation in this model system.     

Cell Cycle-Specific Requirement for Mevalonate, but Not for Cholesterol, for DNA Synthesis in Glial Primary Cultures

The requirement for the sterol biosynthetic pathway for the occurrence of DNA synthesis in glial cells and, in particular, the relative roles of cholesterol and of mevalonate have been studied. Primary cultures of developing glial cells were synchronized by reducing the content of fetal calf serum (FCS) in the culture medium from 10% to 0.1% (vol/vol) for 48 h between days 4 and 6 in culture. Reversal of the resulting quiescent state by the return of the cultures to 10% serum caused after 24 h a marked increase in DNA synthesis, and this increase was prevented by the simultaneous addition of mevinolin, a specific inhibitor of the sterol biosynthetic pathway at the 3-hydroxy-3-methylglutaryl coenzyme A reductase step, at the time of serum repletion. A dose-dependent reversal of the mevinolin inhibition of DNA synthesis occurred with simultaneous addition of mevalonate to the culture medium. The induction of DNA synthesis by serum repletion, its inhibition by mevinolin, and the reversal of the inhibition by mevalonate were unaffected by a 95% reduction in exogenous cholesterol produced by utilization of lipoprotein-poor serum (LPPS) rather than FCS. Similarly, return of quiescent cultures to 10% LPPS containing mevinolin and sufficient low-density lipoprotein (LDL) to raise the cholesterol concentration 80-fold failed to restore DNA synthesis. In addition, reversal of the mevinolin inhibition of DNA synthesis by mevalonate occurred despite the continuous presence of mevinolin if mevalonate was added as late as 12 h after serum repletion, but not if added after 16 h or more.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytoskeletal Structures and Oligodendroglial Differentiation in C-6 Glial Cells

Abstract: The relationship of the cytoskeleton to a biochemical expression of oligodendroglial differentiation was studied in cultured C-6 glial cells. Specifically, we investigated the effect of the cytoskeletal perturbants, colchicine and cytochalasin D, on the induction of the oligodendroglial marker enzyme. 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP), caused by removal of serum from the culture medium. Each drug inhibited CNP induction in a concentration-dependent manner, and essentially complete inhibition of induction was observed with 0.25 μ M colchicine or 2.0μ M cytochalasin D. Detailed study of the effect of colchicine was carried out. This antimicrotubular agent not only totally prevented induction if added at the onset of serum removal, but also prevented further induction when added at various times after serum removal. That the effect of colchicine related to the drug's effect on microtubules was supported by the demonstration that lumicolchicine, a colchicine isomer which has no effect on microtubules, had no effect on the CNP induction. Moreover, colchicine, but not lumicolchicine, prevented the morphological signs of differentiation provoked by serum removal. The effect of colchicine was reversible and relatively specific. Thus, no concomitant effect of colchicine on the activity of another plasma membrane enzyme of C-6 cells, i.e., (Na⁺+ K ⁺)-acti-vated ATPase, or on the rate of incorporation of [³H]leucine into total protein of intact cells could be discerned. The possibility that the site of the effect of colchicine is on intracellular events was suggested by the observation that the drug inhibited the induction of CNP by dibutyryl cyclic AMP. The data suggest that the cytoskeleton is involved in oligodendroglial differentiation.     

Cyclic AMP Induction of the Myelin Enzyme 2'',3''-Cyclic Nucleotide 3''-Phosphohydrolase in Rat Oligodendrocytes

Cyclic AMP (cAMP) is known to induce the activity of the myelin enzyme 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP; EC 3.1.4.37) in C6 rat glioma cells. This report shows that CNP is also inducible in oligodendrocytes explanted from 1-day-old rat cerebrum and grown in tissue culture. Induction was observed after a 1-day treatment with 1 mM N6, O2-dibutyryl cyclic AMP (dbcAMP) and was maximal after 5 days, reaching 200-240% of control. Induction was observed both in mixed cerebral cell cultures containing oligodendrocytes and astrocytes, and in purified cultures of oligodendrocytes prepared by a differential shakeoff procedure. Addition of dbcAMP to the cultures 3-9 days after the cells were explanted from rat brain induced CNP activity, but no induction was observed when dbcAMP treatment was begun 13 or more days after explanation. These results demonstrate that one component of myelin, CNP, is inducible in oligodendrocytes by a cAMP-mediated mechanism, and suggest a role for cAMP in the regulation of the myelin-associated functions of oligodendrocytes.     

Induction of an oligodendroglial enzyme in C-6 glioma cells maintained at high density or in serum-free medium.

The relationship between cell density and the activity of 2':3'-cyclic nucleotide 3'-phosphohydrolase (CNP), an enzyme believed to be specific to oligodendroglial cells and myelin in the brain, has been studied in cultured C-6 glioma cells. Over a 12-day period, the specific activity of CNP underwent a 4-fold increase in conjunction with an increase in the cell density (total protein/flask) and a decline in the growth rate of the cultures. In contrast, the specific activity of Na+,K+-ATPase was not influenced by cell density. Experiments with cultures seeded at different initial densities indicated that the increase in CNP activity coincided with the attainment of a specific cell density rather than with the length of time that the cells were maintained in culture. Arrest of cell proliferation in non-confluent C-6 cells by means of thymidine blockade was not sufficient to cause an increase in the activity of CNP; however, removal of serum from the culture medium resulted in a 3-fold induction of the enzyme in the absence of a high degree of cell contact. The induction of CNP in cells maintained in serum-free medium paralleled the development of a series of distinct morphological changes reminiscent of glial differentiation, which occurred within 48 hours after removal of the serum. Inhibition of protein synthesis by cycloheximide prevented the induction of CNP in serum-free cultures. The demonstration that an enhancement of an oligodendroglial characteristic in C-6 glioma cells can be obtained by growing the cells to high density or by removing serum from the medium, provides further support for the suggestion that these cells may be analogous to the glial stem cells present in the developing brain.     

Purification of Rat 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase

2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP, EC 3.1.4.37) has been isolated from rat brain myelin by chromatography on successive columns of phenyl-Sepharose CL-4B, CM-Sepharose CL-6B, and 8-(6-aminohexyl) amino-2'AMP-Sepharose 4B. From 15 g of rat brain, approximately 400 micrograms of pure CNP was obtained, with a specific activity of 1,200 (2',3'-cyclic AMP) units/mg protein. The Km of the rat enzyme was 3.7 mM, using 2',3'-cAMP as the substrate. Isoelectric focusing of the enzyme indicated a broad isoelectric range of 8.5-9.0. On SDS polyacrylamide gels, rat CNP appears as two protein bands of approximately 48,000 and 50,000 M.W., with an upper band intensity of about 1/10 that of the lower band. The relative intensities of the bands for CNP and the molecular weights correspond to the Wolfgram proteins W1 and W2 described by other investigators. The amino acid analysis of the purified rat enzyme compared favorably with reported determinations for the bovine enzyme and also with reported values for the rat Wolfgram proteins W1 and W2.     

Induction of Myelin Components: Cyclic AMP Increases the Synthesis Rate of 2'',3''-Cyclic Nucleotide 3''-Phosphohydrolase in C6 Glioma Cells

In an effort to determine the factors that stimulate myelin synthesis, we investigated the mechanism by which dibutyryl cyclic AMP induces the activity of the myelin enzyme, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP; EC 3.1.4.37), in C6 glioma cells. Immunotitration experiments and measurements of the accumulation of [35S]methionine-labeled CNP showed that dibutyryl cyclic AMP increased the amount of CNP in the cells but not the catalytic activity per molecule of the enzyme. Moreover, inhibition of protein synthesis with cycloheximide abolished induction of enzyme activity. Dibutyryl cyclic AMP doubled the rate of CNP synthesis but had no effect on the half-life of the enzyme (approximately 33 h). The induction was partially blocked by the inhibitors of mRNA synthesis, cordycepin or alpha-amanitin. Thus, cyclic AMP induces the synthesis of CNP.     

Obligatory Relationship Between the Sterol Biosynthetic Pathway and DNA Synthesis and Cellular Proliferation in Glial Primary Cultures

Primary cultures of newborn rat brain, which are composed predominantly of astroglia, were used to examine the relationship between the sterol biosynthetic pathway and DNA synthesis and cellular proliferation. Reduction of the fetal calf serum content of the culture medium from 10 to 0.1% (vol/vol) for an interval of 48 h between days 4 and 6 in culture resulted in a quiescent state characterized by inhibition of DNA synthesis and cellular proliferation. When 10% fetal calf serum was returned to the medium for these quiescent cells, within 24 h DNA synthesis increased markedly. Preceding the rise in DNA synthesis was an increase in sterol synthesis, which occurred within 12 h of the return of the quiescent cells to the 10% fetal calf serum. Exposure of the quiescent cells to mevinolin, a specific inhibitor of sterol synthesis at the 3-hydroxy-3-methylglutaryl-CoA reductase step, completely inhibited the increase in DNA synthesis that followed serum repletion. The increase in total protein synthesis that followed serum repletion was not similarly inhibited by mevinolin. When mevinolin was removed after causing the 24-h inhibition of DNA synthesis, the cultured cells underwent active DNA synthesis and proliferation. Thus, inhibition of the sterol biosynthetic pathway resulted in a specific and reversible inhibition of DNA synthesis and glial proliferation in developing glial cells. These findings establish a valuable system for the examination of glial proliferation, i.e., primary glial cultures subjected to serum depletion and subsequent repletion. Moreover, the data establish an obligatory relationship between the sterol biosynthetic pathway and DNA synthesis and cellular proliferation in developing glia.     

Steroid synthesis in rat brain cell cultures

Primary cultures derived from neonatal rat forebrains were established and cultured for several weeks. They grow entirely as glial cultures composed of oligodendrocytes and astrocytes. Glial cells undergo maturation and differentiation in culture. This was shown by measuring the oligodendroglial enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), a specific marker for expression of oligodendrocyte differentiation. CNPase activity increased from days 10-21 of culture. Both cell types were characterized by indirect immunofluorescence staining using monoclonal antibodies to galactocerebroside (Gal C) and myelin basic protein (MBP) for oligodendrocytes, and glial fibrillary acidic protein (GFAP) for astrocytes. Using the above criteria, we measured about 60% oligodendrocytes and 40% astrocytes after 3 weeks of culture. Oligodendrocytes, expressing Gal C and MBP, were highly immunoreactive to monospecific polyclonal antibodies to the cytochrome P-450scc, enzyme involved in the synthesis of pregnenolone from cholesterol. After incubation of glial cultures with [3H]mevalonolactone in the presence of mevinoline and trilostane, biosynthesis of [3H]cholesterol, [3H]pregnenolone (P) and [3H]pregn-5-ene-3 beta, 20 alpha-diol (20-OHP) was demonstrated. Steroid biosynthesis was related to oligodendroglial differentiation, as the initial and rapid rate of increase in CNPase activity was found to occur at the same time as the onset of steroid synthesis. Both reached a maximum at 3 weeks of culture and remained stable for several weeks. Steroid synthesis was increased by dibutyryl cAMP (0.2 mM), as well as by dexamethasone (10 nM). When aminoglutethimide, a potent inhibitor of cytochrome P-450scc, was added during the incubation of cells with [3H]mevalonolactone, [3H]cholesterol accumulated in the cells. After the release of aminoglutethimide blockade, [3H]20-OHP was the major steroid produced and released in the culture medium. The demonstration of de novo steroid biosynthesis and of the cholesterol side-chain cleavage cytochrome P-450 in normal rat glial cells brings additional support to the concept of "neurosteroids".     

Alcohol inhibits morphological and biochemical differentiation of C6 glial cells in culture

Cultures of glial cell lines (C6) were exposed to 10-micromilligram Dexamethasone which is known to cause morphological differentiation and induction of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) in these cells. Ethanol in a concentration of 1.5% abolished these responses, and at 1% diminished them.     

A Monoclonal Antibody Raised to Corpus Callosum Extract Reacts with 2'',3''-Cyclic Nucleotide 3''-Phosphohydrolase

Monoclonal antibody against 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) was generated by fusing mouse myeloma cells with spleen cells from BALB/c mice immunized with delipidated white matter from rat corpus callosum. The antibody was characterized by solid-phase radioimmunoassay, immunoblot of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoprecipitation from C6 glioma cells, and indirect immunofluorescence staining of monolayer cultures containing oligodendrocytes. The monoclonal antibody bound specifically to an intracellular antigen of oligodendrocytes, but not to Schwann cells, astrocytes, neurons, or fibroblast cytoplasm. The immunoblot of SDS-PAGE of CNS myelin showed that the antibody identified two protein bands at 48,000 and 50,000 molecular weight. These proteins were not identified in peripheral nervous system myelin. The monoclonal antibody immunoprecipitated CNP enzyme activity from extracts of C6 glioma cells. This monoclonal antibody should prove useful in further study of this myelin-specific enzyme in CNS myelin and in cells responsible for myelin production.     

Induction of N-Glycosylation Activity in Cultured Embryonic Rat Brain Cells

Developmental changes in protein N-glycosylation activity have been studied using cultures of dissociated fetal rat brain cells as an in vitro model system. These cultures undergo an initial phase of neurite outgrowth and cell proliferation (4-6 days in culture), followed by a period of cellular differentiation. N-Glycosylation activity has been measured by assaying the incorporation of [2-3H]mannose into dolichol-linked oligosaccharides and glycoprotein over a period of 1-25 days in culture. This study revealed a marked induction of N-glycosylation activity beginning at approximately 1 week of culture. [2-3H]Mannose incorporation into the oligosaccharide-lipid intermediate fraction and glycoprotein reached maximal values between 12 and 16 days of culture and declined thereafter. The major dolichol-linked oligosaccharide labeled by the brain cell cultures was shown to be Glc3Man9GlcNAc2 by HPLC analysis. Parallel incorporation studies with [3H]leucine showed that the increase in protein N-glycosylation was relatively higher than a concurrent increase in cellular protein synthesis observed during the induction period. Maximal labeling of glycoprotein corresponded to the period of glial differentiation, as indicated by a sharp rise in the marker enzymes, 2',3'-cyclic nucleotide 3'-phosphohydrolase (an oligodendroglial marker) and glutamine synthetase (an astroglial marker). The results describe a developmental activation of the N-glycosylation pathway and suggest a possible relationship between N-linked glycoprotein assembly and the growth and differentiation of glial cells.     

Regulation of sterol 27-hydroxylase and an alternative pathway of bile acid biosynthesis in primary cultures of rat hepatocytes

In man, hepatic mitochondrial sterol 27-hydroxylase and microsomal cholesterol 7-hydroxylase initiate distinct pathways of bile acid biosynthesis from cholesterol, the “acidic” and “neutral” pathways, respectively. A similar acidic pathway in the rat has been hypothesized, but its quantitative importance and ability to be regulated at the level of sterol 27-hydroxylase are uncertain. In this study, we explored the molecular regulation of sterol 27-hydroxylase and the acidic pathway of bile acid biosynthesis in primary cultures of adult rat hepatocytes. mRNA and protein turnover rates were approximately 10-fold slower for sterol 27-hydroxylase than for cholesterol 7-hydroxylase. Sterol 27-hydroxylase mRNA was not spontaneously expressed in culture. The sole requirement for preserving sterol 27-hydroxylase mRNA at the level of freshly isolated hepatocytes (0 h) after 72 h was the addition of dexamethasone (0.1 μM; > 7-fold induction). Sterol 27-hydroxylase mRNA, mass and specific activity were not affected by thyroxine (1.0 μM), dibutyryl-cAMP (50 μM), nor squalestatin 1 (150 nM-1.0 μM), an inhibitor of cholesterol biosynthesis. Taurocholate (50 μM), however, repressed sterol 27-hydroxylase mRNA levels by 55%. Sterol 27-hydroxylase specific activity in isolated mitochondria was increased > 10-fold by the addition of 2-hydroxypropyl-β-cyclodextrin. Under culture conditions designed to maximally repress cholesterol 7-hydroxylase and bile acid synthesis from the neutral pathway but maintain sterol 27-hydroxylase mRNA and activity near 0 h levels, bile acid synthesis from [¹⁴C]cholesterol remained relatively high and consisted of β-muricholate, the product of chenodeoxycholate in the rat. We conclude that rat liver harbors a quantitatively important alternative pathway of bile acid biosynthesis and that its initiating enzyme, sterol 27-hydroxylase, may be slowly regulated by glucocorticoids and bile acids.     

Structural evidence that brain cyclic nucleotide phosphodiesterase is a member of the 2H phosphodiesterase superfamily

2',3'-Cyclic-nucleotide 3'-phosphodiesterase (CNP) is an enzyme abundantly present in the central nervous system of mammals and some vertebrates. In vitro, CNP specifically catalyzes the hydrolysis of 2',3'-cyclic nucleotides to produce 2'-nucleotides, but the physiologically relevant in vivo substrate remains obscure. Here, we report the medium resolution NMR structure of the catalytic domain of rat CNP with phosphate bound and describe its binding to CNP inhibitors. The structure has a bilobal arrangement of two modules, each consisting of a four-stranded beta-sheet and two alpha-helices. The beta-sheets form a large cavity containing a number of positively charged and aromatic residues. The structure is similar to those of the cyclic phosphodiesterase from Arabidopsis thaliana and the 2'-5' RNA ligase from Thermus thermophilus, placing CNP in the superfamily of 2H phosphodiesterases that contain two tetrapeptide HX(T/S)X motifs. NMR titrations of the CNP catalytic domain with inhibitors and kinetic studies of site-directed mutants reveal a protein conformational change that occurs upon binding.     

Bile acid synthesis in the Smith-Lemli-Opitz syndrome: effects of dehydrocholesterols on cholesterol 7alpha-hydroxylase and 27-hydroxylase activities in rat liver.

The Smith-Lemli-Opitz syndrome (SLOS) is a congenital birth defect syndrome caused by a deficiency of 3beta-hydroxysterol Delta(7)-reductase, the final enzyme in the cholesterol biosynthetic pathway. The patients have reduced plasma and tissue cholesterol concentrations with the accumulation of 7-dehydrocholesterol and 8-dehydrocholesterol. Bile acid synthesis is reduced and unnatural cholenoic and cholestenoic acids have been identified in some SLOS patients. To explore the mechanism of the abnormal bile acid production, the activities of key enzymes in classic and alternative bile acid biosynthetic pathways (microsomal cholesterol 7alpha-hydroxylase and mitochondrial sterol 27-hydroxylase) were measured in liver biopsy specimens from two mildly affected SLOS patients. The effects of 7- and 8-dehydrocholesterols on these two enzyme activities were studied by using liver from SLOS model rats that were treated with the Delta(7)-reductase inhibitor (BM15.766) for 4 months and were comparable with more severe SLOS phenotype in plasma and hepatic sterol compositions. In the SLOS patients, cholesterol 7alpha-hydroxylase and sterol 27-hydroxylase were not defective. In BM15.766-treated rats, both enzyme activities were lower than those in control rats and they were competitively inhibited by 7- and 8-dehydrocholesterols. Rat microsomal cholesterol 7alpha-hydroxylase did not transform 7-dehydrocholesterol or 8-dehydrocholesterol into 7alpha-hydroxylated sterols. In contrast, rat mitochondrial sterol 27-hydroxylase catalyzed 27-hydroxylation of 7- and 8-dehydrocholesterols, which were partially converted to 3beta-hydroxycholestadienoic acids. Addition of microsomes to the mitochondrial 27-hydroxylase assay mixture reduced 27-hydroxydehydrocholesterol concentrations, which suggested that 27-hydroxydehydrocholesterols were further metabolized by microsomal enzymes. These results suggest that reduced normal bile acid production is characteristic of severe SLOS phenotype and is caused not only by depletion of hepatic cholesterol but also by competitive inhibition of cholesterol 7alpha-hydroxylase and sterol 27-hydroxylase activities by accumulated 7- and 8-dehydrocholesterols. Unnatural bile acids are synthesized mainly by the alternative pathway via mitochondrial sterol 27-hydroxylase in SLOS.     

Cyclic AMP and serum arrest of the mitotic activity of human diploid fibroblasts.

Mitotic activity in confluent cultures of human diploid fibroblasts was arrested by the reduction of the serum concentration of the incubation medium to 0.5% or by the addition of 0.5 mM 6-N, 2'-O-dibutyryl-adenosine 3':5'-cyclic monophosphate (db cAMP). Under either of these conditions, cultures maintained a constant cell number for 14 days; cultures continuously exposed to medium containing 10% serum doubled their cell number during this 14-day period. The protein cotent per cell decreased by 20% when cells were maintained with 0.5% serum whereas that of cells exposed to db cAMP remained constant. Ultrastructural studies revealed that cells exposed to db cAMP exhibited a morphology typical of cells cultures with 10% serum alone, whereas cells incubated with 0.5% serum showed the ultrastructural changes in mitochondria, endoplasmic reticulum and Golgi complex previously identified with low-serum arrest. Cellular adenosine 3':5'-cyclic monophosphate (cAMP) levels remained constant during the 7-day growth period in which confluency was attained, as well as during the 14-day arrested period with 0.5% serum. These results indicated that the mitotic inhibition induced by reducing the serum concentration of the incubation medium was not mediated by increased intracellular levels of cAMP and differed from that induced by the addition of exogenous db cAMP.