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  • 1
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 49 (1987), S. 0 
    ISSN: 1471-4159
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Medizin
    Notizen: 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. This temporal aspect of the requirement for mevalonate was unaffected by the presence of a 90-fold excess of cholesterol, provided as LDL. Thus, these data establish a requirement for mevalonate or for a presumably nonsterol derivative of mevalonate for the occurrence of DNA synthesis in synchronized glial primary cultures, and demonstrate that this requirement is expressed at a specific time in the cell cycle.
    Materialart: Digitale Medien
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  • 2
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 46 (1986), S. 0 
    ISSN: 1471-4159
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Medizin
    Notizen: Abstract: 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′-dimethyl-ethanolamine. 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′-dimethyl-ethanolamine, 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.
    Materialart: Digitale Medien
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  • 3
    ISSN: 1471-4159
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Medizin
    Notizen: Abstract: In this study we examined the effect on oligodendroglial survival of exogenous cystine deprivation. Oligodendroglia isolated from mixed glial primary cultures derived from brains of 1-day-old rats, and then grown for 3 days, were markedly dependent on extracellular cystine for survival. The EC50 values for cystine for a 24-h exposure ranged from 2 to 65 µM. After 6 h of cystine deprivation, the cellular glutathione level decreased to 21 ± 13% of the control. Free radical scavengers (α-tocopherol, ascorbate, idebenone, and N-tert-butyl-α-phenylnitrone) were protective against cystine deprivation but had no effect on the glutathione level. An iron chelator, desferrioxamine mesylate, also was protective. These findings suggest that intracellular hydroxyl radicals are important for this toxicity. In contrast to the observations in 3-day-old cultures, the dependence on exogenous cystine for cell viability was not observed consistently in oligodendroglia cultured for 6 days before the onset of cystine deprivation. Several observations suggested that this loss of cystine dependence was due to a diffusible factor. Sensitivity to the toxicity of cystine deprivation in day 6 cultures increased as the volume of medium was increased from 0.3 to 2 ml. Furthermore, preincubation of cystine-depleted medium with astrocyte cultures eliminated the toxicity of the cystine deprivation. HPLC assay of the conditioned cystine-depleted medium showed no significant change in cystine or cysteine concentration. We conclude that oligodendroglia are highly susceptible to cystine deprivation in day 3 cultures and that this susceptibility is due to the accumulation of intracellular free radicals in the setting of glutathione depletion. The resistance of day 6 oligodendroglial cultures is caused at least in part by a diffusible factor.
    Materialart: Digitale Medien
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  • 4
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 49 (1987), S. 0 
    ISSN: 1471-4159
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Medizin
    Notizen: Abstract: Primary cultures of newborn rat cerebrum, which are composed of glial cells (principally astroglia), were used for examining the relationship between dolichol-linked glycoprotein synthesis and DNA synthesis in developing cerebral glia. The cells were synchronized by reducing the content of fetal calf serum in the culture medium from 10 to 0.1% (vol/vol) for 48 h between days 4 and 6 in culture. Reversal of the quiescent state by return of the cultures to 10% serum causes a marked increase in DNA synthesis 12–24 h later. A sharp increase in glycoprotein synthesis (incorporation of [3H]mannose) occurred in the first 12 h after serum repletion, preceding the increase in DNA synthesis. Tunicamycin, an inhibitor of the dolichol-linked pathway to glycoprotein synthesis at the first committed step in oligosaccharide formation, promptly and completely prevented the increase in glycoprotein synthesis and, in addition, the subsequent increase in DNA synthesis. The effects of tunicamycin on glycoprotein and DNA syntheses were reversible, and no comparable effect c n total protein synthesis was observed. When tunicamycin was added only during a temporally circumscribed peiod in G1, i.e., from 3 to 9 h after serum repletion, the increase in DNA synthesis between 12 and 24 h after repletion was still markedly inhibited, i.e., to ∼45% of the value in untreated cultures. The data thus show that there is a requirement for dolichol-linked glycoprotein synthesis for the subsequent occurrence of DNA synthesis and that this requirement is expressed late in the G1 phase of the cell cycle.
    Materialart: Digitale Medien
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  • 5
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 46 (1986), S. 0 
    ISSN: 1471-4159
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Medizin
    Notizen: Abstract: 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.
    Materialart: Digitale Medien
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  • 6
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 48 (1987), S. 0 
    ISSN: 1471-4159
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Medizin
    Notizen: Abstract: The oligodendroglial enzyme, 2′,3′-cyclic nucleo-tide 3′-phosphohydrolase (CNP), is a valuable marker for expression of oligodendroglial differentiation in glial primary cultures, and the inducibility of this enzyme by dibu-tyryl-3′,5′-cyclic AMP (dBcAMP) appears to be limited to immature or developing oligodendroglia. To investigate the relationship between the induction of CNP and the sterol biosynthetic pathway, primary cultures of glia dissociated from the brains of newborn rats were maintained in 10% fetal calf serum (FCS) and exposed to 1 mM dBcAMP on day 7 in culture. Cultures so treated for either 48 h or 72 h demonstrated a three- to fourfold induction of CNP specific activity. The magnitude of this induction was not affected when the cholesterol content of the culture medium was reduced by 〉95% by placing the cultures in 10% lipoprotein-poor serum rather than 10% FCS during the exposure to dBcAMP. Mevinolin (10 μM), a specific inhibitor of 3-hy-droxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme of the sterol biosynthetic pathway, completely inhibited the induction of CNP by dBcAMP, while not affecting either the accumulation of cellular protein per flask or rate of protein synthesis. Simultaneous addition of mevalonate (20 mM) prevented the inhibition of the induction of CNP by mevinolin. However, simultaneous addition of low-density lipoprotein sufficient to increase the cholesterol content of the medium 80-fold failed to correct mevi-nolin's inhibition of the induction of CNP. Thus, these results demonstrate that the presence of mevalonate is obligatory for the induction of CNP in primary cultures of developing glia, and suggest that a critical nonsterol derivative of mevalonate is required for this expression of oligodendroglial differentiation.
    Materialart: Digitale Medien
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  • 7
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 44 (1985), S. 0 
    ISSN: 1471-4159
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Medizin
    Notizen: Abstract: Distinct regional differences in dolichol content were defined in human brain from 15 to 76 years of age. Concerning the regional distribution of dolichol, levels were: (1) higher in cortical gray matter than in subcortical white matter, (2) highest among cortical regions in temporal gray matter, (3) highest among all brain regions in thalamus, and (4) lowest among all brain regions in lower brain stem and spinal cord. The developmental changes in the contents of dolichol were found to be different among brain regions. For example, among regions with the highest levels of dolichol, in thalamus there was a six to sevenfold increase, but in parietal gray matter, only a 2.5-fold increase. Regional and developmental changes in the proportions of the individual molecular species (isoprenologues) of dolichol were also observed. The findings indicate that the metabolism of dolichol is not uniform among regions of developing and aging human brain and may have implications for the role of dolichol in normal and diseased human brain.
    Materialart: Digitale Medien
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  • 8
    ISSN: 1471-4159
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Medizin
    Notizen: Abstract: 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.
    Materialart: Digitale Medien
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  • 9
    ISSN: 1471-4159
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Medizin
    Notizen: Abstract : We found that several nitric oxide donors had similar potency in killing mature and immature forms of oligodendrocytes (OLs). Because of the possibility of interaction of nitric oxide with intracellular thiols, we tested the effect of the nitrosonium ion donor S-nitrosylglutathione (SNOG) in OL cultures in the setting of cystine deprivation, which has been shown to cause intracellular glutathione depletion. Surprisingly, the presence of 200 μM SNOG completely protected OLs against the toxicity of cystine depletion. This protection appeared to be due to nitric oxide, because it could be blocked by hemoglobin and potentiated by inclusion of superoxide dismutase. We tested the effect of three additional NO• donors and found that protection was not seen with diethylamine NONOate, a donor with a half-life measured in minutes, but was seen with dipropylenetriamine NONOate and diethylaminetriamine NONOate, donors with half-lives measured in hours. This need for donors with longer half-lives for the protective effect suggested that NO• was required when intracellular thiol concentrations were falling, a process evolving over hours in medium depleted of cystine. These studies suggest a novel protective role for nitric oxide in oxidative stress injury and raise the possibility that intracerebral nitric oxide production might be a mechanism of defense against oxidative stress injury in OLs.
    Materialart: Digitale Medien
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  • 10
    Digitale Medien
    Digitale Medien
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 33 (1979), S. 0 
    ISSN: 1471-4159
    Quelle: Blackwell Publishing Journal Backfiles 1879-2005
    Thema: Medizin
    Notizen: —The distribution of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) relative to that of several biochemical markers has been studied in subcellular fractions prepared from the brains of rats, aged 4 days to adult, by differential centrifugation. In the brains of 10-day-old animals fractions which sedimented at 800 g (P1 and 9000 g (P2) contained 28% and 65% respectively of the total reductase activity. A similar distribulion of the microsomal marker, NADPH-cytochrome c reductase, suggested that the HMG-CoA reductase activity in the low-speed pellets was due to substantial contamination of these fractions with endoplasmic reticulum. When P2 was fractionated on a discontinuous sucrose gradient, the distributions of protein, RNA and NADPH-cytochrome c reductase paralleled that of HMG-CoA reductase, indicaling a non-specific association of endoplasmic reliculum and HMG-CoA reductase with all of the structures sedimenting in P2. As brain maturation proceeded and a greater percentage of total brain protein (primarily associated with myelin) sedimenled in P1, the subcellular distributions of HMG-CoA reductase and the microsomal marker changed in a parallel way. By 21 days P1 contained nearly all of the reductase activity. Because the specific activity of HMG-CoA reductase in P1 decreased steadily between 4 and 21 days, while the specific activity of 2′:3′-cyclic nucleotide 3′-phosphohydrolase in this fraction increased in a coordinate fashion, we conclude that the reductase is not an integral component of myelin, and probably is associated exclusively with the endoplasmic reticulum included in P1. In view of the developmental changes in the distribution of HMG-CoA reductase among subcellular fraclions, we suggest that whole homogenates (or comparable tissue extracts) should be utilized to evaluate reductase activity in the developing brain.
    Materialart: Digitale Medien
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