Membrane Fatty Acid Modifications of PC12 Cells by Arachidonate or Docosahexaenoate Affect Neurite Outgrowth But Not Norepinephrine Release

The relationships between membrane fatty acid modification and neurite outgrowth and norepinephrine release were evaluated in PC12 cells. [³H]Norepinephrine release evoked by carbachol was unaffected by the modifications. Basal spontaneous release was elevated with increases in the degree of unsaturation using cells supplemented with n-3 fatty acids; a reverse correlation was observed for [³H]norepinephrine uptake. Supplementation of PC12 cells with either n-6 fatty acids or 18:1 also increased the basal release and decreased the uptake. Docosahexaenoic acid promoted and arachidonic acid suppressed neurite outgrowth induced by nerve growth factor. Choline acetyltransferase activity was slightly influenced by these fatty acids. Thus, modifications of PC12 cells with arachidonic acid and docosahexaenoic acid had a relatively small effect on the degree of differentiation but had pronounced but opposite effects on neurite elongation. Ethanolamine glycerophospholipid synthesis was elevated during differentiation induced by nerve growth factor and it was suppressed by added arachidonic acid but not by docosahexaenoic acid. Our results raise the possibility that the decreased phospholipid synthesis caused by arachidonate may lead to the suppression of neurite elongation.     

Stability of Lactic Acid Bacteria to Freezing as Related to Their Fatty Acid Composition

The viability of Streptococcus lactis and Lactobacillus sp. A-12 after freezing at -17°C for 48 h was better preserved when the cells were grown in medium supplemented with oleic acid or Tween 80 (polyoxyethylene sorbitan monooleate). A pronounced change in the cellular fatty acid composition was noted when the bacteria were grown in the presence of Tween 80. In S. lactis the ratio of unsaturated to saturated fatty acids increased from 1.18 to 2.55 and in Lactobacillus sp. A-12 it increased from 0.85 to 1.67 when Tween 80 was added to the growth medium. The antibiotic cerulenin markedly inhibited the growth of lactic acid bacteria in tomato juice (TJ) medium but had almost no effect on the growth of the bacteria in TJ medium containing Tween 80 (or oleic acid). The antibiotic inhibited markedly the incorporation of [1-¹⁴C]acetate but had no inhibitory effect on the incorporation of exogenous [1-¹⁴C]oleate (or [1-¹⁴C]palmitate) into the lipid fractions of lactic acid bacteria. Thus, the fatty acid composition of lactic acid bacteria, inhibited by the antibiotic cerulenin, can be modulated by exogenously added oleic acid (or Tween 80) without the concurrent endogenous fatty acid synthesis from acetate. The data obtained suggest that cerulenin inhibits neither cyclopropane fatty acid synthesis nor elongation of fatty acid acyl intermediates. The radioactivity of cells grown in the presence of [1-¹⁴C]oleate and cerulenin was associated mainly with cyclopropane Δ19:0, 20:0 + 20:1, and 21:0 acids. As a consequence, cerulenin caused a decrease in the ratio of unsaturated to saturated fatty acids in lactic acid bacteria as compared with cells grown in TJ medium plus Tween 80 but without cerulenin. Cerulenin caused a decrease in the viability of S. lactis and Lactobacillus sp. A-12 after freezing at -17°C for 48 h only when Tween 80 was present in the growth medium. We conclude that the sensitivity of lactic acid bacteria to damage from freezing can be correlated with specific alterations in the cellular fatty acids.     

RNA-mediated transfer of cellular immunity to a synthetic env antigen of the human immunodeficiency virus (HIV-1)

Evidence is provided in this paper that indicates that fatty acids but not phospholipids are removed from microsomes or artificial membranes (liposomes, unilamellar vesicles) by mouse liver cytosolic preparations enriched with fatty acid binding protein (FABP). The cytosolic proteins can act as acceptors for fatty acids but not for phospholipids of microsomal origin. Direct evidence came when liposomes made of egg yolk phosphatidylcholine, containing both [¹⁴C]labeled phospholipids and [1-¹⁴C] palmitic acid were incubated with FABP. Using sonicated vesicles as fatty acid or phospholipid donors, mouse liver fatty acid binding protein was capable of binding palmitic acid but not phospholipids. These studies suggest that liver fatty acid binding protein can interact with different kinds of membranes increasing specifically the desorption of fatty acids.Abbreviations FABP Fatty Acid Binding Protein - PC Phos phatidylcholine Fellow of the Comisión de Investigaciones Cientificas de la Provincia de Buenos Aires (CIC), ArgentinaMember of Carrera de Investigador Científico, Consejo Nacional de Investigaciones Cientificas y Técnicas de la Republica Argentina (CONICET)     

Modification of membrane phospholipid fatty acyl composition in a leukemic T cell line: effects on receptor mediated intracellular Ca2+ increase

The effect of modifying fatty acyl composition of cellular membrane phospholipids on receptor-mediated intracellular free Ca²⁺ concentration ([Ca²⁺]_i) increase was investigated in a leukemic T cell line (JURKAT). After growing for 72 h in medium supplemented with unsaturated fatty acids (UFAs) and α-tocopherol, the fatty acyl composition of membrane phospholipids in JURKAT cells was extensively modified. Each respective fatty acid supplemented in the culture medium was readily incorporated into phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine in the JURKAT cells. The total n ? 6 fatty acyl content was markedly reduced in phosphatidylinositol and phosphatidylcholine of cells grown in the presence of n ? 3 fatty acids (α-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid). Conversely, in the presence of n ? 6 fatty acids (linoleic acid and arachidonic acid), the total n ? 3 fatty acyl content was reduced in all the phospholipids examined. In n ? 3 and n ? 6 polyunsaturated fatty acid (PUFA) modified JURKAT cells, the total n ? 9 monounsaturated fatty acyl content in the phospholipids were markedly reduced. Changing the fatty acyl composition of membrane phospholipids in the JURKAT cells appear to have no affect on the presentation of the T cell receptor/CD3 complex or the binding of anti-CD3 antibodies (OKT3) to the CD3 complex. However, the peak increase in [Ca²⁺]_i and the prolonged sustained phase elicited by OKT3 activation were suppressed in n ? 3 and n ? 6 PUFA but not in n ? 9 monounsaturated fatty acid modified cells. In Ca²⁺ free medium, OKT3-induced transient increase in [Ca²⁺]_i, representing Ca²⁺ release from the inositol 1,4,5-triphosphate-sensitive Ca²⁺ stores, were similar in control and UFA modified cells. Using Mn²⁺ entry as an index of plasma membrane Ca²⁺ permeability, the rate of fura-2 fluorescence quenching as a result of Mn²⁺ influx stimulated by OKT3 in n ? 9 monounsaturated fatty acid modified cells was similar to control cells, but the rates in n ? 3 and n ? 6 PUFA modified cells were significantly lower. These results suggest that receptor-mediated Ca²⁺ influx in JURKAT cells is sensitive to changes in the fatty acyl composition of membrane phospholipids and n ? 9 monounsaturated fatty acids appears to be important for the maintenance of a functional Ca²⁺ influx mechanism.     

Relationship between fatty acid binding proteins,acetyl-CoA formation and fatty acid synthesis in developing human placenta

The relationship between fatty acid binding proteins, ATP citrate lyase activity and fatty acid synthesis in developing human placenta has been studied. Fatty acid binding proteins reverse the inhibitory efect of palmitoyl-CoA and oleate on ATP citrate lyase and fatty acid synthesis. In the absence of these inhibitors fatty acid binding proteins activate ATP citrate lyase and stimulate [ 1-¹⁴ C] acetate incorporation into placental fatty acids indicating binding of endogenous inhibitors by these proteins. Thus these proteins regulate the supply of acetyl-CoA as well as the synthesis of fatty acids from that substrates. As gestation proceeds and more lipids are required by the developing placenta fatty acid binding protein content, activity of ATP citrate lyase and rate of fatty acid synthesis increase indicating a cause and efect relationship between the demand of lipids and supply of precursor fatty acids during human placental development.     

RADIOLABELING STUDIES OF LIPIDS AND FATTY ACIDS IN NANNOCHLOROPSIS (EUSTIGMATOPHYCEAE), AN OLEAGINOUS MARINE ALGA1

The synthesis of fatty acids and lipids in Nannochloropsis sp. was investigated by labeling cells in vivo with [¹⁴C]-bicarbonate or [¹⁴C]-acetate. [¹⁴C]-bicarbonate was incorporated to the greatest extent into 16:0, 16:1, and 14:0 fatty acids, which are the predominant fatty acids of triacylglycerols. However, more than half of the [¹⁴C]-acetate was incorporated into longer and more desaturated fatty acids, which are constituents of membrane lipids. [¹⁴C]-acetate was incorporated most strongly into phosphatidylcholine, which rapidly lost label during a 5-h chase period. The label associated with phosphatidylethanolamine also decreased during the chase period, whereas label in other membrane lipids and triacylglycerol increased. The dynamics of labeling, along with information regarding the acyl compositions of various lipids, suggests that 1) the primary products of chloroplast fatty acid synthesis are 14:0, 16:0, and 16:1; 2) C₂₀ fatty acids are formed by an elongation reaction that can utilize externally supplied acetate; 3) phosphatidylcholine is a site for desaturation of C₁₈ fatty acids; and 4) phosphatidylethanolamine may be a site for desaturation of C₂₀ fatty acids.     

Lipids of Daucus carota cell suspension cultures

In carrot cell suspension cultures greater amounts of phospholipid were detected than in carrot root material, but the major phospholipid classes were the same in both materials, and their fatty acid composition was very similar. In contrast to the cultured cells, no significant amounts of free fatty acids and monoglycerides, as well as diglycerides, could be detected in the carrot root. The fatty acid composition of the major lipid classes from cell cultures is reported for the first time in this report. The degree of unsaturation was higher in triglycerides and phospholipids than in free fatty acids. In a study of phospholipid biosynthesis, [³H]-glycerol was shown to be incorporated into 4-phospholipids (PE, PC, PG, PS⁷) to different extents. The highest specific activity was observed in PC and PG. Five molecular species were isolated from each of the 4 phospholipids and analyzed by GC-MS and LSC.     

Metabolism of tween-Fatty Acid esters by cultured soybean cells : kinetics of incorporation into lipids, subsequent turnover, and associated changes in endogenous Fatty Acid synthesis

Uptake of Tween-fatty acid esters and incorporation of the fatty acids into lipids by soybean (Glycine max [L.] Merr.) suspension cultures was investigated, together with subsequent turnover of the incorporated fatty acids and associated changes in endogenous fatty acid synthesis. Tween uptake was saturable, and fatty acids were rapidly transferred from Tweens to all acylated lipids. Patterns of incorporation into glycerolipids were similar in cells treated with Tweens carrying [1-¹⁴C]-fatty acids and in cells treated with [1-¹⁴C]acetate, indicating that exogenous fatty acids were used for glycerolipid synthesis essentially as if they had been made by the cell. In Tween-treated cells neutral lipids (which include Tweens) initially accounted for the majority of lipid radioactivity. Radioactivity was then rapidly transferred to glycerolipids. A transient pool of free fatty acids accounting for up to 10% of lipid radioactivity was observed. This was consistent with the hypothesis that fatty acids are transferred from Tweens to lipids by deacylation of the Tweens, creating a pool of free fatty acids which are then used for lipid synthesis. Sterols were only slightly labeled in cells treated with Tweens, but accounted for nearly 50% of lipid radioactivity in cells treated with acetate. This suggested very little degradation and reutilization of the radioactive fatty acids in cells treated with Tweens. In cells treated with either [1-¹⁴C]acetate or Tween-[1-¹⁴C]-18:1, 70% of the initial fatty acid radioactivity remained in fatty acids after a 100 hour chase. By contrast, fatty acids not normally present disappeared more rapidly, suggesting differential treatment of such fatty acids compared with those normally present. Cells which had incorporated large amounts of exogenous fatty acids altered fatty acid synthesis in three distinct ways: (a) amounts of [1-¹⁴C]acetate incorporated into fatty acids were reduced; (b) cells incorporating exogenous unsaturated fatty acids increased the proportion of [1-¹⁴C]acetate partitioned into saturated fatty acids, while the converse was true of cells which had incorporated exogenous saturated fatty acids; (c) desaturation of 18:1 to 18:2 and 18:3 was reduced in cells which had incorporated unsaturated fatty acids. These results suggest that Tween-fatty acid esters will be useful for supplying fatty acids to cells for a variety of studies related to fatty acid or membrane metabolism.     

Enhancement of polyunsaturated fatty acid production by cerulenin treatment in polyunsaturated fatty acid-producing bacteria

When docosahexaenoic acid (DHA)-producing Moritella marina strain MP-1 was cultured in the medium containing 0.5 μ g cerulenin ml⁻¹, an inhibitor for fatty acid biosynthesis, the cells grew normally, but the␣content of DHA in the total fatty acids increased from 5.9–19.4%. The DHA yield of M. marina strain MP-1 cells also increased from 4 to 13.7 mg l⁻¹ by cerulenin treatment. The same effect of cerulenin was observed in eicosapentaenoic acid (EPA)-producing Shewanella marinintestina strain IK-1 grown in the medium containing 7.5 μg cerulenin ml⁻¹, and the cerulenin treatment increased the EPA yield from 1.6 to 8 mg l⁻¹. The use of cerulenin is, therefore, advantageous to increase the content of intracellular polyunsaturated fatty acids (PUFA) in particular PUFA-containing phospholipids in bacterial cells.An erratum to this article can be found at .     

Alterations of phospholipid metabolism by phorbol esters and fatty acids occur by different intracellular mechanisms in cultured glioma, neuroblastoma, and hybrid cells

Differences between the influences of phorbol esters (such as 4 beta-12-O-tetradecanoylphorbol 13-acetate) and of fatty acids (such as oleic acid) on the synthesis and turnover of phosphatidylcholine (PtdCho) and other phospholipids have been studied in glioma (C6), neuroblastoma (N1E-115), and hybrid (NG108-15) cells in culture using [methyl-3H]choline, [32P]Pi, [1,2-14C]ethanolamine, or 1-14C-labeled fatty acids as lipid precursors. 100-500 microM oleic acid stimulated PtdCho synthesis 3- to 5-fold in all three cell lines, but had little influence on chase of choline label following a 24-h pulse. Phorbol ester (50-200 nM) stimulated PtdCho synthesis 1.5- to 3-fold in C6 cells, was without effect in N1E-115 cells, and had intermediate effects on NG108-15 cells. Phorbol ester stimulated both uptake of extracellular choline and synthesis of PtdCho, whereas fatty acid stimulated only synthesis. Release of radioactivity from 24-h pulse-labeled PtdCho to the medium was enhanced by phorbol ester in C6 cells. Incorporation of [32P]Pi, primarily into PtdCho, was stimulated, whereas utilization of [1,2-14C]ethanolamine or 1-14C-fatty acid was little altered by phorbol ester. C6 cells "down-regulated" with phorbol ester lost the stimulatory response of subsequent treatment with phorbol esters on PtdCho synthesis, but the response to fatty acid was enhanced. Fatty acid had little influence on the relative binding of phorbol ester or "translocation" of phorbol ester binding sites. Accordingly, metabolism of phospholipids in these cultured cells of neural origin is markedly influenced by cell type, phospholipid class, condition of incubation medium, and nature of stimulator. Phorbol esters and fatty acids appear to enhance phospholipid synthesis and turnover by distinct intracellular mechanisms.     

Effect of fatty acids on the synthesis and secretion of apolipoprotein B by rat hepatocytes

The modulation of apolipoprotein B synthesis and secretion by fatty acids in rat hepatocytes was studied. Maximum apolipoprotein B production was obtained in the case of oleic acid followed by linoleic, stearic and palmitic/linolenic acid when compared to control which was not supplemented with any fatty acids. Oleic acid was found to exert a concentration dependent increase in the secretion of [³H] apolipoprotein B into the medium while that associated with the cell layer was not affected. Pulse chase experiments in the presence of oleic acid showed that it caused an increase in the secretion of apolipoprotein B into the medium.¹⁴C-acetate incorporation into cholesterol and cholesteryl ester associated with the cell layer and secreted very low density lipoproteins also showed an increase in the presence of oleic acid indicating an increase in cholesterogenesis. The effect of oleic acid on [³H] apolipoprotein B and very low density lipoproteins secretion appeared to be mediated through cholesterol as (i) ketoconazole, an inhibitor of cholesterol synthesis caused significant reduction in the stimulatory effect of oleic acid on apolipoprotein secretion and (ii) mevinolin, another inhibitor of cholesterol synthesis also reversed the stimulatory effect of oleic acid on apolipoprotein B secretion. These results indicated that oleic acid may influence apolipoprotein B synthesis and secretion in hepatocytes probably by affecting cholesterol/cholesteryl ester formation which may be a critical component in the secretion of apolipoprotein B as lipoproteins     

The effect of cadmium on lipid and fatty acid biosynthesis in tomato leaves

This research aims to examine the effect of cadmium uptake on lipid composition and fatty acid biosynthesis, in young leaves of tomato treated seedlings (Lycopersicon esculentum cv. Ibiza F1). Results in membrane lipids investigations revealed that high cadmium concentrations affect the main lipid classes, leading to strong changes in their composition and fatty acid content. Thus, the exposure of tomato plants to cadmium caused a concentration-related decrease in the unsaturated fatty acid content, resulting in a lower degree of fatty acid unsaturation. The level of lipid peroxides was significantly enhanced at high Cd concentrations. Studies of the lipid metabolism using radioactive labelling with [1-¹⁴C]acetate as a major precursor of lipid biosynthesis, showed that levels of radioactivity incorporation in total lipids as well as in all lipid classes were lowered by Cd doses. In total lipid fatty acids, [1-¹⁴C]acetate incorporation was reduced in tri-unsaturated fatty acids (C16:3 and C18:3); While it was enhanced in the palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0) and linoleic (C18:2) acids. [1-¹⁴C]acetate incorporation into C16:3 and C18:3 of galactolipids [monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG)] and some phospholipids [phosphatidylcholine (PC) and phosphatidylglycerol (PG)] was inhibited by Cd stress. Our results showed that in tomato plants, cadmium stress provoked an inhibition of polar lipid biosynthesis and reduced fatty acid desaturation process.     

The effects of high concentrations of sodium or calcium ions on the lipid composition and properties of Tetrahymena membranes

Tetrahymena pyriformis cells have been grown in media varying in NaCl concentration from 3.7 mM (normal medium) to 0.3 M and varying in CaCl₂ from 0.2 mM (normal medium) to 0.1 M. Tetrahymena grown in 0.3 M NaCl showed relatively few alterations in phospholipid composition, with significant changes being found only in the cell surface membranes (pellicle), which increased in phosphatidylethanolamine content from 39% (low Na⁺) to 48% (high Na⁺) of the total phospholipids. The small decrease in fatty acid unsaturation and increase in shorter chain fatty acids in pellicle phospholipids were not statistically significant. No significant changes in phospholipid head group composition or fatty acid distribution were observed in high Ca²⁺-grown cells. Complementary studies of membrane fluidity, as inferred from freeze-fracture electron microscopy analysis, indicated that membranes of high Na⁺-acclimated cells were similar to those of control cells, when each was measured in its respective medium. However, the outer alveolar membrane of the pellicle and the food vacuolar membrane were considerably less fluid in high-Ca²⁺ cells. The lower fluidity in vacuolar membranes may have been responsible for alterations in the cells' capacity to form food vacuoles.     

Differential distribution and metabolism of arachidonic acid and docosahexaenoic acid by human placental choriocarcinoma (BeWo) cells

The time course of incorporation of [¹⁴C]arachidonic acid and [³H]docosahexaenoic acid into various lipid fractions in placental choriocarcinoma (BeWo) cells was investigated. BeWo cells were found to rapidly incorporate exogenous [¹⁴C]arachidonic acid and [³H] docosahexaenoic acid into the total cellular lipid pool. The extent of docosahexaenoic acid esterification was more rapid than for arachidonic acid, although this difference abated with time to leave only a small percentage of the fatty acids in their unesterified form. Furthermore, uptake was found to be saturable. In the cellular lipids these fatty acids were mainly esterified into the phospholipid (PL) and the triacyglycerol (TAG) fractions. Smaller amounts were also detected in the diacylglycerol and cholesterol ester fractions. Almost 60% of the total amount of [3H]Docosahexaenoic acid taken up by the cells was esterified into TAG whereas 37% was in PL fractions. For arachidonic acid the reverse was true, 60% of the total uptake was incorporated into PL fractions whereas less than 35% was in TAG. Marked differences were also found in the distribution of the fatty acids into individual phospholipid classes. The higher incorporation of docosahexaenoic acid and arachidonic acid was found in PC and PE, respectively. The greater cellular uptake of docosahexaenoic acid and its preferential incorporation in TAG suggests that both uptake and transport modes of this fatty acid by the placenta to fetus is different from that of arachidonic acid.     

Fatty acid composition and lipid synthesis in developing safflower seeds

Linoleic acid predominated in every lipid class during the whole period of seed development of safflower, while linolenic acid decreased with increasing maturation and it was not detected in mature seeds. Just before the initiation of triacylglycerol accumulation, the fatty acid composition of triacylglycerols changed more rapidly than those of phospholipids and glycolipids. Saturated fatty acids tended to accumulate at the 1- and 3-positions of the glycerol molecule and the more highly unsaturated acids at the 2-position. The fatty acid compositions at the 1- and 3-positions were similar in all cases investigated, but in none of the triacylglycerols was the distribution completely symmetrical. The positional distribution of linolenic acid in triacylglycerols prepared from the immature seeds 2 days after flowering and from the leaves was unusual; in spite of its highest degree of unsaturation, it was preferentially esterified at the 1- and 3-positions. When triacylglycerol was most rapidly accumulated (14–18 days after flowering), the incorporation of acetate-[U- ¹⁴C] into total lipids was also maximum and dienoic fatty acids were the principal acids labelled. Diacylglycerols and compound lipids reached the highest rate of synthesis 15 days after flowering, and then a maximum incorporation into triacylglycerol occurred 18 days after flowering. Incubation temperature affected the synthesis of individual lipid classes. Triacylglycerol was more rapidly synthesized at 32° than at 10°, while diacylglycerols and compound lipids were accumulated under the low-temperature condition. A rise of incubation temperature caused a depression in dienoic acid synthesis.     

Fatty Acid Acylated Proteins of the Halotolerant Alga Dunaliella salina

The unicellular, wall-less alga Dunaliella salina has been shown to contain an array of proteins modified by the covalent attachment of fatty acids. Myristic acid (14:0) comprised approximately 80% by weight of the protein-linked acyl groups in samples derived from cells cultured in medium containing 1.7 molar NaCl and 93% in samples from cells grown in medium containing 3.0 molar NaCl. Palmitic and stearic acids accounted for most of the remaining protein-bound acyl chains. Approximately 0.2% of the incorporated radioactivity was estimated to be in linkage with protein. The bulk of acyl chains (about 99%) were resistant to cleavage by alkali, indicating a preponderance of amide bonding. The sodium dodecyl sulfate-polyacrylamide electrophoresis labeling pattern of proteins from [³H]myristic-labeled cells was significantly different from that of proteins from cells exposed to [³H]palmitate. The appearance of radioactivity in certain proteins was also influenced by the salinity of the culture medium. Thus growth in moderate (1.7 molar) salt favored the acylation of a 48-kilodalton polypeptide whereas in high (3.0 molar) salt, a 17-kilodalton polypeptide was more heavily labeled.     

Separate binding sites in rat brain synaptic membranes for l-cysteine sulfinate and for l-glutamate

Binding of l-[³H]cysteine sulfinic acid (CSA) and l-[³H]glutamate were compared in various subcellular fractions and in the presence of a variety of pharmacological and ionic manipulations in order to test the possibility that the two amino acids possessed separate binding sites.The specific l-[³H]cysteine sulfinate binding was found to be enriched maximally in medium and high density synaptic membranes, while the crude mitochondrial synaptosomal fraction displayed the highest l-[³H]glutamate binding. The ratio of l-[³H]cysteine sulfinate binding/l-[³H]glutamate binding was variable across brain regions. Several compounds differentially affected l-[³H]cysteine sulfinate and l-[³H]glutamate binding. l-cysteine sulfinate was the most potent displacer regardless of the binding considered. Finally, while cations produced qualitatively similar effects on the binding of the two amino acids, quantitative differences were evident.In sum, these data revealed the complexity of l-[³H]cysteine sulfinate and l-[³H]glutamate binding. They suggest the existence of several binding sites and that some of these are shared by both substances. However, the results also indicate that separate binding sites for the two amino acids exist in synaptic membrane, giving further support to the hypothesis that cysteine sulfinate serves a neurotransmitter role in the central nervous system.     

Compartmentation of NADPH in rat liver.

Rat liver slices were incubated with specifically ³H-labeled glucoses and [2-³H]sorbitol, and the incorporations of ³H into fatty acids and cholesterol were determined. Incorporation of ³H from [1-³H]glucose relative to that from [3-³H]glucose via NADPH formed in the pentose cycle was similar into fatty acids and cholesterol. This indicates (1) the presence of a common pool of NADPH formed via the pentose cycle, from which is derived the reductive hydrogens for fatty acid and cholesterol synthesis; (2) the absence of a major separate pool of NADPH formed from glucose by microsomal glucose dehydrogenase (EC 1.1.1.47) catalysis for use in cholesterol synthesis. ³H from [4-³H]glucose and from [2-³H]sorbitol was incorporated into cholesterol more than into fatty acids relative to the incorporations of ³H from [3-³H]glucose. Assuming that the ³H from [4-³H]glucose and from [2-³H]sorbitol were incorporated via the conversion, catalyzed by malic enzyme, of NADH to NADPH, this indicates the Compartmentation of the NADPH formed via malic enzyme catalysis from that formed via the pentose cycle. Alternatively, NADH provides reductive hydrogens for cholesterol synthesis in greater measure than in fatty acid formation or the stereochemistry of the synthetic processes are such that [A-³H]NADPH has greater excess than [B-³H]NADPH to cholesterol synthesis relative to fatty acid synthesis.     

Changes in sterol biosynthesis accompanying cessation of glial cell growth in serum-free medium

C-6 glioma cells, grown in medium supplemented with 5% delipidated foetal calf serum, were induced to enter a quiescent state by removing serum from the medium. Within 24h there was a 75–80% decline in the rate of incorporation of [¹⁴C]acetate or ³H₂O into digitonin-precipitable sterols. Experiments with [³H]mevalonolactone as a labelled sterol precursor suggested that the decline in sterol synthesis was regulated primarily at a point in the pathway before the formation of mevalonate. The specific activities of 3-hydroxy-3-methylglutaryl-CoA synthase and 3-hydroxy-3-methylglutaryl-CoA reductase decreased sharply in conjunction with the decline in sterol synthesis in the serum-free cultures; however, the activity of acetoacetyl-CoA thiolase was altered only slightly. The magnitude of the initial decline in reductase activity was not affected when 50-mm-NaF was included in the preincubation and assay buffers to prevent activation of physiologically inactive enzyme. However, after 6h of serum deprivation the decline in 3-hydroxy-3-methylglutaryl-CoA reductase activity was due to a decrease in the amount of latent activity. The sterol concentration in C-6 cells was unchanged after 24h in serum-free medium, although a 20% decrease in the sterol/fatty acid molar ratio occurred as a result of a small increase in the fatty-acid concentration. Incorporation of ³H₂O into fatty acids was inhibited in the serum-deprived glial cells; however, this inhibition developed more slowly and was not as pronounced as the diminution in sterol synthesis. The results suggest that in C-6 glia, which resemble the glial stem cells of the developing brain, the decreased demand for membrane sterols in the quiescent state results in a decline in sterol synthesis, mediated primarily through co-ordinate changes in the activities of 3-hydroxy-3-methylglutaryl-CoA synthase and 3-hydroxy-3-methylglutaryl-CoA reductase.     

Control of fatty acid incorporation in membrane phospholipids. X-ray-induced changes in fatty acid uptake by tumor cells

Lymphosarcoma cells isolated from the spleens of tumor-bearing mice were used to study the effect of a low dose of X-rays (5 Gy) on the incorporation of [³H]palmitate and [¹⁴C]arachidonate into the lipids of the tumor cells. Palmitate and arachidonate were rapidly incorporated especially into the phospholipids of the cells. Between one and three hours after the start of the incubation with radiactive palmitate 80–90% of the label of the total lipids was found in the phospholipid fraction. Already after a few minutes of incubation with radioactive arachidonate, about 95% of the label was incorporated in the phospholipids. Irradiation caused a small but significant increase in the rate of fatty acid incorporation for both fatty acids. Concomitantly, a significantly increased amount of fatty acid was removed from the medium by the cells as a result of the irradiation, and the specific radioactivity of the free fatty acids in the cells was found to be enhanced. The radiation effect on the tumor cells could be mimicked by a hypotonic treatment. The magnitude of the radiation-induced stimulation of the fatty acid incorporation was similar to that of the hypotonically induced effect. Cells which had received a hypotonic treatment before the irradiation, did not show an additional radiation-induced enhancement of fatty acid incorporation into the cellular lipids. When the cells were incubated with serum albumin loaded with a relatively large (non-physiological) amount of complexed fatty acids (fatty acid: albumin molar ratio, $\text{ν}\text{= 3.7}$), no radiation effect on the fatty acid incorporation could be detected. It is concluded that hypotonic treatment, irradiation, and increased supply of exogenous fatty acids all lead to an enhanced flux of fatty acids into the cells. These results confirm our previous suggestion that the uptake of fatty acids through the plasma membrane is the rate-limiting step in the fatty acid incorporation into the phospholipids and that ionizing radiation is one of the means to enhance fatty acid uptake through the plasma membrane leading to an increased incorporation into the phospholipids.