Ether lipid studies in mouse C3H/10T1/2 cells and a 3-methylcholanthrene-transformed clone

C3H/10T1/2 mouse embryo cells and a transformed clone were used in these initial experiments to investigate the future application of this model culture system to studies of ether-linked lipids in cancer cells. Clone 8 cells are nontumorigenic, nontransformed, and maintain normal morphology to passages 15–20. Clone 16 cells were derived from morphologically transformed foci of clone 8 cells exposed to the chemical carcinogen, 3-methylcholanthrene, and are highly tumorigenic. The data presented here demonstrate that the high amounts of ether-linked lipids, characteristic of tumors, are likewise elevated in cells that have been oncogenically transformed in vitro. When incubated with labeled fatty alcohols, the transformed cells show a stimulated incorporation of radioactivity into alkyldiacylglycerols (>100% over clone 8), whereas radioactivity in the alkyl moiety of the phospholipids is not altered. Analysis of the lipids formed from [1-¹⁴C]hexadecanol indicates that the nontransformed cells have a greater capacity to oxidize hexadecanol and incorporate the resulting carboxylic acid into acyl groups. Quantitative analysis of cellular lipids shows that in the oncogenically transformed cells alkyldiacylglycerols are increased (123% over clone 8).     

In vivo labeling of myelin lipids and proteolipid protein with [3H]myristate, [14C]linoleate,and [14C]linolenate

To investigate the incorporation of essential fatty acids into myelin components, 24-day-old rabbits were injected intracerebrally with [¹⁴C]linoleate, [¹⁴C]linolenate, or [³H]Myristate for comparison. Animals were killed 22 hr later and myelin was isolated. [³H]myristate labeled all myelin lipids including monogalactosyl diglyceride, with the exception of sulfatides. With¹⁴C-essential fatty acids, only glycerophospholipids were efficiently labeled and their specific activities were in the following decreasing orders: PC>PI>PE>PS with [¹⁴C]linoleate, and PE>PC>PI=PS with [¹⁴C]linolenate. Among myelin proteins, PLP and DM-20 were labeled with all 3 precursors. PLP was purified from myelin labeled with¹⁴C-essential fatty acids. The label was then cleaved from the protein by alkaline methanolysis and was identified as a dienoic ([¹⁴C]linoleate) or a tetraenoic ([¹⁴C]linolenate) fatty acid. MBP was not labeled with [³H]myristate, but was slightly labeled with both¹⁴C-essential fatty acids. The signification of the latter result is discussed.Abbreviations FA fatty acid(s) - HPTLC high-performance thin-layer chromatography - MBP myelin basic protein - PLP proteolipid protein - PC phosphatidylcholine - PE phosphatidylethanolamine and ethanolamine plasmalogens - PI phosphatidylinositol - PS phosphatidylserine - SDS sodium dodecylsulfate     

Distribution and metabolism of arachidonic and docosahexaenoic acids in rat pineal cells. Effect of norepinephrine

The time-course incorporation of 10 μM [¹⁴C]arachidonic (AA) and docosahexaenoic (DHA) acids into glycerolipids was studied in rat pineal cells. The incorporation of both labeled fatty acids into total lipids was approximately equal, but their distribution profiles among the various cell lipids showed marked differences. The esterification of [¹⁴C]DHA in the neutral lipids, triacylglycerols (TAG) and cholesterol esters (CE), was 2-fold higher than that of [¹⁴C]AA whereas the opposite could be observed in total phospholipids (PL). The order of incorporation into PL was phosphatidylcholine (PC) > phosphatidylinositol (PI) = phosphatidylethanolamine (PE) for [¹⁴C]AA and PC = PE for [¹⁴C]DHA, the incorporation of both fatty acids being not detected in phosphatidylserine (PS) and that of DHA not in PI. When using 0.5 μM [³H] fatty acids, the respective distribution patterns resembled that of fatty acids at 10 μM, except for a lower proportion in TAG. The stimulation of ³H-labeled cells by 100 μM norepinephrine induced a 170% increase of basal release of [³H]AA into the medium, while [³H]DHA was virtually not released. However, the analysis of cell labeling revealed that both [³H] fatty acid levels were decreased in PL and increased in TAG. These findings suggest different involvement for AA and DHA in the pineal function. The preferential incorporation of DHA in TAG suggests that TAG might play an important role in the pineal enrichment with DHA. The absence of DHA release after NE stimulation, which however cannot be ascertained, may raise the question of the role of DHA in NE transduction.     

Alterations in the developmental patterns of enzymes in chicken embryos infected with West Nile virus.

Infection of chicken embryos with West Nile (WN) virus, a group B togavirus containing structural lipids, caused a rapidly developing hypertriglyceridemia. Changes in the activity of several hepatic regulatory enzymes in glycolytic and lipogenic pathways occurred during infection. Compared to control values in embryos of the same age (16 days), an 8.8-fold increase in the specific activity of ATP-citrate lyase and a 5.6-fold increase in that of hexokinase were observed on the third day of WN virus infection. Hexose monophosphate shunt dehydrogenase specific activities were elevated twofold in virus-infected livers. Activities of malic enzyme and phosphofructokinase were also elevated in WN virus-infected livers. Malate dehydrogenase and NADP-linked isocitrate dehydrogenase levels showed little or no change during infection. The levels of pyruvate kinase and lactate dehydrogenase were decreased in virus-infected livers. Hepatic acetyl-CoA carboxylase activity was at least twofold higher in virus-infected embryos; however, following removal of low-molecular-weight compounds, the specific activities of this enzyme from infected and control embryos were virtually identical. The results of mixing experiments suggest that the low levels of carboxylase activity in control embryos may be due to the presence of enzyme inhibitor(s) which can be removed by gel filtration.The incorporation of radiolabeled precursors into cellular lipids by liver minces from virus-infected and uninfected embryos was measured. There was a twofold increase in carbohydrate incorporation in virus-infected liver as compared to uninfected liver; [¹⁴C]pyruvic acid was incorporated into lipids to the greatest extent. [1-¹⁴C]acetic acid, [U-¹⁴C]alanine, and [U-¹⁴C]leucine were incorporated very poorly in both infected and control livers. Twice as much [1-¹⁴C]oleic acid or [1-¹⁴C oleic]triolein was incorporated in WN-infected livers as in control. The relative distribution of neutral and polar lipids formed from each precursor was generally similar in infected and uninfected livers as determined by thin-layer chromatography of radiolabeled lipids. Except for a threefold increase in oxidation of [¹⁴C]glucose by virus-infected livers, the oxidations of carbohydrates and fatty acids were similar in infected and uninfected livers. The pentose phosphate pathway appears to be the major pathway utilized in glucose oxidation for both control and virus-infected livers. The results indicate that enhanced flux of metabolites into lipids reflects a virus-induced alteration in embryonic development: The enzyme patterns of infected embryos are more characteristic of older embryos or even newly hatched chicks.     

Lipid synthesis by oligodendrocytes from adult pig brain maintained in long-term culture

Oligodendrocytes were isolated from adult pig brain and cultivated for 18–24 days. [¹⁴C]acetate, [³H]galactose or [³⁵S]sulfate were added to the medium for an additional 24 h. Lipids were extracted and separated by high-performance thin-layer chromatography. The labeled lipids were studied by fluorography and scintillation counting. [¹⁴C]acetate was incorporated in decreasing order into neutral lipids, phosphatidylcholine, ethanolamine phosphatides, galactocerebrosides, phosphatidylinositol, phosphatidylserine, sulfatides and sphingomyelin. From the [¹⁴C]acetate incorporated into ethanolamine and choline phosphatides, 71.6 and 14.8%, respectively, were found in plasmalogens. Among neutral lipids, [¹⁴C]acetate labeled not only cholesterol but also large amounts of triglycerides. No cholesterol esters were synthesized. [³H]galactose primarily labeled galactocerebrosides, sulfatides, and monogalactosyl diglyceride. [³⁵S]sulfate incorporation was restricted to sulfatides. Together with our previous results concerning proteins, these data show that: (1) oligodendrocytes remain highly differentiated in long-term cultures; (2) they are able to synthesize the major components of myelin; (3) they synthesize surprisingly high amounts of triglycerides and of monogalactosyl diglyceride, a marker for myelination.     

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.     

Cell-free transfer of sterols from dictyosome-like structures to plasma membrane vesicles of guinea pig testes

Summary Transfer of radiolabeled lipids from dictyosome-like structures (DLS) from testis tubules of the guinea pig as donor to unlabeled plasma membrane from testis tubules immobilized on nitrocellulose as acceptor was studied in a completely cell-free system. As a general label for lipids of the donor DLS, isolated testis tubules were incubated with [¹⁴C]acetate. Time- and temperature-dependent transfer of [¹⁴C]acetate labeled constituents was observed in the cellfree system. However, despite the fact that phospholipids and other constituents were highly labeled in the donor fraction, primarily radioactive sterols were transferred to the plasma membrane acceptor vesicles. Transfer at 37°C represented 0.4 to 0.7% of the total radiolabeled cholesterol at 37°C but little or no transfer occurred at 4°C. The sterols transferred exhibited Chromatographic mobilities corresponding to those of cholesterol and lanosterol. Similar results were obtained with [¹⁴C]mevalonic acid. In subsequent experiments, cholesterol transfer from DLS to plasma membrane was demonstrated by incubation of DLS with [³H]squalene which was converted into sterol or with [¹⁴C]cholesterol. Transfer of sterols required ATP, but not cytosol, and was both time- and temperature-dependent. DLS were more effective than either endoplasmic reticulum or plasma membrane as the donor fraction. The results from the cell-free analysis suggest a possible functional role of the DLS in sterol biogenesis and transfer to the plasma membrane during spermatid development.Abbreviations DLS dictyosome-like structure(s) - PBS phosphatebuffered saline - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - BSA bovine serum albumin     

Stimulation of hepatic triglyceride synthesis and secretion by clofibrate

Isolated hepatocytes prepared from rat and squirrel monkey livers were used to explore the mechanism of action of clofibrate, a hypolipidemic agent in current use. Addition of sodium clofibrate to cells suspended in Hanks medium stimulated the conversion of [1-¹⁴C]palmitate into esterified lipids and to ¹⁴CO₂. This agent also promoted the incorporation of [2-³H]glycerol into cellular lipids when fatty acids were present in the incubation medium. Triglycerides were the major lipid class increased by the drug. Sodium clofibrate enhanced the discharge of labeled lipids into the medium from liver cells prelabeled with [2-³H]glycerol. These data suggest that clofibrate does not lower plasma triglyceride levels by interference with hepatic triglyceride production or secretion.     

Use of (1 or 3-3H, U-14C)glucose to estimate the synthesis of glycerolipids via acyl dihydroxyacetone phosphate.

Turnover rates of tRNAs in Friend virus-infected mouse leukemia cells are reported. Cells were labeled for one generation with [¹⁴C]- or [³H]uridine. ³H-labeled cells were transferred to nonradioactive medium and allowed to grow exponentially for 72 hours. Low molecular weight cytoplasmic RNAs isolated from ¹⁴C- and ³H-labeled cells were cochromatographed on reverse-phase columns. The results indicate considerable heterogeneity of turnover rates of 4S RNAs, with the most labile species turning over at least 1.75 times as fast as the most stable species.     

Incorporation of [C]Glucosamine and [C]Mannose into Glycolipids and Glycoproteins in Cotyledons of Pisum sativum L

Developing pea cotyledons incorporate radioactivity in vivo from [¹⁴C]glucosamine and [¹⁴C]mannose into glycolipids and glycoproteins. Several different lipid components are labeled including neutral, ionicnonacidic, and acidic lipids. The acidic lipids labeled in vivo appear similar to the polyisoprenoid lipid intermediates formed in vitro in pea cotyledons. Radioactivity from [¹⁴C]glucosamine and [¹⁴C]mannose is also incorporated into glycopeptides. Considerable redistribution of [¹⁴C]mannose into other glycosyl components found in endogenous glycoproteins is observed. An N-acetylglucosamine to asparagine glycopeptide linkage has been isolated from [¹⁴C]glucosamine-labeled glycoproteins.     

Biochemistry of Suberization: Incorporation of [1-C]Oleic Acid and [1-C]Acetate into the Aliphatic Components of Suberin in Potato Tuber Disks (Solanum tuberosum)

Biosynthesis of the aliphatic components of suberin was studied in suberizing potato (Solanum tuberosum) slices with [1-¹⁴C]oleic acid and [1-¹⁴C]acetate as precursors. In 4-day aged tissue, [1-¹⁴C]oleic acid was incorporated into an insoluble residue, which, upon hydrogenolysis (LiA1H₄), released the label into chloroform-soluble products. Radio thin layer and gas chromatographic analyses of these products showed that ¹⁴C was contained exclusively in octadecenol and octadecene-1, 18-diol. OsO₄ treatment and periodate cleavage of the resulting tetraol showed that the labeled diol was octadec-9-ene-1, 18-diol, the product expected from the two major components of suberin, namely 18-hydroxyoleic acid and the corresponding dicarboxylic acid. Aged potato slices also incorporated [1-¹⁴C]acetate into an insoluble material. Hydrogenolysis followed by radio chromatographic analyses of the products showed that ¹⁴C was contained in alkanols and alkane-α,ω-diols. In the former fraction, a substantial proportion of the label was contained in aliphatic chains longer than C₂₀, which are known to be common constituents of suberin. In the labeled diol fraction, the major component was octadec-9-ene-1,18-diol, with smaller quantities of saturated C₁₆, C₁₈, C₂₀, C₂₂, and C₂₄-α,ω-diols. Soluble lipids derived from [1-¹⁴C]acetate in the aged tissue also contained labeled very long acids from C₂₀ to C₂₈, as well as C₂₂ and C₂₄ alcohols, but no labeled ω-hydroxy acids or dicarboxylic acids were detected. Label was also found in n-alkanes isolated from the soluble lipids, and the distribution of label among them was consistent with the composition of n-alkanes found in the wound periderm of this tissue; C₂₁ and C₂₃ were the major components with lesser amounts of C₁₉ and C₂₅. The amount of ¹⁴C incorporated into these bifunctional monomers in 0-, 2-, 4-, 6-, and 8-day aged tissue were 0, 1.5, 2.5, 0.8, and 0.3% of the applied [1-¹⁴C]oleic acid, respectively. Incorporation of [1-¹⁴C]acetate into the insoluble residue was low up to the 3rd day of aging, rapid during the next 4 days of aging, and subsequently the rate decreased. These changes in the rates of incorporation of exogenous oleic acid and acetate reflected the development of diffusion resistance of the tissue surface to water vapor. As the tissue aged, increasing amounts of the [1-¹⁴C]acetate were incorporated into longer aliphatic chains of the residue and the soluble lipids, but no changes in the distribution of radioactivity among the α-ω-diols were obvious. The above results demonstrated that aging potato slices constitute a convenient system with which to study the biochemistry of suberization.     

Synthesis of nuclear lipids in L2C leukemic lymphocytes

We previously reported that propiconazole strongly inhibits cholesterol synthesis, but not cell division in a stimulated cell, the human lymphocyte cultured with phytohemagglutinin, showing that newly synthesized cholesterol is not necessary for cell division. In this study we labeled the L2C leukemic guinea pig lymphocyte, a naturally stimulated cell, with [2-14C]acetate, and compared the composition of newly synthesized lipids isolated from nuclei and whole cells (or microsomes). We observed that the proportion of cholesterol in labeled non-saponifiable lipids extracted from nuclei was lower than in non-saponifiable lipids isolated from whole cells, whereas the proportion of squalene and polar lipids was higher. By analyzing total lipid extracts, the polar lipids were identified as alkylglycerols, and the above mentioned distribution of constituents was confirmed. The identification of alkylglycerols was also supported by the comparison of radioactive lipid composition after labeling cells with three different lipid precursors: [2-14C]mevalonate, [2-14C]acetate and [2-14C]stearate. When cells were labeled in the presence of dodecylimidazole, the percentage of squalene and alkylglycerols decreased in nuclear lipids, but was not altered when cells were cultured in the presence of propiconazole, a cholesterol synthesis inhibitor which does not affect cell division of human stimulated lymphocytes. We have shown that dodecylimidazole inhibited alkylglycerol biosynthesis and squalene uptake by the nucleus, suggesting that these compounds could play a role in the regulation of cell division.     

Biosynthesis of the farnesyl moiety of heme a from exogenous mevalonic acid by cultured chick liver cells

Chick embryo liver cells, when cultured for 41 h in the presence of [2-14C]mevalonic acid, took up label and incorporated radioactivity into heme a, but not into protoheme. Incubation of cells with delta-[4-14C]aminolevulinic acid (ALA) resulted in uptake of label and incorporation of radioactivity into both protoheme and heme a. These results show that both protoheme and heme a are synthesized during the incubation period, and that mevalonic acid is a specific precursor of the farnesyl moiety of heme a. Incubation of cells with [1,2-14C]acetate plus N-methyl mesoporphyrin IX, an inhibitor of heme synthesis, resulted in negligible incorporation of label into protoheme and heme a, although cellular lipids were highly labeled. This result indicates that the heme purification methods employed were capable of separating hemes from lipids, and that the measured incorporation of label into hemes from [14C]mevalonic acid and [14C]ALA was not due to lipid contamination.     

Incorporation of Labeled Precursors into, and Accumulation of, Triglycerides and Phosphoglycerides at Selected Stages of Fungal Growth

There was greater incorporation of [2^?14C] acetate and of [6^?14C] glucose into phosphoglycerides than into triglycerides, of 1 1/2, 2, 3, 4 and 6 day old mycelial sample of the fungus Glomerella cingulata. Maximum incorporation into both classes of lipids occurred in young mycelial samples (2 to 3 days of age) which had a high content of total nitrogen. The five sets of mycelial samples all contained somewhat larger quantities of phosphoglycerides than triglycerides, and changes in content of both classes of lipids were similar in pattern to changes in content of total nitrogen. Incorporation, accumulation and total nitrogen of the mycelial samples, decreased at 4 days but increased again by 6 days. The apparent turnover of the triglycerides and phosphoglycerides was qualitatively similar although there was greater apparent turnover of phosphoglycerides than triglycerides; the similarity in patterns of apparent turnover was inferred to be a consequence of acyl exchange between labeled and unlabeled triglycerides and phosphoglycerides. There was greater incorporation of [2^?14C] acetate into the acyl than into the glyceryl moieties of both classes of lipids but greater incorporation of [6^?14C] glucose into the glyceryl than acyl moieties. With both precursors, the glyceryl and acyl moieties of the phosphoglycerides were more heavily labeled than corresponding moieties of the triglycerides.     

Stimulation of the biosynthesis of membrane glycoproteins from zajdela ascites hepatoma cells by Robinia lectin

Membrane glycoprotein biosynthesis of ascites hepatoma cells is followed by [¹⁴C]glucosamine and [³H]leucine incorporation into cells in culture. The rate of incorporation is strongly increased by the addition of Robinia lectin in culture medium. Labeled glycoproteins are released from lectin stimulated and non-stimulated ceils by trypsin digestion. Studies of labeled trypsinates on sodium dodecyl sulfate gel electrophoresis and Sephadex G-200 filtration exhibit two fractions both labeled with [¹⁴C]glucosamine and [³H]leucine and having different molecular weights, one over 200 000 and the other about 2000. Identical results are obtained when external membrane glycoproteins are solubilized by sodium deoxycholate. Comparison of surface glycoproteins isolated by trypsinization from control cells labeled with [³H]glucosamine and from lectin stimulated cells labeled with [¹⁴C]glucosamine displays no significant qualitative differences between glycoprotein fractions released from both cell groups.     

THE METABOLISM OF LEUCINE BY DEVELOPING RAT BRAIN: EFFECT OF LEUCINE AND 2-OXO-4-METHYLVALERATE ON LIPID SYNTHESIS FROM GLUCOSE AND KETONE BODIES

Abstract— The oxidation of l -[U-¹⁴C]leucine and l -[l-¹⁴C]leucine at varying concentrations from 0.1 to 5mM to CO₂ and the incorporation into cerebral lipids and proteins by brain slices from 1-week old rats were markedly stimulated by glucose. Although the addition of S mM-dl -3-hydroxybutyrate had no effect on the metabolism of [U-¹⁴C]leucine by brain slices from suckling rats, the stimulatory effects of glucose on the metabolism of l -[U-¹⁴C]leucine were markedly reduced in the presence of dl -3-hydroxybutyrate. The stimulatory effect of glucose on leucine oxidation was, however, not observed in adult rat brain. Furthermore, the incorporation of leucine-carbon into cerebral lipids and proteins was also very low in the adult brain. The incorporation of l -[U-¹⁴C]leucine into cerebral lipids by cortex slices was higher during the first 2 postnatal weeks, which then declined to the adult level. During this time span, the oxidation of l -[U-¹⁴C]leucine to CO₂ remained relatively unchanged. The incorporation in vivo of D-3-hydroxy[3-¹⁴C]butyrate into cerebral lipids was markedly decreased by acute hyperleucinemia induced by injecting leucine into 9-day old rats. In in vitro experiments, 5 mM-leucine had no effect on the oxidation of [U-¹⁴C]glucose to CO₂ or its incorporation into lipids by brain slices from 1-week old rats. However, 5 mM-leucine inhibited the oxidation of d -3-hydroxy-[3-¹⁴C]butyrate, [3-¹⁴C]acetoacetate and [1-¹⁴C]acetate to CO₂ by brain slices, but their incorporation into cerebral lipids was not affected by leucine. In contrast 2-oxo-4-methylvalerate, a deaminated metabolite of leucine, markedly inhibited both the oxidation to CO₂ and the incorporation into lipids of labelled glucose, ketone bodies and acetate by cortex slices from 1-week old rats. These findings suggest that the reduction in the incorporation in vivo of d -3-hydroxy[3-¹⁴C]butyrate into cerebral lipids in rats injected with leucine is most likely caused by 2-oxo-4-methylvalerate formed from leucine. Since the concentrations of leucine and 2-oxo-4-methylvalerate in plasma of untreated patients with maple-syrup urine disease are markedly elevated, our findings are compatible with the possibility that an alteration in the metabolism of glucose and ketone bodies in the brain may contribute to the pathophysiology of this disease.     

Lipid Composition and Metabolism of Volvox carteri

The membrane structural lipids of somatic cells and gonidia isolated from Volvox carteri f. nagariensis spheroids have been characterized. The principal polar lipid components of both cell types are sulfoquinovosyl diglyceride, mono- and digalactosyl diglyceride, phosphatidylglycerol, phosphatidylethanolamine, and 1(3), 2-diacylglyceryl-(3)-O-4′-(N,N,N,-trimethyl)homoserine. Light-synchronized cultures of spheroids were shown to incorporate [¹⁴C]bicarbonate, [³⁵S]sulfate, [¹⁴C]palmitic acid, and [¹⁴C]lauric acid into complex lipids. [¹⁴C]Palmitic acid was incorporated mainly into diacylglyceryltrimethylhomoserine and was not significantly modified by elongation or desaturation. In contrast, [¹⁴C]lauric acid was incorporated into a wider variety of complex lipids and was also converted into longer chain saturated and unsaturated fatty acids. Volvox is a promising system for studying the role of membranes in algal cellular differentiation.     

[14C]Acetate incorporation by cultured normal,familial hypercholesterolemia and Down's syndrome fibroblasts

Fibroblasts from patients with homozygous familial hypercholesterolemia (FH), a disease characterized by accelerated atherogenesis, are known to lack functional low-density-lipoprotein receptors, which ultimately results in increased cholesterol biosynthesis in the cultured cells. [¹⁴C]Acetate incorporation in these cells was compared to that of normal fibroblasts and to fibroblasts from patients with Down's syndrome, a disease in which atherosclerosis is rare. Total [¹⁴C]acetate incorporation did not differ significantly between normal and Down's fibroblasts, nor did its partitioning into the hexane-extractable and aqueous fractions of the cell hydrolysates. [¹⁴C]Acetate incorporation was much greater in FH cells in both the aqueous and hexane-extractable fractions. Preincubation in fetal bovine serum increased acetate incorporation only by FH cells, while 50 μg low-density lipoprotein/ml medium depressed acetate incorporation in all three groups. A C₂₇ sterol, identified by gas chromatography-mass spectrometry as a probable isomer of cholesterol, was present in small amounts in FH fibroblasts, but was not detectable in the normal or Down's cells. The absolute amounts of [¹⁴C]acetate incorporated into the non-sterol lipids were greater in the FH fibroblasts, indicating that these cells may have to synthesize, in addition to cholesterol, other required cellular lipids which are delivered to the normal cells by low-density lipoproteins.     

Sorption and Metabolism of Metolachlor by a Bacterial Community

A stable bacterial community absorbed and transformed the herbicide metolachlor [2-chloro-N-(2-ethyl- 6-methylphenyl)-N-(2-methoxy-1-methylethyl)-acetamide] from a liquid medium. About 80% of the added ring-[U-¹⁴C]metolachlor (50 μg/ml) disappeared from the medium and accumulated inside the cells. The ratio of cellular ¹⁴C to ¹⁴C in 1 mg of supernatant reached a value of 1.1 × 10⁴ in a 10-day-old culture. ¹⁴C remaining in the medium consisted primarily of two dechlorinated products of metolachlor with m/z 233 and 263 as determined by mass spectrometry. The ¹⁴C-labeled material absorbed by the cells was strongly bound; only 2% of the ¹⁴C was released into deionized water after shaking for 3 h. Approximately 96% of the ¹⁴C associated with the biomass was extracted with acetone, and high-performance liquid chromatographic analysis of this fraction showed six peaks containing radioactivity. Since no metolachlor was detected by chromatographic analysis, it was concluded that the radioactivity recovered from the cells represented transformed products of metolachlor. Pure cultures isolated from the bacterial mixed culture were less effective in transforming and accumulating metolachlor. These results suggest that it may be advantageous to seed an aquatic environment with a mixture of microorganisms, rather than individual microbial species, as a method for removal or detoxification of metolachlor.     

Synthesis of lipids in isolated nuclei from rat thymus and liver cells

Synthesis of lipids was studied in isolated nuclei from rat thymus and liver cells. On incubation of the isolated nuclei with [2-¹⁴C]acetate and [1-¹⁴C]glycerol, the label was intensively incorporated into phospholipids and with a significantly lower intensity into fatty acids and cholesterol. Only trace amounts of radioactivity were detected in the lipids of chromatin prepared from isolated thymus nuclei after their incubation, and this suggested that lipids were mainly synthesized on the nuclear membrane. On the preincubation of thymus tissue homogenate with [2-¹⁴C]acetate and the subsequent isolation of the nuclei and chromatin, the radioactivity of chromatin lipids was comparable to the radioactivity of nuclear lipids. The findings suggested that in the isolated nuclei the newly synthesized lipids were not transported into chromatin from the nuclear membrane. The specific radioactivities of individual phospholipids and fatty acids were different in the isolated nuclei and in nuclei obtained from preincubated homogenate. Mechanisms of lipid synthesis in isolated nuclei and causes of the different radioactivities of lipids in the isolated nuclei and in the nuclei obtained from the preincubated homogenate are discussed.