Effect of sulphur nutrition on redistribution of sulphur in vegetative barley

Xenobiotics directed against sterol biosynthesis have proved to be useful tools in the determination of which sterol molecules are necessary for successful plant cell growth. However, the exact mode of action by which sterols are able to trigger cell growth remains to be elucidated. Previous studies using the triazole paclobutrazol, demonstrated that in Apium graveolens (cv. New Dwarf White) suspension cultures, sterol and phosphatidylcholine biosynthesis are co-ordinately regulated (C. E. Rolph and L. J. Goad 1991, Physiol. Plant. 83: 605–610). The studies presented herein, were designed to investigate the possible role of phosphatidylcholine in the stimulation of plant cell growth. Sterol biosynthesis, and hence cell growth, was inhibited by the use of the azole xenobiotic miconazole. Treatment of the cultures with miconazole lead to compositional changes in the free sterol content of the cells. For example, 30 μM miconazole treatment led to a reduction in the stigmasterol/sitosterol ratios from 1.53 to 1.24. In contrast, the phospholipid content of the cells remained relatively unchanged with phosphatidylcholine accounting for approximately 25% of the total phospholipids present in both control and miconazole-treated cells. The cytostatic effect of miconazole could be partially counteracted by supplementation of the growth medium with the phytosterol stigmasterol and/or the unsaturated fatty acids oleate and linoleate. The activity of CTP:cholinephosphate cytidylyltransferase (EC 2.7.7.15), a rate-limiting enzyme in phosphatidylcholine biosynthesis, was significantly reduced in cells whose growth had been arrested by miconazole treatment. In miconazole-treated cultures whose growth had been partially restored by supplementation with either specific sterols or unesterified fatty acids, the activity of this key enzyme was increased. In the case of stigmasterol, oleate and linoleate supplementation, the microsomal activity was found to be similar to that exhibited by control cultures. From these studies, it may be concluded that certain phytosterols and unsaturated fatty acids play key roles with respect to phosphatidylcholine biosynthesis and that phosphatidylcholine biosynthesis via the CDP-base pathway is an important pre- and/or co-requisite for successful culture growth.     

Interaction of phosphatidylcholine liposomes and plasma lipoproteins with sheep erythrocyte membranes: Preferential transfer of phosphatidylcholine containing unsaturated fatty acids

The interaction of sheep erythrocyte membranes with phosphatidylcholine vesicles (liposomes) or human plasma lipoproteins is described. Isolated sheep red cell membranes were incubated with liposomes containing [¹⁴C]phosphatidylcholine or [^3H]phosphatidylcholine in the presence of EDTA. A time-dependent uptake of phosphatidylcholine into the membranes could be observed. The content of this phospholipid was increased from 2 to 5%. The rate of transfer was dependent on temperature, the amount of phosphatidylcholine present in the incubation mixture and on the fatty acid composition of the liposomal phosphatidylcholine. A possible adsorption of lipid vesicles to the membranes could be monitored by adding cholesteryl [¹⁴C]oleate to the liposomal preparation. As cholesterylesters are not transferred between membranes [1], it was possible to differentiate between transfer of phosphatidylcholine molecules from the liposomes into the membranes and adsorption of liposomes to the membranes. The phosphatidylcholine incorporated in the membranes was isolated, and its fatty acids were analysed by gas chromatography. It could be shown that there was a preferential transfer of phosphatidylcholine molecules containing two unsaturated fatty acids.     

Control of fatty acid distribution in phosphatidylcholine of spinach leaves

The acylation of lysophosphatidylcholine by enzyme preparations from spinach leaves was studied. The acylation reaction was followed by the incorporation of (14)C-labeled fatty acids from the respective coenzyme A derivatives into phosphatidylcholine. The subcellular fraction with the highest specific activity was the microsomal fraction. Contaminating thioesterase activity which was encountered was inhibited by treatment with sodium dodecyl sulfate. The acyltransferase activity was only mildly inhibited by sulfhydryl reagents. Labeled fatty acid was primarily incorporated into phosphatidylcholine. When saturated and unsaturated fatty acyl CoA derivatives were used, the saturated derivatives were incorporated primarily into the 1-position of the glycerol moiety, and the unsaturated fatty acids went primarily to the 2-position. This pattern of incorporation agrees with the fatty acid distribution in vivo.     

Effect of long chain fatty acids on triacylglycerol accumulation,fatty acid composition and related gene expression in primary cultured bovine satellite cells

Abstract

This study was conducted to determine the effects of long chain fatty acids (LCFAs) on triacylglycerol (TAG) content, as well as on genes associated with lipid synthesis and fatty acid composition in bovine satellite cells. Both saturated (palmitic and stearic) and unsaturated (oleic and linoleic) fatty acids stimulated the TAG accumulation at a concentration of 100?µM and oleate increased it significantly more than stearate and palmitate. The results revealed that the lipid droplet formation was markedly stimulated by linoleate and oleate at 100?µM. Compared to control, the expressions of adipose triglyceride lipase, carnitine acyltransferase 1 and the fatty acid translocase 36 were upregulated by LCFAs. All the fatty acids also significantly increased diacylglycerol acyltransferase 2 than the untreated control (p?<?0.05). The monounsaturated fatty acids significantly increased (p?<?0.05) in response to oleate and linoleate compared to the control as did the polyunsaturated fatty acids (p?<?0.05), in addition to stearate, linoleate and oleate. In contrast, saturated fatty acids were significantly decreased in the oleate and linoleate-treated groups. The study results contribute to our enhanced understanding of LCFAs’ regulatory roles on the bovine cell lipid metabolism.     

PHOSPHOLIPID METABOLISM IN EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS: ACTIVITY OF BRAIN PHOSPHOLIPASE A1, TOWARDS SPECIFICALLY LABELLED GLYCEROPHOSPHOLIPIDS

—The activity of phospholipase A₁ (phosphatide acylhydrolase, EC 3.1.1.4) was measured against phosphatidylcholine, -ethanolamine and -serine, specifically labelled with different fatty acids at either the 1 or 2 position, during experimental allergic encephalomyelitis in rats. In the acute stage of the disease there was a significant increase in enzyme activity in comparison to that from the control animals. The enhanced phospholipase A₁ activity was of the-same order of magnitude for all 1,2-diacyl-glycerophospholipids investigated. The phospholipase A₁-catalysed release of labelled fatty acids from molecular species of phosphatidylcholine decreased slightly with increasing degree of unsaturation of the fatty acids at the 1-position. The K_m-values of the highly purified enzyme were not altered by the demyelinating disorder. The phospholipase A₁ activity returned to the control level in the recovery stage.     

Effect of active dry wine yeast storage upon viability and lipid composition

Alterations in the plasma membrane of Active Dry Wine Yeast (ADWY) produce deleterious effects upon yeast fermentation activity. The aim of this study was to evaluate the changes in viability mediated by plasma membrane alterations induced by poor ADWY storage conditions. Yeast recovery was analyzed after growth in optimal medium. The effects upon vitality were measured by impedance variation. Cell membrane functionality was measured by anisotropy and cell lipid composition was also determined. The results showed a reduction in viability of up to 57–87% in the stored yeast. The storage effect increased saturated fatty acids, and reduced unsaturated fatty acids and phospholipid contents. The stored yeast recovery was related to membrane functionality and significant increases in unsaturated fatty acid, sterol and phospholipid concentrations. Good vitality was positively correlated to high unsaturated fatty acid, phosphatidylserine and phosphatidylethanolamine concentrations and negatively to high saturated fatty acid and phosphatidylcholine contents.     

THE COMPOSITION OF LIPIDS IN CEREBROSPINAL FLUID OF CHILDREN AND ADULTS

Abstract— The proportions of esterified cholesterol and phosphatidyl ethanolamine in lipids of cerebrospinal fluid (CSF) from children were found to be lower than the corresponcling values for adult CSF. The fatty acid patterns of the cholesterol ester, triglyceride + non-esterified fatty acids and phospholipid fractions all displayed low proportions of linoleate; palmitate and oleate were the principal acids present. The fatty acid composition of these lipid classes for CSF derived from children was similar to that from adult subjects. Degradation of CSF lecithin by snake-venom phospholipase A₂ revealed the saturated acids to be located predominantly in the 1-position with the unsaturated ones mainly in the 2-position.     

Phospholipid and fatty acid enrichment of Phanerochaete chrysosporium INA-12 in relation to ligninase production

Summary Ligninase activity of Phanerochaete chrysosporium INA-12 was increased when vegetable oils emulsified with sorbitan polyoxyethylene monooleate (Tween 80) were added to growth medium. Maximal enzyme yield was 22.0 nkat·ml^-1 in olive oil cultures after 4 days incubation. P. chrysosporium INA-12 was also able to utilize tall oil fatty acids for ligninase synthesis. An extracellular lipase activity was detected during the primary phase of growth in culture containing vegetable oils. On the other hand, ligninase production was 1.5-fold enhanced when olive oil cultures were supplemented with soybean asolectin as a phospholipid source. In cultures supplied with olive oil plus asolectin, P. chrysosporium INA-12 mycelium exhibited a preferential enrichment of oleic acid (C18:1), phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) as compared to lipid-free medium. PC and LPC enrichment was associated with an increased ratio of saturated versus unsaturated fatty acids of phospholipids.     

Disaturated and dipolyunsaturated phospholipids in the bovine retinal rod outer segment disk membrane

Thin-layer chromatography was used to separate the major phospholipid headgroup classes of the rod outer segment disk membrane into subfractions which differ markedly in fatty acid composition. At least 18% of the rod outer segment phosphatidylcholine must contain two saturated fatty acids. Furthermore, two unsaturated fatty acids are found in at least 43% of the phosphatidylserine, 24% of the phosphatidylcholine, and 24% of the phosphatidylethanolamine. The unsaturated acids are predominantly polyunsaturated in all cases. A similar separation, but with less resolution, was achieved with silicic acid column chromatography.The temperature dependence of the polarization of the fluorescence of trans-parinaric acid (9,11,13,15-all-trans-octadecatetraenoic acid) showed that the thermal behavior of aqueous dispersions of the phosphatidylcholine subfractions was consistent with their fatty acid compositions.     

Changes of Lipid Composition with Growth Phase of Cryptococcus neoformans

Qualitative and quantitative profiles of phospholipids, neutral lipids, and fatty acid composition in Cr. neoformans during the growth phase were investigated in relation to pyrophosphatidic acid. A marked increase of the total lipid content, which depended on the accumulation of triglyceride in yeast cells with the growth, was observed. The total phospholipid contents in yeast cells remained almostly constant during the exponential phase and slightly decreased in the stationary phase. The major phospholipids of this yeast were phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and cardiolipin, the next groups being pyrophosphatidic acid, phosphatidic acid, lysophos-phatidylcholine, and unidentified components. The amounts of phosphatidylcholine, phosphatidylinositol, and cardiolipin were fairly constant throughout the growth phase, but the amount of phosphatidylethanolamine increased and that of phosphatidylserine decreased with progressive growth. The pyrophosphatidic acid contents were 0.9~0.7% for total phospholipid during the growth phase. The major fatty acids of pyrophosphatidic acid were C_16:0, C_18:1, and C_18:2 acids. The changing patterns of fatty acid composition in pyrophosphatidic acid through the growth phase closely resembled that of phosphatidic acid, which contained larger amounts of C_18:1 acid (35~45%) than C_16:0 acid (30~25%) and C_18:2 acid (30~25%). Phosphatidylserine and phosphatidylinositol contained considerable amounts of saturated fatty acid (C_16:0 acid, more than 55%). On the other hand, phosphatidylcholine, phosphatidylethanolamine, and cardiolipin contained extremely large amounts of unsaturated fatty acid (C_18:1 and C_18:2 acid, 85ç90%).     

Effect of fatty acids and monoglycerides on permeability of lipid bilayer

The effect of fatty acids and monoglycerides on barrier properties of liposomal membranes prepared from egg phosphatidylcholine was investigated. The incorporation of these lipids as liposomal membrane components induced the alteration of the permeability to less permeable liposomally entrapped drugs, sulfanilic acid and procainamide ethobromide (PAEB). Monoolein caused greatly increased permeability of both drugs and unsaturated fatty acids markedly enhanced the release rate of PAEB, while saturated fatty acids caused a small increase in the release rate.Electron spin resonance (ESR) investigation with 5-nitroxide stearic acid showed that fatty acids disordered the hydrophobic region of the lipid bilayer and the disordering effect of unsaturated fatty acids was greater than that of saturated ones. It was demonstrated that the incorporated fatty acids and monoglycerides interacted with the polar region of the membranes by ESR study with cholestane label and ¹H-NMR study. These results indicated that the increase in the membrane permeability caused by fatty acids and monoglycerides associated with the disorder in the membranes' interior and the interaction of the incorporated lipid with the polar head group of phospholipid.     

Oleaginous yeast Meyerozyma guilliermondii shows fermentative metabolism of sugars in the biosynthesis of ethanol and converts raw glycerol and cheese whey permeate into polyunsaturated fatty acids

We studied the biotechnological potential of the recently isolated yeast Meyerozyma guilliermondii BI281A to produce polyunsaturated fatty acids and ethanol, comparing products yields using glucose, raw glycerol from biodiesel synthesis, or whey permeate as substrates. The yeast metabolism was evaluated for different C/N ratios (100:1 and 50:1). Results found that M. guilliermondii BI281A was able to assimilate all tested substrates, and the most efficient conversion obtained was observed using raw glycerol as carbon source (C/N ratio 50:1), concerning biomass formation (5.67 g·L⁻¹) and lipid production (1.04 g·L⁻¹), representing 18% of dry cell weight. Bioreactors experiments under pH and aeration-controlled conditions were conducted. Obtained fatty acids were composed of ~67% of unsaturated fatty acids, distributed as palmitoleic acid (C_16:1, 9.4%), oleic acid (C_18:1, 47.2%), linoleic acid (C_{18:2 n−6}, 9.6%), and linolenic acid (C_{18:3 n−3}, 1.3%). Showing fermentative metabolism, which is unusual for oleaginous yeasts, M. guilliermondii produced 13.7 g·L⁻¹ of ethanol (yields of 0.27) when growing on glucose medium. These results suggest the promising use of this uncommonly studied yeast to produce unsaturated fatty acids and ethanol using cheap agro-industrial residues as substrates in bioprocess.     

Unsaturated fatty acids induce non-canonical autophagy

To obtain mechanistic insights into the cross talk between lipolysis and autophagy, two key metabolic responses to starvation, we screened the autophagy-inducing potential of a panel of fatty acids in human cancer cells. Both saturated and unsaturated fatty acids such as palmitate and oleate, respectively, triggered autophagy, but the underlying molecular mechanisms differed. Oleate, but not palmitate, stimulated an autophagic response that required an intact Golgi apparatus. Conversely, autophagy triggered by palmitate, but not oleate, required AMPK, PKR and JNK1 and involved the activation of the BECN1/PIK3C3 lipid kinase complex. Accordingly, the downregulation of BECN1 and PIK3C3 abolished palmitate-induced, but not oleate-induced, autophagy in human cancer cells. Moreover, Becn1^+/− mice as well as yeast cells and nematodes lacking the ortholog of human BECN1 mounted an autophagic response to oleate, but not palmitate. Thus, unsaturated fatty acids induce a non-canonical, phylogenetically conserved, autophagic response that in mammalian cells relies on the Golgi apparatus.     

The effect of temperature on growth,indole alkaloid accumulation and lipid composition of Catharanthus roseus cell suspension cultures

Cell suspension cultures of Catharanthus roseus were used to study the effect of temperature on plant cell lipids and indole alkaloid accumulation. Lowering the cultivation temperature increased the total fatty acid content per cell dry weight relative to that at higher temperatures, mainly because of increased accumulation of unsaturated C18 acids. In addition, an increase in the relative proportion of phosphatidylcholine and phosphatidylethanolamine was observed. Within individual lipids, the degree of unsaturation was increased and the mean fatty acid chain length decreased with reducing temperature. These changes may be interpreted as modifying the cell membrane fluidity to keep it optimal for growth and metabolism at each temperature. In spite of membrane modifications, the indole alkaloid content of the cells or the medium was not affected by temperature change.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PS phosphatidylserine - PG phosphatidylglycerol - CL cardiolipin - DGD digalactosyldiglyceride - MGD monogalactosyldiglyceride - NL neutral lipids - DU degree of fatty acid unsaturation - TLC thin-layer chromatography - FID-GC flame ionisation detector-gas chromatography - dw dry weight     

Effect of environmental factors on the limpid composition membrane structure and permeability ofMicrosporum gypseum

Supplementation with unsaturated fatty acids, substitution of glucose by glycerol as carbon source and lowered growth temperature (20°C) increased the total phospholipid content ofMicrosporum gypseum spheroplasts. Levels of sterols increased with glycerol substitution and decreased in other growth conditions. Substantial changes were seen in the ratios of unsaturated to saturated fatty acids and phosphatidylcholine to phosphatidylethanolafne under all the experimental conditions. Changed lipid composition resulted in altered uptake of amino acids (L-lysine, L-aspartic acid and L-glycine) and increased number of binding sites for a fluorescent probe, 1-anilinonaphthalene-8-sulfonate.     

Effect of cytochrome c on the linoleic acid-degrading activity of porcine leukocyte 12-lipoxygenase

Hemoproteins are known to have quasilipoxygenase activity that converts linoleic acid (LA) to its hydroperoxides. However, it is not still clear whether, like lipoxygenases, hemoproteins can produce LA hydroperoxides when the LA is part of a mixture containing many different saturated and unsaturated fatty acids. In this study, we found that such hemoprotein as cytochrome c (Cyt c) did not produce LA hydroperoxides from the phospholipase A(2) (PL-A(2)) hydrolysis products of egg yolk phosphatidylcholine (PC). We also found that traces of hydroperoxides and a high concentration of the target unsaturated fatty acid (LA) needs to be present in a fatty acid mixture before the quasi-lipoxygenase activity of Cyt c becomes apparent. We also attempted to elucidate how Cyt c interact with porcine leukocyte 12-lipoxygenase (12-LOX). Hemoproteins are known to possess pseudo-lipohydroperoxidase activity, and can remove the hydroperoxides of unsaturated fatty acids from a reaction mixture. However, we found that Cyt c catalyzed the reaction by which hydroperoxides degrade LA, and thus enhanced the LA-degrading activity of 12-LOX. This hemoprotein-induced promotion of the ability of 12-LOX to degrade LA was observed even when the reaction mixture contained many different saturated and unsaturated fatty acids.     

Oleate stimulation of diacylglycerol formation from phosphatidylcholine through effects on phospholipase D and phosphatidate phosphohydrolase.

Hydrolysis of exogenous phosphatidylcholine (PtdCho) to 1,2-diacylglycerol by rat liver plasma membranes was stimulated by oleate concentrations as low as 0.1 mM. In the presence of 75 mM ethanol, the fatty acid also enhanced phosphatidylethanol (PtdEtOH) formation from PtdCho. These effects were also observed with linoleate and arachidonate, but not with saturated fatty acids or detergents, and were minimal in microsomes or mitochondria. Release of [3H]choline from exogenous Ptd[3H]Cho was stimulated by oleate, whereas phosphoryl[3H]choline formation was inhibited. Oleate and other unsaturated, but not saturated, fatty acids also stimulated the conversion of exogenous [14C]phosphatidic acid to [14C]diacylglycerol. These data are consistent with stimulatory effects of these fatty acids on both phospholipase D and phosphatidate phosphohydrolase in liver plasma membranes. The stimulatory effect of guanosine 5'-O-[3-thio]triphosphate) (20 microM) on PtdEtOH and diacylglycerol formation from PtdCho was enhanced by low concentrations of oleate. Phospholipase A2 also stimulated PtdEtOH and diacylglycerol formation from exogenous PtdCho. It is proposed that unsaturated fatty acids may play a physiological role in the regulation of diacylglycerol production through activation of phospholipase D and phosphatidate phosphohydrolase.     

Lipid Composition of the Yeastlike and Mycelial Mucor hiemalis Cells Grown in the Presence of 4-Chloroaniline

The fungus Mucor hiemalis F-1156, which is commonly thought to be monomorphic, produced two types of cells, yeastlike and mycelial, during growth in a medium containing 4-chloroaniline. Among the polar lipids of yeastlike cells, diphosphatidylglycerol was dominant, while phosphatidylcholine and phosphatidylethanolamine were present in minor amounts. Conversely, mycelial cells mainly contained phosphatidylcholine and phosphatidylethanolamine, whereas the content of diphosphatidylglycerol was low. The neutral lipids of yeastlike cells were dominated by diacylglycerides, sterols, and fatty acids. The content of triacylglycerides and sterol esters was low. Yeastlike cells contained higher amounts of saturated fatty acids and lower amounts of unsaturated fatty acids than the mycelium. The content of stearic acid in the fatty acids of the mycelium grown in the presence of 4-chloroaniline was as high as 25.3–29.9%.     

The role of fatty acid unsaturation in minimizing biophysical changes on the structure and local effects of bilayer membranes

Studying the effects of saturated and unsaturated fatty acids on biological and model (liposomes) membranes could provide insight into the contribution of biophysical effects on the cytotoxicity observed with saturated fatty acids. In vitro experiments suggest that unsaturated fatty acids, such as oleate and linoleate, are less toxic, and have less impact on the membrane fluidity. To understand and assess the biophysical changes in the presence of the different fatty acids, we performed computational analyses of model liposomes with palmitate, oleate, and linoleate. The computational results indicate that the unsaturated fatty acid chain serves as a membrane stabilizer by preventing changes to the membrane fluidity. Based on a Voronoi tessellation analysis, unsaturated fatty acids have structural properties that can reduce the lipid ordering within the model membranes. In addition, hydrogen bond analysis indicates a more uniform level of membrane hydration in the presence of oleate and linoleate as compared to palmitate. Altogether, these observations from the computational studies provide a possible mechanism by which unsaturated fatty acids minimize biophysical changes and protect the cellular membrane and structure. To corroborate our findings, we also performed a liposomal leakage study to assess how the different fatty acids alter the membrane integrity of liposomes. This showed that palmitate, a saturated fatty acid, caused greater destabilization of liposomes (more “leaky”) than oleate, an unsaturated fatty acid.     

Growth temperature affects accumulation of exogenous fatty acids and fatty acid composition in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis>

The incorporation of exogenously supplied fatty acids, palmitic acid, palmitoleic acid, oleic acid and linoleic acid, was examined in the yeast Schizosaccharomyces pombe at two growth temperatures, 20 °C and 30 °C. Fatty acids supplied to S. pombe in the growth medium were found to be preferentially incorporated into the cells, becoming a dominant species. The relative increase in exogenous fatty acids in cells came at the expense of endogenous oleic acid as a proportion of total fatty acids. Lowering the temperature at which the yeast were grown resulted in decreased levels of incorporation of the fatty acids palmitic acid, palmitoleic acid and linoleic acid compared to cells supplemented at 30 °C. In addition, the relative amount of the endogenously produced unsaturated fatty acid oleic acid, while greatly reduced compared to unsupplemented cells, was increased in cells supplemented with fatty acids at 20 °C compared to supplemented cells at 30 °C. The differential production of oleic acid in S. pombe cells indicates that regulation of unsaturated fatty acid levels, possibly by control of the stearoyl-CoA desaturase, is an important control point in membrane composition in response to temperature and diet in this species.