Incorporation Of Ethanolamine Research Articles

Impact of surfactants covering ZnO nanoparticles on solution-processed field-effect transistors: From dispersion state to solid state

Solution-processed n-channel oxide semiconductor thin-film transistors (TFTs) were fabricated using zinc oxide (ZnO) nanoparticles. The solution-processable semiconductor dispersions based on ZnO were prepared by dispersing the synthesized ZnO nanospheres in solution. The dispersion state was strongly affected by either the surfactant type or the solvent nature. Two small organic molecules commercially available, ethanolamine (EA) and benzoic acid (BA), were used as surfactant in alcohol solvent. The introduction of EA reduced the size of nanocrystal agglomerates and improved their dispersion properties simultaneously. Spin-coating of EA-modified ZnO dispersion following by a low-temperature annealing produced uniform polycrystalline nanoparticulate thin films. The resulting solid state of thin films demonstrated that the incorporation of EA has not a direct impact on the morphology but slightly decrease the field-effect mobility by less than one order of magnitude due to the insulating properties of EA organic molecules grafted on ZnO particles. As contrary, BA generated the existence of large-size cohesive ZnO agglomerates in solution which persist in the solid state to generate a less efficient charge transport. The modification of the particles surface by capping agent and the resulting particle–particle interactions in thin films were attributed as the main factors to impact on the field-effect mobility. To surfactant-free dispersions, the cohesive forces in ZnO agglomerates have been strongly influence by the solvent nature for a balance between a suitable dispersity of solution state and an effective solid state to charge transport.

Ammonium polyphosphate chemically-modified with ethanolamine as an efficient intumescent flame retardant for polypropylene

Ammonium polyphosphate (APP) is not an efficient flame retardant for polypropylene (PP) when it is used alone. In order to improve its flame-retardant efficiency, ethanolamine (ETA) was used to chemically modify APP via ion exchange reaction. The resulting ethanolamine-modified ammonium polyphosphate (ETA-APP) was alone applied to flame retard PP, the limiting oxygen index (LOI) value could reach 35.0% and the vertical burning test (UL-94) could pass the V-0 rating at a loading of 35 wt% ETA-APP. Moreover, cone calorimeter (CC) test results showed that the heat release rate (HRR), the total heat release (THR), the mass loss rate (MLR), the smoke production rate (SPR) and the total smoke production (TSP) of PP/35 wt% ETA-APP composite largely decreased, for example, by 77.2%, 88.5% and 77.9% for THR, TSP and the fire growth rate (FGR), respectively, compared with PP containing an equal amount of APP. In addition, the residual char of PP/35 wt% ETA-APP increased by 195.6% compared with that of PP/35 wt% APP. Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) were used to analyze the flame retardant mechanism of ETA-APP. It was confirmed that the formation of P–O–C and P–N–C structures, resulting from the incorporation of ETA could greatly improve the stability of intumescent char layer and consequently lead to the much better flame retardancy of ETA-APP than that of unmodified APP. Experimental results demonstrated that the prepared ETA-APP acted not only as the acid source and blowing source as efficient as unmodified APP, but also as an excellent charring agent beyond the unmodified APP.

Reliability of Antimalarial Sensitivity Tests Depends on Drug Mechanisms of Action

In vitro antimalarial activity tests play a pivotal role in malaria drug research or for monitoring drug resistance in field isolates. We applied two isotopic tests, two enzyme-linked immunosorbent assays (ELISA) and the SYBR green I fluorescence-based assay, to test artesunate and chloroquine, the metabolic inhibitors atovaquone and pyrimethamine, our fast-acting choline analog T3/SAR97276, and doxycycline, which has a delayed death profile. Isotopic tests based on hypoxanthine and ethanolamine incorporation are the most reliable tests provided when they are applied after one full 48-h parasite cycle. The SYBR green assay, which measures the DNA content, usually requires 72 h of incubation to obtain reliable results. When delayed death is suspected, specific protocols are required with increasing incubation times up to 96 h. In contrast, both ELISA tests used (pLDH and HRP2) appear to be problematic, leading to disappointing and even erroneous results for molecules that do not share an artesunatelike profile. The reliability of these tests is linked to the mode of action of the drug, and the conditions required to get informative results are hard to predict. Our results suggest some minimal conditions to apply these tests that should give rise to a standard 50% inhibitory concentration, regardless of the mechanism of action of the compounds, and highlight that the most commonly used in vitro antimalarial activity tests do not have the same potential. Some of them might not detect the antimalarial potential of new classes of compounds with innovative modes of action, which subsequently could become promising new antimalarial drugs.

Phosphatidylethanolamine synthesized by four different pathways is supplied to the plasma membrane of the yeast Saccharomyces cerevisiae

In this study, we examined the contribution of the four different pathways of phosphatidylethanolamine (PE) synthesis in the yeast Saccharomyces cerevisiae to the supply of this phospholipid to the plasma membrane. These pathways of PE formation are decarboxylation of phosphatidylserine (PS) by (i) phosphatidylserine decarboxylase 1 (Psd1p) in mitochondria and (ii) phosphatidylserine decarboxylase 2 (Psd2p) in a Golgi/vacuolar compartment, (iii) incorporation of exogenous ethanolamine and ethanolamine phosphate derived from sphingolipid catabolism via the CDP-ethanolamine pathway in the endoplasmic reticulum (ER), and (iv) synthesis of PE through acylation of lyso-PE catalyzed by the acyl-CoA-dependent acyltransferase Ale1p in the mitochondria associated endoplasmic reticulum membrane (MAM). Deletion of PSD1 and/or PSD2 led to depletion of total cellular and plasma membrane PE level, whereas mutation in the other pathways had practically no effect. Analysis of wild type and mutants, however, revealed that all four routes of PE synthesis contributed not only to PE formation but also to the supply of PE to the plasma membrane. Pulse-chase labeling experiments with L[(3)H(G)]serine and [(14)C]ethanolamine confirmed the latter finding. Fatty acid profiling demonstrated a rather balanced incorporation of PE species into the plasma membrane irrespective of mutations suggesting that all four pathways of PE synthesis provide at least a basic portion of "correct" PE species required for plasma membrane biogenesis. In summary, the PE level in the plasma membrane is strongly influenced by total cellular PE synthesis, but fine tuned by selective assembly mechanisms.

Increased phospholipid methylation and glycogen phosphorylase activation after a single dose of dexamethasone in mouse liver.

Two different effects on phospholipid methylation and glycogen phosphorylase a activation were accomplished by treating mice livers with dexamethasone. A biphasic effect on the glycogen phosphorylase a was determined in the homogenated liver of dexamethasone-treated animals: The enzymatic activity increased between 45 to 90 min and decreased about 3 h. Moreover, the increased activity of glycogen phosphorylase a was also found in adrenalectomized mice. Also, the incorporation of ethanolamine into PE1) and PC1) after 15 min of hormone-administration was 77% and 270%, respectively, over the control values. The increase in phospholipid methylation was maintained during the first hour. After 4 h the hormone-treated animals also showed significant differences compared to the controls. Finally, studies of total lipid phosphorus content in the liver fail to show any differences between control and hormone-treated mice.

Uptake and kinetic properties of choline and ethanolamine in <i>Plasmodium falciparum</i>

Purpose: The asexual proliferation of Plasmodium, inside the erythrocyte, is accompanied by the synthesis of huge quantities of phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn). These needful phospholipids for the cytoplasmic membrane of the merozoites are provided by the precursors choline and ethanolamine. PtdCho and PtdEtn are synthesized by the parasite because the erythrocyte is unable to do it. In order to assess the dynamism of the phospholipid pathways, we aimed to investigate the respective shape of the uptake of choline and ethanolamine by Plasmodium falciparum. Method: Time-course experiments and kinetic assays were performed respectively with fixed and ranged concentrations of radioactively-labelled choline and ethanolamine. The labelled-precursors were added in the culture of P. falciparum infected-erythrocytes and the incorporated molecules in phospholipids were measured with a scintigraph counter. Result: The results showed that the incorporation of precursors in the infected-erythrocyte occurred with a Michaelis-Menten\'s kinetic shape. According to the maximum rate (Vmax), the pathway of ethanolamine incorporation was faster than that of choline. Similarly, affinity for ethanolamine was greater than that of choline. Conclusion: Although PtdCho is the major phospholipid in the membrane, this study rules out that the influx of ethanolamine in the infected-erythrocyte, in vivo conditions, is more dynamic than choline. Keywords: Plasmodium, phospholipids, kinetics, metabolism, choline, ethanolamine Tropical Journal of Pharmaceutical Research Vol. 7 (2) 2008: pp. 953-959

Alternative splicing of CTP:phosphoethanolamine cytidylyltransferase produces two isoforms that differ in catalytic properties

CTP:phosphoethanolamine cytidylyltransferase (Pcyt2) catalyzes the rate-controlling reaction of the CDP-ethanolamine (Kennedy) pathway. We have previously established that Pcyt2 is encoded by a single gene that can be alternatively spliced from an internal exon into two transcripts, designated Pcyt2alpha and Pcyt2beta. Little is currently known about the regulation of Pcyt2. Here, we functionally express both murine Pcyt2 (mPcyt2) transcripts and investigate the roles of the two proteins in the regulation of mPcyt2 activity. We demonstrate that the tagged and purified alpha and beta proteins differ significantly in their kinetic properties. The K(m) of mPcyt2alpha for phosphoethanolamine was 318.4 microM, compared with 140.3 microM for mPcyt2beta. The maximal velocities of the alpha and beta isoforms at saturating conditions for both substrates were 138.0 and 114.4 nmol/min/mumol enzyme, respectively. When phosphoethanolamine was used at a fixed concentration of 1 mM, the K(m) of mPcyt2alpha for CTP was 102.0 microM and that of mPcyt2beta was 84.09 microM. Using a combination of nondenaturing PAGE, gel filtration chromatography, and immunoprecipitation, we provide evidence that mPcyt2alpha and mPcyt2beta proteins can form both homodimeric and heterodimeric complexes. We show that alternative splicing of the mPcyt2 transcript is ubiquitous but could also be regulated in a tissue-specific manner, producing a variable ratio of mPcyt2alpha/mPcyt2beta mRNAs. The expression of two distinct protein isoforms maybe an important mechanism by which Pcyt2 activity is regulated.

Commonality and Biosynthesis of the O-Methyl Phosphoramidate Capsule Modification in Campylobacter jejuni

In this study we investigated the commonality and biosynthesis of the O-methyl phosphoramidate (MeOPN) group found on the capsular polysaccharide (CPS) of Campylobacter jejuni. High resolution magic angle spinning NMR spectroscopy was used as a rapid, high throughput means to examine multiple isolates, analyze the cecal contents of colonized chickens, and screen a library of CPS mutants for the presence of MeOPN. Sixty eight percent of C. jejuni strains were found to express the MeOPN with a high prevalence among isolates from enteritis, Guillain Barré, and Miller-Fisher syndrome patients. In contrast, MeOPN was not observed for any of the Campylobacter coli strains examined. The MeOPN was detected on C. jejuni retrieved from cecal contents of colonized chickens demonstrating that the modification is expressed by bacteria inhabiting the avian gastrointestinal tract. In C. jejuni 11168H, the cj1415-cj1418 cluster was shown to be involved in the biosynthesis of MeOPN. Genetic complementation studies and NMR/mass spectrometric analyses of CPS from this strain also revealed that cj1421 and cj1422 encode MeOPN transferases. Cj1421 adds the MeOPN to C-3 of the beta-d-GalfNAc residue, whereas Cj1422 transfers the MeOPN to C-4 of D-glycero-alpha-L-gluco-heptopyranose. CPS produced by the 11168H strain was found to be extensively modified with variable MeOPN, methyl, ethanolamine, and N-glycerol groups. These findings establish the importance of the MeOPN as a diagnostic marker and therapeutic target for C. jejuni and set the groundwork for future studies aimed at the detailed elucidation of the MeOPN biosynthetic pathway.

Regulation of the Yeast EKI1-encoded Ethanolamine Kinase by Inositol and Choline

Regulation of the EKI1-encoded ethanolamine kinase by inositol and choline was examined in Saccharomyces cerevisiae. Transcription of the EKI1 gene was monitored by following the expression of beta-galactosidase activity driven by a P(EKI1)-lacZ reporter gene. The addition of inositol to the growth medium resulted in a dose-dependent decrease in EKI1 expression. Supplementation of choline to inositol-containing growth medium brought about a further decrease in expression, whereas choline supplementation alone had no effect. Analysis of EKI1 expression in ino2Delta, ino4Delta, and opi1Delta mutants indicated that the transcription factors Ino2p, Ino4p, and Opi1p played a role in this regulation. Moreover, mutational analysis showed that the UAS(INO) element in the EKI1 promoter was required for the inositol-mediated regulation. The regulation of EKI1 expression by inositol and choline was confirmed by corresponding changes in ethanolamine kinase mRNA, protein, and activity levels. The repression of ethanolamine kinase by inositol supplementation correlated with a decrease in the incorporation of ethanolamine into CDP-ethanolamine pathway intermediates and into phosphatidylethanolamine and phosphatidylcholine.

Control of the CDPethanolamine pathway in mammalian cells: effect of CTP:phosphoethanolamine cytidylyltransferase overexpression and the amount of intracellular diacylglycerol.

For an insight regarding the control of PtdEtn (phosphatidylethanolamine) synthesis via the CDPethanolamine pathway, rat liver cDNA encoding ECT (CTP:phosphoethanolamine cytidylyltransferase) was transiently or stably transfected in Chinese-hamster ovary cells and a rat liver-derived cell line (McA-RH7777), resulting in a maximum of 26- and 4-fold increase in specific activity of ECT respectively. However, no effect of ECT overexpression on the rate of [3H]ethanolamine incorporation into PtdEtn was detected in both cell lines. This was explored further in cells overexpressing four times ECT activity (McA-ECT1). The rate of PtdEtn breakdown and PtdEtn mass were not changed in McA-ECT1 cells in comparison with control-transfected cells. Instead, an accumulation of CDPethanolamine (label and mass) was observed, suggesting that in McA-ECT1 cells the ethanolaminephosphotransferase-catalysed reaction became rate-limiting. However, overexpression of the human choline/ethanolaminephosphotransferase in McA-ECT1 and control-transfected cells had no effect on PtdEtn synthesis. To investigate whether the availability of DAG (diacylglycerol) limited PtdEtn synthesis in these cells, intracellular DAG levels were increased using PMA or phospholipase C. Exposure of cells to PMA or phospholipase C stimulated PtdEtn synthesis and this effect was much more pronounced in McA-ECT1 than in control-transfected cells. In line with this, the DAG produced after PMA exposure was consumed more rapidly in McA-ECT1 cells and the CDPethanolamine level decreased accordingly. In conclusion, our results suggest that the supply of CDPethanolamine, via the expression level of ECT, is an important factor governing the rate of PtdEtn biosynthesis in mammalian cells, under the condition that the amount of DAG is not limiting.

Brevican isoforms associate with neural membranes.

Brevican is a neural-specific proteoglycan of the brain extracellular matrix, which is particularly abundant in the terminally differentiated CNS. It is expressed by neuronal and glial cells, and as a component of the perineuronal nets it decorates the surface of large neuronal somata and primary dendrites. One brevican isoform harbors a glycosylphosphatidylinositol anchor attachment site and, as shown by ethanolamine incorporation studies, is indeed glypiated in stably transfected HEK293 cells as well as in oligodendrocyte precursor Oli-neu cells. The major isoform is secreted into the extracellular space, although a significant amount appears to be tightly attached to the cell membrane, as it floats up in sucrose gradients. Flotation is sensitive to detergent treatment. Brevican is most prominent in the microsomal, light membrane and synaptosomal fractions of rat brain membrane preparations. The association with the particulate fraction is in part sensitive to chondroitinase ABC and phosphatidylinositol-specific phospholipase C treatment. Furthermore, brevican staining on the surface of hippocampal neurons in culture is diminished after hyaluronidase or chondroitinase ABC treatment. Taken together, this could provide a mechanism by which perineuronal nets are anchored on neuronal surfaces.

Nature of glycosylphosphatidylinositols produced by mouse embryonic stem cells

Incorporation of [(3)H]inositol into mouse embryonic stem cells of the CCE cell line leads to the labelling of the three common phosphoinositides, phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, and a fourth unknown lipid (lipid X). Incubation with [(3)H]glucosamine results in the labelling of lipid X and at least one other lipid that co-migrates with phosphatidylinositol (lipid Y), indicating that both of these lipids are putative glycosylphosphatidylinositols. In this study, the incorporation of other possible glycosylphosphatidylinositol precursors, ethanolamine, mannose and galactose, into lipids X and Y was examined. Galactose was incorporated into lipids X and Y, and ethanolamine and mannose into lipid Y only. Inhibitors of glycosylphosphatidylinositol biosynthesis pathways, mannosamine and 2-fluoro-2-deoxyglucose, both significantly inhibited ethanolamine incorporation into lipid Y. A high glucose concentration (25 mmol l(-1)) abolished the action of both inhibitors. Phospholipase C treatment of embryonic stem cells that had been labelled in culture with [(3)H]ethanolamine caused a large release of ethanolamine label into the incubation medium and markedly decreased the amount of ethanolamine-labelled lipid Y remaining in the cell membranes. These effects were almost totally abolished by incubation with mannosamine before ethanolamine labelling. These studies strongly indicate that lipid Y is a member of the protein anchor class of glycosylphosphatidylinositol, whereas lipid X is a member of the signal transduction inositol phosphoglycan class of glycosylphosphatidylinositol.

Nature of glycosylphosphatidylinositols produced by mouse embryonic stem cells.

Incorporation of [(3)H]inositol into mouse embryonic stem cells of the CCE cell line leads to the labelling of the three common phosphoinositides, phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, and a fourth unknown lipid (lipid X). Incubation with [(3)H]glucosamine results in the labelling of lipid X and at least one other lipid that co-migrates with phosphatidylinositol (lipid Y), indicating that both of these lipids are putative glycosylphosphatidylinositols. In this study, the incorporation of other possible glycosylphosphatidylinositol precursors, ethanolamine, mannose and galactose, into lipids X and Y was examined. Galactose was incorporated into lipids X and Y, and ethanolamine and mannose into lipid Y only. Inhibitors of glycosylphosphatidylinositol biosynthesis pathways, mannosamine and 2-fluoro-2-deoxyglucose, both significantly inhibited ethanolamine incorporation into lipid Y. A high glucose concentration (25 mmol l(-1)) abolished the action of both inhibitors. Phospholipase C treatment of embryonic stem cells that had been labelled in culture with [(3)H]ethanolamine caused a large release of ethanolamine label into the incubation medium and markedly decreased the amount of ethanolamine-labelled lipid Y remaining in the cell membranes. These effects were almost totally abolished by incubation with mannosamine before ethanolamine labelling. These studies strongly indicate that lipid Y is a member of the protein anchor class of glycosylphosphatidylinositol, whereas lipid X is a member of the signal transduction inositol phosphoglycan class of glycosylphosphatidylinositol.

Overexpression of a Mammalian Ethanolamine-specific Kinase Accelerates the CDP-ethanolamine Pathway

Ethanolamine kinase (EKI) is the first committed step in phosphatidylethanolamine (PtdEtn) biosynthesis via the CDP-ethanolamine pathway. We identify a human cDNA encoding an ethanolamine-specific kinase EKI1 and the structure of the EKI1 gene located on chromosome 12. EKI1 overexpression in COS-7 cells results in a 170-fold increase in ethanolamine kinase-specific activity and accelerates the rate of [3H]ethanolamine incorporation into PtdEtn as a function of the ethanolamine concentration in the culture medium. Acceleration of the CDP-ethanolamine pathway does not result in elevated cellular PtdEtn levels, but rather the excess PtdEtn is degraded to glycerophosphoethanolamine. EKI1 has negligible choline kinase activity in vitro and does not influence phosphatidylcholine biosynthesis. Acceleration of the CDP-ethanolamine pathway also does not change the rate of PtdEtn formation via the decarboxylation of phosphatidylserine. The data demonstrate the existence of separate ethanolamine and choline kinases in mammals and show that ethanolamine kinase can be a rate-controlling step in PtdEtn biosynthesis.

Incorporation of ethanolamine into insulin-sensitive glycosylated phosphatidylinositol of chick embryo fibroblasts

Insulin sensitive glycosylated phosphatidylinositol (GPI) from chick embryo fibroblasts was isolated and partially characterized. [ 3H]Ethanolamine was incorporated into lipids different from phosphatidylethanolamine, as shown by two sequential thin layer chromatographies (TLC) using an acidic solvent system followed by a basic solvent system. Other isotopes, myo-[ 3H]inositol, [ 3H]glucosamine, [ 3H]galactose, and [ 3H]palmitic acid were also incorporated into these lipids. These lipids were separated into two peaks on the second basic TLC, designated as peaks I and II from the origin. Insulin stimulation of cells caused a rapid breakdown of these two lipids. These two lipids were treated by nitrous acid and phosphatidylinositol-specific phospholipase C (PI-PLC). The radioactivity of peak I lipid was decreased by both treatments, and that of peak II lipid was also decreased by PI-PLC treatment but not significantly by nitrous acid treatment. Peak II lipid did not fulfill the criteria for GPI. Tritium released by the treatment of PI-PLC of peak I lipid was recovered in the aqueous phase. [ 3H]Ethanolamine-labeled peak I lipid was hydrolyzed by acid treatment and the hydrolysis products were analyzed by TLC and high performance liquid chromatography (HPLC). Tritium label was recovered as native label at the rate of 95%. [ 3H]Ethanolamine of peak I lipid was reductively methylated completely with formaldehyde and cyanoborohydride, as shown by HPLC analysis. The results indicate that peak I lipid contains primary ethanolamine as a glycan component and is insulin-sensitive free GPI.

Dengue virus nonstructural protein 1 is expressed in a glycosyl-phosphatidylinositol-linked form that is capable of signal transduction.

Dengue virus nonstructural protein 1 (NS1) is expressed on the surface of infected cells and is a target of human antibody responses to dengue virus infection. We show here that dengue virus uses the cellular glycosyl-phosphatidylinositol (GPI) linkage pathway to express a GPI-anchored form of NS1 and that GPI anchoring imparts a capacity for signal transduction in response to binding of NS1-specific antibody. This study is the first to identify GPI linkage of a virus-encoded protein. The GPI anchor addition signal for NS1 was identified, by transfection of HeLa cells with dengue cDNA constructs, as a downstream hydrophobic domain in NS2A. GPI linkage of NS1 in both transfected and infected cells was demonstrated by cleavage of NS1 from the surface by PI-specific phospholipase C and by metabolic incorporation of the GPI-specific components ethanolamine and inositol. In common with other GPI-anchored proteins, addition of specific antibody resulted in signal transduction, as evidenced by tyrosine phosphorylation of cellular proteins. Antibody-induced signal transduction by GPI-linked NS1 suggests a mechanism of cellular activation that may contribute to the pathogenesis of human dengue disease. Signal transduction by a GPI-anchored viral antigen interacting with a specific antibody that it induces is a new concept in the pathogenesis of viral disease.

Inhibition of phosphatidylcholine and phosphatidylethanolamine biosynthesis in rat‐2 fibroblasts by cell‐permeable ceramides

Phospholipids and sphingolipids are important precursors of lipid-derived second messengers such as diacylglycerol and ceramide, which participate in several signal transduction pathways and in that way mediate the effects of various agonists. The cross-talk between glycerophospholipid and sphingolipid metabolism was investigated by examining the effects of cell-permeable ceramides on phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) synthesis in Rat-2 fibroblasts. Addition of short-chain C6-ceramide to the cells resulted in a dose- and time-dependent inhibition of the CDP-pathways for PtdCho and PtdEtn synthesis. Treatment of cells for 4 h with 50 microM C6-ceramide caused an 83% and a 56% decrease in incorporation of radiolabelled choline and ethanolamine into PtdCho and PtdEtn, respectively. Exposure of the cells for longer time-periods (>/= 16 h) to 50 microM C6-ceramide resulted in apoptosis. The structural analogue dihydro-C6-ceramide did not affect PtdCho and PtdEtn synthesis. In pulse-chase experiments, radioactive choline and ethanolamine accumulated in CDP-choline and CDP-ethanolamine under the influence of C6-ceramide, suggesting that synthesis of both PtdCho and PtdEtn were inhibited at the final step in the CDP-pathways. Indeed, cholinephosphotransferase and ethanolaminephosphotransferase activities in membrane fractions from C6-ceramide-treated cells were reduced by 64% and 43%, respectively, when compared with control cells. No changes in diacylglycerol mass levels or synthesis of diacylglycerol from radiolabelled palmitate were observed. It was concluded that C6-ceramide affected glycerophospholipid synthesis predominantly by inhibition of the step in the CDP-pathways catalysed by cholinephosphotransferase and ethanolaminephosphotransferase.

Effects of Docosahexaenoic and Arachidonic Acids on the Synthesis and Distribution of Aminophospholipids during Neuronal Differentiation of PC12 Cells

We have shown previously that docosahexaenoic acid (DHA) promotes and arachidonic acid (AA) suppresses neurite outgrowth of PC12 cells induced by nerve growth factor (NGF) and that incorporation of [3H]ethanolamine into phosphatidylethanolamine (PE) is suppressed in PC12 cells by AA while DHA has no effect. In the present study, the effects of these fatty acids on PE synthesis via decarboxylation of phosphatidylserine (PS), another pathway of PE synthesis, and distribution of aminophospholipids were examined. Incorporation of [3H]serine into PS and PE was elevated in the course of NGF-induced differentiation and was further stimulated significantly by DHA, but not by AA. [3H]Ethanolamine uptake by PC12 cells was significantly suppressed by AA but not by DHA while these fatty acids did not affect [3H]serine uptake, indicating that the suppression by AA of [3H]ethanolamine incorporation into phosphatidylethanolamine is attributable, at least in part, to a reduction in [3H]ethanolamine uptake. The distribution of PE in the outer leaflet of plasma membrane decreased during differentiation, which is known to be accompanied by an increase in the surface area of plasma membrane. Supplementation of PC12 cells with DHA or AA did not affect the distribution of aminophospholipids. Thus, DHA and AA affected aminophospholipid synthesis and neurite outgrowth differently, but not the transport and distribution of aminophospholipids, while the PE concentration in the outer leaflet of the plasma membrane decreased in association with morphological changes in PC12 cells induced by NGF.

Uptake and incorporation of choline and ethanolamine into lipoteichoic acid and teichoic acid by the choline-independent mutant JY2190 of Streptococcus pneumoniae

Lipoteichoic acid- and teichoic acid-containing muropeptides were isolated from choline- or ethanolamine-grown cells of the choline-independent mutant JY2190 of Streptococcus pneumoniae. Choline was taken up and incorporated into lipoteichoic acid and teichoic acid with 81% efficiency, compared with the parent strain Rx1. With similar efficiency, ethanolamine was incorporated. Accordingly, the mutant is a valuable tool for identifying the individual genes encoding the enzymes of choline utilisation, because any of these genes can be deleted without affecting viability and growth rate.

Different mechanisms regulate phosphatidylserine synthesis in rat cerebral cortex.

Transduction of extracellular signals through the membrane involves both the lipid and protein moiety. Phosphatidylserine participates to these processes as a cofactor for protein kinase C activity and thus the existence of a regulatory mechanism for its synthesis ought to be expected. In plasma membranes from rat cerebral cortex, the activity of serine base exchange enzyme, that is mainly responsible for phosphatidylserine synthesis in mammalian tissues, was reduced by the addition to the incubation mixture of AlF4- or GTP-gamma-S, known activators of G proteins, whereas ATP was almost uneffective. GTP-gamma-S inhibited the enzyme activity only at relatively high concentration (> 0.5 mM). When the synthesis of phosphatidylserine in the same cerebral area was investigated by measuring the incorporation of labelled serine into the phospholipid in the homogenate buffered at pH 7.6, ATP had an inhibitory effect as GTP-gamma-S and AlF4-. Heparin activated both serine base exchange enzyme in plasma membranes and phosphatidylserine synthesis in the homogenate. The preincubation of plasma membranes in the buffer without any other addition at 37 degrees C for 15 min reduced by 30% serine base exchange enzyme activity. The remaining activity responded to the addition of GTP-gamma-S but was insensitive to 5 mM AlF-4, a concentration that inhibited by 60% the enzyme assayed without preincubation. These results indicate the existence of different regulatory mechanisms, involving ATP and G proteins, possibly acting on different enzymes responsible for the synthesis of phosphatidylserine. Since previous studies have shown that hypoxia increases the synthesis of this phospholipid in brain slices or homogenate (Mozzi et al. Mol Cell Biochem 126: 101-107, 1993), it is possible that hypoxia may interfere with at least one of these mechanisms. This hypothesis is supported by the observation that in hypoxic homogenate 20 mM AlF-4 was not able to reduce the synthesis of phosphatidylserine as in normoxic samples. A similar difference between oxygenated and hypoxic samples, concerning their response to AlF4-, was observed when the incorporation of ethanolamine into phosphatidylethanolamine was studied. The incorporation of choline into phosphatidilcholine was, on the contrary, inhibited at a similar extent in both experimental conditions.