Phosphatidylethanolamine from phosphatidylserine decarboxylase2 is essential for autophagy under cadmium stress in Saccharomyces cerevisiae.

Cadmium (Cd) is a potent toxic element used in several industries and in the process contaminates air, soil, and water. Exposure of Saccharomyces cerevisiae to Cd increases the major phospholipids, and profound increase was observed in phosphatidylethanolamine (PE). In yeast, there are four different pathways contributing to the biosynthesis of PE, and contribution to PE pool through phosphatidylserine decarboxylase2 (psd2) is not significant in normal conditions. Upon Cd exposure, psd2Δ strain showed a significant decrease in major phospholipids including PE. When exposed to Cd, wild-type (WT) cells depicted an increase in ER stress and autophagy, whereas in psd2, ER stress was noted but autophagy process was impaired. The supplementation of ethanolamine did not overcome the Cd stress and also the autophagy process, whereas overexpression of PSD2 in psd2Δ increased the cellular tolerance, PE levels, and the autophagy process against Cd stress. From our studies, we can suggest that PSD2 of S. cerevisiae has an important role in PE synthesis and in autophagy process under Cd stress.

Functional characterization of the fission yeast phosphatidylserine synthase gene, pps1, reveals novel cellular functions for phosphatidylserine.

To investigate the contributions of phosphatidylserine to the growth and morphogenesis of the rod-shaped fission yeast Schizosaccharomyces pombe, we have characterized the single gene in this organism, pps1, encoding a predicted phosphatidylserine synthase. S. pombe pps1Delta mutants grow slowly in rich medium and are inviable in synthetic minimal medium. They do not produce detectable phosphatidylserine in vivo and possess negligible in vitro phosphatidylserine synthase activity, indicating that pps1 encodes the major phosphatidylserine synthase activity in S. pombe. Supplementation of growth medium with ethanolamine partially suppresses the growth-defective phenotype of pps1Delta cells, reflecting the likely importance of phosphatidylserine as a precursor for phosphatidylethanolamine in S. pombe. In medium lacking ethanolamine, pps1Delta mutants exhibit striking cell morphology, cytokinesis, actin cytoskeleton, and cell wall remodeling and integrity defects. Overexpression of pps1 likewise leads to defects in cell morphology and cytokinesis, thus implicating phosphatidylserine as a dosage-dependent regulator of these processes. During log-phase growth, green fluorescent protein-Pps1p fusion proteins are concentrated at the cell and nuclear peripheries as well as presumptive endoplasmic reticulum membranes, while in stationary-phase cells, they are redistributed to unusual cytoplasmic structures of unknown origin. Moreover, stationary-phase pps1Delta cultures retain very poor viability relative to wild-type S. pombe cells, even in medium containing ethanolamine, demonstrating a role for phosphatidylserine in the physiological adaptations required for stationary-phase survival. Our findings reveal novel cellular functions for phosphatidylserine and emphasize the usefulness of S. pombe as a model organism for elucidating potentially conserved biological and molecular functions of this phospholipid.

Dietary eritadenine and ethanolamine depress fatty acid desaturase activities by increasing liver microsomal phosphatidylethanolamine in rats.

The effects of eritadenine, a constituent of the Lentinus edodes mushroom, and ethanolamine, the base constituent of phosphatidylethanolamine (PE), on fatty acid desaturase activities and lipid profiles were investigated comparatively in rats. Rats were fed a control diet or a diet supplemented with either eritadenine (0.05 g/kg) or ethanolamine (8 g/kg) for 14 d. Eritadenine and ethanolamine had marked hypocholesterolemic effects. The concentration of liver microsomal PE was significantly increased and the ratio of phosphatidylcholine (PC) to PE was significantly decreased by both eritadenine and ethanolamine. These changes in phospholipid profile were also observed in the mitochondria and plasma membranes in the liver. The activities of the Delta5-, Delta6- and Delta9-desaturases in liver microsomes were significantly decreased by eritadenine and ethanolamine; there was a significant correlation between the activity of Delta5- or Delta6-desaturase and the proportion of PE in the total phospholipids or the PC/PE ratio. Reflecting decreased Delta5- and Delta6-desaturase activities, the 20:4(n-6)/18:2(n-6) ratio was significantly decreased by eritadenine and ethanolamine in PC of the liver microsomes, mitochondria and plasma membranes. Although the 20:4(n-6)/18:2(n-6) ratio of liver microsomal PE was also significantly decreased by eritadenine and ethanolamine, the fatty acid composition of phosphatidylinositol and phosphatidylserine was less affected by these compounds. Eritadenine and ethanolamine increased the proportion of 16:0-18:2 and decreased the proportion of 18:0-20:4 in liver PC. The results suggest that dietary eritadenine and ethanolamine might lead to decreases in desaturase activities and changes in fatty acid and molecular species composition of PC through an increase in liver microsomal PE.

Beta 3 agonists. Part 1: evolution from inception to BMS-194449.

Screening of the BMS collection identified 4-hydroxy-3-methylsulfonanilidoethanolamines as full beta 3 agonists. Substitution of the ethanolamine nitrogen with a benzyl group bearing a para hydrogen bond acceptor promoted beta(3) selectivity. SAR elucidation established that highly selective beta(3) agonists were generated upon substitution of C(alpha) with either benzyl to form (R)-1,2-diarylethylamines or with aryl to generate 1,1-diarylmethylamines. This latter subset yielded a clinical candidate, BMS-194449 (35).(1)

A genetic screen for ethanolamine auxotrophs in Saccharomyces cerevisiae identifies a novel mutation in Mcd4p, a protein implicated in glycosylphosphatidylinositol anchor synthesis.

A genetic screen for ethanolamine auxotrophs has identified a novel mutant allele of the morphogenesis checkpoint dependent (MCD)-4 gene, designated mcd4-P301L. In the presence of a null allele for the phosphatidylserine (PtdSer) decarboxylase 1 gene (psd1 Delta), the mcd4-P301L mutation confers temperature sensitivity for growth on minimal medium. This growth defect is reversed by either ethanolamine or choline supplementation. Incubation of mutant cells with [(3)H]serine followed by analysis of the aminoglycerophospholipids demonstrated a 60% decrease in phosphatidylethanolamine (PtdEtn) formation compared to parental cells. Chemical analysis of phospholipid content after culture under non-permissive conditions also demonstrated a 60% decrease in the PtdEtn pool compared to the parental strain. Although the morphogenesis checkpoint dependent (MCD)-4 gene and its homologues have been shown to play a role in glycosylphosphatidylinositol (GPI) anchor synthesis, the mcd4-P301L strain displayed normal incorporation of [(3)H]inositol into both proteins and lipids. Thus, a defect in GPI anchor synthesis does not explain either the ethanolamine auxotrophy or biochemical phenotype of this mutant. We also examined the growth characteristics and PtdSer metabolism of a previously described mcd4-174 mutant strain, with defects in GPI anchor synthesis, protein modification and cell wall maintenance. The mcd4-174, psd1 Delta strain is a temperature sensitive ethanolamine auxotroph that requires osmotic support for growth, and displays normal PtdEtn formation compared to parental cells. These results reveal important genetic interactions between PSD1 and MCD4 genes, and provide evidence that Mcd4p can modulate aminoglycerophospholipid metabolism, in a way independent of its role in GPI anchor synthesis.

N-methyl bases of ethanolamine prevent apoptotic cell death induced by oxidative stress in cells of oligodendroglia origin.

A major reason for brain tissue vulnerability to oxidative damage is the high content of polyunsaturated fatty acids (PUFAs). Oligodendroglia-like OLN 93 cells lack PUFAs and are relatively insensitive to oxidative stress. When grown in serum-free defined medium in the presence of 0.1 mM docosahexaenoic acid (DHA; 22:6 n-3) for 3 days, OLN 93 cells release in the medium 2.6-fold more thiobarbituric acid-reactive substances (TBARS) after a 30-min exposure to 0.1 mM H2O2 and 50 microM Fe2+. Release of TBARS was substantially decreased by approximately 20 and 30% on coincubation with either 1 mM N-monomethylethanolamine or N,N'-dimethylethanolamine (dEa), respectively. The protective effect of dEa was concentration- and time-dependent and was still visible after dEa removal, suggesting a long-lasting mechanism of protection. After 24 h following H2O2-induced stress, cell death monitored by cell sorting showed 16% of the cells in the sub-G1 area, indicative of apoptotic cell death. DHA-supplemented cultures showed 35% cell death, whereas cosupplements with dEa reduced cell death to 12%, indicating cell rescue. Although the exact mechanism for this protection is not known, the nature of the polar head group and the degree of unsaturation may determine the ultimate resistance of nerve cells to oxidative stress.

Enhancement of free fatty acid incorporation into phospholipids by choline plus cytidine.

Cytidine and choline, present in cytidine 5'-diphosphate choline (CDP-choline), are major precursors of the phosphatidylcholine found in cell membranes and important regulatory elements in phosphatide biosynthesis. Administration of CDP-choline to rats increases blood and brain cytidine and choline levels; this enhances the production of endogenous CDP-choline which then combines with fatty acids (as diacylglycerol), to yield phosphatidylcholine. We examined the effect of providing cytidine and choline on incorporation of free fatty acids into phosphatidylcholine and other major phospholipids in PC12 cells. Addition of equimolar cytidine and choline (100-500 microM) to [3H]-arachidonic acid (50 microM, 0.2 microCi, bound to bovine serum albumin) dose-dependently increased the accumulations of [3H]-phosphatidylcholine (PtdCho), [3H]-phosphatidylinositol (PtdIno) and [3H]-phosphatidylethanolamine (PtdEtn) (by up to 27+/-3%, 16+/-3% and 11+/-3%, respectively, means+/-S.E.M.). This effect was seen with 8-18 h of incubation. The incorporation of [3H]-oleic acid into [3H]-PtdCho was even more enhanced (by up to 42+/-3%) as were the incorporations of [14C]-choline and [3H]-glycerol. The effects of choline and cytidine were enhanced by 12-O-tetradecanoylphorbol-13-acetate (TPA, 1 microM), which activates CTP:phosphocholine cytidylyltransferase (CT) and facilitates choline uptake. Replacing choline by ethanolamine also enhanced the incorporation of [3H]-arachidonic acid into [3H]-PtdEtn, [3H]-PtdIno and [3H]-PtdCho. Arachidonic acid (10-200 microM) alone failed to affect the incorporation of [14C]-choline into phosphatidylcholine. We suggest that the increases in phospholipid synthesis caused by concurrent cytidine and choline supplementation enhance the incorporation of arachidonic acid and certain other fatty acids into the major glycerophospholipids. Removing these fatty acids as source of potentially toxic oxidation products could contribute to the beneficial effects of CDP-choline in treating stroke or other brain damage.