Induction of glycoprotein biosynthesis in activated B lymphocytes

Resting murine splenic B lymphocytes (B cells) can be stimulated to proliferate by exposure to a variety of polyclonal activators. To investigate changes in glycoprotein synthesis that occur during the activation process, N-glycosylation activity was assessed by following the incorporation of [2-3H]mannose into dolichol-linked oligosaccharide intermediates and glycoprotein after B cells were exposed to anti-immunoglobulin M (anti-mu). Stimulation of B cells by anti-mu resulted in a dramatic induction of N-glycosylation activity. The incorporation of radiolabeled mannose into oligosaccharide-lipid increased 9-fold while the rate of labeling of glycoprotein increased 27-fold between 18 and 38 h after exposure to anti-mu. Maximal stimulation of N-glycosylation activity was observed at an anti-mu concentration of 20-50 micrograms/ml. Similar results were obtained when B cells were activated by bacterial lipopolysaccharide (LPS), another polyclonal activating agent. The major dolichol-bound oligosaccharide labeled during the induction period was determined to be Glc3Man9GlcNAc2 by HPLC analysis. Nearly full induction of oligosaccharide-lipid synthesis and protein N-glycosylation was also seen when DNA synthesis was suppressed by activating B cells with anti-mu in a serum-free medium, or by activating with anti-mu or LPS in the presence of hydroxyurea. The results suggest that the N-glycosylation pathway is induced during the G0 to G1 transition or during the G1 period, and that entry into S phase is not required. These studies describe a striking developmental increase in N-glycosylation activity and extend the information on biochemical changes occurring during the activation of B cells.     

Inhibition of hepatic phosphatidylcholine synthesis by 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside is independent of AMP-activated protein kinase activation

5-Aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAr), a commonly used indirect activator of AMP-activated protein kinase (AMPK), inhibits phosphatidylcholine (PC) biosynthesis in freshly isolated hepatocytes. In all nucleated mammalian cells, PC is synthesized from choline via the Kennedy (CDP-choline) pathway. The purpose of our study was to provide direct evidence that AMPK regulates phospholipid biosynthesis and to elucidate the mechanism(s) by which AMPK inhibits hepatic PC synthesis. Incubations of hepatocytes with AICAr resulted in a dose-dependent activation of AMPK and inhibition of PC biosynthesis. Surprisingly, adenoviral delivery of constitutively active AMPK did not alter PC biosynthesis. In addition, expression of dominant negative mutants of AMPK was unable to block the AICAr-dependent inhibition of PC biosynthesis, indicating that AICAr was acting independently of AMPK activation. Determination of aqueous intermediates of the CDP-choline pathway indicated that choline kinase, the first enzyme in the pathway, was inhibited by AICAr administration. Flux through the CDP-choline pathway was directly correlated to the level of intracellular ATP concentrations. Therefore, it is possible that inhibition of PC biosynthesis is another process by which the cell can reduce ATP consumption in times of energetic stress. However, unlike cholesterol and triacylglycerol biosynthesis, PC production is not regulated by AMPK.     

The protein phosphatase inhibitor, okadaic acid, inhibits phosphatidylcholine biosynthesis in isolated rat hepatocytes.

There is evidence that phosphatidylcholine (PC) biosynthesis in hepatocytes is regulated by a phosphorylation-dephosphorylation mechanism. The phosphatases involved have not been identified. We, therefore, investigated the effect of okadaic acid, a potent protein phosphatase inhibitor, on PC biosynthesis via the CDP-choline pathway in suspension cultures of isolated rat hepatocytes. Okadaic acid caused a 15% decrease (P less than 0.05) in [Me-3H]choline uptake in continuous-pulse labeling experiments. After 120 min of treatment, the labeling of PC was decreased 46% (P less than 0.05) with a corresponding 20% increase (P less than 0.05) in labeling of phosphocholine. Cells were pulsed with [Me-3H]choline for 30 min and subsequently chased for up to 120 min with choline in the absence or presence of okadaic acid. The labeling of phosphocholine was increased 86% (P less than 0.05) and labeling of PC decreased 29% (P less than 0.05) by 120 min of chase in okadaic acid-treated hepatocytes. The decrease of label in PC was quantitatively accounted for in the phosphocholine fraction. Incubation of hepatocytes with both okadaic acid and CPT-cAMP did not produce an additive inhibition in labeling of PC. Choline kinase and cholinephosphotransferase activities were unaltered by treatment with okadaic acid. Hepatocytes were incubated with digitonin to cause release of cytosolic components. Cell ghost membrane cytidylyltransferase (CT) activity was decreased 37% (P less than 0.005) with a concomitant 33% increase (P less than 0.05) in released cytosolic cytidylyltransferase activity in okadaic acid-treated hepatocytes. We postulate that CT activity and PC biosynthesis are regulated by protein phosphatase activity in isolated rat hepatocytes.     

Choline Deficiency in Cultured Adrenal Medullary Cells: Effect on Phosphatidylcholine Biosynthesis

The effect of choline deficiency on the composition and biosynthesis of the major membrane phospholipids was examined in adrenal medullary cells maintained in suspension cultures. The amount and proportions of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in these cells were not affected by the removal of choline from the culture media. However, the rate of biosynthesis of choline at the phosphatide level by the stepwise methylation of PE increased twofold within 24 h after choline was removed from the culture media, while ethanolamine incorporation into PE was increased by 50%. In contrast, the rate of incorporation of labeled choline into PC, presumably via CDP-choline, was virtually identical in cells that had been preincubated in the presence or absence of 1 mM choline. These results demonstrate that cultured cells of neural origin are capable of compensating for lack of exogenous choline by forming choline at the phosphatide level through the sequential methylation of PE. The hypolipidemic drug, DH-990, when added to the culture media, inhibited conversion of phosphatidylmonomethylethanolamine (PME) to PC, but had no effect on the N-methylation of PE. This differential effect indicates that the initial N-methylation of PE is catalyzed by an enzyme that is distinguishable from the enzyme(s) catalyzing the conversion of PME to PC.     

Uptake of Exogenous PhosphatidyJserine by Human Neuroblastoma Cells Stimulates the Incorporation of [methyl-l4C]Cholne into Phosphatidylcholine

The phosphatidylserine (PtdSer) content of human cholinergic neuroblastoma (LA-N-2) cells was manipulated by exposing the cells to exogenous PtdSer, and the effects on phospholipid content, membrane composition, and incorporation of choline into phosphatidylcholine (PtdCho) were investigated. The presence of liposomes containing PtdSer (10-130 microM) in the medium caused time- and concentration-dependent increases in the PtdSer content of the cells, and smaller and slower increases in the contents of other membrane phospholipids. The PtdSer levels in plasma membrane and mitochondrial fractions prepared by discontinuous sucrose density gradient centrifugation increased by 50 and 100%, respectively, above those in control cells after 24 h of exposure to PtdSer (130 microM). PtdSer caused a concomitant, concentration-dependent increase of up to twofold in the incorporation of [methyl-14C]choline chloride into PtdCho at a choline concentration (8.5 microM) compatible with activation of the CDP-choline pathway, suggesting that the levels of PtdSer in membranes may serve as a stimulus to regulate overall membrane composition. PtdSer caused a mean increase of 41% in PtdCho labeling, but the phorbol ester, phorbol 12-myristate 13-acetate (PMA), which stimulates PtdCho synthesis in a number of cell lines, increased [14C]PtdCho levels by only 14% in LA-N-2 cells, at a concentration (100 nM) which caused complete translocation of the calcium- and phospholipid-dependent enzyme protein kinase C to the membrane. The translocation was inhibited by prior exposure of the cells to PtdSer. Treatment with PMA for 24 h diminished protein kinase C activity by 80%, but increased the labeling of PtdCho in both untreated and PtdSer-treated cells. These data suggest that uptake of PtdSer by LA-N-2 cells alters both the phospholipid composition of the membrane and synthesis of the major membrane phospholipid PtdCho; the latter effect does not involve activation of protein kinase C.     

神经节苷脂GM_3对人白血病J6－2细胞磷脂酰胆碱代谢的影响

神经节苷脂ＧＭ３诱导人单核样白血病Ｊ６－２细胞沿单核／巨噬细胞途径分化．在ＧＭ３诱导分化同时,Ｊ６－２细胞磷脂代谢发生了显著变化．采用（（３２）Ｐ）Ｐｉ、［ＧＨ３－３Ｈ］胆碱和［ＣＨ３－３Ｈ］ＳＡＭ参入实验对ＧＭ３影响Ｊ６－２细胞ＰＣ代谢的机制进行了初步的探讨．ＧＭ３促进［（３２）Ｐ］Ｐｉ参入Ｊ６－２细胞ＰＣ;抑制［ＣＨ３－３Ｈ］胆碱参入ＰＣ及ＰＣ合成的前体磷酸胆碱及ＣＤＰ－胆碱;ＧＭ３促进［ＣＨ３－３Ｈ］ＳＡＭ参入ＰＣ,但抑制［ＣＨ３－３Ｈ］ＳＡＭ参入ＰＣ合成的前体胆碱、磷酸胆碱和ＣＤＰ－胆碱．上述结果提示,ＧＭ３抑制Ｊ６－２细胞ＰＣ合成的ＣＤＰ－胆碱途径,促进ＰＣ合成的ＰＥ甲基化途径．     

Evidence for a unique profile of phosphatidylcholine synthesis in late mitotic cells

Evidence is presented that the structural rearrangements in late mitosis are accompanied by an alteration in membrane lipid synthesis. This evidence was derived from analyzing phospholipid classes after rapid-labeling, as well as from determining the intracellular site of incorporation of choline by HeLa S3 cells as they progressed from metaphase into early interphase (G1). Compared with postmitotic cell data, the recent mitotic cell data indicate a specific two- to threefold increase in the net synthesis of phosphatidylcholine (PC) species, which appeared to contain the more saturated fatty acids. Since this was observed with glycerol, choline, and orthophosphate labelings, and not with methyl labeling, it appears that the CDP-choline plus diacylglycerol pathway rather than the phosphatidylethanolamine to PC pathway was augmented. Electron microscope autoradiography of anaphase, telophase, and early G1 cells demonstrated that the reformed nuclear envelope was the incorporation site of a significant proportion of the newly synthesized PC. This incorporation occurred by early telophase prior to chromosome decondensation. The potential significance of PC metabolism with regard to membrane rearrangements, such as nuclear envelope reformation, is discussed.     

Uptake and utilization of CDP-choline in primary brain cell cultures from fetal brain

The utilization of double-labeled CDP-choline by cultured brain cells has been studied. CDP-choline is demonstrated to be rapidly hydrolysed into CMP and choline phosphate. The fragments, or their hydrolysis products, penetrate into the cells and are utilized for lipid synthesis. At short times after the isotope administration a rapid labeling of phosphatidylcholine was detected, when cells were incubated with CDP-choline. The same was not seen when cells were incubated with labeled choline. From these observations it can be inferred that either CDP-choline can penetrate the cell membrane or that some mechanism involving CDP-choline and leading to phospholipid synthesis can work at the external surface of the plasma membranes.     

Intracellular processing of cytidylyltransferase in Krebs II cells during stimulation of phosphatidylcholine synthesis. Evidence that a plasma membrane modification promotes enzyme translocation specifically to the endoplasmic reticulum

After a 3-h incubation of Krebs II ascitic cells in the presence of phospholipase C from Clostridium welchii under nonlytic conditions, the incorporation of [3H] choline into phosphatidylcholine was increased 1.7-fold as compared to untreated cells. The total amounts of phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin were unchanged up to 3 h of incubation. The limiting step in phosphatidylcholine biosynthesis was the formation of CDP-choline catalyzed by CTP:choline-phosphate cytidylyltransferase (EC 2.7.7.15) as monitored by the decrease in phosphocholine labeling following phospholipase C treatment of cells prelabeled with [3H]choline. The specific activity of homogenate cytidylyltransferase was increased about 1.6-fold in phospholipase C-treated cells. Specific activity of the membrane fraction was increased 2-fold, whereas cytosolic specific activity decreased in phospholipase C-treated cells. The activation of cytidylyltransferase was concomitant with translocation of the enzyme from the cytosol to the membrane fraction. The latter was further fractionated using a Percoll gradient that allowed an efficient separation between endoplasmic reticulum and other subcellular membranes. In control cells, particulate cytidylyltransferase activity co-migrated with the endoplasmic reticulum and ribosome markers and not with the plasma membrane. Also, in treated cells, the stimulation of cytidylyltransferase activity occurred at the endoplasmic reticulum level and did not involve either the external cell membrane or other cellular organelles including the Golgi apparatus, lysosomes, or mitochondria. Thus, our results demonstrate that a stimulus acting on the plasma membrane promotes the translocation of the soluble form of cytidylyltransferase specifically to the endoplasmic reticulum.     

Channeling of intermediates in the CDP-choline pathway of phosphatidylcholine biosynthesis in cultured glioma cells is dependent on intracellular Ca2+

The major route of phosphatidylcholine (Ptd-choline) biosynthesis in mammalian cells is the CDP-choline pathway which involves stepwise conversion of choline to phosphocholine (P-choline), cytidine diphosphate choline (CDP-choline), and Ptd-choline. Our previous studies with electropermeabilized (EP) rat glioma (C6) cells have indicated that the intermediates of this pathway are not freely diffusible in the cell but are channeled toward synthesis of Ptd-choline (George, T.P., Morash, S.C., Cook, H.W., Byers, D.M., Palmer, F. B. St.C., and Spence, M.W. (1989) Biochim. Biophys. Acta 1004, 283-291). In this study, Ca(2+)-[ethylene-bis(oxyethylenenitrilo)]tetraacetic acid buffers were used to investigate the role of intracellular free Ca2+ levels in functional organization of this pathway in EP glioma cells. In EP cells reduction of free Ca2+ in the medium from 1.8 mM to less than 200 nM resulted in 2-3-fold stimulation of exogenous [3H]choline and [14C]P-choline incorporation into Ptd-choline whereas incorporation of exogenous CDP-[14C]choline was augmented 100-fold; there was no uptake or incorporation of labeled P-choline or CDP-choline in intact cells. In EP cells incubated at 1.8 mM Ca2+ the water-soluble products of choline metabolism (choline, P-choline, CDP-choline, and glycerophosphocholine) were retained at 37 degrees C; in contrast, in the presence of 100 nM Ca2+ there was uniform leakage of these metabolites. Experiments with hemicholinium-3, an inhibitor of choline transport, and EP cells at 100 nM Ca2+ show that linkage of choline transport and Ptd-choline biosynthesis is also dependent on Ca2+. These results suggest that channeling of intermediates in the CDP-choline pathway of Ptd-choline biosynthesis in glioma cells is mediated by intracellular Ca2+ levels that may coordinately regulate the steps involved in conversion of choline to Ptd-choline.     

Glycoprotein biosynthesis in B lymphocytes: induction of protein N-glycosylation, RNA synthesis, and DNA synthesis by phorbol ester plus ionomycin is blocked by protein kinase inhibitors

The combination of phorbol 12-myristate 13-acetate (PMA) and ionomycin produces a dramatic increase in the incorporation of [2-3H]mannose into Glc3Man9GlcNAc2-P-P-dolichol and glycoprotein, and the induction of RNA and DNA synthesis in murine splenic B lymphocytes (B cells). The kinetics of the induction processes and the concentrations of PMA and ionomycin required for the optimal response have been defined. While the levels of induction of RNA and DNA synthesis by PMA + ionomycin were similar to the mitogenic response to bacterial lipopolysaccharide, activation by PMA and the calcium ionophore resulted in a threefold higher stimulation in dolichol-linked oligosaccharide biosynthesis and protein N-glycosylation. These results indicate that all signalling mechanisms that trigger RNA and DNA synthesis may not be sufficient to produce maximal induction of the N-glycosylation apparatus. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H-7), a potent protein kinase C inhibitor, prevented the induction of protein N-glycosylation activity (IC50 = 11 microM), as well as RNA (IC50 = 18 microM) and DNA synthesis (IC50 = 12 microM), two common indices of B cell activation. N-[2-(Methylamino)ethyl]-5-isoquinolinesulfonamide (H-8) also inhibited the induction of oligosaccharide-lipid intermediate, glycoprotein, RNA, and DNA synthesis, but required higher concentrations than H-7 for 50% inhibition. N-(2-Guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), a potent inhibitor of cyclic nucleotide-dependent protein kinases, had little effect on the activation of the B cell metabolic processes. The H-7-sensitive reactions involved in the induction of RNA and DNA synthesis occurred within 4 h, but induction of lipid intermediate and glycoprotein biosynthesis remained sensitive to H-7 for 10 h after exposure to PMA and ionomycin. Direct in vitro assays in the presence of 0.6% Brij 58 reveal that a cytosolic, phospholipid-dependent protein kinase activity is translocated to a membrane site(s) after treatment with PMA and ionomycin, and the translocated protein kinase is sensitive to H-7. The relative order of potency of the protein kinase inhibitors on the metabolic processes strongly supports the hypothesis that protein kinase C, acting synergistically with Ca2+ mobilization, plays a key regulatory role in the early stages of B cell activation. The synthesis of oligosaccharide-lipid intermediates and protein N-glycosylation are also shown to be induced in B cells activated by PMA + ionomycin.     

Hexadecylphosphocholine inhibits phosphatidylcholine biosynthesis and the proliferation of HepG2 cells.

Hexadecylphosphocholine (HePC) is a synthetic lipid representative of a new group of antiproliferative agents, alkylphosphocholines (APC), which are promising candidates in anticancer therapy. Thus we have studied the action of HePC on the human hepatoblastoma cell line HepG2, which is frequently used as a model for studies into hepatic lipid metabolism. Non-toxic, micromolar concentrations of HePC exerted an antiproliferative effect on this hepatoma cell line. The incorporation into phosphatidylcholine (PC) of the exogenous precursor [methyl-14C]choline was substantially reduced by HePC. This effect was not due to any alteration in choline uptake by the cells, the degradation rate of PC or the release of PC into the culture medium. As anaccumulation of soluble choline derivatives points to CTP:phosphocholine cytidylyltransferase (CT) as the target of HePC activity we examined its effects on the different enzymes involved in the biosynthesis of PC via CDP-choline. Treatment with HePC altered neither the activity of choline kinase (CK) nor that of diacylglycerol cholinephosphotransferase (CPT), but it did inhibit CT activity in HepG2 cells. In vitro HePC also inhibited the activity of cytosolic but not membrane-bound CT. Taken together our results suggest that HePC interferes specifically with the biosynthesis of PC in HepG2 cells by depressing CT translocation to the membrane, which may well impair their proliferation.     

Ha-ras-transformation alters the metabolism of phosphatidylethanolamine and phosphatidylcholine in NIH 3T3 fibroblasts

Cultured NIH 3T3 fibroblasts were employed to investigate the changes in the phospholipid metabolism induced by Ha-ras transformation. All phospholipid fractions were reduced in ras-transformed fibroblasts except phosphatidylethanolamine (PE). The incorporation of labeled choline and ethanolamine into phosphatidylcholine (PC), PE and their corresponding metabolites were elevated in a similar manner in the transformed cells. The enhanced uptake of choline and ethanolamine correlated with the activation of choline kinase and ethanolamine kinase. Similarly, the uptake of arachidonic, oleic and palmitic acids by PC and PE was higher in ras-cells. Acyl-CoA synthetases, which esterify fatty acid before their incorporation into lysophospholipids, were also activated. However, both CTP:phosphocholine-cytidylyltransferase and CTP:phosphoethanolamine-chytidyltransferase were inhibited in the transformed cells. This fact, taken together with the observed activation of choline- and ethanolamine kinases, led to accumulation of phosphocholine and phosphoethanolamine, which have been presumed to participate in the processes of tumor development. PC biosynthesis seemed to be carried out through the CDP-choline pathway, which was stimulated in the oncogenic cells, whereas PE was more likely, a product of phosphatidylserine decarboxylation rather than the CDP-ethanolamine pathway.     

Synthesis of phosphatidylcholine by rat lung during choline deficiency

The effect of choline deficiency on the de novo pathway for phosphatidylcholine (PC) synthesis in the lung was investigated in rats fed a washed soy protein (lipotrophic) diet deficient in choline and methionine for 2-3 wk. Lungs from lipotrophic rats showed a decreased content of choline and choline-phosphate (P less than 0.05) compared with control but no change in content of cytidine 5'-diphosphocholine or PC. Isolated perfused lungs from lipotrophic rats were evaluated for choline and fatty acid utilization for PC synthesis. Lipotrophic lungs perfused with 5 microM [14C-methyl]-choline chloride showed increased incorporation into PC while there was no significant effect at saturating levels of choline (100 microM). There was increased incorporation of [1-14C]-palmitic acid into PC and diglyceride and increased incorporation of D-[U-14C]glucose into fatty acids of PC. Increased choline and glucose incorporation was not due to alteration of intracellular specific activity of these substrates. This study indicates the utilization of choline and fatty acid for PC synthesis is stimulated as a result of choline deficiency while lung CDP-choline concentration is maintained, possibly through regulation of choline phosphate cytidyl transferase activity. These mechanisms compensate for decreased choline availability to maintain the PC content of lungs.     

Stimulatory effect of n-alkanes on CTP: Cholinephosphate cytidyltransferase activity in rat liver microsomal membranes in vitro.

To assess the effect of alteration of membrane structure on the enzymic activities related to phospholipid synthesis in microsomal membrane, the effects of several organic solvents have been studied in an in vitro system, in which the cytoplasmic extract prepared from rat liver incorporated [14C]choline or [14C]CDP-choline into phosphatidycholine (lecithin). The optimum conditions for the incorporation were determined. Among several organic solvents examined, n-alkanes such as n-hexane, n-octane, and n-tetradecane stimulated the incorporation. It was shown that n-alkanes stimulated one of three enzymic steps of lecithin biosynthesis from choline; that is, the formulation of CDP-choline catalyzed by CTP: cholinephosphate cytidyltransferase [EC 2.7.7.15], an enzyme on the microsomal membrane. It was further shown that the same enzyme was also stimulated by preincubation of microsomes in the absence of substrate. It is suggested that alteration of the lipid environment of the microsomal membrane induced by n-alkanes caused activation of this enzymic step.     

Pseudomonas aeruginosa synthesizes phosphatidylcholine by use of the phosphatidylcholine synthase pathway

Phosphatidylcholine (PC) is a ubiquitous membrane lipid in eukaryotes but has been found in only a limited number of prokaryotes. Both eukaryotes and prokaryotes synthesize PC by methylating phosphatidylethanolamine (PE) by use of a phospholipid methyltransferase (Pmt). Eukaryotes can synthesize PC by the activation of choline to form choline phosphate and then CDP-choline. The CDP-choline then condenses with diacylglycerol (DAG) to form PC. In contrast, prokaryotes condense choline directly with CDP-DAG by use of the enzyme PC synthase (Pcs). PmtA was the first enzyme identified in prokaryotes that catalyzes the synthesis of PC, and Pcs in Sinorhizobium meliloti was characterized. The completed release of the Pseudomonas aeruginosa PAO1 genomic sequence contains on open reading frame predicted to encode a protein that is highly homologous (35% identity, 54% similarity) to PmtA from Rhodobacter sphaeroides. Moreover, the P. aeruginosa PAO1 genome encodes a protein with significant homology (39% amino acid identity) to Pcs of S. meliloti. Both the pcs and pmtA homologues were cloned from PAO1, and homologous sequences were found in almost all of the P. aeruginosa strains examined. Although the pathway for synthesizing PC by use of Pcs is functional in P. aeruginosa, it does not appear that this organism uses the PmtA pathway for PC synthesis. We demonstrate that the PC synthesized by P. aeruginosa PAO1 localized to both the inner and outer membranes, where it is readily accessible to its periplasmic, PC-specific phospholipase D.     

Turnover of phosphatidylcholine in Saccharomyces cerevisiae. The role of the CDP-choline pathway

The regulation of phosphatidylcholine degradation as a function of the route of phosphatidylcholine (PC) synthesis and changing environmental conditions has been investigated in the yeast Saccharomyces cerevisiae. In the wild-type strains studied, deacylation of phosphatidylcholine to glycerophosphocholine is induced when choline is supplied to the culture medium and, also, when the culture temperature is raised from 30 to 37 degrees C. In strains bearing mutations in any of the genes encoding enzymes of the CDP-choline pathway for phosphatidylcholine biosynthesis (CKI1, choline kinase; CPT1, 1, 2-diacylglycerol choline phosphotransferase; PCT1, CTP:phosphocholine cytidylyltransferase), no induction of phosphatidylcholine turnover and glycerophosphocholine production is seen in response to choline availability or elevated temperature. In contrast, the induction of phosphatidylcholine deacylation does occur in a strain bearing mutations in genes encoding enzymes of the methylation pathway for phosphatidylcholine biosynthesis (i.e. CHO2/PEM1 and OPI3/PEM2). Whereas the synthesis of PC via CDP-choline is accelerated when shifted from 30 to 37 degrees C, synthesis of PC via the methylation pathway is largely unaffected by the temperature shift. These results suggest that the deacylation of PC to GroPC requires an active CDP-choline pathway for PC biosynthesis but not an active methylation pathway. Furthermore, the data indicate that the synthesis and turnover of CDP-choline-derived PC, but not methylation pathway-derived PC, are accelerated by the stress of elevated temperature.     

Cerebellar metabolism of phosphatidylcholine and its hydrosoluble precursors during bicuculline-induced convulsive seizures

The cerebellar incorporation of labeled choline into phosphatidylcholine (PC) and its hydrosoluble choline-containing precursors has been examined during the course of bicuculline-induced convulsive seizures. The labeling of phosphocholine and of PC diminished in these conditions whereas that of cytidine-5-diphosphate choline (CDP-choline) was practically unaffected. Moreover, the cerebellar pools of phosphocholine and CDP-choline increased by 75–100% after 6 min of convulsions; these compounds were formed from lipid through the action of phospholipases or through the reverse action of choline phosphotransferase. From the data reported in this paper it should also be inferred that the cytidylyltransferase reaction was activated. It is therefore concluded that the cerebellar metabolism of PC and its precursors was affected in various ways by the bicuculline-induced convulsive seizures.     

Biosynthesis of phosphatidylcholine in bacteria

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and can be synthesized by either of two pathways, the methylation pathway or the CDP-choline pathway. Many prokaryotes lack PC, but it can be found in significant amounts in membranes of rather diverse bacteria and based on genomic data, we estimate that more than 10% of all bacteria possess PC. Enzymatic methylation of phosphatidylethanolamine via the methylation pathway was thought to be the only biosynthetic pathway to yield PC in bacteria. However, a choline-dependent pathway for PC biosynthesis has been discovered in Sinorhizobium meliloti. In this pathway, PC synthase, condenses choline directly with CDP-diacylglyceride to form PC in one step. A number of symbiotic (Rhizobium leguminosarum, Mesorhizobium loti) and pathogenic (Agrobacterium tumefaciens, Brucella melitensis, Pseudomonas aeruginosa, Borrelia burgdorferi and Legionella pneumophila) bacteria seem to possess the PC synthase pathway and we suggest that the respective eukaryotic host functions as the provider of choline for this pathway. Pathogens entering their hosts through epithelia (Streptococcus pneumoniae, Haemophilus influenzae) require phosphocholine substitutions on their cell surface components that are biosynthetically also derived from choline supplied by the host. However, the incorporation of choline in these latter cases proceeds via choline phosphate and CDP-choline as intermediates. The occurrence of two intermediates in prokaryotes usually found as intermediates in the eukaryotic CDP-choline pathway for PC biosynthesis raises the question whether some bacteria might form PC via a CDP-choline pathway.