Secretory immunoglobulins in colonic neoplasms

1. The phosphonium analogues of choline, phosphorylcholine, CDPcholine and phosphatidylcholine were synthesized chemically and characterized by 1H-NMR and 31P-NMR; in 1,2-distearoyl-DL-glycero-3-phosphorylphosphocholine, the 31P-NMR chemical shift of phosphonium relative to phosphate was--28.2 ppm. 2. A comparison was made of the rates of reaction of choline kinase, cholinephosphate cytidyltransferase, cholinephosphotransferase and phospholipase C on natural and phosphonium substrates. Enzyme reaction rates were similar for all but the cytidyltransferase, which exhibited a 3-fold preference for the normal substrate. 3. Weanling rats were maintained for 6 weeks on a diet in which choline was fully replaced by phospho[1,2-14C2]choline mixed with a trace of [Me-3H] choline. Incorporation of phosphocholine into liver lipids was detectable by 31P-NMR even in crude tissue homogenates. Choline-based phospholipids of liver, kidney, lung and brain were extracted, and phosphocholine incorporation calculated from 31P-NMR peak area ratios. The phosphatidylcholine analogues were separated by preparative thin-layer chromatography. Incorporation of phosphocholine ranged from 33% in lung phosphatidylcholine to 6% in kidney sphingomyelin. Variations in 14C/3H ratio between feed and phospholipid extracts indicated preferences for exogenous choline over phosphocholine varying from 1.3: 1 in brain to 3.2: 1 in liver. The results indicated that phosphocholine is a potentially useful 31P-NMR probe for the study of membrane lipids.     

TNF-induced modulations of phospholipid metabolism in human breast cancer cells

Tumor necrosis factor alpha (TNF) is a cytokine that is cytocidal for certain tumor cells and induces necrotic and apoptotic forms of cell death. Flow cytometry and transmission electron microscopy analysis demonstrated that in human breast cancer cells (MCF7) TNF induces cell cycle arrest in G0+G1/S, accompanied by apoptosis. 31P and 13C NMR spectroscopy was applied to study cellular metabolism of MCF7 cells during TNF-induced signal to apoptosis. Deuterated choline and 2H NMR spectroscopy were utilized to monitor the kinetics of the rate limiting reactions in phosphocholine metabolism. The NMR measurements revealed that immediately after administration of TNF, choline transport was inhibited by 52+/-6%. Later (approximately 15 h), the activity of phosphocholine:cytidine triphosphate cytidylyltransferase, a key enzyme in the biosynthesis of phosphatidylcholine, was enhanced two-fold. These two opposing changes led to a decrease in the level of phosphocholine. Throughout these changes the energetic state of the cells, determined by the level of nucleoside triphosphates and the rate of glucose metabolism via glycolysis, remained constant. The results indicate that TNF specifically modulates the kinetics of membrane-bound enzymes of the rate determining steps in phosphatidylcholine biosynthesis, possibly as part of early events involved in apoptosis.     

Hepatic betaine-homocysteine methyltransferase activity in the chicken is influenced by dietary intake of sulfur amino acids, choline and betaine

There is much interest in the metabolism of homocysteine, because elevated plasma homocysteine [hyperhomocyst(e)inemia] is an independent risk factor for the development of cardiovascular disease. Four chick assays were conducted to determine the effects of varying dietary sulfur amino acids, choline and betaine on the activity of hepatic betaine-homocysteine methyltransferase (BHMT), an enzyme likely to be important in modulating plasma homocysteine. In Experiment 1, chicks were fed a purified crystalline amino acid diet containing adequate sulfur amino acids and choline. Excess dietary methionine, or the combination of excess cystine with choline or betaine, caused a small increase (P < 0.05) in BHMT activity. In Experiment 2, use of a methionine-deficient purified diet resulted in a threefold increase (P < 0.05) in BHMT activity, and addition of choline or betaine further increased (P < 0.05) BHMT activity. In Experiment 3, use of a methionine-deficient corn-peanut meal diet increased BHMT (P < 0.05) relative to that of chicks supplemented with adequate methionine, and addition of surfeit choline to the methionine-deficient basal diet caused a further increase (P < 0.05). In Experiment 4, addition of both surfeit choline and surfeit betaine to the methionine-deficient corn-peanut meal diet caused an increase (P < 0.05) in BHMT activity relative to that observed in chicks fed the methionine-deficient basal diet. These assays show that large increases in BHMT activity can be produced under methionine-deficient conditions, especially in the presence of excess choline or betaine.     

Characterization of the T cells in aged rat bone marrow

The purpose of this study was to examine the effect of exogenous CDP-choline on choline metabolism and phosphatidylcholine biosynthesis in adult rat ventricular myocytes. Choline uptake and metabolism were examined, using [methyl3H] choline. CDP-choline in the medium produced a concentration dependent reduction in the amount of radio-label in phosphocholine and phospholipid but it did not alter choline uptake into the myocytes. CDP-choline also did not antagonize the effect of hypoxia on phosphatidylcholine synthesis; rather it accentuated the hypoxia-induced reductions in cellular phosphocholine and phosphatidylcholine biosynthesis. These results indicate that the exogenous administration of CDP-choline alters choline metabolism in the heart by reducing the formation of phosphocholine and phosphatidylcholine without altering choline uptake and suggest an effect of a CDP-choline metabolite on choline metabolism which is not effective in opposing the effect of hypoxia on phosphatidylcholine biosynthesis.     

Radioactive choline metabolism in guinea pig gallbladder. Is there measurable acetylcholine release?

Acetylcholine may be released from gallbladder intrinsic nerves in response to cholecystokinin stimulation. This study characterized metabolites of [14C]choline produced in the gallbladder and released during incubation, with or without cholecystokinin-octapeptide. Radiolabeled [14C]choline was applied to the mucosal or muscle surface of intact guinea pig gallbladders in an organ bath. After radiolabeling, gallbladders were incubated with or without the contractile agonist cholecystokinin-octapeptide. Metabolites of [14C]choline were identified in gallbladder tissue and incubation buffers using HPLC and thin-layer chromatography. The major metabolites of [14C]choline were betaine and phosphocholine. [14C]Phosphocholine was incorporated slowly into [14C]phosphatidylcholine. [14C]Choline was released into buffers during incubation. [14C]Acetylcholine constituted less than 1% of radiolabel in the gallbladder. There was no identifiable [14C]acetylcholine released in buffers. Cholecystokinin-octapeptide did not affect choline metabolism. These studies showed that choline in the gallbladder is metabolized along pathways similar to those in the liver. Gallbladders released mostly choline, rather than acetylcholine, even during hormonally induced contraction.     

Phosphorus metabolism during growth of lymphoma in mouse liver: a comparison of 31P magnetic resonance spectroscopy in vivo and in vitro

Large phosphomonoester (PME) signals are detected in the phosphorus magnetic resonance spectra (31P MRS) of many neoplastic and rapidly dividing tissues. In addition, alterations in phosphodiester (PDE) signals are sometimes seen. The present study of a murine lymphoma growing in liver showed a positive correlation between the hepatic PME/PDE ratio measured in vivo by 31P MRS at 4.7 T and the degree of lymphomatous infiltration in the liver, quantified by histology. High-resolution 31P MRS of liver extracts at 9.7 T showed that the PME peak consists largely of phosphoethanolamine (PE) and to a lesser extent of phosphocholine (PC). The concentration of both PE and PC increased positively with lymphomatous infiltration of the liver. In vivo, the PDE peak contains signals from phospholipids (mostly phosphatidylethanolamine and phosphatidylcholine) and the phospholipid breakdown products glycerophosphoethanolamine (GPE) and glycerophosphocholine (GPC). Low levels of GPE and GPC were detected in the aqueous extracts of the control and infiltrated livers; their concentrations remained unchanged as the infiltration increased. The total concentration of phospholipids measured by 31P MRS of organic extracts decreased about 3-fold as the infiltration increased to 70%. Thus, our data showed that the increased PME/PDE ratio in vivo is due to both an increase in the PME metabolites and a decrease in the PDE metabolites. We propose that this ratio can be used as a non-invasive measure of the degree of lymphomatous infiltration in vivo.     

Utilization of phosphocholine from extracellular complex polysaccharide as a source of cytoplasmic choline derivatives in Penicillium fellutanum

Penicillium fellutanum produces a phosphorylated, choline-containing extracellular polysaccharide, peptidophosphogalactomannan (pP(x)GM) [where x is the number of phosphodiester residues]). The 13C-methyl-labeled pP(x)GM ([methyl-13C]pP(x)GM) was prepared from the cultures supplemented with L-[methyl-13C]methionine and was used as a probe to monitor the fate of phosphocholine in this polymer. The addition of [methyl-13C]pP(x)GM to growing cultures in low-phosphate medium resulted in the disappearance within 5 days of [methyl-13C]phosphocholine and N,N'-dimethylphosphoethanolamine from the added [methyl-13C]pP(x)GM. Two 13C-methyl-enriched cytoplasmic solutes, choline-O-sulfate and glycine betaine, were found in mycelial extracts, suggesting that phosphocholine-containing extracellular pP(x)GM of P. fellutanum is a precursor of intracellular choline-O-sulfate and glycine betaine. The mycelia cultured in low-phosphate (2 mM) medium contained glycine betaine and 1.5-fold more choline-O-sulfate than those grown in high-phosphate (20 mM) medium. The high levels of extracellular nonspecific phosphocholine:phosphocholine hydrolase and acid phosphomonoesterase observed in the low-phosphate culture medium are likely related to the release of phosphocholine from pP(x)GM and hydrolysis of phosphocholine, respectively. These results suggest that extracellular pP(x)GM of P. fellutanum provides phosphate needed as the environment becomes depleted of this nutrient. Choline, in excess of that needed immediately, is stored in the cytoplasm in forms that can be reutilized.     

Phospholipid head-group conformations; intermolecular interactions and cholesterol effects

The predominant orientation of the phosphorylcholine polar head group in phosphatidylcholine and sphingomyelin bilayers and cholesterol perturbations of that orientation have been identified by exploiting the 31P (1H) nuclear Overhauser effect (NOE) in the 31P NMR spectra of phospholipid bilayers. In pure egg phosphatidylcholine bilayers, a NOE of 40% is observed. The magnitude of the NOE has been measured as a function of continuous-wave proton-decoupler frequency in order to identify the proton source of the NOE. In pure egg phosphatidylcholine bilayers, the maximum NOE occurs at the N-methyl proton resonance position of the choline moiety. In a modified phosphatidylcholine in which all the N-methyl protons were replaced by deuterium, the NOE arose from methylene protons next to the phosphate. In mixed systems of phosphatidylcholine and phosphatidylethanolamine, and phosphatidylcholine and diphosphatidylglycerol, both phospholipid resonances attained maximum NOE at the position of the N-methyl proton resonance of phosphatidylcholine. An analogous result was obtained with pure sphingomyelin. These results are explained by orienting the phosphorylcholine portion of the molecule parallel to the surface of the bilayer so that the positively charged N-methyl moiety is located close to the negatively charged phosphate on a neighboring phospholipid in an intermolecular interaction. Addition of cholesterol is shown to disrupt the intermolecular interaction in phosphatidylcholine bilayers.     

Interactive effects of betaine, crude protein, and net energy in finishing pigs

Two experiments were conducted to determine the effect of betaine on growth and carcass characteristics of finishing pigs. In Exp. 1, 32 gilts were fed one of two diets: 1) a corn-soybean meal basal (B) diet or 2) B + .125% betaine diet. In Exp. 2, 122 gilts were allotted to one of eight dietary treatments in a 2 x 2 x 2 factorial arrangement with two levels of betaine (0 or .125%), crude protein (adequate [ACP] or inadequate [ICP]), and net energy (NE; 0 or 6% added fat). In Exp. 1, betaine did not affect (P > .10) growth performance or carcass traits other than an increased (P < .05) dressing percentage. In Exp. 2, betaine tended to decrease ADFI during the overall experimental period (P = .11). In the late finishing period (LF), betaine increased ADG in inadequate CP low-NE diets and adequate CP high-NE diets, but decreased ADG in inadequate CP high-NE and adequate CP low-NE diets (betaine x CP x NE, P < .04). Betaine increased (P < .04) carcass length and decreased (P < .01) color score for pork quality. Other carcass measurements were unaffected (P > .10) by betaine. Betaine decreased (P < .02) serum urea N (SUN) in fed pigs during the LF period. Betaine decreased fasting SUN and albumin in pigs fed the ACP diets, but it increased fasting SUN and albumin in pigs fed the ICP diets during the LF period (betaine x CP, P = .10). Betaine increased serum total protein in the low-NE diets, but not in the high-NE diets (betaine x NE, P < .08). The serum metabolite data suggest that betaine may affect protein status of pigs, and these effects may depend on the crude protein and energy content of the diet.     

Biochemical effects of gadolinium chloride in rats liver and kidney studied by ^1H NMR metabolomics

The biochemical effects of gadolinium chloride were studied using high-resolution IH nuclear magnetic resonance (NMR) spec-troscopy to investigate the biochemical composition of tissue (liver and kidney) aqueous extracts obtained from control and gadolinium chlo-ride (GdCl3) (10 and 50 mg/kg body weight, intraperitoneal injection, i.p.) treated rats. Tissue samples were collected at 48, 96 and 168 h p.d. after exposure to GdCl3, and extracted using methanol/chloroform solvent system. 1H NMR spectra of tissue extracts were analyzed by pat-tern recognition using principal components analysis. The liver damages caused by GdCl3 were characterized by increased succinate and de-creased glycogen level and elevated lactate, alanine and betaine concentration in liver. Furthermore, the increase of creatine and lactate, and decrease of glutamate, alanine, phosphocholine, glycophosphocholine (GPC), betaine, myo-inositoi and trimethylamine N-oxide (TMAO)levels in kidney illustrated kidney disturbance induced by GdCl3.     

Effect of fasting and feeding a high-sucrose, fat-free diet on the synthesis of hepatic glycerolipids in vivo and in isolated hepatocytes

1. The synthesis of glycerolipids from a number of radioactive precursors, such as [2-3H] glycerol, [32P]phosphate, [Me-14C]choline and [1,2-14C2]ethanolamine proceeds in enzymatically isolated hepatocytes with a specificity that agrees very well with that observed in the liver in vivo. 2. The nutritional state of the rat has a profound influence on the glycerolipid metabolism of isolated hepatocytes. Fasting strongly decreased the incorporation of glycerol via sn-glycerol 3-phosphate into triacylglycerols whereas the formation of phosphatidylcholine and, particularly, phosphatidylethanolamine was much less affected by food deprivation. Refeeding a high-sucrose, fat-free diet caused a tremendous increase in the synthesis of diacylglycerols and triacylglycerols to values exceeding those found in hepatocytes from normally fed animals. The formation of phosphatidylcholine increased more slowly and the synthesis of phosphatidylethanolamine was even depressed by refeeding a high-sucrose, fat-free diet. 3. Alteration of the nutritional state resulted in similar changes in the amounts and metabolism of hepatic glycerolipids in vivo. The formation and, consequently, the amount of diacyglycerol and triacylglycerol were decreased by fasting and increased to values above normal in rats refed a high-sucrose, fat-free diet. The formation and the amounts of phospholipids in the liver decreased slightly by fasting which was mainly due to a decrease in phosphatidylcholine. Refeeding caused a significant increase in the formation and amount of phosphatidylcholine. The amount of phosphatidylethanolamine was even further diminished by feeding a high-sucrose, fat-free diet to 48-h-fasted rats. 4. These results show that the alterations, induced in the in vivo metabolism of hepatic glycerolipids, by changes of the dietary state, are also reflected in the isolated hepatocytes. This finding strengthens the potential significance of isolated hepatocytes in studies on the regulation of hepatic lipid metabolism.     

Effect of betaine on the growth performance of chicks inoculated with mixed cultures of avian Eimeria species and on invasion and development of Eimeria tenella and Eimeria acervulina in vitro and in vivo

At 7 d postinoculation (DPI) with a mixed culture of avian Eimeria species, 21-d-old chicks maintained in batteries and floor pens on a diet containing 0.15% (3 lb/ton) betaine plus 66 ppm (60 g/ton) salinomycin were significantly heavier and had significantly lower feed conversion ratios and mortality than chicks fed diets containing 0.15% betaine or 66 ppm salinomycin alone, or the control diet. At 31 DPI, when the chicks were 45 d old, the differences between the diet groups were not as great as at 7 DPI. In vitro, except at high concentrations, betaine was nontoxic to sporozoites of Eimeria tenella or Eimeria acervulina and had little effect on their invasion and development in cultured cells. In vivo, invasion by E. tenella and E. acervulina sporozoites was significantly reduced in all chicks fed diets containing betaine or salinomycin compared with that in control chicks. There was a significant interaction between betaine and salinomycin that impacted on invasion by both species. Overall development of E. tenella did not appear to be adversely affected by addition of betaine to diets containing salinomycin. Conversely, development of E. acervulina was reduced in chicks fed diets containing 0.075% (1.5 lb/ton) betaine plus 66 ppm salinomycin as compared with that in chicks fed salinomycin alone.     

Administration of phosphatidylcholine increases brain acetylcholine concentration and improves memory in mice with dementia

Studies on the effect of phosphatidylcholine administration on memory are limited. We administered egg phosphatidylcholine to mice with dementia and to normal mice and compared the differences in memory and serum choline concentration, and choline and acetylcholine concentrations and choline acetyltransferase activities of three forebrain regions (cortex, hippocampus and the remaining forebrain). Mice with dementia were produced by mating sibling mice who had impaired memory for > 20 generations. These mice had poor memory and low brain acetylcholine concentration. We administered 100 mg of egg phosphatidylcholine (phosphatidylcholine group) or water (control group) by gavage to each mouse daily for about 45 d. Control mice with dementia had poorer memory in passive avoidance performance and lower brain choline (cortex and hippocampus) and acetylcholine (hippocampus and forebrain excluding cortex and hippocampus) concentrations and lower cortex choline acetyltransferase activity than the control normal mice (P < 0.05). The administration of phosphatidylcholine to mice with dementia improved memory and generally increased brain choline and acetylcholine concentrations to or above the levels of the control normal mice. In normal mice, phosphatidylcholine treatment did not affect memory or acetylcholine concentrations in spite of the great increase in choline concentrations in the three brain regions. Serum choline concentration in mice treated with phosphatidylcholine increased to a similar level in both strains of mice, indicating that the absorption of phosphatidylcholine was not impaired in mice with dementia. The results suggest that administration of egg phosphatidylcholine to mice with dementia increases brain acetylcholine concentration and improves memory.     

Experimental allergic encephalomyelitis raises betaine levels in the spinal cord of strain 13 guinea-pigs

Chronic relapsing experimental allergic encephalomyelitis, an animal model of multiple sclerosis, was induced in Strain 13 guinea-pigs by subcutaneous injection of spinal cord homogenate and Freund's incomplete adjuvant supplemented with Mycobacterium tuberculosis. High resolution 1H NMR spectra of CNS tissue extracts indicated that the levels of choline metabolites, particularly betaine, were elevated in the spinal cord tissue, the principal site of lesion formation in this guinea-pig strain. The spectra also show that N-acetylated compounds are slightly depleted in the disease. The results are discussed in relation to the biochemical interpretation of NMR spectra obtained in vivo from patients with multiple sclerosis.     

Transverse redistribution of phospholipids during human platelet activation: evidence for a vectorial outflux specific to aminophospholipids

The redistribution kinetics of phospholipids during human platelet activation by calcium ionophore were investigated to determine the specificity of the observed phospholipid outflux [Bassé et al. (1993) Biochemistry 32, 2337]. (1) Two double-labeling experiments were performed with a combination of equal amounts of spin- and fluorescently-labeled phosphatidylserine and phosphatidylcholine. During A23187-induced activation, 50% of the internal phosphatidylserine analogs were rapidly (t1/2 < 1 min) reexposed on the platelet surface while no reciprocal influx of the external phosphatidylcholine analogs was observed. (2) Treatment with chlorpromazine allowed the internalization of 20% of external spin-labeled sphingomyelin or spin-labeled phosphatidylcholine, without either inducing platelet activation or interfering with aminophospholipid translocase activity or with A23187-induced activation (dense granule secretion and vesicle shedding). During A23187-induced activation, none of the previously internalized choline head phospholipids were exposed externally, while spin-labeled phosphatidylserine outward movements were similar irrespective of whether platelets were pretreated or not pretreated with chlorpromazine. Our results demonstrated that during strong platelet activation (1) the PL excess in the internal leaflet, due to the probe addition, is not responsible for their outflux; (2) the rapid aminophospholipid outflux is definitely a vectorial outflux not counterbalanced by a rapid reciprocal influx of choline head phospholipids (i.e., not scrambling); and (3) the vectorial outflux is specific for aminophospholipids since previously internalized sphingomyelin and phosphatidylcholine did not move outward. This suggests that the specific vectorial outflux of aminophospholipids could be catalyzed by a "reverse aminophospholipid translocase" activity.     

Health indices as predictors of cognition among older African Americans: MacArthur studies of successful aging

The purpose of this study was to visualize muscarinic receptors and their distribution on cardiomyocytes and to examine the effects of muscarinic cholinergic receptor (mACh-R) stimulation with carbachol on phosphatidylcholine hydrolysis. Cardiomyocytes were prepared as primary culture from 7-day-old chick embryo hearts. Cardiomyocytes, grown on cover slips, were labelled with BODIPY PZ, a fluorescent analog of the muscarinic receptor antagonist pirenzepine, and examined with a laser scanning confocal microscope, mACh-R clusters were visualized and were fairly homogeneous in size with diameters ranging from 0.5 to 1.0 micron. The number of receptor clusters per cell was 83.5 +/- 6.8 (mean +/- SEM) and clusters were found at the periphery of the cell. Cardiomyocytes, grown as a monolayer in dishes, were treated with the 10(-4) M carbachol, a mACh-R agonist, and the effects on phosphatidylcholine hydrolysis were ascertained in cells preincubated with [methyl-3H]choline for 18 h. Cells were washed, lysed, and subjected to thin-layer chromatography to separate [3H]choline in various metabolites of phosphatidylcholine. Carbachol significantly (p < 0.05) increased intracellular free choline and decreased cellular phospholipid consistent with phosphatidylcholine hydrolysis. Carbachol increased the amount of [3H]choline that effluxed out of the cardiomyocyte into the medium. Carbachol-induced choline efflux was not prevented by pretreatment with n-butanol, a phospholipase D inhibitor, suggesting that other lipases such as phospholipase C are the major enzyme involved in phosphatidylcholine hydrolysis. Pertussis toxin prevented carbachol-induced choline efflux and the changes in intracellular free choline and phospholipid. An action of carbachol through G proteins was supported by the ability of pertussis toxin to antagonize the carbachol-induced reduction in cAMP generation from isoproterenol. In summary, mACh-Rs, visualized in living cardiomyocytes, were peripheral to the nucleus. Phosphatidylcholine hydrolysis induced by mACh-R stimulation may be a signal transduction pathway for mACh-R in the cardiomyocyte, operating through inhibitory G proteins sensitive to pertussis toxin.     

Biochemical and evolutionary significance of phospholipid methylation

All nucleated mammalian cells synthesize phosphatidylcholine from choline via the CDP-choline pathway. Hepatocytes have a second pathway for the synthesis of phosphatidylcholine, a stepwise methylation of phosphatidylethanolamine, catalyzed by phosphatidylethanolamine N-methyltransferase and encoded by the Pempt gene. We report that when Pempt-deficient mice were fed a choline-deficient diet for 3 days, severe liver pathology occurred apparently due to a lack of phosphatidylcholine biosynthesis. The hepatic concentration of phosphatidylcholine decreased by 50% compared with wild type mice on the diet. The levels of plasma triacylglycerols and cholesterol were decreased by greater than 90% in the Pempt-deficient mice. We suggest that the Pempt gene has been maintained during evolution to provide phosphatidylcholine when dietary choline is insufficient, as might occur during starvation or pregnancy.     

Dietary choline supplementation in pregnant rats increases hippocampal phospholipase D activity of the offspring

Supplementation with choline during pregnancy in rats causes a long-lasting improvement of visuospatial memory of the offspring. The biochemical mechanism of this effect may be related to the function of choline as a precursor of phosphatidylcholine (PC), the substrate of a receptor-stimulated enzyme, phospholipase D (PLD). PLD activation initiates the sequential formation of two intracellular messengers, phosphatidic acid and 1,2-sn-diacylglycerol. We hypothesized that prenatal choline status may cause long-term modulation of PLD-catalyzed PC hydrolysis in the hippocampus, a brain region implicated in visuospatial memory functions. PLD activity was determined in hippocampal slices prelabeled with [3H]glycerol or [3H]oleic acid by measuring the PLD-catalyzed formation of [3H]phosphatidylpropanol in the presence of 1-propanol. Slices were obtained from male pups born to mothers consuming a control diet, a choline-supplemented diet, or a choline-free diet from days 11 to 17 of pregnancy. The radiolabeling of phospholipid classes was unaffected by the treatments. Prenatal choline supplementation significantly increased basal PLD activity in [3H]glycerol-labeled slices [by 46% of controls on postnatal day (P) 7 and by 36% on P21], and [3H]oleate-labeled slices (by 91% on P7), as well as glutamate-stimulated PLD activity in [3H]oleate-labeled slices (by 60% on P7). Prenatal choline deficiency failed to alter PLD activity. The actions of choline apparently required intact cells because in vitro assays of PLD activity in hippocampal homogenates, using fluorescent NBD-PC as substrate, revealed no differences between groups. The results show that prenatal choline supplementation up-regulates basal and receptor-stimulated PLD activity in the hippocampus during postnatal development.     

Competitive inhibition of choline phosphotransferase by geranylgeraniol and farnesol inhibits phosphatidylcholine synthesis and induces apoptosis in human lung adenocarcinoma A549 cells

We have previously shown that, among various isoprenoids, farnesol and geranylgeraniol specifically induced actin fiber disorganization, growth inhibition, and apoptosis in human lung adenocarcinoma A549 cells (Miquel, K., Pradines, A., and Favre, G. (1996) Biochem. Biophys. Res. Commun. 225, 869-876). Here we demonstrate that isoprenoid-induced apoptosis was preceded by an arrest in G0/G1 phase. The isoprenoid effects were independent of protein prenylation and of mitogen-activated protein kinase activity. Moreover, geranylgeraniol and farnesol induced a rapid inhibition of phosphatidylcholine biosynthesis at the last step of the CDP-choline pathway controlled by choline phosphotransferase and not at the level of CTP:phosphocholine cytidylyltransferase, the key enzyme of the pathway. Inhibition of choline phosphotransferase was confirmed by in vitro assays on microsomal fractions, which clearly showed that the isoprenoids acted by competitive inhibition with the diacylglycerol binding. Exogenous phosphatidylcholine addition prevented all the biological effects of the isoprenoids, including actin fiber disorganization and apoptosis, suggesting that inhibition of phosphatidylcholine biosynthesis might be the primary event of the isoprenoid action. These data demonstrate the molecular mechanism of geranylgeraniol and farnesol effects and suggest that the mevalonate pathway, leading notably to prenylated proteins, might be linked to the control of cell proliferation through the regulation of phosphatidylcholine biosynthesis.     

The influence of medium formulation on phosphomonoester and UDP-hexose levels in cultured human colon tumor cells as observed by 31P NMR spectroscopy

High-resolution 31P NMR spectroscopy at 11.7 T was used to examine the influence of medium formulation (medium and serum type, and concentrations of glucose and inositol) on the cellular phosphate metabolism of CX-1 cells, a human colon cancer cell line derived from HT-29 cells. Striking differences in the 31P spectra of harvested CX-1 cells were observed. The largest variation was seen in the phosphocholine and UDP-hexose levels (up to seven-fold changes), with smaller differences in the levels of other phosphate metabolites. The major UDP-hexose species were found to be UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine (ca 2:1 ratio), which have been proposed in the literature to be markers of cell differentiation status. Medium-induced alterations in metabolite levels were much greater than the normal variations seen in CX-1 control samples grown under identical conditions. They even exceeded the characteristic differences observed between different human tumor cell lines grown under one set of culture conditions. The remarkable sensitivity of CX-1 cellular phosphate metabolism to the culture environment has implications for the comparison of in vitro vs in vivo spectra, and for the interpretation of effects due to growth and therapy.