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D E Vance

Publications and source records attributed to D E Vance.

At least 55 records · Page 3Linked to original sources
PubMed1996

Expression of phosphatidylethanolamine N-methyltransferase-2 is markedly enhanced in long term choline-deficient rats.

When rats are fed a choline-deficient (CD) diet, acute fatty liver develops along with other biochemical changes. However, when choline deficiency is prolonged, the growth rate of CD rats is similar to that of control rats fed a choline-supplemented diet. Furthermore, CD rats maintain their levels of choline-containing lipids, such as phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin. The mechanism for this compensation in CD rats was investigated. We screened the major tissues for the activities of two important enzymes involved in the biosynthesis of phosphatidylcholine, CTP:phosphocholine cytidylyltransferase (CT) and phosphatidylethanolamine N-methyltransferase (PEMT). Only the livers of CD rats had higher specific enzyme activities of PEMT and CT than control animals. The amount of PEMT2, one of two PEMTs in liver, increased 5-fold in CD rats after 6 weeks on the CD diet. A similar increase in the level of PEMT2 mRNA suggested that this activation was due to enhanced expression of the PEMT2 gene in CD livers. The labeling of phosphatidylcholine in isolated hepatocytes from CD rats was consistent with the conversion of PE to PC being increased as a result of a higher expression of liver PEMT. We conclude that activation of PE methylation at the level of gene expression may be the mechanism by which CD rats compensate for the lack of dietary choline.

Animals↗
PubMed1996

Phosphatidylethanolamine N-methyltransferase: unexpected findings from curiosity-driven research.

In the process of investigating basic questions about the function of the enzyme phosphatidyl-ethanolamine N-methyltransferase (PEMT), we have made several unexpected findings. PEMT catalyzes the conversion of phosphatidylethanolamine to phosphatidylcholine in hepatocytes. A novel isoform, PEMT2, has been cloned, expressed and localized to a mitochondria-associated membrane in rat liver. Expression of PEMT2 in cultured rat hepatoma cells decreased the rate of cell division. Mechanistic studies suggest that the slower growth of transfected hepatoma cells is due to down regulation of CTP:phosphocholine cytidylyltransferase and the CDP-choline pathway for phosphatidylcholine biosynthesis. A role for PEMT2 in the regulation of hepatocyte cell division is also indicated by PEMT2 down-regulation in regenerating rat liver. Another unexpected finding was the discovery that PEMT2 is a liver specific tumor suppressor. Also surprising was the finding that expression of PEMT2 does not rescue mutant Chinese hamster ovary cells that have a temperature sensitive defect in the CDP-choline pathway even though the levels of phosphatidylcholine are returned to normal. Thus, curiosity-driven research has resulted in unexpected findings about PEMT and its function in liver.

Animals↗
PubMed1996

CTP: phosphocholine cytidylyltransferase is both a nuclear and cytoplasmic protein in primary hepatocytes.

CTP:phosphocholine cytidylyltransferase (CT) has recently been reported to be a predominantly intranuclear enzyme in several cell lines (Wang et al., J. Biol. Chem. 268, 5899-5904 (1993)). This contrasts with previous reports that CT was a cytosolic protein that translocated to the endoplasmic reticulum upon activation. The aim of the present study was to compare the localization of CT in CHO cells and in primary rat hepatocytes. Indirect immunofluorescence of CHO cells revealed a largely nuclear localization of the CT. On the other hand, immunogold electron microscopy and biochemical studies showed a similar density of distribution of CT between the nucleus and cytoplasm. In primary rat hepatocytes immunofluorescence studies indicated that CT was largely cytoplasmic. Studies by immunogold electron microscopy of rat hepatocytes demonstrated that the enzyme was homogeneously distributed throughout all cytoplasmic regions and the nucleoplasm. This result was confirmed by biochemical studies using digitonin and streptolysin O, which permeabilizes the plasma membrane of cells. Enucleation studies indicated that in CHO cells 76% of the CT activity was in the nuclear fraction, whereas in hepatocytes only 32% was recovered in this fraction. The data indicate that CT is found both in nuclear and cytoplasmic fractions of primary hepatocytes and is not predominantly a nuclear enzyme.

Animals↗
PubMed1996

Characterization of the murine phosphatidylethanolamine N-methyltransferase-2 gene.

Phosphatidylethanolamine N-methyltransferase (PEMT) catalyzes the conversion of phosphatidylethanolamine to phosphatidylcholine in the mammalian liver via three sequential methylations. In the present studies, we cloned and characterized the murine gene for PEMT2, the isoform of the enzyme that localized to the mitochondria-associated membrane. The structure of the gene was determined by analysis of two lambda and three P1 genomic clones, and compared to the known rat PEMT2 cDNA sequence. Southern blotting of mouse genomic DNA indicated that PEMT2 is a single-copy gene. The gene spans at least 35 kb, with seven exons and six introns. Two transcription start sites, 139 and 148 base pairs upstream of the translation start site, were detected by primer extension and reverse transcriptase-polymerase chain reaction. These experiments indicated that the PEMT2 gene is transcribed from a single promoter. Finally, the PEMT2 gene was localized to mouse chromosome 11 by interspecific backcrossing. These experiments represent the first cloning and characterization of a full-length mammalian gene involved in phospholipid biosynthesis.

Animals↗
PubMed1995

Expression of phosphatidylethanolamine N-methyltransferase-2 in McArdle-RH7777 hepatoma cells inhibits the CDP-choline pathway for phosphatidylcholine biosynthesis via decreased gene expression of CTP:phosphocholine cytidylyltransferase.

Phosphatidylethanolamine N-methyltransferase-2 (PEMT2) of rat liver was expressed in McArdle-RH7777 rat hepatoma cells, which lack endogenous PEMT activity. Expression of the enzyme was confirmed by assay of PEMT activity and immunoblotting. There was no change in the amount of phosphatidylcholine in the transfected cells [Cu, Houweling and Vance (1994) J. Biol. Chem. 269, 24531-24533], even though the expression of PEMT2 caused an increased incorporation of [methyl-3H]methionine and [3H]ethanolamine into phosphatidylcholine. In contrast, [3H]serine incorporation into phosphatidylcholine was only marginally enhanced by PEMT2 expression. Incorporation of [methyl-3H]choline into phosphatidylcholine was decreased by greater than 60%, suggesting that the CDP-choline pathway was inhibited as a result of PEMT2 expression. CTP:phosphocholine cytidylyltransferase (CT) activities in transfected cell lines were decreased in proportion to the level of expression of PEMT2. Immunoblot analyses showed a decrease in CT mass as a function of PEMT2 expression. In contrast, there was no change in the mass of protein disulphide-isomerase or the relative amounts of most proteins expressed in the PEMT2-transfected, compared with control, cells. Similarly, the expression of CT mRNA was decreased in PEMT2-expressing cells, whereas the mRNAs for protein disulphide-isomerase and actin were unchanged. When cell growth was slowed by incubating McArdle-RH7777 cells at 25 degrees C, compared with 37 degrees C, there was no difference in the specific activity of the CT. These results argue that PEMT2 expression down-regulates the CDP-choline pathway by decreasing the expression of the gene for the CT. The decreased activity of the CDP-choline pathway might contribute to the slower rate of cell division in PEMT2-transfected hepatoma cells.

Animals↗
PubMed1995

Alkylphosphocholines inhibit choline uptake and phosphatidylcholine biosynthesis in rat sympathetic neurons and impair axonal extension.

At least 50% of the major axonal membrane lipid, phosphatidylcholine, of rat sympathetic neurons is synthesized in situ in axons [Posse de Chaves, Vance, Campenot and Vance (1995) J. Cell Biol. 128, 913-918]. In the same study we reported that, in a choline-deficient model for neuron growth, phosphatidylcholine synthesis in cell bodies is neither necessary nor sufficient for growth of distal axons. Rather, the local synthesis of phosphatidylcholine in distal axons is required for normal axon growth. We have now used three alkylphosphocholines (hexadecylphosphocholine, dodecylphosphocholine and octadecylphosphocholine) as inhibitors of PtdCho biosynthesis in a compartmented model for culture of rat sympathetic neurons. The experiments reveal that alkylphosphocholines decrease the uptake of choline into these neurons and inhibit PtdCho synthesis, but not via an effect on the activity of the enzyme CTP: phosphocholine cytidylyltransferase. We also show that when the distal axons, but not the cell bodies, are exposed to alkylphosphocholines, axonal elongation is inhibited, which is consistent with the hypothesis that phosphatidylcholine synthesis in axons, but not in cell bodies, is required for axonal elongation. The inhibitory effect of alkylphosphocholines on axon growth is most likely not mediated via a decrease in the activity of protein kinase C, since when this enzyme activity is down-regulated by treatment of the cells with phorbol ester, the alkylphosphocholines retain their ability to inhibit axonal growth.

Animals↗
PubMed1995

Reciprocal regulation of choline acetyltransferase and choline kinase in sympathetic neurons during cholinergic differentiation.

The regulation of the synthesis of acetylcholine and phosphatidylcholine in rat sympathetic neurons was examined in the context of cholinergic differentiation. We demonstrate that the activities of choline acetyltransferase (ChAT) and choline kinase (CK) are inversely affected by treatment of sympathetic neurons with retinoic acid, utilized as an agent that induces cholinergic differentiation. Whereas ChAT specific activity increased 2- to 4-fold after 12 days of treatment with 5 microM retinoic acid, CK specific activity decreased by 25-30%. These changes in enzyme activities were essentially reflected in the incorporation of [methyl-3H]choline into ACh and the metabolites of the CDP-choline pathway for phosphatidylcholine synthesis. When sympathetic neurons were treated under high potassium conditions (50 mM) for 12 days, the specific activity of CK increased 1.3-fold whereas the activity of ChAT decreased by up to 90%. Furthermore, experiments in which the incorporation of [methyl-3H]choline into ACh and the metabolites of the CDP-choline pathway was measured in the absence of Na+ or in the presence of hemicholinium-3 (HC-3), demonstrate that CK has access to the same pool of choline utilized by ChAT. These results provide evidence that the activities of ChAT and CK may be inversely regulated during the process of cholinergic differentiation.

Acetylcholine↗
PubMed1995

Nascent VLDL phospholipid composition is altered when phosphatidylcholine biosynthesis is inhibited: evidence for a novel mechanism that regulates VLDL secretion.

Previous work has shown that inhibition of phosphatidylcholine biosynthesis inhibits very low density lipoprotein (VLDL) secretion by causing a decrease in the number of particles in the Golgi but not in the endoplasmic reticulum of rat liver (Verkade et al. (1993) J. Biol. Chem. 268, 24,990-24,996). One explanation for this observation was that VLDL from choline deficient livers was degraded in a post-endoplasmic reticulum compartment. This hypothesis was supported by experiments in which choline deficient (CD) or choline supplemented (CS) rat hepatocytes were incubated +/- Brefeldin A. In the presence of Brefeldin A, VLDL secretion was blocked, but no difference was observed in the degradation of apolipoprotein B (apoB) within the CD or CS cells. If increased catabolism of apoB were occurring in the endoplasmic reticulum of CD hepatocytes, enhanced degradation of apoB in CD cells might have been expected. Inhibition of phosphatidylcholine biosynthesis also caused decreases in the phosphatidylcholine content of membranes of the secretory pathway. The lipids of nascent VLDLs from the lumina of endoplasmic reticulum and Golgi prepared from CD rat liver were relatively enriched in phosphatidylethanolamine and depleted of phosphatidylcholine when compared to samples from CS liver. The changes in nascent VLDL phospholipid composition mimicked that of the organelle membranes from which the VLDLs were isolated. Possibly the phospholipid composition of the organelles is a factor in determining the final phospholipid composition of VLDLs. One hypothesis is that when phosphatidylcholine biosynthesis is impaired, nascent VLDL is assembled incorrectly and degraded by a quality control protease in a post-endoplasmic reticulum compartment.

Animals↗
PubMed1995

Expression of phosphatidylethanolamine N-methyltransferase-2 cannot compensate for an impaired CDP-choline pathway in mutant Chinese hamster ovary cells.

Phosphatidylcholine is a product of the CDP-choline pathway and the pathway that methylates phosphatidylethanolamine. We have asked the question: are the two pathways functionally interchangeable? We addressed his question by investigating the expression of phosphatidylethanolamine N-methyltransferase-2 (PEMT2) of rat liver in mutant Chinese hamster ovary cells (MT-58) (Esko, J. D., Wermuth, M.M., and Raetz, C. R. H. (1981) J. Biol. Chem. 256, 7388-7393) defective in the CDP-choline pathway for phosphatidylcholine biosynthesis. Cell lines stably expressing different amounts of PEMT2 activity (up to 700 pmol/min.mg protein) were isolated. A positive correlation between the amount of PEMT2 activity expressed and the incorporation of [3H]methionine into phosphatidylcholine at both the permissive and restrictive temperatures showed that PEMT2 was functional in the Chinese hamster ovary MT-58 cells. In contrast to mutant cell lines stably expressing transfected CTP:phosphocholine cytidylyltransferase, the cell lines stably expressing PEMT2 did not survive at the restrictive temperature. Determination of the phosphatidylcholine mass in wild type cells, mutant MT-58 cells, and cells with the highest level of PEMT2 expression showed that PEMT2 was functional and synthesized the same amount of phosphatidylcholine as did wild type cells at the restrictive temperature. Indirect immunofluorescence studies showed that localization of the over-expressed cytidylyltransferase in MT-58 cells was largely nuclear, whereas PEMT2 was predominantly located outside the nucleus. Our data show that methylation of phosphatidylethanolamine to phosphatidylcholine cannot substitute for the CDP-choline pathway.

Animals↗
PubMed1995

Sphingolipid biosynthesis de novo by rat hepatocytes in culture. Ceramide and sphingomyelin are associated with, but not required for, very low density lipoprotein secretion.

Sphingolipids are constituents of liver and lipoproteins, but relatively little is known about their synthesis and secretion. Incubation of rat hepatocytes with [14C]- or [3H]serine labeled the long-chain base backbones of mainly ceramide and sphingomyelin. Most of the labeled sphingolipids were associated with the cells; however, 1-5% (the majority of which was ceramide) was released into the medium as part of very low density lipoproteins (VLDL). Since this is the first report that lipoproteins contain ceramide, lipoproteins were isolated from rat plasma, and the ceramide contents were (per mg of protein): 6.5 nmol for VLDL (d < 1.018), 0.6 nmol for low density lipoproteins (1.018 < d < 1.063), 0.2 nmol for high density lipoproteins (1.063 < d < 1.18), and 0.1 nmol for the albumin fraction; the lipoproteins also contained 0.1-0.4 nmol of free sphingosine/mg of protein. A number of factors affected the secretion of radiolabeled sphingolipids: 1) addition of palmitic acid, but not stearic or oleic acid, enhanced secretion due to an increase in long-chain base synthesis de novo. 2) Choline deficiency caused a 42% reduction in the secretion of radiolabeled sphingomyelin, but this was due to an effect on VLDL secretion rather than a decrease in sphingolipid synthesis. Removal of choline was examined because previous studies (Yao, Z. M., and Vance, D. E. (1988) J. Biol. Chem. 263, 2998-3004) have shown that choline deficiencies depress phosphatidylcholine synthesis and lipoprotein secretion. 3) Incubation of the cells with fumonisin B1, a mycotoxin inhibitor of sphinganine (sphingosine) N-acyltransferase, reduced overall sphingolipid synthesis and secretion by 90%, but had no effect on the secretion of apoB, phosphatidylcholine, or cholesterol. All together, these findings demonstrate that rat hepatocytes synthesize ceramide and sphingomyelin de novo and incorporate them into both cellular membranes and secreted VLDL, but normal sphingolipid synthesis is not required for lipoprotein secretion.

Animals↗
PubMed1995

Axonal synthesis of phosphatidylcholine is required for normal axonal growth in rat sympathetic neurons.

The goal of this study was to assess the relative importance of the axonal synthesis of phosphatidylcholine for neurite growth using rat sympathetic neurons maintained in compartmented culture dishes. In a double-labeling experiment [14C]choline was added to compartments that contained only distal axons and [3H]choline was added to compartments that contained cell bodies and proximal axons. The specific radioactivity of labeled choline was equalized in all compartments. The results show that approximately 50% of phosphatidylcholine in distal axons is locally synthesized by axons. The requirement of axonal phosphatidylcholine synthesis for neurite growth was investigated. The neurons were supplied with medium lacking choline, an essential substrate for phosphatidylcholine synthesis. In the cells grown in choline-deficient medium for 5 d, the incorporation of [3H]palmitate into phosphatidylcholine was reduced by 54% compared to that in cells cultured in choline-containing medium. When phosphatidylcholine synthesis was reduced in this manner in distal axons alone, growth of distal neurites was inhibited by approximately 50%. In contrast, when phosphatidylcholine synthesis was inhibited only in the compartment containing cell bodies with proximal axons, growth of distal neurites continued normally. These experiments imply that the synthesis of phosphatidylcholine in cell bodies is neither necessary nor sufficient for growth of distal neurites. Rather, the local synthesis of phosphatidylcholine in distal axons is required for normal growth.

Animals↗
PubMed1995

Belief in lunar effects on human behavior.

Questionnaires sent to 325 people indicated that 140 people (43%) held the personal belief that lunar phenomena alter individual behavior. Specifically, mental health professionals (social workers, master's clinical psychologists, nurses' aides, LPNs) held this belief more strongly than other occupational groups.

Adolescent↗
PubMed1994

Suppression of rat hepatoma cell growth by expression of phosphatidylethanolamine N-methyltransferase-2.

The expression of rat liver phosphatidylethanolamine N-methyltransferase-2 (PEMT2) in McA-RH7777 rat hepatoma cells resulted in the unexpected inhibition of cell growth. There was a strict correlation (r = 0.973) between the level of expression of the enzyme activity and the generation time for hepatoma cell division. Expression of other foreign proteins via the same vector did not inhibit McA-RH7777 cell growth; thus, retardation of cell division was specific for the methyltransferase. Addition of 1 microM 3-deazaadenosine, which causes inhibition of phosphatidylethanolamine methylation, reversed the PEMT2-mediated inhibition of cell division. Transfection of a line of Chinese hamster ovary cells with PEMT2 had no effect on the division of these cells. Induction of hepatic tumors in rats with N-nitrosodiethylamine coincided with a striking decrease in methyltransferase activity and immunoreactive protein in the tumor nodules. Thus, data from studies in cell culture and intact rats suggest a regulatory role for PEMT2 in hepatocyte cell growth and possibly in the development of liver cancer.

Animals↗
PubMed1994

Dephosphorylation of CTP-phosphocholine cytidylyltransferase is not required for binding to membranes.

The sequence of the reversible phosphorylation and activation of CTP:phosphocholine cytidylyltransferase was investigated. Treatment of primary rat hepatocytes with oleic acid or phospholipase C caused a significant increase in the activity and amount of particulate cytidylyltransferase which correlated with decreased cytidylyltransferase activity and protein in the cytosol. The increase in membrane-associated cytidylyltransferase is accompanied by a decrease in the phosphorylation of the enzyme. Reversal of membrane association resulted in an increased amount of phosphorylated cytidylyltransferase in the cytosol. We wished to determine if dephosphorylation of the enzyme were a prerequisite for its translocation from the cytosol to the membranes. In vitro studies with membranes from oleic acid- or phospholipase C-treated cells showed that phosphorylated cytosolic cytidylyltransferase associated with these membranes with negligible dephosphorylation. Incubation of hepatocytes with oleic acid for different periods of time demonstrated that cytidylyltransferase associated with membranes in an active, phosphorylated form and was subsequently dephosphorylated. This result was supported by comparison of phosphopeptide maps of 32P-labeled cytidylyltransferase obtained from cytosolic, as well as membrane fractions of control, oleic acid-treated, or phospholipase C-treated cells. These studies revealed dephosphorylation on some sites and phosphorylation on other sites. Our data strengthen the hypothesis that a change in the lipid composition of membranes can mediate the initial binding of cytidylyltransferase to the membrane and that subsequently the enzyme becomes dephosphorylated.

Animals↗
PubMed1994

Evidence that the major membrane lipids, except cholesterol, are made in axons of cultured rat sympathetic neurons.

Membrane lipids and proteins required for axonal growth and regeneration are generally believed to be synthesized in the cell bodies of neurons and transported into the axons. However, we have demonstrated recently that, in cultured rat sympathetic neurons, axons themselves have the capacity to synthesize phosphatidylcholine, sphingomyelin, and phosphatidylethanolamine. In these experiments, we employed a compartment model of neuron culture in which pure axons grow in a fluid environment separate from that containing the cell bodies. In the present study, we again used compartmented cultures to confirm and extend the previous results. We have shown that three enzymes of phosphatidylcholine biosynthesis via the CDP-choline pathway are present in axons. We have also shown that the rate-limiting step in the biosynthesis of phosphatidylcholine by this route in neurons, and locally in axons, is catalyzed by the enzyme CTP:phosphocholine cytidylytransferase. The biosynthesis of other membrane lipids, such as phosphatidylserine, phosphatidylethanolamine derived by decarboxylation of phosphatidylserine, phosphatidylinositol, and fatty acids, also occurs in axons. However, the methylation pathway for the conversion of phosphatidylethanolamine into phosphatidylcholine appears to be a quantitatively insignificant route for phosphatidylcholine synthesis in neurons. Moreover, our data provided no evidence for the biosynthesis of another important membrane lipid, cholesterol, in axons.

Acetates↗
PubMed1994

Requirement of phosphatidylcholine for normal progression through the cell cycle in C3H/10T1/2 fibroblasts.

We have investigated the possible requirement of phosphatidylcholine for normal progression through the cell cycle of C3H/10T1/2 fibroblasts. Incubation of the cells in a medium with 0.5% serum synchronized the cells in the G0 stage of the cell cycle. Supplementation of the cells with 10% dialyzed and delipidated serum +/- choline resulted in normal cell division and growth for cells with choline, whereas cell division was markedly impaired in the absence of choline. Flow cytofluorometry analysis indicated that after 4 days in the absence of choline, 85% of the fibroblasts were in the G1 phase. Addition of choline resulted in synchronous synthesis of DNA with a peak occurring after 14 h. Incubation of cells with 0.5% serum had no effect on phosphatidylcholine (PC) levels in cells supplemented with 28 microM choline, but the concentration of PC was reduced from 32 to 20 nmol/10(6) cells after 1 day of incubation in the absence of choline. Supplementation with dialyzed serum, but not dialyzed and delipidated serum, allowed choline-deficient cells to replicate normally. This was attributed to the presence of lysophosphatidylcholine in dialyzed serum as this lipid, but not other lipids (e.g., phosphatidylcholine or mitogenic lipids) was able to replace the choline requirement. The choline-deficient effect was not complete; some DNA synthesis occurred in the absence of choline in the medium, and approximately 30% of the cells completed mitosis in 35 h compared to 100% in the presence of choline. The data suggest that phosphatidylcholine is required for normal progression of the cell cycle beyond the G1 phase and is unrelated to the induction of G0 to G1 transition. Choline deficiency should be a useful method for synchronizing cells in the G1 phase.

Animals↗
PubMed1993

Impaired biosynthesis of phosphatidylcholine causes a decrease in the number of very low density lipoprotein particles in the Golgi but not in the endoplasmic reticulum of rat liver.

We have investigated the mechanism by which inhibition of phosphatidylcholine biosynthesis in rat hepatocytes by choline deprivation causes a reduction in the secretion of very low density lipoprotein (VLDL) (Yao, Z., and Vance, D. E. (1988) J. Biol. Chem. 263, 2998-3004). Rats ingested a choline-deficient or control diet for 3 days, and subcellular fractions of liver were prepared. No change in the amount of apolipoprotein B in the lumina of the endoplasmic reticulum was observed, but there was a 40-50% decrease of apolipoprotein B in the lumina of the Golgi from choline-deficient compared with control rats. Incubation of microsomes, derived from choline-deficient and -supplemented hepatocytes, with trypsin showed similar degradation of apolipoprotein B, indicating similar quantities of this protein are present on the surface and within the lumina. The VLDL particles in the Golgi of liver cells and in plasma, on average, were larger in samples derived from choline-deficient compared with choline-supplemented animals. Incubation of plasma VLDL with proteases demonstrated that the apolipoprotein B of plasma VLDL particles from choline-deficient animals had a different susceptibility to digestion than did VLDL from choline-supplemented animals. These data indicate that the number of VLDL particles assembled in the endoplasmic reticulum of liver is similar in choline-deficient and -supplemented rats, but the number of particles is decreased in the Golgi from choline-deficient animals.

Animals↗