Radio-sensitizing effect of MEK inhibition in glioblastoma in vitro and in vivo.

INTRODUCTION: Glioblastoma (GBM) is an incurable cancer type. New therapeutic options are investigated, including targeting the mitogen-activated protein kinase (MAPK) pathway using MEK inhibitors as radio-sensitizers. In this study, we investigated whether MEK inhibition via PD0325901 leads to radio-sensitization in experimental in vitro and in vivo models of GBM. MATERIALS AND METHODS: In vitro, GBM8 multicellular spheroids were irradiated with 3 fractions of 2 Gy, during 5 consecutive days of incubation with either PD0325901 or MEK-162. In vivo, we combined PD0325901 with radiotherapy in the GBM8 orthotopic mouse model, tumor growth was measured weekly by bioluminescence imaging and overall survival and toxicity were assessed. RESULTS: Regrowth and viability of spheroids monitored until day 18, showed that both MEK inhibitors had an in vitro radio-sensitizing effect. In vivo, PD0325901 concentrations were relatively constant throughout multiple brain areas and temporal PD0325901-related adverse events such as dermatitis were observed in 4 out of 14 mice (29%). Mice that were treated with radiation alone or combined with PD0325901 had significantly better survival compared to vehicle (both P < 0.005), however, no significant interaction between PD0325901 MEK inhibition and irradiation was observed. CONCLUSION: The difference between the radiotherapy-enhancing effect of PD0325901 in vitro and in vivo urges further pharmacodynamic/pharmacokinetic investigation of PD0325901 and possibly other candidate MEK inhibitors.

Effects of ß-hydroxybutyrate and isoproterenol on lipolysis in isolated adipocytes from periparturient dairy cows and cows with clinical ketosis.

An in vitro model was used to investigate effects of ß-hydroxybutyrate and isoproterenol (ß-adrenergic receptor agonist) on lipolysis in isolated adipocytes from late pregnant and recently calved dairy cows (n=5) and cows with clinical ketosis (n=3). Incubation with 3.0 mmol/L ß-hydroxybutyrate reduced lipolysis in isolated adipocytes. This inhibitory effect was lower in the first lactation week (47%±16%) compared with late pregnancy (71%±6.5%). Incubation with 0.3 µmol/L isoproterenol stimulated lipolysis in isolated adipocytes from periparturient dairy cows. Basal lipolysis resulted in non-esterified fatty acid to glycerol ratios in the incubation media of 2.0±0.23 in prepartum samples, 2.1±0.23 in the first lactation week and 2.2±0.09 in cows with clinical ketosis. ß-Hydroxybutyrate reduced lipolysis by 45%±9.6% in isolated adipocytes from cows with clinical ketosis, indicating that impaired feedback of ß-hydroxybutyrate may not play a role in the disease etiology.

Protein and fat mobilization and associations with serum ß-hydroxybutyrate concentrations in dairy cows.

The objective of this study was to obtain information on variation between dairy cows in muscle and fat tissue mobilization around parturition and to study the association between protein and fat mobilization and serum ß-hydroxybutyrate (BHBA) concentrations (hyperketonemia) in this period. Thirty-four cows kept under similar conditions at a university dairy farm (no experimental treatments) were monitored from 4 wk before until 8 wk after calving. Mobilization of muscle protein was investigated by analysis of plasma 3-methylhistidine concentrations (3-MH, analyzed by a recently developed HPLC tandem mass spectrometry method) and ultrasound measurements of longissimus muscle thickness. Mobilization of fat tissue was monitored by serum nonesterified fatty acid (NEFA) concentrations and ultrasound measurements of backfat thickness. Large variation was observed between cows in onset and duration of periparturient protein and fat mobilization. Plasma 3-MH concentrations and muscle thickness profiles indicated that protein mobilization started, on average, before parturition and continued until approximately wk 4 of lactation. Serum NEFA concentrations and backfat thickness profiles showed that fat mobilization occurred from parturition until the end of the study. Thus, muscle protein mobilization occurred in advance of fat mobilization in most cows from this study. We hypothesized that this might be due to a prepartum amino acid deficiency in the absence of negative energy balance. The incidence of hyperketonemia in this study was 16/34 = 47%. With the exception of 3 cows defined as having severe hyperketonemia, cows with lower 3-MH concentrations had higher serum BHBA concentrations. A possible explanation for this observation might be that higher mobilization of protein around calving might restrict ketone body production due to the higher availability of glucogenic precursors in the period of most severe negative energy balance and highest fat mobilization. The validity of this hypothesis needs to be confirmed, but data from this study indicate that further research on the role of protein mobilization in the etiology of hyperketonemia in dairy cows is needed.

Technical note: quantification of plasma 1- and 3-methylhistidine in dairy cows by high-performance liquid chromatography-tandem mass spectrometry.

To improve monitoring of protein mobilization in dairy cows, we developed and evaluated a method to quantify 1-methylhistidine and 3-methylhistidine in plasma by HPLC-tandem mass spectrometry. The analytical method described is (1) sensitive: both histidine derivates can be detected in the picomole range; (2) accurate: intra- and interassay coefficients of variation were < 5% for all standard solutions of 1-methylhistidine and 3-methylhistidine measured (31 to 500 pmol); (3) specific: 1-methylhistidine is clearly separated from 3-methyl-histidine in plasma samples from dairy cows; and (4) flexible: can be easily adapted to measure other amino acids or compounds containing a primary amine. 1-Methylhistidine is present in plasma of dairy cows at concentrations of 5.0 ± 1.7 µM, similar to concentrations of 3-methylhistidine (4.4 ± 2.4 µM). Analytical separation of both histidine metabolites is essential when plasma 3-methylhistidine is used as indicator for muscle breakdown in dairy cows. Specific quantification of the concentration of 3-methylhistidine in bovine plasma samples by HPLC tandem mass spectrometry can improve monitoring of protein mobilization in dairy cows.

Iliopsoas impingement after total hip replacement: the results of non-operative management, tenotomy or acetabular revision.

We have reviewed a group of patients with iliopsoas impingement after total hip replacement with radiological evidence of a well-fixed malpositioned or oversized acetabular component. A consecutive series of 29 patients (30 hips) was assessed. All had undergone a trial of conservative management with no improvement in their symptoms. Eight patients (eight hips) preferred continued conservative management (group 1), and 22 hips had either an iliopsoas tenotomy (group 2) or revision of the acetabular component and debridement of the tendon (group 3), based on clinical and radiological findings. Patients were followed clinically for at least two years, and 19 of the 22 patients (86.4%) who had surgery were contacted by phone at a mean of 7.8 years (5 to 9) post-operatively. Conservative management failed in all eight hips. At the final follow-up, operative treatment resulted in relief of pain in 18 of 22 hips (81.8%), with one hip in group 2 and three in group 3 with continuing symptoms. The Harris Hip Score was significantly better in the combined groups 2 and 3 than in group 1. There was a significant rate of complications in group 3. This group initially had better functional scores, but at final follow-up these were no different from those in group 2. Tenotomy of the iliopsoas and revision of the acetabular component are both successful surgical options. Iliopsoas tenotomy provided the same functional results as revision of the acetabular component and avoided the risks of the latter procedure.

Luteinizing hormone inhibits Fas-induced apoptosis in ovarian surface epithelial cell lines.

Gonadotrophins including LH have been suggested to play an important role in the etiology of epithelial ovarian cancers. The goal of the present study was to obtain more insight in the mechanism of gonadotrophin action on ovarian surface epithelium (OSE) cells. As the Fas system is known to be a major player in the regulation of the process of apoptosis in the ovary, we investigated whether LH interfered with Fas-induced apoptosis in the human OSE cancer cell lines HEY and Caov-3. Activation of Fas receptor by an agonistic anti-Fas receptor antibody induced apoptosis, as was evaluated by caspase-3 activation, poly(ADP-ribose) polymerase fragmentation, phosphatidylserine externalization and morphological changes characteristic of apoptosis. Co-treatment with LH reduced the number of apoptotic cells following activation of Fas in a transient manner, while LH by itself did not affect apoptosis or cell proliferation. The anti-apoptotic effect of LH could be mimicked by the membrane-permeable cAMP analog 8-(4-chlorophenylthio) cAMP (8-CPT-cAMP), and blocked by H89, a specific inhibitor of protein kinase A (PKA). In conclusion, these findings suggest that LH protects HEY cells against Fas-induced apoptosis through a signaling cascade involving PKA. Although it is plausible that in vivo LH might also enhance OSE tumor growth through inhibition of apoptosis, further research is necessary to confirm this hypothesis.

Effects of a single dose of dexamethasone-21-isonicotinate on the metabolism of heifers in early lactation.

Eight Swedish crossbred heifers, about two-and-a-half years old, were given a single intramuscular dose of dexamethasone-21-isonicotinate between nine and 15 days after they had calved and eight similar heifers were left untreated. The treatment had no significant effects on the lipolytic activity of the heifers' fat tissues, and no effect on the concentrations of non-esterified fatty acids and beta-hydroxybutyrate in blood or the triacylglycerol content of the liver. However, there were significant increases in plasma glucose concentrations two days after the injection and in plasma insulin concentrations two and four days after the injection.

8-year survivorship analysis and subjective results of 687 primary Balgrist hip sockets.

The Balgrist hip socket consists of an outer split ring in the form of a truncated cone, made of titanium, which is expanded by a tapered HDPE insert during implantation, thus ensuring firm primary press-fit and the possibility of retightening in the postoperative remodelling phase. Between November 1987 and October 1996, 687 primary Balgrist hip sockets were implanted in 555 patients. Five hundred and thirty-seven patients were investigated. Of these patients, 71.1% never had pain in the operated hip, 88.1% had no problems putting on their shoes, 76.2% were able to walk one or more hours. Furthermore, 91.7% are very or mostly content with the postoperative result. Nineteen hip sockets had to be revised until April 1997. With a 92.1% Kaplan-Meier survivorship rate after 8 years the Balgrist hip socket ranks among the most successful noncemented acetabular components.

Cloning and expression of CTP:phosphoethanolamine cytidylyltransferase cDNA from rat liver.

CTP:phosphoethanolamine cytidylyltransferase (ET) is a key regulatory enzyme in the CDP-ethanolamine pathway for phosphatidylethanolamine synthesis. As a first step in the elucidation of the structure-function relationship and the regulation of ET, an ET cDNA was cloned from rat liver. The cloned cDNA encodes a protein of 404 amino acid residues with a calculated molecular mass of 45.2 kDa. The deduced amino acid sequence is very similar to that of human ET (89% identity). Furthermore, it shows less, but significant, similarity to yeast ET as well as to other cytidylyltransferases, including rat CTP:phosphocholine cytidylyltransferase and Bacillus subtilis glycerol-3-phosphate cytidylyltransferase. Like human and yeast ET, rat ET has a large repetitive internal sequence in the N- and C-terminal halves of the protein. Both parts of the repeat contain the HXGH motif, the most conserved region in the N-terminal active domain of other cytidylyltransferases, indicating the existence of two catalytic domains in ET. The hydropathy profile revealed that rat ET is largely hydrophilic and lacks a hydrophobic stretch long enough to span a bilayer membrane. There was no prediction for an amphipathic alpha-helix. Transfection of COS cells with the cDNA clone resulted in an 11-fold increase in ET activity, corresponding to an increase in the amount of ET protein as detected on a Western blot. Determination of the ET activity during liver development showed a 2. 5-fold increase between day 17 of gestation and birth (day 22) and the amount of ET protein changed accordingly. Northern blot analysis showed that this was accompanied by an increase in the amount of ET mRNA. Between day 17 of gestation and birth, the amount of mRNA in fetal rat liver increased approx. 6-fold, suggesting the regulation of ET at both pretranslational and post-translational levels during rat liver development.

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

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

Induction of hepatocyte proliferation after partial hepatectomy is accompanied by a markedly reduced expression of phosphatidylethanolamine N-methyltransferase-2.

Expression of phosphatidylethanolamine N-methyltransferase (PEMT)-2 in rat hepatoma cells caused an increase in the time for cell division from 18 to 50 h [Cui et al. (1994) J. Biol. Chem. 269, 24531-24533]. We investigated whether or not a similar inverse relationship might exist for liver proliferation in vivo. Thus, partial hepatectomized rats were used to investigate the expression of PEMT2 during liver regeneration. Enhanced biosynthesis of phosphatidylcholine after partial hepatectomy was due to increased activity and amount of CTP:phosphocholine cytidylyltransferase. On the other hand the total activity of PEMT was markedly decreased during the first days of rat liver regeneration. Maximal decrease of total PEMT activity (45%) and loss of PEMT2 protein (90%) coincided with maximal DNA synthesis and CTP:phosphocholine cytidylyltransferase activity 24 h after partial hepatectomy in both male and female rats. Supplementing dietary choline in the diets of female rats shifted this pattern from 24 h to 36 h after partial hepatectomy, whereas the pattern in male rats was not affected. Northern blot studies showed that the amount of PEMT2 mRNA was decreased accordingly, suggesting regulation of the amount and activity of PEMT2 at a pre-translational level. Thus, our data show a reciprocal regulation of CTP:phosphocholine cytidylyltransferase and PEMT2 at the level of gene expression in regenerating rat liver. These results implicate PEMT2 in the regulation of hepatocyte cell growth in a physiologically relevant model.

A genetic defect in phosphatidylcholine biosynthesis triggers apoptosis in Chinese hamster ovary cells.

We have investigated the cell death of a Chinese hamster ovary mutant (MT-58) with a thermo-sensitive CTP:phosphocholine cytidylyltransferase, the rate-limiting enzyme of the CDP-choline pathway for phosphatidylcholine biosynthesis (Esko, J. D., Wermuth, M. M., and Raetz, C. R. H. (1981) J. Biol. Chem. 256, 7388-7393). After MT-58 cells were shifted to the restrictive temperature of 40 degrees C, the cytidylyltransferase was inactivated immediately leading to a decrease in phosphatidylcholine biosynthesis and cell death. DNA content and number of cells in the S phase decreased significantly in the dying MT-58 cells according to flow cytometrical analyses. The fragmentation of genomic DNA was detected by DNA ladders in agarose gel and release of the prelabeled genomic DNA into cytosolic fractions 14 h after the temperature shift. The dying cells underwent a dramatic reduction of cellular volume while maintaining the membrane containment of cellular contents. These events indicated that the inactivation of cytidylyltransferase triggered apoptosis in Chinese hamster ovary cells. This is the first report that apoptosis was induced in cultured cells, not by an added agent, but by a mutation in phospholipid biosynthesis.

Phosphatidylethanolamine methylation and hepatoma cell growth.

Phosphatidylethanolamine is converted to phosphatidylcholine in hepatocytes via the enzyme phosphatidylethanolamine N-methyltransferase (PEMT). An isoform, PEMT2 has been cloned, expressed and localized to a mitochondria-associated membrane in rat liver. Expression of PEMT2 caused a decreased rate of cell division of cultured rat hepatoma cells. Mechanistic studies suggest that the slower growth of transfected hepatoma cells may be 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.

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.

Stimulation of CTP:phosphocholine cytidylyltransferase by free cholesterol loading of macrophages involves signaling through protein dephosphorylation.

Free cholesterol-loaded macrophages in atheromata synthesize excess phosphatidylcholine (PC), which may be an important adaptive response to the excess free cholesterol (FC) load. We have recently shown that FC loading of macrophages leads to 2-4-fold increases in PC mass and biosynthesis and to the post-translational activation of the membrane-bound form of CTP:phosphocholine cytidylyltransferase (CT), a key enzyme in PC biosynthesis. Herein, we explore further the mechanism of CT activation in FC-loaded macrophages. First, enrichment of membranes from control macrophages with FC in vitro did not increase CT activity, and PC biosynthesis in vivo is up-regulated by FC loading even when CT and FC appear to be mostly in different intracellular sites. These data imply that FC activates membrane-bound CT by a signaling mechanism. That the proposed signaling mechanism involves structural changes in the CT protein was suggested by data showing that two different antibodies against synthetic CT peptides showed increased recognition of membrane-bound CT from FC-loaded cells despite no increase in CT protein. Since CT is phosphorylated, two-dimensional maps of peptides from 32P-labeled control and FC-loaded macrophages were compared: six peptide spots from membrane-bound CT, but none from soluble CT, were dephosphorylated in the FC-loaded cells. Furthermore, incubation of FC-loaded macrophages with the phosphatase inhibitor, calyculin A, blocked increases in both PC biosynthesis and antipeptide-antibody recognition of CT. Last, treatment of membranes from control macrophages with lambda phage protein phosphatase in vitro increased both CT activity (2-fold) and antipeptide-antibody recognition of CT; soluble CT activity and antibody recognition were not substantially affected by phosphatase treatment. In summary, FC loading of macrophages leads to the partial dephosphorylation of membrane-bound CT, and possibly other cellular proteins, which appears to be important in CT activation. This novel regulatory action of FC may allow macrophages to adapt to FC loading in atheromata.

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.

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.

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.

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.

Phospholipid-transfer proteins and their mRNAs in developing rat lung and in alveolar type-II cells.

Gene expression of non-specific lipid-transfer protein (nsL-TP; identical with sterol carrier protein 2) and phosphatidylinositol-transfer protein (PI-TP) was investigated in developing rat lung. During the late prenatal period (between days 17 and 22) there is a 7-fold increase in the level of nsL-TP and a 2-fold rise in that of PI-TP. The prenatal increases in the levels of nsL-TP and PI-TP are accompanied by parallel increases in the levels of their mRNAs, indicating pretranslational regulation. Compared with whole lung, isolated alveolar type-II cells are enriched in nsL-TP and its mRNA, but not in PI-TP and its mRNA. The observation that the levels of nsL-TP and its mRNA in rat lung show a pronounced increase in the period of accelerated surfactant formation, together with the observation that the surfactant-producing type-II cells are enriched in nsL-TP and its mRNA, suggest that nsL-TP plays a role in the metabolism of pulmonary surfactant.