Inhibition of myogenesis by the H-ras oncogene: implication of a role for protein kinase C.

Expression of the oncogenic form of H-ras p21 in the mouse myogenic cell line, 23A2, blocks myogenesis and inhibits expression of the myogenic regulatory factor gene, MyoD1. Previous studies from a number of laboratories have demonstrated that the activation of ras p21 is associated with changes in phospholipid metabolism that directly, or indirectly, lead to elevated levels of intracellular diacylglycerol and the subsequent activation of protein kinase C (PKC). To assess the importance of PKC activity to the ras-induced inhibition of skeletal myogenesis, we examined the levels of PKC activity associated with the terminal differentiation of wild-type myoblasts and with the differentiation-defective phenotype of 23A2 ras cells. We demonstrate that there is a 50% reduction in PKC activity during normal myogenesis and that PKC activity is required for myoblast fusion, but not for the transcriptional activation of muscle-specific genes. In contrast, we found that the differentiation-defective 23A2 ras cells possess two- to threefold more PKC activity than wild-type myofibers and that reducing the PKC activity in these cultures does not reverse their non-myogenic phenotype. On the other hand, if PKC activity is downregulated in 23A2 cells before the expression of activated ras p21, myogenesis is not inhibited. These results suggest that activated ras p21 relies on a PKC-dependent signal transduction pathway to initiate, but not to sustain, its negative effects on 23A2 skeletal myogenesis and underscore the potential importance of PKC activity to the proper control of skeletal muscle differentiation.

Geometric structure of the femoral neck measured using dual-energy x-ray absorptiometry.

An algorithm was developed to estimate the strength of the femoral neck from data generated by the dual-energy x-ray absorptiometry (DXA). This algorithm considers shape of the proximal femur as well as cross-sectional moment of inertia (CSMI) in the estimate. Proximal femora (10) from cadavers of white adults and an aluminum step wedge were scanned with the Lunar DPX to validate the calculation of CSMI. After scanning, each femoral neck was sectioned at its narrowest portion for direct measurement of CSMI. Three healthy young women were scanned five times each to evaluate the reproducibility of geometric measurements using DXA. There was a strong linear association between the CSMI measured directly and using DXA in both cadaver bones (r2 = 0.96) and the aluminum step wedge (r2 = 0.99). The coefficient of variation for CSMI from repeated measurements using DXA was less than 3%. This indicates that it is possible to estimate reproducibly the bending rigidity of bone from DXA measurements. The data from 306 normal subjects were analyzed to investigate geometric changes in the femoral neck with age. Although there was no strong correlation between CSMI and age in normal subjects of either sex, safety factor (SF, an index of strength of the femoral neck during walking) and fall index (FI, an index of the strength of the femoral neck during a fall) decrease with age in both sexes. We observed an alteration of the geometric structure of the femoral neck with age that may increase the stress on the femoral neck and decrease SF and FI.

Peak bone mass in young women.

Increasing peak bone mineral density (BMD) or content (BMC) in young women may help to reduce the incidence of osteoporosis. Identifying the age when peak bone content or density is attained is essential to develop strategies aimed at optimizing peak BMD and BMC. Total body bone mineral density (TBBMD) and content (TBBMC) were measured by a dual X-ray absorptiometer in healthy females (n = 247, aged 11-32 years). TBBMD and TBBMC were modeled separately as a nonlinear function of age. By age 22.1 +/- 2.5 years, 99% of peak BMD is attained, and by age 26.2 +/- 3.7 years, 99% of peak BMC is attained. Nonlinear relationships between weight and TBBMD or TBBMC were also modeled. In this model, the influence of several parameters, including age, weight, and height, on BMC and BMD were simultaneously assessed. A model with age and weight described the best fit for TBBMD, whereas age, weight, and height described the best fit for total body TBBMC.

Previous milk consumption is associated with greater bone density in young women.

BACKGROUND: Dietary calcium and milk intakes at specific ages may influence bone mineral measures at specific sites during development of peak bone mass. OBJECTIVE: Relations of previous milk intake and current calcium intake to current bone mineral measures were investigated in young women. DESIGN: A food-frequency interview and recall of previous milk intake from early childhood to 12 y of age and during adolescence (13-19 y) were completed in a cross-sectional analysis in young women (age 18-31 y; n = 224). Three levels of previous milk intake were defined: 1) infrequently or never, 2) sometimes, and 3) at every or almost every meal. Total body (TB), femoral neck, radius (R), and spine (S) bone mineral density (BMD) and bone mineral content (BMC) were determined by using dual-energy X-ray absorptiometry. RESULTS: Childhood and adolescent milk intakes were positively correlated (r = 0.66). Childhood and adolescent milk intakes correlated with current calcium intakes (r = 0.26 and 0.33, respectively). Adolescent milk intake correlated with RBMD (r = 0.16). When weight was controlled for, adolescent milk intake correlated with TBBMD (r = 0.16), TBBMC (r = 0.21), SBMC (r = 0.16), RBMD (r = 0.18), and RBMC (r = 0.15). Current calcium intakes correlated with SBMC (r = 0.17). Regression analyses supported these results. CONCLUSIONS: Results were consistent with the hypothesis that higher milk intake during adolescence is associated with greater total body, spine, and radial bone mineral measures during development of peak bone mass, whereas current calcium intakes may influence SBMC. In addition, milk intake at a younger age may contribute to similar habits of milk intake later in life.

Dietary calcium, protein, and phosphorus are related to bone mineral density and content in young women.

BACKGROUND: Dietary factors have been implicated in modifying bone health, although the results remain controversial, particularly in young women. OBJECTIVE: The objective of the study was to determine relations of selected dietary factors and anthropometric measurements to bone mineral density (BMD) of the spine, femoral neck, trochanter, Ward's triangle, radius, and total body and the bone mineral content (BMC) of the spine, radius, and total body. DESIGN: The study was a cross-sectional analysis of 215 women aged 18-31 y. RESULTS: Weight, height, and lean mass were correlated with bone mineral measures at every site (r = 0.17-0.78). Postmenarcheal age (years since onset of menses) was positively correlated with total-body BMD and BMC, radius BMD and BMC, and spine BMC, and negatively correlated with Ward's triangle BMD. Radius BMD was correlated with protein, calcium, and phosphorus intakes, and spine BMD and BMC were correlated with energy, protein, calcium, and phosphorus intakes. These correlations remained significant when postmenarcheal age, lean mass, and fat mass were controlled. A pattern emerged in multiple regression analyses that showed a complex relation among calcium, protein or phosphorus, and the calcium-protein or calcium-phosphorus ratio and spine or total-body BMC and BMD. All 3 variables (calcium, protein or phosphorus, and calcium-protein or calcium-phosphorus ratio) were required in the model for significance. CONCLUSIONS: Anthropometric measures were predictors of bone mass. A single ratio of calcium to phosphorus or protein did not optimize bone mass across the range of calcium intakes.

Exercise and oral contraceptive use suppress the normal age-related increase in bone mass and strength of the femoral neck in women 18-31 years of age.

Women who exercise during their second and third decades may increase their peak bone mass and lower their eventual risk for postmenopausal fracture. However, the effects of exercise in younger women can be modulated by the use of oral contraceptives, which may prevent the normal accretion of bone mass that would otherwise occur. We hypothesized that exercise intervention in young adult women would significantly increase both bone mass and the bending rigidity of the femoral neck. We further hypothesized that exercise intervention in the presence of oral contraceptive use would have a negative effect on bone mass and bending rigidity. Women 18-31 years of age (n = 123) were classified by oral contraceptive use (OC, NOC) and age (18-23, 24-31 years), and then randomized into exercise or nonexercise groups. The exercise protocol consisted of three sessions/week of aerobic and nonaerobic exercises, and continued for 2 years. Each 6 months, the femoral neck of each subject was scanned using a Lunar dual-energy X-ray absorptiometry (DEXA) scanner, and bone mineral content, density and geometric information were used to calculate estimated stresses and bending rigidity at the hip. Percent changes from baseline were analyzed using two-way analysis of variance (ANOVA) at 6, 12, 18, and 24 months. Women who neither exercised nor took oral contraceptives (NE/NOC) had the greatest percentage increases in cross-sectional area (4.98 +/- 2.29%), cross-sectional moment of inertia (9.45 +/- 2.37%), total bone mineral density (2.07 +/- 2.09%), fracture index (8.03 +/- 2.03%), and safety factor (20.03 +/- 5.79%) over the 24 month exercise program. Women who exercised and did not take oral contraceptives (E/NOC) declined on most variables related to femoral strength and bone mass, whereas those women who took oral contraceptives were usually intermediate between NE/NOC and E/NOC, whether they exercised or not. These data show that either exercise or OC use is associated with a suppression of the normal increase in bone mass and mechanical strength in the femoral neck in women 18-31 years old, but the combination of exercise and OC use appears to have a less suppressive effect.

Peak spine and femoral neck bone mass in young women.

Achievement of higher peak bone mass early in life may play a critical role against postmenopausal bone loss. Bone mineral density (BMD) of the spine, femoral neck, greater trochanter, Ward's triangle, and spine bone mineral content (BMC) and bone surface area (BSA) were assessed by dual energy x-ray absorptiometry in 300 healthy females (age 6-32 years). Bone measurements were described by using nonlinear models with age, weight, height, or dietary calcium intake as the explanatory variables. At the spine, femoral neck, greater trochanter, and Ward's triangle, the highest BMD level was observed at 23.0 +/- 1.4, 18.5 +/- 1.6, 14.2 +/- 2.0, and 15.8 +/- 2.1 years, respectively. The age of attaining peak spine BMC and BSA cannot be estimated, as significant increases in these two measures were observed through this age group. Age, weight, and height were all significant predictors of all these bone measurements. Weight was a stronger predictor than age for all sites. Dietary calcium intake was not a significant predictor for any of these bone measurements. We conclude that age of attaining peak bone mass at the hip is younger than at the spine, and BMC and BSA at the spine continue to increase through the early thirties in females.

Transfection of C3H10T1/2 cells with the Harvey-ras oncogene reduces cytosolic phospholipase A2 function.

Several lines of evidence suggest that phospholipase A2 may play a role in the activated ras-mediated transformation process. In the present study, phospholipase A2 activity and expression were assessed in a murine fibroblast cell line (C3H10T1/2 cells) that was stably transfected with the Harvey ras oncogene, a cellular model used for studying multistage carcinogenesis. Reduced levels of fatty acids were released from the ras-transfected cells compared to untransfected controls. The in vitro phospholipase A2 activity apparent in the C3H10T1/2 showed preference for sn-2 arachidonyl phosphatidylcholine compared to dipalmitoyl phosphatidylcholine. The activity, as well as the cytosolic phospholipase A2 immunoreactive protein, was reduced by 50% in the ras-transfected cells compared to control cells. These results suggest that the cytosolic phospholipase A2 is the predominant form of this enzyme family expressed in C3H10T1/2 cells and that the activity and protein amount is reduced by 50% in these cells when stably transfected with the Harvey ras oncogene.

Increased prostaglandin H synthase-1 expression and activity level in stably Harvey-ras transfected C3H10T1/2 cells.

In these studies, prostaglandin H synthase activity was increased in stably Harvey-ras transfected C3H10T1/2 cells. The level of total prostaglandin H synthase protein was two-fold higher in stably Harvey-ras transfected C3H10T1/2 cells than in control cells with no difference in prostaglandin H synthase-2 level. Prostaglandin H synthase-1 mRNA level was two-fold higher in transfected than in control cells, while prostaglandin H synthase-2 was not significantly different. Thus, prostaglandin H synthase-1, but not prostaglandin H synthase-2, expression was increased in Harvey-ras transfected C3H10T1/2 cells.

Calcium supplementation increases bone density in adolescent girls.

In a longitudinal calcium intervention study, bone density was assessed in pubertal girls for 18 months. Significant additional increases in total body bone mineral density (1.3%) and spine bone mineral density (2.9%) and content (4.7%) were noted in the calcium-supplemented group. Increasing bone mass during adolescence with adequate calcium intake, if maintained into adulthood, could decrease the risk of osteoporosis later in life.

Extensive cutaneous burn complicated by severe necrotizing amebic enterocolitis. Pathogenic factors resulting in severe amebic disease.

A 59-yr-old Mexican-American man developed fatal necrotizing amebic enterocolitis following an extensive cutaneous thermal injury. Those factors in the severely burned patient which predispose to amebic disease are discussed.

Previous physical activity relates to bone mineral measures in young women.

Exercise may increase accretion of bone, potentially reducing the risk of osteoporosis. Previous physical activity was assessed in 204 minimally active young women (18-31 yr). Bone mineral content (BMC) and bone mineral density (BMD) for the total body, femoral neck, and spine were assessed by a dual x-ray absorptiometer, and the radius by a single photon absorptiometer. Self-reported occupation and leisure activity for the 5 yr before enrollment in the study, as well as high school and college sports participation, were assigned energy expenditure (EE) values. From this information, EE variables were created as follows: 1) occupation EE + leisure EE + high school sport and/or college sport EE if within prior 5 yr (5-yr EE); 2) occupation EE + leisure EE (occupation + leisure EE); and 3) high school sport EE (high school EE). These variables were correlated with bone mineral measures and significant results follow (P < 0.05). Five-year EE and occupation + leisure EE correlated with all measures of bone health (r from 0.13 to 0.39). High school EE correlated with total body BMD (r = 0.25) and BMC (r = 0.28), femoral neck BMD (r = 0.28), radius BMC (r = 0.20), as well as spine BMD (r = 0.20) and BMC (r = 0.27). When weight was controlled, 5-yr EE and occupation + leisure EE remained correlated with all BMC measures (r from 0.14 to 0.22). When controlled for weight, high school EE remained associated with femoral neck BMD (r = 0.24), total body BMD (r = 0.20) and BMC (r = 0.26), and spine BMC (r = 0.17). To partially control for selection bias, data were also controlled for total body BMD. Five-year EE and occupation + leisure EE remained positively correlated with all measures of BMC. High school EE remained correlated both with femoral neck BMD and total body BMC. In multiple regression analyses, 5-yr EE or occupation + leisure EE were significant predictors of all measures of bone health, except femoral neck BMD. High school EE was a significant predictor for total body BMD and BMC, femoral neck BMD, and spine BMC.

Impact of exercise on bone health and contraindication of oral contraceptive use in young women.

PURPOSE: The effect of quantified resistance and high impact exercise training on bone mass as modified by age and oral contraceptive (OCont) use in young women was studied. METHODS: Women were categorized by age (18-23 vs 24-31 yr) and OCont use, and were then randomized into either three sessions of resistance exercise plus 60 min.wk-1 of jumping rope or a control group for 24 months. Total body, spine, femoral neck, greater trochanter, Ward's area, and radial bone mineral density (BMD) and/or content (BMC), biochemical markers of bone turnover, dietary intake of calcium, lean body mass, maximal oxygen uptake, and strength were determined at baseline and every 6 months. RESULTS: Total body (TB) BMC percent change from baseline was higher in exercisers compared with nonexercisers at 6 and 24 months. OCont users had lower bone turnover at baseline and a decrease in TBBMC from baseline compared with non-OCont users at 24 months. Spine BMC and BMD decreased in the exercise and OCont group at 6 months and remained significantly below nonexercisers who used oral contraceptives at 2 yr. Femoral neck BMD also decreased in the exercise and oral contraceptive group at 6 months. CONCLUSIONS: Exercise prevented a decline in TBBMC seen in the nonexercisers. On the other hand, exercise in oral contraceptive users prevented the increase observed in the spine of the nonexercise plus OCont group.

A self referencing platinum nanoparticle decorated enzyme-based microbiosensor for real time measurement of physiological glucose transport.

Glucose is the central molecule in many biochemical pathways, and numerous approaches have been developed for fabricating micro biosensors designed to measure glucose concentration in/near cells and/or tissues. An inherent problem for microsensors used in physiological studies is a low signal-to-noise ratio, which is further complicated by concentration drift due to the metabolic activity of cells. A microsensor technique designed to filter extraneous electrical noise and provide direct quantification of active membrane transport is known as self-referencing. Self-referencing involves oscillation of a single microsensor via computer-controlled stepper motors within a stable gradient formed near cells/tissues (i.e., within the concentration boundary layer). The non-invasive technique provides direct measurement of trans-membrane (or trans-tissue) analyte flux. A glucose micro biosensor was fabricated using deposition of nanomaterials (platinum black, multiwalled carbon nanotubes, Nafion) and glucose oxidase on a platinum/iridium microelectrode. The highly sensitive/selective biosensor was used in the self-referencing modality for cell/tissue physiological transport studies. Detailed analysis of signal drift/noise filtering via phase sensitive detection (including a post-measurement analytical technique) are provided. Using this highly sensitive technique, physiological glucose uptake is demonstrated in a wide range of metabolic and pharmacological studies. Use of this technique is demonstrated for cancer cell physiology, bioenergetics, diabetes, and microbial biofilm physiology. This robust and versatile biosensor technique will provide much insight into biological transport in biomedical, environmental, and agricultural research applications.

Parathyroid hormone is associated with decreased fat mass in young healthy women.

OBJECTIVE: To investigate the relationship of parathyroid hormone (PTH) with dietary calcium and changes in body composition. DESIGN: Cross-sectional and 1-year longitudinal trial. SUBJECTS: Normal-weight young women (age: 18-31), 155 subjects analyzed at baseline, and data for 41 subjects analyzed prospectively between baseline and 12 months. MEASUREMENTS: Levels of fasting serum calcium and PTH, intakes of calcium (3-day diet records), and total body weight and body composition (dual energy X-ray absorptiometry). RESULTS: Baseline dietary calcium, regardless of whether unadjusted or adjusted for energy intake, did not predict baseline levels of fasting serum PTH. Change in dietary calcium also did not predict change in serum PTH. However, log PTH was significantly correlated with body fat mass (R = 0.27), but not lean mass at baseline (n = 155), independent of serum calcium (corrected R = 0.25). Further, 12-month changes (n = 41) in log PTH positively predicted the 12-month change in body weight (R = 0.32) and body fat (R = 0.32), but not lean mass even when controlled for age or change in serum calcium. CONCLUSION: Fasting serum PTH was associated with increased fat mass, in both cross-sectional and prospective analysis. Thus, serum PTH may play a role in the regulation of body fat mass in young women.

Ceramide conversion to sphingosine-1-phosphate is essential for survival in C3H10T1/2 cells.

Ceramide and sphingosine-1-phosphate (S1P) are important dietary lipids involved in cell growth, differentiation, apoptosis and cell survival. Treatment of C3H10T1/2 murine fibroblast cells (10T1/2) with ceramide did not induce apoptosis, a commonly observed effect of ceramide treatment. To determine whether the metabolism of ceramide played a role in this resistance to apoptosis, inhibitors of ceramidase and sphingosine kinase, two important enzymes in sphingolipid metabolism, were used. Treatment of 10T1/2 cells both without or with ceramide plus N-oleoyl-ethanolamine (NOE) and (1S,2R)-D-erythro-s-(N-myristoylamino)-1-phenol-1-propanol (MAPP), two ceramidase inhibitors, resulted in fourfold and eightfold increases, respectively, in apoptosis. Cells treated without or with ceramide plus N,N-dimethylsphingosine (DMS), a potent sphingosine kinase inhibitor, resulted in fourfold and sixfold increases, respectively, in apoptosis. In all treatments the induction of apoptosis was prevented by the addition of S1P. With the addition of S1P with NOE and MAPP as well as with ceramide, treatments reduced the apoptotic response by 30 and 35%, respectively; whereas the addition of S1P with the DMS only and ceramide with DMS treatments reduced the apoptotic response by 60 and 70%, respectively. Studies using labeled ceramide demonstrated ceramide was metabolized to S1P. In addition, a 14-fold increase in apoptosis occurred in cells treated with a nonhydrolyzable analog of ceramide, ceramine, compared with vehicle control. Because inhibiting the conversion of ceramide to S1P resulted in apoptosis, the lack of an apoptotic response to ceramide alone for C3H10T1/2 cells is attributable to the conversion of this pro-apoptotic sphingolipid to the anti-apoptotic metabolite S1P, which is essential for cell survival.

Characterization of 25-hydroxyvitamin D binding protein from intestinal cells.

We have previously purified a cytosolic vitamin D metabolite binding protein (cDBP) from rat enterocytes, which has characteristics distinct from other vitamin D binding proteins. In these studies, we demonstrate that cDBP in a semi-purified fraction from human intestinal cells (Caco-2 cells) binds 25-hydroxyvitamin D (25OHD) with at least a 1000-fold greater affinity than 1, 25-dihydroxyvitamin D (1,25(OH)(2)D) or 24,25-dihydroxyvitamin D. Treatment of cells with 1,25(OH)(2)D reduced 25OHD binding to approximately one third that of the untreated cells (0.42 CPM/mg total protein vs 1.34 CPM/mg total protein, respectively). Finally, the cDBP is not immunoreactive to antibodies prepared against the C-terminus of the nuclear vitamin D receptor (VDR). In summary, cDBP bound 25OHD with greater affinity than either 1,25(OH)(2)D or 24,25 dihydroxyvitamin D, the cytosolic binding activity was down-regulated by 1,25(OH)(2)D and cBDP is distinct from the nuclear VDR.

Amino acid-mediated stimulation of renal phospholipid biosynthesis after acute tubular necrosis.

The mechanism by which amino acid infusion stimulates membrane physpholipid biosynthesis during renal regeneration after mercuric-chloride-induced acute tubular necrosis was studied in the rat. Amino acids can act directly on regenerating renal tissue to enhance net phospholipid synthesis because preincubation of cortical slices with amino acids induced an increase in [14C]-choline incorporation into phospholipid without altering the rate of breakdown. This amino acid stimulation of phospholipid biosynthesis was studied further by measuring [14C]-choline accumulation and its sequential conversion to phosphorylcholine, cytidine diphosphocholine (CDP-choline), and phosphatidylcholine via the Kennedy pathway in regenerating renal tissue. [14C]-Choline accumulation was increased after amino acid infusion, compared to glucose infusion. There were also increments in the Vmax of the choline kinase reaction, which converts entering [14C]-choline into [14C]-phosphorylcholine, and of the cholinephosphotransferase reaction in which [14C]-CDP-choline is incorporated into [14C]-phosphatidylcholine, whereas the apparent Km of each reaction was unchanged. Thus, amino acids infused after tubular necrosis can act directly on regenerating renal cells to increase precursor availability and augment two reactions of the phospholipid biosynthetic pathway.

Determination of the affinity of vitamin D metabolites to serum vitamin D binding protein using assay employing lipid-coated polystyrene beads.

We have developed an assay to measure the affinity of serum vitamin D binding protein for 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3, and vitamin D3, using uniform diameter (6.4 microns) polystyrene beads coated with phosphatidylcholine and vitamin D metabolites as the vitamin D donor. The lipid metabolite coated beads have a solid core, and thus all of the vitamin D metabolites are on the bead surface from which transfer to protein occurs. After incubating these beads in neutral buffer for 3 h, essentially no 3H-labeled vitamin D metabolites desorb from this surface. Phosphatidylcholine/vitamin D metabolite-coated beads (1 microM vitamin D metabolite) were incubated with varying concentrations of serum vitamin D binding protein under conditions in which the bead surfaces were saturated with protein, but most of the protein was free in solution. After incubation, beads were rapidly centrifuged without disturbing the equilibrium of binding and vitamin D metabolite bound to sDBP in solution was assayed in the supernatant. All three vitamin D metabolites became bound to serum vitamin D binding protein, and after 10 min of incubation the transfer of the metabolites to serum vitamin D binding protein was time independent. The transfer followed a Langmuir isotherm, and the Kd for each metabolite binding to serum vitamin D binding protein was derived by nonlinear least-squares fit analysis. From this analysis the following values for the Kd were obtained: 5.59 x 10(-6) M, 25-hydroxyvitamin D; 9.45 x 10(-6) M, 1,25-dihydroxyvitamin D; and 9.17 x 10(-5) M, vitamin D. In conclusion, we have developed a method which avoids problems encountered in previous assays and allows the precise and convenient determination of binding affinities of vitamin D metabolites and serum vitamin D binding protein.

Altered phosphatidylcholine metabolism in C3H10T1/2 cells transfected with the Harvey-ras oncogene.

The effect of expression of the Harvey-ras oncogene on phosphatidylcholine metabolism in C3H10T1/2 mouse fibroblast cells was examined. There were multiple changes in the CDP-choline pathway for phosphatidylcholine biosynthesis in the ras-expressing cells. The activity of the first enzyme in the pathway, choline kinase, was stimulated 1.9-fold, while the activity of the second enzyme, CTP:phosphocholine cytidylyltransferase, was decreased by one-half. High levels of intracellular phosphocholine measured in the ras cells were consistent with the altered activities of choline kinase and cytidylyltransferase. The overall rate of phosphatidylcholine synthesis appeared to be increased because the turnover rate of phosphocholine from the intracellular pool was higher in the ras-transfected cells. There also appeared to be an increased rate of phosphatidylcholine degradation in ras-expressing C3H10T1/2 cells. Very high levels of glycerophosphocholine (6-fold increased over control cells) suggested that phospholipase A was activated in these cells. These results indicate that the ras oncogene product directly or indirectly causes an increased turnover of phosphatidylcholine in C3H10T1/2 cells.