bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparative energy metabolism in cultured heart muscle and hela cells
Authors: Stanisz Jolanta , Wice Burton M. , Kennell David E.
Published: February 2005

The yields of energy from oxidation of fatty acids, glucose, and glutamine were compared in cultures of chick embryo heart muscle (heart) and HeLa cells. Aerobic energy production, as measured by oxygen utilization, was comparable in the two cell types. In media containing dialyzed sera, the rates of incorporation of fatty acids directly into lipids were similar in both cells and accounted for > 97% of fatty acid metabolism in HeLa cells. However, in heart cells only 45% ended in lipid, 42% in protein, and 13% was released as CO2; the latter two products probably reflect the oxidation of fatty acids to acetyl‐coenzyme A (‐CoA) and its subsequent metabolism in the citrate cycle. Increased serum concentration in the medium did not affect fatty acid metabolism in HeLa cultures, but resulted in greater oxidation by heart cells (> 100 times that by HeLa cells).

The metabolisms of both glucose and glutamine were similar in heart and HeLa cells with ⩾ 60% of glucose carbon ending as medium lactate and only 3–5% converted to acetyl‐CoA. About 25% of glutamine carbon ended as CO2 and increased utilizations with increasing serum concentrations was accountable in both cells by increased lactate from glucose and glutamate from glutamine. CO2 production (and energy) from glutamine was independent of glutamine concentration within a tenfold range of physiological concentrations.

The yields of energy have been calculated. In 10% dialyzed calf serum, oxidation of glutamine carbon provided about half of the total energy in heart cells, glucose about 35–45%, with most coming from glycolysis; oxidation of fatty acid carbon provided only 5–10%. That > 90% of the aerobic energy comes from glutamine in both cells can account for the comparable rates of oxygen utilization. HeLa cells derived little or no energy from fatty acids.
Hybrids between irradiated and unirradiated mammalian cells: Survival and chromosome segregation
Authors: Zelesco Paula A. , Graves Jennifer A. Marshall
Published: February 2005

We have studied the effect of X or γ irradiation, of one parent of a cell hybrid, on hybrid viability and chromosome segregation. The hybrid types studied were mouse‐Chinese hamster (which spontaneously lose a few hamster chromosomes) and Chinese hamster‐human (which spontaneously lose most of the human complement). Preirradiation of the segregated and retained cell parent resulted in highly asymmetric hybrid survival curves; survival was greatly reduced when the retained parent was irradiated, especially for hamster‐human fusions. Preirradiation of the parents of mouse‐hamster hybrids modified both the direction and the extent of chromosome segregation, but no consistent effect on elimination was observed for hamster‐human hybrids, and reversal of the direction of loss was never observed. These results are more consistent with the hypothesis that chromosome segregation from hybrids results from an intracellular chromosome selection, than with the hypothesis that cellular selection acts on randomly generated chromosome variants.
Isolation and characterization of a Chinese hamster ovary cell mutant which accumulates UDP glucuronic acid and requires uridine for growth
Authors: Patterson David , Vannais Diane B. , Laas William
Published: February 2005

The isolation and characterization of a new pyrimidine requiring mutant of Chinese hamster ovary cells (CHO‐KI, pro) is described. This mutant (P192) shows an absolute requirement for uridine for growth but is fully capable of pyrimidine nucleotide synthesis. P192 cells accumulate exceedingly large amounts of a compound identified by a variety of chromatographic, chemical, and labelling criteria as UDP glucuronic acid (UDPglcUA). This compound comprises approximately 60% of the acid‐soluble nucleotides of P192 cells. Possible genetic mechanisms leading to the phenotype of P192 are examined, and the role of UDPglcUA in cellular metabolism is discussed.
Mitogenic effect of prostaglandin E1 in Swiss 3T3 cells: Role of cyclic AMP
Authors: Rozengurt Enrique , Collins Mary K. L. , Keehan Margaret
Published: February 2005

Addition of prostaglandin E1 (PGE1) to quiescent cultures of Swiss 3T3 cells rapidly elevates the intracellular levels of cAMP and increases the activity of adenylate cyclase in particulate fractions of these cells. In the presence of insulin, PGE1 stimulates the reinitiation of DNA synthesis. Both effects (increase in cellular cAMP and stimulation of DNA synthesis) are markedly potentiated by 1‐methyl‐3‐isobutyl xanthine (IBMX) or by 4‐(3‐butoxy‐4 methoxy benzyl)‐2‐imidazolidine (Ro 20–1724), both of which are potent inhibitors of cyclic nucleotide phosphodiesterase activity. In the presence of 50 μM IBMX, PGE1 caused a dose‐dependent increase in cAMP levels and in [3H]thymidine incorporation into acid‐insoluble material at concentrations (5–50 ng/ml) that are orders of magnitude lower than those used in previous studies (50 μg/ml) to demonstrate growth‐inhibitory effects. Thus, the inhibitory effects produced by adding high concentrations of PGE1 on the initiation of DNA synthesis in Swiss 3T3 cells are not mediated by cAMP and should be regarded as nonspecific. In contrast, the mitogenic activity of PGE1 parallels its ability to increase the intracellular levels of cAMP. The findings support the propostion that a sustained increase in the level of this cyclic nucleotide acts as a mitogenic signal for confluent and quiescent Swiss 3T3 cells.
Change in water proton relaxation time during erythrocyte maturation
Authors: Cameron I. L. , Dung H. C. , Hunter K. E. , Hazlewood C. F.
Published: February 2005

Erythrocyte populations from newborn and mature mice were characterized according to: size; ultrastructural features; water content; concentration of intraerythrocyte elements including Na, Cl, K, P, S, Mg, and Fe; and the spin‐lattice (T1) and spin‐spin relaxation times of water protons as measured by nuclear magnetic resonance (NMR) spectroscopy. A significant increase in the T2 time from 142 ± 3 msec to 184 ± 3 msec occurred during erythrocyte maturation. This change in T2 time was correlated with a change from a polyribosome‐rich hemoglobin‐poor cell type to a polyribosome‐absent hemoglobin‐rich cell type. The change in T2 time could also be correlated to a significantly higher K and P concentration in the mature erythrocytes. The change in T2 time was not correlated to a change in cellular water content or to the concentration of any of the other elements measured by electron probe X‐ray microanalysis. If the NMR relaxation times of water molecules truly reflect their average motional freedom, then the findings suggest that greater water ordering interaction occurs in the ribosome containing immature erythrocyte.
Properties of a cell growth inhibitor produced by mouse embryo fibroblasts
Authors: Wells Valerie , Mallucci Livio
Published: February 2005

Secondary mouse embryo fibroblasts produce a growth inhibitor with the character of a thermolabile, nondialysable protein. The inhibitor was harvested from conditioned medium, and following G‐75 Sephadex fractionation it was isolated in one peak which consisted of two fractions eluting at approximately two thirds of the bed volume of the column where approximately 80 percent of the original activity was recovered with an increase in specific activity of about tenfold. Polyacrylamide gradient gel electrophoresis of fractions from L‐[35S] methionine‐labelled conditioned medium showed that the two fractions with growth inhibitory activity contained some 4–5 bands and shared the two major components. Cell cycle studies showed that the growth inhibitory effect was exerted after addition during early and late G1 and during S phase, and morphological studies showed that where growth was inhibited the morphological expression of the cells was altered.
Optimal conditions for proliferation of bone marrow‐derived mouse macrophages in culture: The roles of CSF‐1, serum, Ca2+, and adherence
Authors: Hume David A. , Gordon Siamon
Published: February 2005

A method is described for the analysis of [3H]‐thymidine incorporation in microtitre cultures of bone marrow‐derived mouse macrophage responding to macrophage colony‐stimulating factor (CSF‐1). [3H]‐thymidine incorporation depends on cell density, culture medium, and the concentration of CSF‐1 and serum, but is independent of Ca2+. Bone marrow‐derived macrophages are strongly adherent, but adherence can be dissociated from [3H]‐thymidine incorporation.
Serum stimulates na entry and the Na‐K pump in quiescent cultures of epithelial cells (MDCK)
Authors: Reznik Vivian M. , Villela Javier , Mendoza Stanley A.
Published: February 2005

The growth of an epithelial canine kidney line (MDCK) was reversibly arrested by gradually lowering the serum concentration in the medium over a 3‐day period. The cells were demonstrably quiescent by autoradiography after an additional 24 hours in serum‐free media. Addition of fresh serum produced DNA synthesis after an 18‐hour lag period. The quiescent cells then grew to confluency retaining their transport capacities as seen by the formation of “domes”. This system allows for measurement of monovalent ion fluxes and its relationship to growth regulation. The addition of fresh serum to quiescent MDCK cells increased the uptake of 86Rb, a measure of Na‐K pump activity. This stimulation was mediated by increased uptake of Na into the cells. Serum‐stimulated DNA synthesis was blocked by the addition of ouabain in concentrations that inhibit the Na‐K pump. Serum appears to stimulate growth in epithelial cells by increasing the amount of intracellular Na available to the Na‐K pump. Monovalent ion transport may play a role in the regulation of epithelial cell proliferation.
Glycerol transport and phosphorylation by rat hepatocytes
Authors: Li Chien‐Chung , Lin E. C. C.
Published: February 2005

The entry of glycerol into isolated rat hepatocytes appears to be catalyzed by a specific carrier. At a physiological concentration of 0.1 mM, glycerol utilization is rate limited by the permeation step. Intracellular glycerol is trapped by an excess of glycerol kinase, which has a higher apparent affinity for the substrate than that of the membrane carrier. The entry of glycerol into the hepatocytes is highly sensitive to inhibition by monoacetin and cytochalasin B, but not by DL‐1,2‐propanediol, erythritol, D‐glucose, D‐galactose, D‐mannose, or D‐fructose.
Autocrine and paracrine growth stimulation of cells derived from human skin
Authors: Gilchrest Barbara A. , Karassik Rita L. , Wilkins Leon M. , Vrabel Michael A. , Maciag Thomas
Published: February 2005

A sensitive serum‐free culture system was used to demonstrate that cells derived from normal human skin release soluble mediators which can modulate keratinocyte, melanocyte, and fibroblast growth in vitro. In M199 supplemented with epidermal growth factor, hydrocortisone, insulin, transferrin, triidothyronine, bovine serum albumin, and an extract of bovine hypothalamus, keratinocytes underwent approximately three to five cumulative population doublings (CPD) over a 7‐day period. Addition of 20% keratinocyte‐conditioned medium more than doubled keratinocyte growth and increased fibroblast growth 25–40% above controls. Dermal fibroblasts maintained in the same serum‐free hormone‐supplemented medium (SFHSM) alone underwent approximately 4.5–5.5 CPD over a 7‐day period; 20% fibroblast‐conditioned medium increased fibroblast growth 50–80% and increased keratinocyte growth 30–50%. Melanocytes maintained in the same SFHSM underwent approximately 1 CPD over a 14‐day period and 2 CPD if the medium was supplemented with 2% fetal bovine serum (FBS). Addition of 20% melanocyte‐conditioned medium more than doubled melanocyte growth in either SFHSM or in medium containing 2% FBS, but decreased both keratinocyte and fibroblast growth up to 30–60%. None of the conditioned media altered cellular morphology. These data provide the first demonstration of mutual growth modulation by cell types normally contiguous in vivo and expand existing evidence for autocrine and paracrine growth regulation by normal human cells.
Regulation and drug insensitivity of f‐actin association with adhesion areas of transformed cells
Authors: Carley William W. , Lipsky Mary G. , Webb Watt W.
Published: February 2005

F‐actin aggregates have been found near the substrate attachments in a variety of transformed cells (Carley et al., 1981). Interference reflection microscopy shows that these aggregates are present in central close adhesion areas in Rous sarcoma virus (RSV)‐transformed rat kidney cells. If these transformed cells are incubated with N6, O2‐dibutyryl 3′:5′‐cyclic monophosphoric acid (db‐cAMP), adenosine (5′‐monophosphoric acid (5′‐cAMP) or adenosine, the F‐actin aggregates and their associated close adhesion areas disappear, and the cells flatten out. Treatment of untransformed cells with db‐cAMP spreads their local adhesion plaques and thickens microfilament bundles. Furthermore, f‐action aggregates are substantially more resistant to cytochalasin B and the Ca2+ ionophore A23187 than microfilament bundles in untrasformed cells. These differences between F‐acttin complexes in untransformed and in RSV‐transformed cells, with respect to morphology and sensitivities to db‐cAMP and cytoskeleton‐disrupting drugs, define properties of the change in F‐actin regulation and association with the plasma membrane due to transformation.
Immunofluorescent visualization of specifically bound thrombin reveals cellular heterogeneity in number and density of preclustered receptors
Authors: Carney Darrell H.
Published: February 2005

Thrombin binding to formaldehyde‐fixed mouse embryo (ME) cells was visualized by indirect immunofluorescence as a dot‐like pattern with dots of approximately 500 nm diameter located over the entire cell surface. Experiments comparing the binding of 125I‐thrombin and dot appearance on parallel cultures indicate that the immunofluorescent pattern is specific for thrombin‐binding to high‐affinity receptors. Similar patterns were observed on cells fixed in ethanol or glutaraldehyde prior to thrombin binding and on cells maintained at 4°C. These patterns were also observed in a number of established cell lines. Thus, thrombin receptors may be clustered prior to thrombin binding on all cells with these receptors. Comparing the amount of 125I‐thrombin bound to CHO cells with the number of fluorescent dots per cell indicated that each dot represents a cluster of over 1000 receptors. On ME cells, the number of thrombin receptor clusters per cell ranged from fewer than 50 to over 5,000. Based on previous studies, this indicates that on ME cells each cluster contains an average of approximately 200 thrombin binding sites with some cells having few, if any, receptors and others having more than a million receptors per cell.
Rat esophageal and epidermal keratinocytes: Intrinsic differences in culture and derivation of continuous lines
Authors: Heimann Ruth , Rice Robert H.
Published: February 2005

Serially cultivated with 3T3 feeder layer support as colonies of stratified squamous epithelium, rat epidermal and esophageal epithelial cells were readily distinguishable by three critera. First, the epidermal colonies, exhibiting extensive piling up of squarnes in the centers, were more stratified than esophageal colonies. Second, in sparse culture 70 to 90% of the esophageal cells but as few as 1 to 5% of the epidermal cells were competent in cross‐linked envelope formation upon treatment with the ionophore X537A. After reaching confluence, up to 90% of the cells of both types formed envelopes upon ionophore treatment. Third, epidermal cells in suspension culture reached maximal levels of spontaneously cross‐linked envelopes in 1 day or less, while esophageal cells required about 4 days in suspension to reach maximal levels. A reproducible finding with both cell types was that initial colony‐forming efficiencies of less than 1% increased to about 40% upon serial passage with consequent derivation of continuous lines. Sparse cultures of esophageal cells with high colony‐forming ability retained a high degree of envelope competence (70 to 90%), indicating these two properties are not mutually exclusive. The derived lines exhibited reduced dependence upon feeder layer support at clonal density, but in suspension culture the cells did not grow and lost colony‐forming ability with a half‐time of several hours. We conclude that cells from these keratinized rat epithelia exhibit intrinsic differences in culture and become continuous lines expressing characteristic regulation of envelope competence and loss of germinative capability in suspension.
Metabolic requirements for maintenance of the chlortetracycline‐labeled pool of membrane‐bound calcium in human neutrophils
Authors: Smolen James E. , Noble Paul , Freed Richard , Weissmann Gerald
Published: February 2005

Human neutrophils labeled with chlortetracycline (CTC), commonly used as a probe of membrane‐bound calcium, release lysosomal enzymes and exhibit a rapid decrease in fluorescence when exposed to the chemotactic peptide fMet‐Leu‐Phe or the lectin Con A. This decrease has been attributed to the release of calcium from a membrane‐associated “trigger pool.” The nature of this putative pool has been further characterized by examining the effects of various inhibitors on the CTC fluorescence response and lysosomal enzyme release from stimulated neutrophils. These agents included inhibitors of glycolysis (2‐deoxyglucose and iodoacetate), an uncoupler of oxidative‐phosphorylation (KCN), and a sulfhydryl inhibitor (N‐ethylmaleimide). Resting neutrophils, labelled with CTC demonstrated an enhanced decay of baseline fluorescence when exposed to 2‐deoxyglucose or iodoacetate. This suggested that the pool of membrane‐bound calcium labeled by this probe was maintained by glycolytic metabolism. Furthermore, 2‐deoxyglucose and iodoacetate inhibited both the stimulated decrease in CTC fluorescence and lysosomal enzyme release induced by fMet‐Leu‐Phe and Con A in a time‐dependent manner. KCN did not inhibit either response to stimulation, but did retard the recovery of CTC fluorescence observed when fMet‐Leu‐Phe was used as the stimulus. High concentrations of N‐ethylmaleimide (100 μM) completely inhibited both the CTC fluorescence response and lysosomal enzyme release almost immediately; low concentrations of N‐ethylmaleimide (30 μM) inhibited lysosomal enzyme release in a time‐dependent manner without significantly affecting changes in CTC fluorescence. These results are consistent with the hypothesis that CTC serves as a probe of membrane‐bound “trigger” calcium, the release of which is dependent upon intact glycolysis and is a requirement for lysosomal enzyme release.
Histone protein and DNA synthesis in HeLa cells after thermal shock
Authors: Warters Raymond L. , Lee Stone O.
Published: February 2005

Exposure of suspension‐cultured HeLa cells to a 45° thermal shock resulted in cell inactivation and inhibition of both protein and DNA synthesis. DNA synthesis was inhibited in a biphasic manner with a more sensitive (D0 = 7 min) and a less sensitive (D0 = 20 min) phase. The less sensitive process was demonstrated to be DNA chain elongation. Transport of thymidine into intracellular pools was significantly less sensitive to thermal shock (D0 in excess of 200 min). When HeLa cells were heated at 45° for 15 min there was an 80% inhibition of incorporation of precursors into both DNA and protein with little effect on precursor transport into cellular pools. While the rate of synthesis of whole cell and histone protein (H2a, H2b, H3, and H4) and DNA chain elongation recovered by 6 h after cell heating, total precursor incorporation into DNA was only 0.4 of control levels. The long‐term depression of the DNA synthetic rate could not be explained by a cell cycle redistribution, a depression in the total fraction of S phase cells synthesizing DNA, or by a depression in the rate of DNA chain elongation. We conclude that thermal shock results in a long‐term depression in the fraction of cell replicons involved in DNA replication.
Colchicine prevents the translation of mRNA molecules transcribed immediately after proliferative activation of hepatocytes in regenerating rat liver
Authors: Walker P. Roy , Whitfield J. F.
Published: February 2005

A dual‐labelling technique has been used to establish that partial hepatectomy has no effect on the degradation of poly (A)+ mRNA and confirms that the increased incorporation of precursor into mRNA during early prereplicative development reflects an actual increase in mRNA biosynthesis. Simultaneous studies on the changes in nuclear RNA metabolism support the conclusion that an increase in gene transcription does occur. Colchicine, at concentrations known to disrupt microtubules, has no effect on this increase in gene transcription but prevents the translation of the gene products by promoting polysome disaggregation transiently during a critical stage of prereplicative development. Studies with mefenamic acid and hydrocortisone, specific inhibitors of prostaglandin metabolism, have ruled out any involvement of prostaglandins in the induction of prereplicative mRNA synthesis.
A kinetic and clonal analysis of heterogeneity in K562 cells
Authors: Alder Susan , Ciampi A. , McCulloch E. A.
Published: February 2005

Expression of markers of differentiation was measured in a clone of the continuous cell line K562, derived originally from the cells of a patient with leukemia. Three of the markers were lineage specific, R18 for erythropoiesis and 80H.5 and My‐1 for granulopoiesis. The fourth marker was the self‐renewal capacity of clonogenic cells. The markers were measured as a function of time in pooled colonies from day 2 to day 12, and at a point of time in individual colonies. Evidence of an orderly pattern of marker appearance and disappearance was not seen. Rather, their expression appeared to occur at random during growth.
Prereplicative changes in the soluble calmodulin of isoproterenol‐activated rat parotid glands
Authors: Gonzales R. Campos , Whitfield J. F. , Boynton A. L. , MacManus J. P. , Rixon R. H.
Published: February 2005

Cells of rat parotid glands were maximally stimulated to initiate DNA synthesis by injecting into the animal a single dose of 25 to 150 mg of isoproterenol/ kg of body weight. During the 18‐ to 21‐hr prereplicative period following injection of the highest dose of the drug, there were two predominant and transient redistributions of calmodulin from the bound to the soluble form, which tripled the level of soluble calmodulin at 3 hr and again at 18 hr just before the initiation of DNA synthesis. A small (50%) increase in total calmodulin was observed only during the early (3‐h) prereplicative surge of soluble calmodulin. The late, pre‐DNA‐synthetic surge of soluble camodulin and the initiation of DNA synthesis were both prevented in rats that lacked their parathyroid‐thyroid gland complex and had been hypocalcemic for 48 or 72 hr. Unlike the effect of high doses of isoproterenol, low doses (e.g., 25 mg/kg body weight) of the β‐adrenergic drug could maximally stimulate DNA synthetic activity without the later pre‐DNA‐synthetic surge of soluble calmodulin, suggesting that any apparent correlation between the level of calmodulin and DNA synthesis may be spurious and that an actual increase in the level of soluble calmodulin just before the onset of DNA synthesis was not a prerequisite for DNA synthetic activity in parotid cells.
The role of Protein breakdown in growth, quiescence, and starvation of vascular smooth muscle cells
Authors: Libby Peter , O'Brien Kathleen V.
Published: February 2005

Protein accumulation in growing cells may be due in part to a reduction in the rate of protein breakdown. Previous studies of the relation of cell proliferation to protein degradation often produced growth arrest by conditions that may involve nutritional deprivation. However, nutrient lack can itself accelerate proteolysis and produce negative protein balance. We therefore reexamined the relation between growth and protein breakdown using a more selective method for limiting cell growth. We produced quiescent cell cultures using a chemically defined, serum‐free medium supplemented with hormones and nutrients. Such media can maintain viability and near neutral protein balance in cultured vascular smooth muscle cells, in part because of reduced breakdown of cellular protein. We then compared rates of protein degradation in these quiescent but not starving cells, to those of cultures stimulated to grow by addition of mitogenic substances. Platelet‐derived growth factor, fibroblast growth factor, or fetuin added to insulin‐containing medium stimulated growth of smooth muscle cells, but further reduced protein breakdown only slightly. Contrary to the implications of certain previous studies, our results show that proliferating cells can accumulate protein without an appieciable reduction in the rates of protein breakdown. Thus, while accelerated proteolysis appears to be an important adaptation to adverse nutritional conditions, growth of smooth muscle cells does, not require changes in overall protein breakdown, but occurs primarily through an increase in protein synthesis.
Galactosyltransferase activity and cell growth: Uridine diphosphate (UDP)galactose inhibition of murine leukemic
Authors: Klohs Wayne D. , Wilson James R. , Weiser Milton M. , Frankfurt Oscar , Bernacki Ralph J.
Published: February 2005

UDPgalactose inhibits the growth of mouse leukemic L1210 cells. In calf serum supplemented Dulbecco's medium (CS‐DMEM), 1.2 mM UDPgalactose (UDPgal) inhibited cell growth by 50% (IC50), and 5 mM UDPgalactose inhibited cell growth by 92%. Other nucleotide sugars as well as galactose, glucose, and galactose‐1‐phosphate had little or no effect on cell growth. Uridine nucleotides, which inhibit galactosyltransferase activity, protected L1210 cells from the growth inhibitory effect of UDPgalactose when both were added simultaneously to culture media. Unlike mouse 3T12 cells, in which no inhibition of cell growth was observed with heat‐inactivated calf serum (HICS)‐DMEM, 5 nM UDPgalactose inhibited L1210 cell growth in HICS‐DMEM to the same degree as that observed in CS‐DMEM. In contrast to 3T12 cells, L1210 cells secrete significant galactosyltransferase activity into the media. Complete inhibition of 3T12 cell growth by UDPgal was observed if HICS‐DMEM medium was first conditioned by L1210 cells for 48 hours. No difference in cell growth or [3H]thymidine uptake was detected after 6 hours of exposure to UDPgalactose, but both were significantly decreased at 24 and 48 hours. Flow cytometric analysis of UDPgalactose effects on L1210 cells revealed no differences in the distribution of cells in G1, S, or G2‐M of the cell cycle after 6 hours of incubation, but after 16 hours of UDPgalactose treatment, L1210 cells were arrested in early S phase. These cells were completely viable and morphologically similar to control L1210 cells. Normal growth was resumed when UDPgal was removed. The data suggest that UDPgalactose inhibition of cell growth requires extracellular galactosyltransferase activity and that the effect is mediated via the cell membrane.
Altered methionine metabolism occurs in all members of a set of diverse human tumor cell lines
Authors: Stern Peter H. , Wallace C. Douglas , Hoffman Robert M.
Published: February 2005

Methionine dependence is a metabolic defect found thus far only in transformed and malignant cells. The defect is manifested as the inability of cells to grow in media in which methionine (Met) is replaced by its immediate precursor homocysteine (Hcy). We have termed this Met − Hcy + media. We demonstrate here that methionine‐dependent cells derived from human tumors, compared to normal methionine‐independent cells, have low levels of free Met, low levels of S‐adenosylmethionine (AdoMet) and elevated levels of S‐adenosylhomocysteine (AdoHcy) when incubated in Met − Hcy + medium. Methionine‐independent human tumor cells also have very low levels of free Met compared to normal cells but generally have levels of AdoMet and AdoHcy comparable to normal cells in Met − Hcy+ medium. All tumor cell types incorporate amounts of Met into protein similar to normal methionine‐pindependent human fibroblasts when incubated in Met − Hcy+ medium, thereby indicating apparently normal levels of Met synthesis in the tumor cells. The methionine‐independent tumor cell lines in Met − Hcy+ medium seem able to regulate their AdoMet/AdoHcy ratios normally despite this defect in having very low levels of free Met. Thus, in a diverse set of human tumor cell lines, all are defective in at least one aspect of Met metabolism, giving rise to the possibility of a general metabolic defect in cancer.
Prostaglandin F2α stimulates phosphatidylinositol turnover and increases the cellular content of 1,2‐diacylglycerol in confluent resting swiss 3T3 cells
Authors: Macphee Colin H. , Drummond Alan H. , Otto Angela M. , de Asua Luis Jimenez
Published: February 2005

Prostaglandin F2α(PGF2α); which stimulates DNA synthesis in resting 3T3 cells, also stimulates the incorporation of [32P]PO4 into phosphatidylinositol. The effect is selective for PGF2α when compared with PGE1, PGE2, and PGF2α. Epidermal growth factor (EGF) also stimulates DNA synthesis but does not affect phosphatidylinositol turnover. PGE1, which acts synergistically with PGF2α to enhance DNA synthesis, does not affect the ability of PGF2α to enhance the incorporation of [32P]PO4 into phosphatidylinositol. PGF2α also causes a small increase in the cellular content of 1,2‐diacylglycerol. This effect is not shared by EGF or PGE1. Stimulation of phosphatidylinositol metabolism resulting in an increase in the cellular content of 1,2‐diacylglycerol may thus constitute an event in the pathway leading to the initiation of DNA synthesis in which PGF2α differs in its action from EGF.
Fibroblast spreading and phagocytosis: Similar cell responses to different‐sized substrata
Authors: Grinnell Frederick
Published: February 2005

Experiments were carried out to test the hypothesis that cell spreading and phagocytosis are similar cell responses to different‐sized substrata. The following morphological and biochemical studies provided evidence for this supposition. Cells phagocytosed 1.09‐μm and 5.7‐μm latex beads, but were unable to completely ingest 15.8‐μm or 25.7‐μm beads. With the larger beads, the cells spread around the bead surfaces with an appearance typical of cells spread on culture dishes. Biochemical studies with cytochalasin D, azide, and iodoacetate, as well as temperature‐dependence studies, demonstrated similar responses of cell spreading and phagocytosis to these treatments. Similar cell surface receptors were involved in cell spreading and phagocytosis based upon experiments using antibodies to baby hamster kidney (BHK) cell wheat germ agglutinin receptors. And finally, BHK cell variants with defective plasma fibronectin (pFN) receptors were unable to spread on pFN‐coated dishes or ingest pFN‐coated beads. Evidence also is presented concerning the “contact” process in cell adhesion. It was found that azide and low temperature inhibited cell attachment per se but did not block fibronectin‐receptorinter actions based upon cell binding of pFN‐coated beads. A possible explanation for the contact process is presented based upon the resistance of cells and beads to shear forces.
Phenothiazines cause a shift in the cAMP dose‐response: Selection of resistant variants in a murine thymoma line
Authors: Gruol Donald J. , Dalton Dyana K.
Published: February 2005

Cyclic AMP and glucocorticoid hormones each promote a cytolytic response in the murine lymphoid cell line WEHI‐7.1 (W7). The sensitivity to dibutyryl cyclic AMP (dbcAMP), but not dexamethasone, can be enhanced by several psychotropic drugs that have the capacity to interfere with a variety of calcium‐regulated functions. We have characterized the response of W7 cells to the phenothiazine trifluoperazine (TFP) and found that TFP concentrations, which decreased the growth rate by twofold, shifted the dose response to dbcAMP approximately tenfold. A similar but smaller shift was seen with the phosphodiesterase inhibitor methylisobutylxanthine (MIX). The effects of TFP and MIX on the dbcAMP response were additive, suggesting that TFP may act to increase the sensitivity of W7 cells to the action of cAMP‐dependent protein kinase, and that the increased sensitivity may be due to altered lytic functions coregulated by both cAMP and calcium. The change in the dose response to dbcAMP caused by TFP provided a means of selection to obtain variants that were altered in their capacity to respond to dbcAMP, TFP, or the combination of the two drugs. Eight (out of 36) of the lines that were obtained after a mutagenesis and combined drug selection have been partially characterized. This has revealed the existence of several new phenotypes. Most have an altered response to dbcAMP in the presence of TFP and are likely to represent variants that have not been observed before.
An improved procedure for the isolation of glia maturation factor
Authors: Lim Ramon , Miller Joyce F.
Published: February 2005

A procedure for the bulk isolation of glia maturation factor (GMF) in high yield and high purity from bovine brains is outlined. The method involves extraction by homogenization and centrifugation, followed by ammonium sulfate precipitation and column chromatography with diethylaminoethyl (DEAE) Sephacel, Sephadex G‐75, and hydroxylapatite. The method results in a 10,000‐fold purification, a purity exceeding that of previously published procedures, and enables us to handle as much as 2.8 kg brain tissue or eight brains/week. The ability to mass‐produce GMF with this method greatly facilitates its biological studies, further purification, and chemical characterization. The isolated GMF shows a molecular weight of 13,000 on Bio‐gel P‐30 column and an isoelectric point of about 5.4 on isoelectric focusing. The isolated GMF is heat labile and susceptible to papain and ficin but relatively resistant to trypsin, neuraminidase, and endoglycosidase.
Relationship of muscle growth in vitro to sodium pump activity and transmembrane potential
Authors: Vandenburgh Herman H.
Published: February 2005

Serum stimulates embryonic avian skeletal muscle growth in vitro and the growth‐related processes of amino acid transport and protein synthesis. Serum also stimulates myotube Na pump activity (measured as ouabain‐sensitive rubidium‐86 uptake) for at least 2 h after serum addition. Serum‐stimulated growth depends on this Na pump activity since ouabain added at the same time as serum totally inhibits the growth responses. The relationship of myotube growth, Na pump activity, and transmembrane potential was studied to determine whether serum‐stimulated Na pump activation and growth are coupled by long‐term membrane hyperpolarization. When myotube amino acid transport and protein synthesis are prestimulated by serum, ouabain was found to have little inhibitory effect, indicating that the already stimulated growth‐related processes are not tightly coupled to continued Na pump activity. Serum‐stimulated protein synthesis is tightly coupled to Na pump activity, but only during the first 5–10 min after serum addition. When myotube transmembrane potentials were measured using the lipophilic cation tetraphenylphosphonium, serum at concentrations that stimulate myotube growth and Na pump activity was found to have little effect on the cell's transmembrane potential. Furthermore, partial depolarization of the myotubes with 12‐ to 55‐mM extracellular potassium does not prevent serum stimulation of myotube growth. Monensin was found to hyperpolarize the myotubes, but causes myotube atrophy. These results indicate that although Na pump activity is associated with initiation of serum‐stimulated myotube growth, continued Na pump activity is not essential, and there is little relationship between myotube growth and the myotube's transmembrane potential.
Heterogeneity of endothelial cells of adult rat liver as resolved by sedimentation velocity and flow cytometry
Authors: Morin O. , Patry P. , Lafleur L.
Published: February 2005

Sinusoidal cells isolated from adult rat liver were fractionated by velocity sedimentation at 1 × g (primarly on the basis of size) and the various cell fractions were further analysed by flow cytometry on the basis of forward and perpendicular light scattering and autofluorescence. Cell volume was also measured electronically using a Coulter counter. At least four enriched cell populations were resolved after velocity sedimentation. They corresponded to cells having a modal diameter of 6.5, 7.5, 9, and 11 μm, respectively. Transmission electron microscopy (TEM) analysis of the various cell populations revealed that the 7.5‐ and 9‐μm cell fractions represented two distinct classes of endothelial cells while the 11‐μm cells corresponded to Kupffer cells. The 6.5‐μm cells were identified as lymphocytes. Fat‐storing cells, identified by their autofluorescence and lipid content, were included in the Kupffer population. Further information about the nature of the two physically distinct endothelial cell populations was obtained by TEM. It demonstrated that the smaller endothelial cells possessed quantitatively and relatively less retracted sieve plates than the larger ones. This ultrastructural feature can be possibly correlated to a differential localization of the two classes of endothelial cells within the liver acinus.
Cultured fetal rat myoblasts release peptide growth factors which are immunologically and biologically similar to somatomedin
Authors: Hill David J. , Grace Caroline J. , Fowler Lindsay , Holder Andrew T. , Milner R. D. G.
Published: February 2005

The production of immunologically and biologically active somatomedin activity from isolated myoblasts and fibroblasts from fetal rats of 21 days gestational age was investigated. Myoblast‐rich cell populations were derived from primary cultures of dispersed muscle cells by the tendency of myoblasts to become detached from the culture dish in the presence of cytochalasin B. Fibroblasts were obtained from fetal muscle. Culture medium conditioned by exposure to myoblasts for 48 hours produced an increased incorporation of both [35S]sulphate and [3H]thymidine by explants of fetal rat costal cartilage in vitro compared to fresh medium. Myoblast‐conditioned medium also contained somatomedin‐C‐like immunoreactivity which diluted in parallel with partially purified human somatomedin‐C (3,271 ± 446 mU/mg cell protein; mean ± SEM, seven experiments). Medium conditioned by exposure to fetal rat fibroblasts did not promote isotope uptake by fetal rat cartilage above control values, and contained only low levels of somatomedin‐C‐like immunoreactivity (343 ± 89 mU/mg cell protein, three experiments). The release of both somatomedin bioactivity and immunoreactivity into conditioned medium was significantly reduced by the incubation of myoblasts in the presence of rat growth hormone (100 ng/ml and 500 ng/ml). We conclude that fetal rat myoblasts released growth factor activity during culture which exhibited biological and immunologic characteristics of somatomedin. Since the bioactivity was demonstrated on skeletal tissues from rat fetuses of the same gestational age as those that yielded myoblasts such growth factor release may be physiological.
Rapid changes in bidirectional K+ fluxes preceding DMSO‐induced granulocytic differentiation of HL‐60 human leukemic cells
Authors: Gargus J. J. , Adelberg E. A. , Slayman C. W.
Published: February 2005

When grown in medium containing 5 mM potassium and 140 mM sodium, HL‐60, a human promyelocytic cell line, maintained a steady‐state intracellular K+ concentration of 145 mmol/L cells and a steady‐state intracellular Na+ concentration of 30 mmol/L cells. Nearly 90% of the unidirectional 42K+ influx could be inhibited by the cardiac glycoside ouabain with a Ki of 5 × 10−8 M. This ouabain‐sensitive component of influx rose as a saturating function of the extracellular K+ concentration with a K1/2 of 0.85 mM. The component of 42K+ influx resistant to ouabain inhibition was a linear function of the extracellular K+ concentration and was insensitive to inhibition by the diuretic furosemide. Unidirectional K+ efflux followed first order kinetics with a half‐time of 55 min. Addition of 1.5% dimethyl sulfoxide (DMSO) to a culture of HL‐60 cells allowed two population doublings followed by the cessation of growth without an impairment of cell viability. Beginning 2 to 3 days after DMSO addition, the cells underwent a dramatic reduction in volume (from 925 μm3 to 500 μm3) and began to take on the morphological features of mature granulocytes. Throughout this process of differentiation there was no change in the intracellular sodium or potassium concentration. However, immediately following the addition of DMSO to a culture of cells, there began an immediate, coordinated reduction in bidirectional K+ flux. The initial rate of the ouabain‐sensitive component of K+ influx fell with a half‐time of 11 h to a final rate, at 6 days induction, equal to one ninth that of the uninduced control, and over the same period, the rate constant for K+ efflux fell with a half‐time of 14 h to a final value one fourth that of the uninduced control. The rapidity with which these flux changes occur raises the possibility that they play some role in the control of subsequent events in the process of differentiation.
Control of enzyme activity levels by serum and hydrocortisone in neonatal rat heart cells cultured in serum‐free medium
Authors: Freerksen Deborah L. , Schroedl Nancy A. , Hartzell Charles R.
Published: February 2005

A serum‐free, hormone‐supplemented medium (SFHM) for maintaining neonatal rat heart cells in culture has been developed in this laboratory (Mohamed et al., 1983). Morphological assessment of heart cells grown in SFHM show it to be similar to commonly used serum‐supplemented media. To quantitatively compare cell behavior in SFHM with serum‐supplemented media, the activities of ten regulatory enzymes which represent four metabolic pathways were studied in heart cells cultured in SFHM. The enzyme activities which were measured included hexokinase, glucose‐6‐phosphate dehydrogenase, 6‐phosphogluconate dehydrogenase, phosphofructokinase, pyruvate kinase, NAD+‐linked sn‐glycerol‐3‐phosphate dehydrogenase, malate dehydrogenase, NAD+‐linked isocitrate dehydrogenase, NADH‐cytochrome c reductase, and succinic cytochrome c reductase. Rat heart cells maintained in culture on SFHM are not only qualitatively and quantitatively similar to those maintained in serum‐supplemented medium but also provide a more suitable model system for metabolic studies of neonatal cardiac tissue for several reasons: (1) many enzyme activities that may represent dedifferentiation are elevated by serum; (2) NAD‐linked glycerol‐3‐phosphate dehydrogenase activity in cells maintained on SFHM is similar to the in vivo activity; (3) cells beat at or near the in vivo frequency and can be maintained 3 months on SFHM; (4) the SFHM is chemically defined and thus can be completely manipulated by the investigator. The effects of three concentrations of hydrocortisone (HC) (5,000 ng/ml, 50 μg/ml, 0 ng/ml) on heart cells cultured in SFHM supported our previous conclusion that function (beating) and growth (protein accumulation) are inversely related in cultured neonatal rat heart cells.
25‐Hydroxycholesterol‐induced elevation in 45Ca uptake: Correlation with depressed DNA synthesis
Authors: Boissonneault Gilbert A. , Heiniger Hans‐Jörg
Published: February 2005

The mechanism whereby 25‐hydroxycholesterol, an inhibitor of the synthesis of cholesterol, depresses DNA synthesis in cycling P815 mastocytoma cells was investigated. The uptake of 45Ca into P815 cells treated with 1 μg/ml 25‐hydroxycholesterol began to rise above control levels by 6 hours after initiation of treatment and was increased tenfold by 15 hours. Kinetic data of calcium uptake indicated the presence of at least two components of calcium uptake, fast and slow. The fast phase of calcium exchange at the cell surface was changed little by treatment with 25‐hydroxycholesterol. The slow phase of calcium exchange with the intracellular compartment was markedly affected by treatment with the inhibitor, there being a large increase in the flux and half‐time of uptake, and a fall in the rate constant. This resulted in a large elevation of the intracellular compartment size. Incorporation of [3H]thymidine into DNA began to decline between 9 and 12 hours posttreatment in these cultures. Uptake of calcium and depression of DNA synthesis were shown to be directly reiated to the dose of 25‐hydroxycholesterol used. The changes in 45Ca uptake and DNA synthesis due to 25‐hydroxycholesterol treatment were abolished by addition of exogenous cholesterol to the incubation medium. The results are consistent with the hypothesis that 25‐hydroxycholesterol, by inhibiting cholesterol production, depresses DNA synthesis via an elevation in the uptake of calcium into the cell to a level incompatible with continued DNA replication.
Thermal down‐regulation of exportable rRNA in nuclei
Authors: Wunderlich Frank , Giese Günter , Herlan Gerhard
Published: February 2005

The export of rRNP particles from nuclei isolated from Tetrahymena was investigated after preincubating the nuclei at different temperatures under nonpermissive export‐conditions. We observed a new phenomenon: Temperature elevation from the sublethal cells' growth temperature, 8°C, to the optimal temperature, 28°C, lead to a gradual down‐regulation in the maximal proportion of rRNP particles subsequently exported from nuclei at 28°C. This thermal down‐regulation is apparently not due to qualitative changes in the exported rRNP particles, a derangement in the gross nuclear organization, a degradation and/or nicking of the nuclear rRNA, a gross decomposition of the major nuclear proteins, a random cross‐linking of nuclear components by disulfide bonds, or an elution of nuclear factors possibly required for rRNP export. Moreover, there is a corresponding thermal down‐regulation in nuclear envelope‐free nuclei. Our data indicate that nuclei possess a mechanism that regulates the number of potentially exportable rRNP particles at a level preceding the rRNP passage through the nuclear envelope.
Characterization and partial purification of keratinocyte growth factor from the hypothalamus
Authors: Gilchrest B. A. , Marshall W. L. , Karassik R. L. , Weinstein R. , Maciag T.
Published: February 2005

An extract of bovine hypothalamus is known to be mitogenic for human keratinocytes in vitro. In order to identify the'responsible substance(s), biochemical characterization and subsequent bioassay of the extract in a serum‐free culture system were performed. The keratinocyte growth‐promoting activity of the hypothalamic extract was unaffected by heating (100 C, 10 min); acidification to pH 3.3; or by exposure to lipase, RNAase, or proteolytic enzymes; but was abolished by alkalinization to pH 11. An approximate molecular weight of 1,700 daltons was determined by elution on a calibrated Sephadex G‐25 column, and an approximate pI of 3.5 was determined by isoelectric focusing. Optimal concentrations of the crude extract (150‐300 μg/ ml) increased keratinocyte growth approximately 50‐fold compared to control cultures lacking the extract. Partial purification resulted in a preparation biologically active at 30 ng/ml protein equivalent and was consistent with the presence of a single mitogen which we have termed keratinocyte growth factor (KGF). Mitogenic activity for human melanocytes, dermal fibroblasts, and endothelial cells, present in the crude hypothalamic extract, was lacking in heat‐treated preparations that contained KGF. Optimal concentrations of purified epidermal growth factor and ethanolamine, the only remotely similar substances previously reported to augment keratinocyte growth in vitro, could not substitute for KGF in the serum‐free culture system. Keratinocyte growth‐promoting activity comparable to that observed in bovine hypothalamic extracts was present in human hypothalamic extracts prepared in the same manner.