bluuebunny/crossref_metadata_2025_split huggingface dataset:
Irreversible reduction in potassium fluxes accompanies terminal differentiation of human myoblasts to myotubes
Authors: Panet Rivka , Digregorio Debra M. , Brown Robert H. 
Published: February 2005

AbstractPotassium and sodium fluxes believed to be important in the cellular response to serum and growth factors have not been widely investigated in cells which have undergone terminal differentiation. In this study we have analyzed two main K+ transport systems—the ouabain‐sensitive Na+/K+ pump and the bumetanide‐sensitive transporter—in human muscle in vitro at two developmental stages: proliferating myoblasts and differentiated myotubes. Myoblast differentiation to myotubes was accompanied by a marked decrease in both the ouabain‐sensitive and the bumetanide‐sensitive K+ (Rb+) influxes. The addition of serum to the terminally differentiated myotubes had no effect on these K+ transporters. However, serum addition to serum‐deprived, undifferentiated myoblasts produced a marked stimulation of these K+ fluxes.The bumetanide‐sensitive K+ transporter in human myoblasts and myotubes has the following properties: (1) It carries 30% and 40% of the total K+ influx in myoblasts and myotubes, respectively. (2) It performs net efflux of K+ in the undifferentiated myoblasts and zero net flux (self‐exchange) in terminally differentiated myotubes. (3) It is dependent on extracellular Na+ and Cl− in addition to K+. (4) In myoblasts, the Km value for K+ is 1.36 mM, similar to the Km for K+ of the Na+/K+ pump. (5) It is resistant to ouabain (up to 2 mM) and sensitive to furosemide (K0.5 = 5 × 10−6 M) and bumetanide (K0.5 = 10−7 M). These data indicate that following terminal differentiation of proliferating myoblasts to mitotically inactive myotubes there is an irreversible reduction of K+ fluxes with a change in the net flux of K+ carried by the bumetanide‐sensitive transporter.
Parallel changes in amino acid transport and protein kinase C localization in LLC‐PK1 cells treated with TPA or diradylglycerols
Authors: Dawson W. David , Cook John S. 
Published: February 2005

AbstractProtein kinase C is considered to be a major target for tumor promoting phorbol esters such as 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA). We have analyzed the correlation between A‐system amino acid transport and the distribution of protein kinase C (PKC) between a membrane‐rich fraction (100,000 g pellet) and cytosol (supernatant) from homogenized LLC‐PK1 cells, a pig kidney epithelial cell line grown in culture. During log growth 1 day after seeding the cells onto culture plates, PKC activity is high in the membrane fraction and low in the cytosol. As the cells become confluent the PKC distribution shifts to a cytosolic pool. Concomitantly, A‐system amino acid transport, as measured by methylaminoisobutyric acid [14C]MeAIB uptake, decreases. TPA (0.01–1.0 μM) induces a shift of PKC activity from the cytosol back to the membrane‐rich fraction in post‐confluent cells with a concomitant 2–3 fold stimulation of MeAIB uptake. The same responses can be achieved by treating cells with certain diradylglycerols, either diacylglycerols such as 1‐oleyl‐2‐acetyl‐sn‐glycerol (OAG) or alkylacylglycerols such as 1‐hexadecenyl‐2‐oleyl‐sn‐glycerol. Both responses to TPA are blocked by cytochalasin B, but cycloheximide inhibits the transport response without affecting PKC redistribution. It is suggested that the redistribution may be a necessary but not sufficient concomitant to the transport activation.
Novel regulatory actions of 1α,25‐dihydroxyvitamin D3 on the metabolism of polyphosphoinositides in murine epidermal keratinocytes
Authors: Tang Wilson , Ziboh Vincent A. , Isseroff R. Rivkah , Martinez D. 
Published: February 2005

AbstractThe in vitro incubation of murine keratinocytes in the presence of 1α,25‐dihydroxyvitamin D3 enhanced the rapid hydrolysis of the prelabeled keratinocyte polyphosphoinositides (polyPtdIns) when compared to untreated cells. The rapid hydrolysis of the polyPtdIns and the release of the inositol phosphosphates (particularly InsP3 and InsP2) precede the onset of differentiation of these cells. These data therefore suggest that 1α,25‐didydroxyvitamin D3 functions in vitro to initiate the rapid generation of InsP3 from cellular polyPtdIns; this in turn may mobilize intracellular Ca2+, thus providing the signal which program the murine keratinocytes from a proliferating mode into a differentiating mode.
Dexamethasone inhibits feedback regulation of the mitogenic activity of tumor necrosis factor, interleukin‐1, and epidermal growth factor in human fibroblasts
Authors: Kohase Masayoshi , Henriksen‐Destefano Dorothy , Sehgal Pravinkumar B. , Vilĉek Jan 
Published: February 2005

AbstractTumor necrosis factor (TNF), interleukin‐1 (IL‐1), and epidermal growth factor (EGF) were mitogenic for human diploid FS‐4 fibroblasts. Dexamethasone amplified the growth‐stimulating action of all three agents. Amplification of the growth‐stimulating action was maximal when dexamethasone was added along with TNF or EGF; no amplification was seen if the addition of dexamethasone was delayed for more than 3 hr. Prolonged simultaneous treatment with TNF and EGF resulted in less growth stimulation than treatment with EGF alone. Dexamethasone abolished this apparent antagonistic interaction between TNF and EGF. Dexamethasone also inhibited the antiviral action of TNF against encephalomyocarditis (EMC) virus in FS‐4 cells. TNF and IL‐1 increased the steady state level of interferon (IFN)‐β2 mRNA but failed to induce detectable levels of IFN‐β1 mRNA in FS‐4 cells. Dexamethasone inhibited the increase of IFN‐β2 mRNA levels by IL‐1 or TNF. Inhibition of IFN‐β synthesis is likely to be responsible for the inhibition of the TNF‐induced antiviral state by dexamethasone. Since IFNs suppress cell growth, inhibition of endogenous IFN‐β synthesis may also be responsible for the amplification by dexamethasone of the growth‐stimulating action of TNF and IL‐1. Amplification of the mitogenic action of EGF by dexamethasone appears to be mediated by a different mechanism.
Effects of EGF and calcium on adult parenchymal hepatocyte proliferation
Authors: Eckl Peter M. , Whitcomb Wendi R. , Michalopoulos George , Jirtle Randy L. 
Published: February 2005

AbstractAdult rat hepatocytes were grown in serum‐free medium containing 0.05‐4 mM Ca+ + and 40 ng/ml EGF. After 48 hours of cultivation the mitotic index and the percentage of second division metaphases were determined. The results demonstrated a maximum proliferation response to EGF at a Ca+ + concentration of 0.4 mM. With lower and higher external Ca+ + concentrations the fraction of cells undergoing more than one cell division decreased. At lower Ca+ + concentrations this decrease appears to result from a reduced viability. In contrast, the low response to EGF at higher Ca+ + concentrations—especially in the physiological range—may reflect the influence of Ca+ + on the state of hepatocyte differentiation.
Free sterol metabolism and low density lipoprotein receptor expression as differentiation markers of cultured human keratinocytes
Authors: Williams Mary L. , Mommaas‐Kienhuis Anne‐Marieke , Rutherford Suzanne L. , Grayson Stephen , Vermeer Bert Jan , Elias Peter M. 
Published: February 2005

AbstractIn contrast to most tissues, epidermis and its derivatives appear to lack low density lipoprotein (LDL) receptors and exhibit sterologenesis rates unaffected by circulating lipoprotein (LP) cholesterol content. Since LDL receptors have been demonstrated in both cultured squamous cell carcinoma cells and human foreskin keratinocytes, when maintained in low‐calcium media, LDL receptor expression may be related to keratinocyte differentiation. We compared receptor binding and internalization of LDL‐gold in normal keratinocytes at different stages of growth at physiological calcium concentrations (early, 3–5 days; preconfluent, 6–10 days; postconfluent, 12–17 days), and correlated receptor expression with sterologenesis in LP‐replete vs.‐depleted media. Whereas in early cultures about 60% of sterologenesis was LP dependent, this fraction declined in preconfluent and confluent cultures despite continued culture growth and little decline in total sterologenesis. Accordingly, LDL receptors were most evident in early cultures, declining in preconfluent cultures in parallel with the decrease in LP‐dependent sterol synthesis. In contrast, sterologenesis in human foreskin fibroblasts was profoundly influenced by exogenous LP at all stages of confluence; total and LP‐dependent sterologenesis declined in parallel with growth cessation. These studies represent the first demonstration that normal keratinocytes express functional LDL receptors at physiologic calcium concentrations. Moreover, they demonstrate that LDL receptor expression in keratinocytes, in contrast to fibroblasts, can only in part be attributed to growth requirements. Instead, loss of LDL receptor expression serves as a distinctive marker of keratinocyte differentiation and may reflect the specific functional requirements of the epidermis in vivo.
Effects of epidermal growth factor on transferrin receptor phosphorylation and surface expression in malignant epithelial cells
Authors: Castagnola Jan , MacLeod Carol , Sunada Hironobu , Mendelsohn John , Taetle Raymond 
Published: February 2005

AbstractThe transferrin (Tf) receptor is a major transmembrane protein which provides iron for normal and malignant cell growth. Epidermal growth factor (EGF) has been reported to rapidly and transiently alter the number of surface Tf receptors in normal and transformed epithelial cells. To investigate mechanisms of EGF‐induced changes in surface Tf display, EGF effects on surface Tf receptors were compared in two cell lines which differ in their number of EGF receptors and growth responses to EGF. In cloned A431 cells with high receptor numbers which are growth‐inhibited by EGF, EGF caused a 50% decrease in Tf receptor expression after 30 min. In contrast, EGF induced a rapid, transitory increase (within 5 min) in the number of surface Tf receptors on KB carcinoma cells which returned to basal levels by 15 min. The observed changes in Tf receptor display were due to altered receptor distribution and not changes in ligand affinity or total cellular transferrin receptor pools. Anti‐EGF receptor monoclonal antibody blocked effects of EGF on transferrin receptor expression. Since the antibody is internalized and causes EGF receptor down‐regulation, effects on transferrin receptor expression were independent of these events. EGF‐induced alterations in Tf receptor display occurred even when cells were pretreated with colchicine, suggesting that changes in surface Tf binding were not mediated by cytoskeletal components. Na orthovanadate, which mimics some early cellular effects of EGF, duplicated EGF's effects on A431 Tf receptors, but had no effect on KB cells, suggesting these responses occur by differing mechanisms. To determine whether EGF caused changes in Tf receptor phosphorylation, 32P‐labelled Tf receptors were immunoprecipitated after EGF treatment. After exposure to EGF, A431 cells showed no change in Tf phosphorylation, but KB cells showed a transient, 6‐fold increase in transferrin receptor phosphorylation on serine residues. In both A431 and KB cells, phorbol ester (PMA) also increased phosphorylation on transferrin receptors, but had little effect on surface Tf receptor expression. In malignant cell lines, EGF induces rapid, variable changes in transferrin receptor expression and phosphorylation which differ from the effects of PMA. These early responses to EGF appear to differ with the cell type and correlate poorly with alterations in Tf receptor phosphorylation. These results suggest Tf receptor phosphorylation does not regulate Tf receptor display in all cells.
Effect of dexamethasone on parathyroid hormone stimulation of cyclic AMP in an opossum kidney cell line
Authors: Rizzoli R. , Bonjour J.‐Ph. 
Published: February 2005

AbstractThe influence of the glucocorticoid dexamethasone on the cAMP response to parathyroid hormone (PTH) and various agonists was studied in epithelial monolayers of opossum kidney (OK) cells. The incubation with dexamethasone for 72 hours led to a dose‐dependent higher cAMP response to PTH or forskolin in intact cells as well as in digitonin‐permeabilized cells. This effect did not appear to result from changes in phosphodiesterase (PDE) activity nor from alterations in cAMP efflux from the cells. Moreover, dexamethasone increased the formation of domes by OK cell epithelium. Thus, dexamethasone seems to promote a more differentiated renal epithelial phenotype as suggested by enhanced hormonal response.
Comparison of oxidative metabolism in vitro in endothelial cells from different species and vessels
Authors: Kjellström B. Thomas , Örtenwall Per , Risberg Bo 
Published: February 2005

AbstractOxygen consumption was compared in confluent cultures of endothelial cells from human umbilical cord veins, rat pulmonary arteries, and bovine aortas. A microrespirometric method utilizing oxygenated hemoglobin as oxygen supply and indicator of respiration was used. Respiratory rate was equal in human and murine cells (2.0 × 10−6 and 2.2 × 10−6 μl O2/cell/hour respectively), compared on a cell‐to‐cell basis, while respiratory rate of the bovine endothelium was significantly lower (0.4 × 10−6 μl O2/cell/hour) (P < .001).
Lipid transfer between endothelial and smooth muscle cells in coculture
Authors: Stoll Lynn L. , Spector Arthur A. 
Published: February 2005

AbstractA coculture system was employed to study the interactions between endothelium and vascular smooth muscle cells in arachidonic acid metabolism. Bovine aortic endothelial cells grown on micropore filters impregnated with gelatin and coated with fibronectin are mounted on polystyrene chambers and suspended over confluent smooth muscle cultures. The endothelial basal laminae are oriented toward the underlying smooth muscle, and the two layers are separated by only 1 mm. Each cell layer was assayed individually: apical and basolateral fluid also was collected separately for assay. Fatty acids, including arachidonic acid, are readily transferred between the endothelial and smooth muscle cells in this system. Distribution of the incorporated fatty acids among the lipids of each cell is the same as when the fatty acid is added directly to the culture medium. Arachidonic acid released from endothelial cells is available as a substrate for prostaglandin production by smooth muscle. In addition, fatty acids released from the smooth muscle cells can pass through the endothelium and accumulate in the fluid bathing the endothelial apical surface. These fatty acid interchanges may be involved in cell‐cell signaling within the vascular wall, the clearance of lipids from the vascular wall, or the redistribution of arachidonic acid and other polyunsaturated fatty acids between adjacent cell types. Furthermore, the findings suggest that prostaglandin production by smooth muscle cells can occur in response to stimuli that cause arachidonic acid release from endothelial cells.
Insulin‐like growth factor‐I: Specific binding to high and low affinity sites and mitogenic action throughout the life span of WI‐38 cells
Authors: Phillips Paul D. , Pignolo Robert J. , Cristofalo Vincent J. 
Published: February 2005

AbstractInsulin‐like growth factor‐I (IGF‐I) (13 nM) can replace insulin (0.8 μM) in a serum‐free medium containing epidermal growth factor (EGF) (16 nM) and dexamethasone (DEX) (140 nM) and stimulate DNA synthesis in young cultures of WI‐38 cells, similar to the stimulation of serum‐supplemented medium. By contrast, senescent cells become unresponsive to all of these hormones. The effect of IGF‐I, EGF, and DEX is synergistic in stimulating multiple rounds of low density cell division. Total specific binding of [125]IGF‐I per cell in monolayer culture does not change with age, which indicates, in light of increased cell size with age, an actual decrease in specific binding per μM2 of cell surface area. Binding can be traced to two separate cell proteins. Binding to the α subunit of the IGF‐I transmembrane receptor may increase slightly with age while the 50% displacement remains unchanged. The remainder of the IGF‐I specific binding (five‐ to thirty‐fold more) is to a low molecular weight, cell‐associated binding protein whose 50% displacement is 10 times higher, but also remains unchanged with age. Specific binding to the lower affinity sites decreases slightly with age at equal cell densities. IGF‐I binding to the α subunit of the transmembrane receptor is independent of cell density, while binding to the low molecular weight binding protein is inversely proportional to cell density and may vary by as much as tenfold.
Contact‐inhibition‐induced quiescent state is marked by intense nuclear expression of Statin
Authors: Wang Eugenia 
Published: February 2005

AbstractStatin, a nuclear protein of 57,000 daltons, is found in in vitro aged, nonproliferating human fibroblasts but not in their young, replicating counterparts or transformed derivatives; it is also found in the nuclei of young fibroblasts when their growth is arrested but rapidly disappears from the cells once the restriction to growth is removed. We reported earlier that as cells leave the quiescent state, the loss of Statin from the nucleus precedes the initiation of DNA synthesis; here we report that in a confluent culture, as cells leave the traverse of the replicative cycle and assume the quiescent phenotype, Statin is not expressed maximally until total contact inhibition of growth is achieved. This full manifestation of Statin occurs in monolayer culture with cells forming the typical swirling pattern and fibronectin organized into large intercellular cables. The late expression of Statin in cells approaching the quiescent state is also verified biochemically by immunoblotting assays. The present results, taken together with those reported earlier, indicate that the nuclear appearance of Statin occurs only after the complete cessation of DNA synthesis and that the full manifestation of this protein can be used as a marker for the G0 quiescent state.
Superoxide dismutase activity and glutathione concentration during the calcium‐induced differentiation of Physarum polycephalum microplasmodia
Authors: Nations C. , Allen R. G. , Balin A. K. , Reimer R. J. , Sohal R. S. 
Published: February 2005

AbstractMicroplasmodia of Physarum polycephalum differentiate into spherules when the CaCl2 concentration of their nutrient medium is increased to 54mM (high‐calcium). The salts starvation medium routinely used to induce differentiation contains 8mM CaC12. This medium will not induce spherulation in the absence of a calcium salt; no other metal is essential. High‐calcium also induces the spherulation of a strain of Physarum that had not been previously observed to spherulate. The striking increase in superoxide dismutase activity (SOD) and the decrease in glutathione concentration (GSH) that are characteristic of salts‐induced spherulation do not occur in salts media containing high‐calcium. In the absence of calcium, no significant change in SOD is observed and very little change in GSH occurs. The immediate effect of the oxidative stress associated with spherulation may be the release of calcium stores into the cytosol. The parameters modulating this stress are, in turn, sensitive to exogenous calcium concentrations.
Differentiation of human keratinocytes: Changes in lipid synthesis, plasma membrane lipid composition, and 125I‐EGF binding upon administration of 25‐hydroxycholesterol and mevinolin
Authors: Ponec Maria , Kempenaar Johanna , Weerheim Ari , Boonstra Johannes 
Published: February 2005

AbstractWe have studied the relationship between differentiation capacity, plasma membrane composition, and epidermal growth factor (EGF) receptor expression of normal keratinocytes in vitro. The plasma membrane composition of the cells was modulated experimentally by cholesterol depletion, using specific inhibitors of cholesterol synthesis, such as 25‐hydroxycholesterol and mevinolin. Exposure of the cells towards these inhibitors resulted in a drastic decrease of cholesterol biosynthesis, as determined from 14C‐acetate incorporation into the various lipid fractions. This effect on cholesterol biosynthesis was reflected by changes in plasma membrane composition, as determined by lipid analysis of isolated plasma membrane fractions, these resulting in a decreased cholesterol‐phospholipid ratio. The experimental modulation of plasma membrane composition by 25‐hydroxycholesterol or mevinolin were accompanied by a decreased cornified envelope formation and by high expression of EGF binding sites. These phenomena were more pronounced in cells induced to differentiate by exposure of cells grown under low Ca2+ to normal Ca2+ concentrations, as compared to cells grown persistantly under low Ca2+ concentrations. These results suggest a close correlation between plasma membrane composition, differentiation capacity, and EGF receptor expression.
Insulin‐like growth factor‐II receptors in cultured rat hepatocytes: Regulation by cell density
Authors: Scott Carolyn D. , Baxter Robert C. 
Published: February 2005

AbstractInsulin‐like growth factor‐II (IGF‐II) receptors in primary cultures of adult rat hepatocytes were characterized and their regulation by cell density examined. In hepatocytes cultured at 5 × 105 cells per 3.8 cm2 plate, [125I]IGF‐II bound to specific, high affinity receptors (Ka = 4.4 ± 0.5 × 109 I/mol). Less than 1% cross‐reactivity by IGF‐I and no cross‐reactivity by insulin were observed. IGF‐II binding increased when cells were permeabilized with 0.01% digitonin, suggesting the presence of an intracellular receptor pool. Determined by Scatchard analysis and by polyacrylamide gel electrophoresis after affinity labeling, the higher binding was due solely to an increase in binding sites present on 220 kDa type II IGF receptors. In hepatocytes cultured at low densities, the number of cell surface receptors increased markedly, from 10–20,000 receptors per cell at a culture density of 6 × 105 cell/well to 70–80,000 receptors per cell at 0.38 × 105 cells/well. The increase was not due simply to the exposure of receptors from the intracellular pool, as a density‐related increase in receptors was also seen in cells permeabilized with digitonin. There was no evidence that IGF binding proteins, either secreted by hepatocytes or present in fetal calf serum, had any effect on the measurement of receptor concentration or affinity. We conclude that rat hepatocytes in primary culture contain specific IGF‐II receptors and that both cell surface and intracellular receptors are regulated by cell density.