bluuebunny/crossref_metadata_2025_split huggingface dataset:
Growth regulation and amino acid transport in epithelial cells: Influence of culture conditions and transformation on A, ASC, and l transport activities
Authors: Boerner Paula , Saier Milton H. 
Published: February 2005

AbstractAmino acid transport in Madin‐Darby canine kidney (MDCK) cells, grown in a defined medium, was investigated as a function of cell density, exposure to specific growth factors, and transformation. MDCK cells were found to transport neutral amino acids by systems similar to the A, ASC, L, and N systems which have been characterized using other cell lines. Experimental conditions were developed for MDCK cells which allowed independent measurement of A, ASC, and L transport activities. The activity of the L system was measured as Na+‐independent leucine or methionine uptake at pH 7.4. The activity of the A system was measured as Na+‐dependent α(methylamino)isobutyric acid (mAIB) uptake at pH 7.4, the activity of the ASC system was measured as Na+‐dependent alanine uptake in the presence of 0.1 mM mAIB at pH 6.0, and the activity of system N was observed by measuring Na+‐dependent glutamine uptake at pH 7.4 in the presence of high concentrations of A and ASC system substrates. The L transport system responded minimally to changes in growth state, but Na+‐dependent amino add transport responded to regulation by growth factors, cell density, and transformation. The activities of the A and ASC systems both decreased at high cell density, but these activities responded dissimilarly under other conditions. The activity of the A system was stimulated by insulin, was inhibited by PGE1, and was elevated 3–7 fold in the transformed cell line, MDCK‐T1. The activity of the ASC system was slightly stimulated by insulin and by PGE1, but was unchanged after chemical transformation. Changes in cellular growth were monitored and were found to correlate best with the activity of the A system. These results suggested that MDCK cell growth may be more closely related to the activity of the A than of the ASC system.
Cytochalasin B binding by human platelets
Authors: Zobel C. Richard , Jung Chan Y. 
Published: February 2005

AbstractIntact human platelets bind cytochalasin B (CB) with a capacity of 100– 120 p mols CB/mg protein or approximately 7 × 104 molecules/cell and dissociation constants (KD) ranging from 2 × 10−8 to 10−6 M. Up to 85% of this saturable binding is displaced by 10−5 M cytochalasin E (CE). This CE‐sensitive binding also appears heterogeneous with KD similar to those of the overall binding. The CE‐insensitive binding, however, appears as a single component with KD ≌ 4 × 10−7 M. The sedimentable constituents from frozen, thawed, and washed cells also bind CB with KD ranging from 2.4 × 10−8 to 1.5 × 10−6 M and a total capacity of approximately 39 p mols/mg protein which accounts for only 4% of the ligand binding to the intact cell. The major portion (60–80%) of this CB binding is displaceable by 500 mM D‐glucose and has a KD of 1.5 × 10−6M, while only 10–15% is CE‐sensitive with a KD of 2.4 ± 10−8 M. It is concluded that 95% of the saturable CB binding in platelets is associated with the cytosol of which 80–85% is sensitive to CE and that only 3% of the cellular binding is glucose sensitive, membrane‐associated binding. If the CE‐sensitive binding associated with the cytosol is entirely to actin, the stoichiometry of this binding is approximately one CB to 30 actin monomers, which is greater by an order of magnitude than that for CB binding to muscle actin.
Involvement of Na+ and HCO in receptor‐mediated endocytosis of α2‐macroglobulin, epidermal growth factor, and vesicular stomatitis virus
Authors: Dickson Robert B. , Schlegel Richard , Willingham Mark C. , Pastan Ira H. 
Published: February 2005

Abstractα2‐Macroglobulin (α2M), epidermal growth factor (EGF), and vesicular stomatitis virus (VSV) each enter cultured fibroblasts by receptor‐mediated endocytosis. The present study defines some basic ionic requirements in the cell culture medium which are necessary for the maximal rate of endocytosis of these three ligands. Na+ and HCO were both necessary for maximal endocytosis of 125I‐α2M, 125I‐EGF, and 35S‐VSV at 37°C. The ion specificities for both the anion and cation requirements were established. The binding of 125I‐α2M to its cellular receptors at 4°C was unaffected by the absence of Na+ and HCO in the culture medium. In addition, the absence of Na+ and HCO in the culture medium did not reduce cellular uptake of horseradish peroxidase by fluid phase endocytosis. Na+ and HCO may be general requirements in receptor‐mediated endocytosis.
The role of protein metabolism in glucocorticoid‐lnduced prolongation of G1 phase in human NHIK 3025 cells
Authors: Bakke Oddmund , Rønning Øystein W. 
Published: February 2005

AbstractIt has previously been found that human NHIK 3025 cells have a glucocortiocoid‐sensitive restriction point in mid‐G1 phase of the cell cycle. When these cells were synchronized by mitotic selection and exposed to dexamethasone before the restriction point, G1 phase was prolonged whereas the rest of the cell cycle was unperturbed by the hormone. These observations were confirmed by flowcytometric mesurements of synchronized cells in the present study. Cells that received dexamethasone (10−6 M) just after mitotic selection had a 4 hour prolongation of both G1 and the total cell cycle. However, the general rates of both protein synthesis and protein degradation were found not to be altered by the hormone, i.e., the rate of protein accumulation in dexamethasone exposed cells was equal to that of control cells. Dexamethasone exposed NHIK 3025 cells were found to be larger than control cells at the time of cell division. This is a direct consequence of a prolonged cell cycle duration with no change in general protein metabolism. It thus appears that the dexamethasone‐induced prolongation of G1 phase is the result of a steroid‐regulated G1 specific process(es) leading toward DNA replication, a process that does not alter general protein accumulation.
Rat serum albumin synthesis in variant rat hepatoma cells
Authors: Wolf Joyce , Golden Jonathan J. , Chasin Lawrence A. 
Published: February 2005

AbstractVariant subclones of the rat hepatoma cell line FU5‐5 have been isolated that are altered in their production of rat serum albumin. Three of these variants, isolated in a random screening, have been categorized as high, intermediate, and low producers. They secrete albumin into the culture medium at different rates: 16, 1.7, and 0.3 μg/mg cell protein/48 h. A fourth variant, isolated on the basis of altered morphology, secretes no detectable albumin. Unlike the albumin‐producing variants, this null variant is also deficient in the level and inducibility of tyrosine aminotransferase activity. Albumin biosynthesis as determined in pulse‐labeling experiments is affected similarly in the four variants, yielding albumin synthetic rates of 0.24, 0.035, 0.006, and < 0.002% of total protein synthesis. The translatable albumin messenger RNA content in these variants was measured using a rabbit reticulocyte lysate system. The null variant contains no detectable mRNA, and the three quantitative variants contain levels of translatable albumin messenger RNA corresponding to 0.07, 0.03, and 0.005% of total stimulated polypeptide synthesis. The highest producing variant contains less translatable albumin mRNA than expected on the basis of cellular biosynthetic measurements, suggesting a translation efficiency difference in this clone. Cell hybrids constructed by fusing the high‐producing clone and the null variant produce little or no albumin. This extinction indicates that the null variant contains a diffusible regulatory factor capable of decreasing albumin gene expression. The relatively stable and discrete heritable phenotypic changes exhibited by these clones may serve as a model for similar changes that occur during hepatic differentiation.
Specific protein production during melanogenesis in B16/C3 melanoma cells
Authors: Laskin Jeffrey D. , Piccinini Linda , Engelhardt Dean L. , Weinstein I. Bernard 
Published: February 2005

AbstractThe mouse melanoma cell line B16/C3 offers an excellent in vitro model for studying melanocyte differentiation. Melanogenesis can be induced by serum, a hormone‐supplemented serum‐free medium, melanocyte stimulating hormone, and dibutyryl cAMP. The tumor promoter, 12‐O‐tetradecanoyl‐phorbol‐13‐acetate, 5‐bromodeoxyuridine, and acidic pH inhibit this process. Using two‐dimensional polyacrylamide gel electrophoresis, we have identified four cellular proteins whose production is modulated during melanogenesis, a process which includes concomitant increases in levels of ty‐rosinase, the rate limiting enzyme for melanin biosynthesis, melanization, and ultimately, cell death. The production of these proteins are coordinately expressed or inhibited in response to the diverse inducers and inhibitors of melanogenesis. We conclude from these studies that these specific proteins are intimately involved in the differentiation of B16/C3 melanoma cells.
Production of colony stimulating factor in long‐term bone marrow cultures
Authors: Shadduck Richard K. , Waheed Abdul , Greenberger Joel S. , Dexter T. Michael 
Published: February 2005

AbstractPrevious studies have shown no detectable colony‐stimulating factor (CSF) in media harvested from long‐term bone marrow cultures. In the present experiments supernatants from long‐term cultures established in three laboratories were assayed for CSF by colony assay and by radioimmunoassay (RIA). Most samples were devoid of biologic activity but all contained CSF as judged by RIA. Biologic activity was found in the majority of samples after diafiltration to remove low molecular weight inhibitors or 5‐fold concentration by ultrafiltration. Samples that remained inactive in the colony assay were subjected to gel filtration on Sephadex G‐150 to remove potential high molecular weight inhibitors. Biologic activity remained lower than that by RIA in two of three samples tested. Thus, most long‐term cultures appear to contain biologically active CSF but this activity is masked by various types of inhibitors. In addition some media appear to contain material that is only detected by RIA.
Dexamethasone can modulate glucose‐regulated and heat shock protein synthesis
Authors: Kasambalides E. J. , Lanks K. W. 
Published: February 2005

AbstractWhen L929 cells are cultured in the abssence of glucose the rate of synthesis of two polypeptides (95,000 and 82,000 Mr) is increased while that of an 85,000 Mr polypeptide is correspondingly decreased. It has been shown recently that the 85,000 Mr polypeptide is identical with the heat shock protein that is associated with the Rous sarcoma virus transforming gene product (pp60src). The present study shows that dexamethasone completely inhibits the alterations in the pattern of protein synthesis that would normally ensue following glucose deprivation. In fact, synthesis of the 85,000 Mr polypeptide and another polypeptide (69,000 M,) that is not glucose regulated are dramatically increased. In contrast, when L929 cells are maintained in ample glucose the effects of dexamethasone are much less dramatic with only one major SDS polyacrylamide gel band (90,000 Mr) being affected. This effect is specific for glucocorticoids. One‐dimensional peptide mapping did not demonstrate any obvious structural relatedness among the 95,000, 90,000, 85,000, and 82,000 Mr polypeptides. Therefore, the observed changes appear to result from alterations in the rate of protein synthesis rather than from changes in precursor‐product relationships. It is suggested on the basis of this data that glucocorticoids may play a role in modulating the response of cultured cells to glucose deprivation by eliciting a heat shock‐like response.
Loss of the PGE1 requirement for MDCK cell growth associated with a defect in cyclic AMP phosphodiesterase
Authors: Taub Mary , Saier Milton H. , Chuman Lorraine , Hiller Sue 
Published: February 2005

AbstractProstaglandin E1(PGE1), one of the components in the hormone‐supplemented, serum‐free medium for Madin Darby Canine Kidney (MDCK) cells (Medium K‐1), is required for both long‐term growth and for dome formation. Variant cells have been isolated from MDCK populations, which lack the PGE1, requirement for long‐term growth in Medium K‐1. These variants will be useful in identifying the molecular events initiated by PGE1 which are necessary for the growth response to be observed. The growth and functional properties of five independently isolated PGE1 independent clones have been examined. Normal MDCK cells grew at an equivalent rate in Medium K‐1 and in serum‐supplemented medium; the growth rate was lower in Medium K‐1 lacking PGE1. In contrast, PGE1 independent clone 1 grew at an equivalent rate in Medium K‐1 minus PGE1, and in serum‐supplemented medium. When PGE1 was added to K‐1 minus PGE1, less growth of PGE1 independent clone 1 was observed. A similar observation was made with one other PGE1 independent clone which was studied. A hormone deletion study indicated that PGE1 independent clone 1 still retained growth responses to the other four supplements in Medium K‐1 (insulin, transferrin, T3, and hydrocortisone). The molecular alterations associated with loss of the PGE1 requirement for long‐term growth were examined. At confluency, all of the PGE1 independent clones studied had higher intracellular cyclic AMP levels following PGE1 treatment, as compared with normal MDCK cells. The increased cyclic AMP levels in the variant cells could result from a number of different types of defects, including reduced cyclic adenylic acid (cyclic AMP) efflux, an increased affinity of PGE2 for the PGE1 receptor, or a defect in cyclic AMP metabolism. However, in all of the variant clones studied a decreased rate of cyclic AMP degradation by cyclic AMP phosphodiesterase was observed. Thus, the increased cyclic AMP levels in the PGE1 independent variants may result from alterations which affect cyclic AMP metabolism. The effect of PGE1 on dome formation by the variant cells was also examined. The frequency of dome formation by PGE1 independent clone 1 was enhanced in a dosage‐dependent manner, like normal MDCK cells. This observation suggests that PGE1 affects MDCK cell growth and dome formation by different mechanisms.
Hexose uptake as an indicator of JB6 mouse epidermal cell resistance to the mitogenic activity of TPA
Authors: Copley M. , Gindhart T. , Colburn N. 
Published: February 2005

AbstractJB6 mouse epidermal cells have been selected for resistance to the tumor‐promoting phorbol diester TPA for (1) the plateau density mitogenic (M) response, and (2) the promotion of tumor cell phenotype (P) response. The purpose of this study was to determine the relationship of hexose uptake to the two TPA‐dependent processes. Monolayers of JB6 mouse epidermal cells showing one of four different phenotypes (M + P+, M + P−, M− P+, M− P−) were exposed to 60 nM [3H(G)]2 deoxy‐D‐glucose (2DG) with or without TPA (10 ng/ml) stimulation. The TPA mitogen‐sensitive (M + P +/−) cells, when in logarithmic growth, had a lower basal 2DG uptake rate than TPA mitogen‐resistant (M− P +/−) cells. At plateau density, however, only the M+P+ cells had a significantly lower basal rate. The M + (TPA mitogen‐sensitive) cells (with low basal rates), when preincubated with TPA, exhibited a two to threefold increase in 2DG uptake, while the M− (TPA mitogen‐resistant) lines, which already showed elevated rates, remained unchanged. There was also a positive association between TPA mitogen sensitivity and slower growth rate. These results suggest that low hexose sugar uptake is related to TPA mitogen sensitivity, but not to promotion sensitivity. Hence the cell's ability to increase its uptake rate may be required for the cells to respond to mitogenic stimulation by TPA.
Glycerol‐3‐phosphate dehydrogenase is induced by glucocorticoids in hepatocytes and hepatoma cells in vitro
Authors: Meyer Robert D. , Preston Sheryl L. , McMorris F. Arthur 
Published: February 2005

AbstractPrevious studies have shown that cytosolic glycerol‐3‐phosphate dehydrogenase (GPDH; EC 1.1.1.8) can be induced by glucocorticoids in mammalian brain, mammary gland, and thymus, but it was thought that no induction occurred in liver. We report here that GPDH is induced by glucocorticoids in several lines of hepatoma cells and in rat hepatocytes cultured in vitro. When rat hepatoma cells of clone FU5AH were exposed to 3 μM hydrocortisone (HC) for 3 days, GPDH specific activity increased greater than sixfold over control. The rate and extent of induction were similar in exponentially growing and stationary‐phase cultures of cells. Four other hepatoma cell lines were inducible to a lesser extent, and three lines were not inducible. GPDH was also induced by glucocorticoids in cultures of hepatocytes isolated from livers of 6‐day‐old rats. The enzyme was induced threeto fourfold by the synthetic glucocorticoid, dexamethasone, in the presence of 1 nM insulin, but the induction was not observed in the absence of insulin.
Ionic behaviors and neuronal survival in developing ganglia. III. Studies with embryonic chick sympathetic neurons
Authors: Selak Ivan , Skaper Stephen D. , Varon Silvio 
Published: February 2005

AbstractWe have shown in the past that (1) Nerve Growth Factor (NGF) controls the Na+,K+‐pump in its ganglionic neuronal targets and (2) the NGF requirement for pump control is developmentally regulated in the chick embryo dorsal root ganglion. We report here that NGF is fully competent to insure the control of intracellular Na+ concentrations (as expression of pump control) in intact chick sympathetic ganglia and enriched suspensions of sympathetic neurons from embryonic day 8 (E8) through 13. At later stages (E13–E18), NGF becomes less and less required for that control as the neurons gain a self‐sustained ionic pump competence. In monolayer cultures of enriched sympathetic neurons, an increasing neuronal survival in the absence of NGF occurs. These data demonstrate that the ability of developing sympathetic neurons to survive without NGF increases with the same temporal pattern as does their independence from NGF for ionic pump control, stressing the importance of ionic events for neuronal survival.
Biochemical properties of the endothelium‐derived growth factor: Comparision to other growth factors
Authors: Dicorleto Paul E. , Gajdusek Corinne M. , Schwarth Stephen M. , Ross Russell 
Published: February 2005

AbstractCultured bovine aortic endothelial cells (BAEC) can be maintained at saturation density for several weeks in the absence of serum. These cells retain viability and normal culture morphology, and continuously produce a growth factor for mesenchymally derived cells–the endothelium‐derived growth factor (EDGF). The amount and specific activity of EDGF that is produced by BAEC under serum‐free conditions remains constant for weeks. The levels of EDGF produced under these serum‐free conditions is equivalent to levels produced in medium containing 5% plasma‐derived serum. EDGF has been found to be trypsin sensitive, acetone and ammonium sulfate precipitable, and resistant to heat and sodium dodecyl sulfate treatment. Gel filtration on Sephacryl S‐200 in the presence of formic acid (1%) yields two major peaks of activity corresponding to proteins of apparent molecular weights of approximately 24,000 and 14,000 daltons. This chromatographic step affords a ten‐to 12‐fold purification with a combined recovery of greater than 85%. Unlike brain or pituitary fibroblast growth factor, EDGF activity is destroyed by dithiothreitol or periodic acid. EDGF is not a somatomedin since it exhibits no detectable sulfation activity in a porcine cartilage assay. EDGF is not inhibited by antiserum to epidermal growth factor and is capable of stimulating DNA synthesis in a 3T3 variant cell line that is nonresponsive to and lacks receptors for epidermal growth factor. The majority of EDGF activity does not behave like the platelet‐derived growth factor during ion exchange chromatography. Antisera prepared in rabbits and in mice to human platelet‐derived growth factor has little effect on bivine or human EDGF activity. These biochemical and immunological properties of EDGF indicate that it is distinct from several other well‐characterized polypeptide growth factors.
Production of growth factors by type 5 adenovirus trannsformed rat embryo cells
Authors: Fisher Paul B. , Boersig Michael R. , Graham Gary M. , Bernard Weinstein I. 
Published: February 2005

AbstractTransformation of Sprague‐Dawley rat embryo (RE) cell and a cloned Fischer rat embryo cell line (CREF) with wild‐type (Ad5) or a temperature‐sensitive DNA‐minus mutant (H5ts125) of type 5 adenovirus results in a reduction in binding of epidermal growth factor (EGF) to cell surface receptors. A reduction in EGF binding is also seen in a Syrian hamster embryo cell line transformed by a hexon mutant of Ad5. In constrast, a human embryonic kidney cell line (293) transformed by sheared Ad5 DNA or transfected clones of KB cells expressing the E1 transforming region of Ad5 do not show a decrease in receptor binding. When cocultivated, the adenovirus transformed rat cells were able to induce the growth in agar of normal CREF cells. Medium from Ad5 transformed RE cells stimulated the growth in agar of CREF cells and also inhibited [125I]‐EGF binding in CREF cells. When fractionated by gel filtration, two peaks of [125I]‐EGF inhibiting activities were obtained with apparent molecular weights of 35,000 and 16,000. These results provide the first evidence that cells transformed by an adenovirus can produce a growth factor(s) that inhibits EGF‐receptor binding and induces anchorage‐independent growth of normal cells.
Analysis of growth factor “relaxation” in chinese hamster lung fibroblasts required for tumoral expression
Authors: van Obberghen‐Schilling E. , Pérez‐Rodríguez R. , Franchi A. , Chambard J. C. , Pouysségur J. 
Published: February 2005

AbstractThe Chinese hamster lung fibroblast line, CCI39, displays the properties characteristic of normal secondary cultures of Chinese hamster fibroblasts including: reversible GO growth arrest (<2% labeled nuclei), anchorage dependence, and high serum‐growth factor dependence. Injection of CCI39 cells, or anchorage‐independent variants, in nude mice leads to tumor formation; however, as we have previously shown (Pérez‐Rodriguez et al., 1981b), the resulting tumor clones no longer possess the high serum dependence of injected CCI39 cells. Hormonal growth restraints imposed by the host create an in vivo selection for diminished, or “relaxed,” growth factor requirement. To characterize this growth factor “relaxation” further, we have analyzed the mitogenic response of parental CCI39 cells, anchorage‐independent clones, and selected tumoral derivatives, to purified growth factors. Two highly purified growth factors, thrombin and insulin, together fulfill the growth factor requirements of CCI39 cells; thrombin (1 U/ml) stimulates the reinitiation of DNA synthesis in GO‐arresed CCI39 cells, and insulin (10 μ/ml) maximally potentiates this stimulation to the level obtained with 10% fetal calf serum. First, we found no correlation between loss of anchorage dependence and growth factor relaxation. Second, we found that A71 (anchorage independent), a tumoral variant of CCI39 capable of growth arrest, and tumor‐derived cells all display an increased sensitivity to thrombin and a diminished requirement for the potentiating action of insulin. Examination of thrombin binding to CCI39, A51 (nontumoral, anchorage independent), and A71 cells revealed that the increased sensitivity to thrombin of A71 cells is not attributable to an alteration in thrombin cell surface receptor number or affinity for thrombin. Rather, under standard conditions of serum or growth factor removal (30 hr), A71 cells maintain a metabolically elevated growth‐arrested state, different from that of their nontumoral counterparts. Consequently, much lower concentrations of growth factors are needed to induce a proliferative response in these tumoral cells.