bluuebunny/crossref_metadata_2025 huggingface dataset:
Glucocorticoid influence on growth of vascular wall cells in culture
Authors: Longenecker John P. , Kilty Laura A. , Johnson Lorin K.
Published: February 2005

Primary mass cultures and cloned strains of bovine aortic endothelial and smooth muscle cells were investigated with respect to their growth responses to glucocorticoid hormones. The growth of primary endothelial cells was not influenced by glucocorticoid treatment in the absence of fibroblast growth factor (FGF) but was inhibited by about 30% in the presence of FGF; with cloned endothelial cells, glucocorticoids were also growth inhibitory only in the presence of FGF. In contrast, smooth muscle cell growth was inhibited 30%–70% by glucocorticoid treatment in both primary cultures and in the cloned strains in the absence of FCF, and this inhibition was totally abolished by the addition of FGF for both cultures. The corticosteroid influences on cell growth were glucocorticoid specific, concentration dependent, and were observed to be independent of the serum concentration. The results indicate that glucocorticoid hormones have direct and pronounced growth inhibiting effects on aortic smooth muscle cells but only minimal effects on endothelial cells when these components of the vascular wall are analyzed under identical conditions in vitro.
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

Amino 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

Intact 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

α2‐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

It 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

Variant 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

The 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

Previous 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

When 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

Prostaglandin 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

JB6 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

Previous 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

We 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

Cultured 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

Transformation 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

The 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.
The use of HgCl2 to evaluate the cosubstrate: Amino acid transport stoichiometry in ehrlich ascites tumor cells
Authors: David Dawson W. , Robinson Susan C. , Smith Thomas C.
Published: February 2005

Recent investigations have indicated that cellular rheogenic properties may interfere with the correct estimation of Na+ and amino transport stoichiometry. We have reevaluated the stoichiometry of Na+ and α‐aminoisobutyric acid (α‐AIB) cotransport in Ehrlich ascites tumor cells depleted of Na+ and ATP by incubation in Na+ ‐free HEPES‐buffered medium (pH 7.2) containing 160 mM K+ and 2.5 μM valinomycin. Transfer of the cells to a medium with 10 mM 22Na+, 10 mM 3H‐AIB, and 150 mM K+ resulted in an enhancement of Na+ flux above basal levels, which represents 0.6 of the AIB uptake. Under these conditions the membrane potential, −7.0 ± 0.1 mV (SEM), does not change with the addition of AIB, −7.3 ± 0.6 mV (SEM). HgCl2 (10 m̈M) added to the medium inhibited AIB flux and AIB‐stimulated Na+ flux by 45–50% but did not change the coupling ratio. HgCl2 (10 m̈M) does not inhibit the basal Na+ flux nor does it affect cellular Na+ or K+ content. In physiological medium cotransport is electrogenic. The membrane potential of Ehrlich cells in physiological medium is −22.3 ± 0.8 mV (SEM) and depolarizes to −16.7 ± 0.7 mV (SEM) upon addition of AIB. Under these conditions the coupling ratio was highly variable but the ratio of codepression is 0.90 ± 0.02 (SEM) in the presence of HgCl2 (10 m̈M). These results are consistent with a model (Smith and Robinson, 1981) in which the stoichiometry is one cosubstrate molecule per molecule of α‐AIB. We suggest that H+ provides the alternative cosubstrate in this low Na+ environment and that in high Na+ medium the Na+ :AIB stoichiometry approaches 1:1.
Coordinate secretion of rat and mouse albumin by mouse hepatoma × rat hepatoma hybrid cells directly reflects the intracellular concentration of the corresponding mRNAs
Authors: Sellem Carole H. , Cassio Doris , Sala‐Trepat Joséa M. , Weiss Mary C.
Published: February 2005

The ratio of mouse to rat albumin secreted by mouse hepatoma × rat hepatoma hybrid cells is constant (of the order of 5.0) irrespective of the total amounts produced. The present results establish for seven independent hybrid clones that the coordination in the ratio of mouse to rat product applies also at the level of accumulation of albumin mRNAs of the two species. The interpretation that coordinate synthesis reflects coordinate transcription of the relevant genes is thus reinforced.
Comparative biochemical and cytochemical studies on superoxide and peroxide in mouse macrophages
Authors: Badwey J. A. , Robinson J. M. , Lazdins J. K. , Briggs R. T. , Karnovsky M. J. , Karnovsky M. L.
Published: February 2005

Maximal rates of superoxide (O) release, and the cytochemical locales of peroxide staining in resident, elicited, and activated macrophages have been determined. Macrophages elicited into the peritoneum with either casein (1.2% w/v) or proteose‐peptone (10.0% w/v) release about twice as much O as macrophages activated by infection of the animals with either Listeria monocytogenes, or Bacille Calmette‐Guerin (BCG) followed by immune boosting with Purified Protein Derivative (PPD) (i.e., about 35 vs. 14–18 nmol O/min/107 cells). Macrophages elicited with thioglycollate (3.0% w/v) and resident macrophages produce negligible amounts of O upon stimulation with PMA. These data are compared with those reported by other investigators who used different procedures. A cytochemical procedure for localizing peroxide has been modified for use with murine macrophages. No production of H2O2 by macrophages is detected cytochemically in the absence of stimulation. Upon exposure to PMA, resident macrophages are still largely unresponsive. Approximately 20% of the casein elicited macrophages and BCG‐PPD activated macrophages exhibit H2O2 staining, which is largely restricted to the cytoplasmic vesicles and channels induced by PMA in these cells. The only exception to this staining pattern is a small population (about 2%) of activated macrophages which exhibits H2O2 staining in the cytoplasmic vesicles and channels and on the plasmalemma as well.
Properties of growth hormone receptors in relation to the adipose conversion of 3T3 cells
Authors: Nixon Tracy , Green Howard
Published: February 2005

Cultured preadipose 3T3 cells undergo a process of differentiation in which they convert to adipose cells. Growth hormone promotes this conversion. Since 3T3 sublines vary in their susceptibility to adipose conversion, it was of interest to examine the properties of the growth hormone receptors in relation to that susceptibility. It was found that preadipose 3T3‐F442A cells, which are able to convert to adipose cells with high frequency, are able to bind about 104 growth hormone molecules per cell with Kd ∼︁ 10−9M. After adipose conversion, no appreciable change in hormone binding was detected. The binding of growth hormone to 3T3‐C2 cells (a line virtually insusceptible to adipose conversion) was indistinguishable from that to 3T3‐F442A cells. Internalization and degradation of the hormone were also similar in the two cell lines. Susceptibility to adipose conversion is therefore not determined by the relative ability of the cells to bind or degrade the hormone, but must instead depend on some response, as yet unidentified, that follows binding of the hormone.
The formation of capillary‐like tubes by calf aortic endothelial cells grown in vitro
Authors: Feder J. , Marasa J. C. , Olander J. V.
Published: February 2005

Cloned, large vessel endothelial cells derived from fetal bovine and bovine calf aortas formed three‐dimensional structures in vitro without tumor‐conditioned medium or special substrata. Transmission electron microscopy showed the structures to be hollow tubes composed of typical endothelial cells with overlapping and interdigitating cytoplasmic processes typical of those seen in in vivo capillaries. The putative lumen of these tubes generally contained abundant electron‐dense fibrous material, which by ruthenium red and indirect immunofluorescent staining appeared to be extracellular matrix. This suggests that the endothelial cell orientation in the tubes is the reverse of that normally found in in vivo vessels.
The role of the mannose/N‐acetylglucosamine receptor in the pinocytosis of horseradish peroxidase by mouse peritoneal macrophages
Authors: Sung Sun‐Sang J. , Nelson Rolf S. , Silverstein Samuel C.
Published: February 2005

Saccharomyces cerevisiae mannan inhibits the pinocytosis of horseradish peroxidase (HRP) by resident, thioglycollate‐, proteose peptone‐, and Co‐rynebacterium parvum‐elicited macrophages from 30 to 70% when 1 mg/ml HRP is used, and 65 to 87% when 250 μg/ml HRP is used. In contrast, HRP uptake by J774 cells, a macrophage cell line reported to have little mannose receptor activity, is inhibited only about 25% by mannan. HRP uptake by resident and thioglycollate‐elicited (thio) macrophages is also inhibited 34 and 66% by addition of EGTA to the medium and 55 and 79% by trypsin treatment of the macrophages, respectively. The inhibitory effect of EGTA can be reversed by 1 mM excess Ca2+. High extracellular concentrations of Ca2+, in the range of 10–20 mM, however, inhibit pinocytosis in resident macrophages by about 50%. Sucrose uptake by resident macrophages is not appreciably affected by mannan. These results support the hypothesis that HRP uptake is mediated by the macrophage mannose/N‐acetylgluco‐samine receptor. PMA stimulates fluid‐phase pinocytosis of HRP by thio‐macrophages but does not affect receptor‐mediated uptake of HRP, while the combination of adenosine, homocysteine, and erythro‐9‐(2‐hydroxy‐3‐nonyl) adenine (EHNA) selectively inhibits bulk‐phase uptake by thio macrophages.
Calcium‐dependent activation of lymphocytes by ionophore, A23187, and a phorbol ester tumor promoter
Authors: Mastro Andrea M. , Smith M. C.
Published: February 2005

The phorbol ester tumor promoter 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) and the calcium ionophore, A23187, have similar effects on many different cells. For example, both show mitogenic and comitogenic activities for lymphocytes. It had been suggested that some of TPA's effects are due to its ability to act as a calcium ionophore. In order to test this idea, we compared the ability of TPA and ionophore to synergize with concanavalin A (Con A) in a two‐phase system of lymphocyte mitogenesis. We found that ionophore was most comitogenic with Con A when present in the early phase of stimulation. TPA was only comitogenic when present in the late phase, lonophore and TPA could not replace one another in the system. However, both ionophore and TPA together could replace Con A and stimulate DNA synthesis when they were presented to the cells in the sequential order of ionophore followed by TPA. Both compounds required the presence of external calcium to be effective.
Metabolic characteristics of cells cultured from human umbilical blood vessels: Comparison with 3T3 fibroblasts
Authors: Bruttig Stephen P. , Joyner William L.
Published: February 2005

Little metabolic information is available for cells cultured from umbilical vascular tissue. These studies were undertaken to compare the metabolism of cells isolated from human umbilical arteries and veins with that of umbilical vascular segments. These studies also compared umbilical vascular cells to standard fibroblast preparations. Oxygen consumption by umbilical venous cells or tissues was greater than that for either arterial cells or tissues. Cellular adenosine triphosphate (ATP) content was greater in umbilical venous than arterial cells. However, an oxidation‐phosphorylation ratio (R) was constant for either arterial or venous cells. Oxygen consumption by vascular cells was greater than that by nonvascular cells, as was cellular ATP content. R for nonvascular cells was much greater than that for vascular cells, indicating loose coupling between oxygen consumption and cellular ATP content. Finally, cellular oxygen consumption was dependent upon cell density, and upon media serum content in vascular endothelial cells. We conclude therefore that the metabolism of umbilical vascular cells in culture reflects that of the parent tissue but is different from that of either vascular or nonvascular fibroblasts.
Tumor‐promoting phorbol esters inhibit the binding of colony‐stimulating factor (CSF‐1) to murine peritoneal exudate macrophages
Authors: Chen Ben D.‐M. , Lin Hsiu‐San , Hsu Shin
Published: February 2005

L‐cell colony‐stimulating factor (CSF‐1) is a sialoglycoprotein of molecular weight 70,000 daltons that specifically stimulates macrophage colony formation by single committed cells from normal mouse bone marrow and by various classes of more differentiated tissue‐derived mononuclear phagocyte colony‐forming cells (Stanley et al., 1978). CSF‐1 interacts with target cells by direct and specific binding to membrane receptors (CSF‐1 receptors) that are present only on cells of the mononuclear phagocyte series and their precursors. We studied the effect of tumor‐promoting phorbol esters on the binding of 125I‐labeled CSF‐1 (125‐CSF‐1) to murine peritoneal exudate macrophages (PEM). Biologically active TPA (12‐O‐tetradecanoyl phorbol‐13‐acetate) inhibits the binding of 125I‐CSF‐1 to its receptor on PEM. This inhibition exhibits temperature, time, and concentration dependence. At 37°C, maximum inhibition occurred at about 10−7 M; inhibition was 50% at 5 × 10−9 M. At 0°C, the inhibitory activity of TPA is diminished. The action of TPA on PEM is transient. Treated cells recover their 125I‐CSF‐1‐binding activity whether TPA is later removed or not. The process of recovering CSF‐1‐binding activity is completely blocked by the addition of cycloheximide. When several phorbol derivatives were tested for their inhibitory activities, only biologically active phorbol esters were found to possess such activities. Furthermore, the inhibitory activities of various phorbol esters are proportional to their tumor‐promoting activities. Inhibition appears to be due to a reduction in the total number of available CSF‐1 receptors rather than a decrease in receptor affinity.
Characteristics of an amiloride‐sensitive sodium entry pathway in cultured rodent glial and neuroblastoma cells
Authors: Benos Dale J. , Sapirstein Victor S.
Published: February 2005

We have studied the induction of an amiloride‐sensitive sodium influx into C6 glioma, NIE, and NB2A neuroblastoma cell lines. In late log phase, cells grown continuously in the presence of 10% fetal calf serum showed Na+ influxes of approximately 25–30 nmol/mg protein min; < 5% of this flux was inhibited by amiloride. Removal of serum for 24 h caused a decrease in the total Na+ influx to 15–20 nmol/mg protein/min. Upon readdition of serum to the incubation medium, there was an increase in total Na+ influx, depending on the cell type, of 20–400% within 2 min. This increment in Na+ influx represented an increase in amiloride‐sensitive Na+ transport with an apparent K′, of 0.4 mM. By adding serum back at various times after serum deprivation, it was determined that 4 h was required to observe a detectable increase in the amiloride‐sensitive Na+ flux. Thus, serum removal results in the induction of the amiloride transport system which, however, remains latent until the reintroduction of serum to the medium. Addition of 5 μg/ml of cycloheximide blocked the increase in Na+ transport, indicating that de novo protein synthesis mediated this serum deprivation–induced increase in Na+ transport. Moreover, inhibition of de novo lipid synthesis by 0.1 mM fenfluramine also blocked the induction of this transport activity, suggesting that a coordinated synthesis of lipid and protein is required for the expression of this sodium transport site. We have also found that this serum stimulated Na+ influx did not saturate with Na+ concentration, up to 140 mM. Also, among commonly used inhibitors of passive Na+ entry into epithelial tissues, only amiloride was capable of inhibiting this transport system in these neural cell lines.
Adenosine metabolism in wild‐type and enzyme‐deficient variants of Chinese hamster ovary and Novikoff rat hepatoma cells
Authors: Plagemann Peter G. W. , Wohlhueter Robert M.
Published: February 2005

Variants of Chinese hamster ovary and Novikoff rat hepatoma cells resistant to tubercidin and 2,5‐diaminopurine, or to both drugs, were isolated, and their ability to convert adenosine and various adenosine analogs to nucleotides was compared to that of wild‐type cells, both in intact cells and cell‐free extracts. Adenosine deamination, and thus its conversion to nucleotides via inosine‐hypoxanthine‐inosine monophosphate, was inhibited by pretreatment of the cells or cell extracts with 2‐deoxycoformycin. Cell‐free extracts of the tubercidin‐resistant variants, as well as of two adenosine‐resistant mutants of Chinese hamster ovary cells, phosphorylated adenosine, tubercidin, pyrazofurin, or tricyclic nucleoside in the presence of ATP at < 1% of the rate of extracts of wild‐type cells. However, addition of phosphoribosyl pyrophosphate stimulated the conversion of adenosine to nucleotides 40‐fold. Similarly, intact adenosine kinase‐deficient cells failed to phosphorylate the adenosine analogs, but still converted adenosine to nucleotides at 5–10% the rate observed with wild‐type cells. Phosphorylation of adenosine and tubercidin in wild‐type cells was inhibited by substrate at concentration above 5–10 μM. In contrast, the rate of conversion of adenosine to nucleotides by adenosine kinase‐deficient cells increased linearly up to a concentration of 400 μM adenosine, with the consequence that, at this concentration, these cells took up adenosine almost as rapidly as wild‐type cells. Adenosine uptake by these kinase‐deficient cells was inhibited by adenine and 5′‐deoxyadenosine, and was largely abolished in mutants devoid also of adenine phosphoribosyltransferase. We conclude that adenosine is converted to nucleotides in adenosine kinase‐deficient cells via adenine. Indirect evidence implicates 5′‐methylthioadenosine phosphorylase as the enzyme responsible for the degradation of adenosine to adenine.
Adenosine and tubercidin binding and transport in Chinese hamster ovary and Novikoff rat hepatoma cells
Authors: Plagemann Peter G. W. , Wohlhueter Robert M.
Published: February 2005

The uptake of adenosine and tubercidin by control and ATP‐deleted wild‐type and adenosine kinase‐deficient cells was measured by rapid kinetic techniques. Adenosine deamination was inhibited by pretreatment with 2‐deoxy‐coformycin. Control wild‐type cells phosphorylated adenosine so rapidly that the kinetics of transport per se could not be assessed unambiguously. ATP depletion and adenosine kinase deficiency did not abolish the conversion of adenosine to nucleotides, but reduced it to such an extent that initial velocities of uptake could be safely construed as transport velocities in both zerotrans and equilibrium exchange modes. The same was true for tubercidin, which was not phosphorylated in adenosine kinase‐deficient cells. It accumulated intracellularly, however, to concentrations 50 to 120% higher than those in the extracellular space, apparently due to binding to some intracellular component(s). Binding was not saturated up to a concentration of 200 μM, but seemed to be slow relative to transport. Fits of appropriate integrated rate equations based on the simple carrier model to uptake time courses obtained under these conditions yielded Michaelis‐Menten constants for adenosine and tubercidin transport of 100 to 200 μM and maximum velocities of 10 to 30 pmol/μl cell H2O ċ sec, whereas the rate of intracellular phosphorylation was maximal at concentrations between 2 and 8 μM. The first‐order rate constant (Vmax/Km) for adenosine phosphorylation, however, seemed to be appreciably higher than that for its transport. This indicates that at physiological concentrations, which fall in the first‐order range for both processes, adenosine trapping is very efficient. Adenosine, tubercidin, tricyclic nucleoside, 2′‐deoxyadenosine, and 3′‐deoxyadenosine all inhibited uridine and thymidine transport to about the same extent, whereas pyrazofurin was signficantly less effective.
Efflux of 45CA2+ from human fibroblasts in response to serum or growth factors
Authors: Owen Nancy E. , Villereal Mitchel L.
Published: February 2005

Previous studies have indicated that intracellular Ca2+is involved in fetal bovine serum (FBS)‐ or growth factor (GF)‐stimulated Na+ influx in human foreskin fibroblasts (HSWP). In the present study, 45Ca2+ efflux from serum‐deprived HSWP cells was measured in response to 10% FBS or GF [lys‐bradykinin, vasopressin, epidermal growth factor, and insulin]. Efflux data were analyzed using a computer program and the best fit indicated the presence of three Ca2+ compartments: a compartment (C1) with a very fast turnover rate, one (C2) with a fast turnover rate, and one (C3) with a slow turnover rate. When serum‐deprived cells were treated with 10% FBS, efflux from C2 and C3 increased significantly (p>0.05). Similar effects on efflux were observed when serum‐deprived cells were treated with individual GFs. Combination of the four GFs produced a higher stimulation than any single factor and a response that was equal to that for FBS. On the other hand, when cells were serum‐treated in the presence of the intracellular Ca2+ antagonist, 8‐(N‐N, diethylamino)‐octyl 3,4,5‐trimethoxybenzoate (TMB‐8), 45Ca2+ efflux from C2 was substantially reduced. Finally, when cells were treated with the Na+ transport inhibitor amiloride, there was no significant effect on serum‐stimulated Ca2+ efflux. These results are consistent with a FBS‐ or GF‐induced mobilization of Ca2+ that can be blocked by intracellular Ca2+ antagonists, and support the hypothesis that the action of these agents on Na+ influx may be via their effects on intracellular Ca2+.
Growth stimulatory precipitates of CA2+ and pyrophosphate
Authors: Bowen‐Pope D. F. , Rubin H.
Published: February 2005

Inorganic pyrophosphate (PPi) forms an insoluble precipitate with calcium in growth medium when its concentration exceeds about 0.1 mM. This PPi precipitate can reproduce the effects of 10% calf serum on all cell processes examined in Balb/c 3T3 cells, including hexose uptake and metabolism to lactate, 3H‐uridine, and 3H‐choline uptake, and the incorporation of 3H‐leucine and 3H‐thymidine into trichloroacetic acid (TCA)‐insoluble material. Concentrations of PPi insufficient to form a precipitate are without effect on cell metabolism. The precipitates are most effective when prepared with concentrations of PPi just sufficient to result in precipitate formation and become considerably less effective as the PPi concentration increases, even though the quantity of precipitate formed continues to increase with PPi concentration up to 1 mM PPi. Precipitates formed at low PPi concentrations consist largely of Ca2+ (81% of cations). PPi (77% of anions), and Pi (23% of anions). Precipitates formed with higher concentrations of PPi contain proportionately less Ca2+ and Pi and more monovalent cations and PPi. We have distinguished cell surface‐bound PPi from intracellular PPi by differential extraction. The quantity of surface‐bound PPi increases sharply when the PPi concentration reaches the point of precipitate formation. If the precipitate is prevented from binding to the cell surface by inverting monolayer cultures in precipitate‐containing medium, the cells are not stimulated. These findings suggest that the binding of PPi precipitate to the cell surface is involved in the stimulation of cell metabolism by PPi. PPi precipitates do not absorb serum mitogens or inhibitors from the culture medium, nor do they affect the binding of 125I‐platelet‐derived growth factor to its specific cell‐surface receptor, suggesting that PPi precipitates do not act directly through either of these mitogen‐receptor systems. In analogy to cell stimulation by epidermal growth factor and by antigens, we suggest that PPi may be active only in the form of a precipitate because multivalent binding of receptors with formation of clusters is required for stimulation. The inhibitory effects of high concentrations of PPi may be due to interference by free PPi with formation of active receptor clusters.