bluuebunny/crossref_metadata_2025 huggingface dataset:
Granulocyte‐macrophage colony formation in serum‐free culture: Effects of purified colony‐stimulating factors and modulation by hydrocortisone
Authors: Eliason James F.
Published: February 2005

The effects of three purified colony‐stimulating factors (CSFs) with different specificities for the granulocyte (G) and macrophage (M) lineages (G‐CSF, CSF‐1 and GM‐CSF) were studied in a serum‐free clonal assay system. The results were compared with those obtained in similar cultures containing fetal calf serum (FCS). Total clone (≥ 10 cells) and colony (≥ 50 cells) numbers were enhanced by FCS under most conditions. However, the extent of enhancement was highly dependent on the concentration and type of CSF. In some instances, FCS also altered the proportions of G, M, and mixed GM clones induced by the CSFs. In cultures stimulated with GM‐CSF, enhancement by FCS was significant only at low CSF concentrations, primarily due to increased numbers of M clones. In contrast, clonal growth was increased by FCS only at high concentrations of CSF‐1. Clone and colony numbers induced by G‐CSF were greatly increased in cultures with FCS at all CSF concentrations tested. Virtually all clones developing in serum‐free medium with G‐CSF were pure G, whereas, M and GM clones were usually present in serum‐containing cultures with high doses of G‐CSF. The effects of hydrocortisone (HC) were also examined in these experiments. Like modulation by FCS, modulation of clonal growth by HC depended on the CSF used as stimulus, having no effect in cultures with G‐CSF, inhibitory effects with CSF‐1, and variable effects with GM‐CSF related to CSF concentration.
Parathyroid hormone stimulation of alkaline phosphatase activity in cultured neonatal mouse calvarial bone cells: Involvement of cyclic AMP and calcium
Authors: Yee John A. , Sutton Julie K. , Shew Ronald L. , Olansky Leann
Published: February 2005

The involvement of cAMP and calcium in the rise in alkaline phosphatase (AP) activity observed when confluent, serum‐free primary cultures of neonatal mouse calvarial cells are treated with parathyroid hormone (PTH) has been studied. Synthetic bovine PTH [bPTH‐(1–34)] increased cellular cAMP at concentrations (10−9 to 10−7 M) previously found to elevate AP activity. Other substances that increase cAMP in these cells (forskolin, prostaglandin E2, 8‐bromoadenosine cAMP and 3‐isobutyl‐1‐methylxanthine) also increased enzyme activity. By comparison, increasing the concentration of calcium in the culture medium from 1.8 to 3.8 or 5.8 mM lowered the magnitude of the maximal AP response. In addition, treatment of cultures with the divalent cation ionophore A23187 caused a significant decrease in AP activity. These results suggest that: (1) cAMP mediates the rise in the specific activity of AP in cultured neonatal mouse calvarial cells treated with bPTH‐(1–34) and (2) the concentration of calcium in the environment significantly influences the responsivity of bone cells to the hormone.
Protein phosphorylation in cultured endothelial cells
Authors: Mackie Ken , Lai Yvonne , Nairn Angus C. , Greengard Paul , Pitt Bruce R. , Lazo John S.
Published: February 2005

We have investigated the protein phosphorylation systems present in cultured bovine aortic and pulmonary artery endothelial cells. The cells contain cyclic AMP‐dependent protein kinase, three calcium/calmodulin‐dependent protein kinases, protein kinase C, and at least one tyrosine kinase. No cyclic GMP‐dependent protein kinase activity was found. The cells also contained numerous substrates for cyclic AMP‐dependent protein kinase and protein kinase C. Fewer substrates were found for the calcium/calmodulin‐dependent protein kinases. There was little difference between either protein kinase activities or substrates when pulmonary artery endothelium was compared to aortic endothelium grown under similar culture conditions. It is likely that these various protein kinases and their respective substrate proteins are involved in mediating several of the actions of the hormones and drugs which affect the vascular endothelium.
Density‐dependent inhibition of expression of syncytiotrophoblastic markers by cultured human choriocarcinoma (BeWo) cells
Authors: Burres Neal S. , Cass Carol E.
Published: February 2005

In the presence of methotrexate, cultured human choriocarcinoma (BeWo) cells undergo a differentiative response that resembles normal trophoblastic development. In the current study, the effects of cell number and population density on drug‐induced conversion of BeWo cells from the cytotrophoblast‐like to the syncytiotrophoblastlike phenotype were investigated using as markers of differentiation formation of “giant” cells, a process shown to require exogenous purines, and expression of placental (heat‐stable) alkaline phosphatase. Giant cell formation, assessed by determination of cell volumes, was reduced in crowded cultures, and addition of hypoxanthine to growth media partially restored methotrexate‐induced cell enlargement. Cellular uptake of methotrexate, assessed by following the loss of methotrexate from cell culture fluids during drug exposures, was two‐threefold greater in sparsely populated than in densely populated cultures. Although the concentration of methotrexate in culture fluids of crowded cultures declined during exposures of 48 hr, the amount of extracellular drug remaining at 48 hr was well above the threshold for induction of the differentiative response. When culture population was held constant and population density was manipulated by varying the substratum available to cells, methotrexate‐induced cell enlargement was inversely related to population density. Expression of placental alkaline phosphatase, salvage of exogenous hypoxanthine, and synthesis of RNA were also reduced at high population densities. These results indicate that expression of markers of methotrexate‐induced differentiation of BeWo cells was inhibited in a density‐dependent manner that may have been related to reduced cellular uptake of the inducing agent and of exogenous nutrients (purines) from culture fluids.
Prominent glutamine oxidation activity in mitochondria of avian transplantable hepatoma induced by MC‐29 virus
Authors: Matsuno Tetsuya , Satoh Teikichi , Suzuki Haruo
Published: February 2005

Well coupled mitochondria were isolated from transplantable chicken hepatoma induced by MC‐29 virus. The mitochondrial phosphate‐dependent and phosphate‐independent glutaminase activities were increased compared with those from normal chicken liver. Glutamate dehydrogenase was undetectable in the tumor mitochondria. Oxypolarographic tests showed the following: (1) glutamine oxidation was prominent in the tumor mitochondria and was mediated through an NAD‐linked reaction, while mitochondria from the liver showed a feeble glutamine oxidation; (2) glutamine oxidation by tumor mitochondria was inhibited either by aminooxyacetate, inhibitor of transaminases, or prior incubation of mitochondria with DON (6‐diazo‐5‐oxonorleucine), which inhibited mitochondrial glutaminases. Bromofuroate, inhibitor of glutamate dehydrogenase, had little or no effect; and (3) glutamate oxidation was also inhibited by aminooxyacetate, while it was not affected by DON. These findings clearly show a high glutamate oxidation activity in the hepatoma and indicate that the product of glutamine hydrolysis, glutamate, is catabolized via transamination in the mitochondria to supply ATP.
Down‐modulation of receptors for granulocyte colony‐stimulating factor on human neutrophils by granulocyte‐activating agents
Authors: Nicola N. A. , Vadas M. A. , Lopez A. F.
Published: February 2005

Purified human blood neutrophils were able to bind radioiodinated murine granulocyte‐colony‐stimulating factor (G‐CSF) in a specific manner. This factor has previously been shown to stimulate functional activities of human and murine neutrophilic granulocytes and to be functionally analogous to human‐derived CSFβ. The binding of 125I G‐CSF to human neutrophils was competed for equally by unlabeled G‐CSF and CSFβ but not by other CSF's. Saturation analysis indicated that human neutrophils displayed about 700–1,500 receptors for G‐CSF/CSFβ per cell. Three other agents (N‐formyl‐methionine‐leucine phenylalanine, bacterial lipopolysaccharide, and human CSFα) known to activate neutrophils did not compete directly for G‐CSF binding sites but, in preincubation experiments at 37°C, were able to down‐modulate the expression of G‐CSF receptors on human neutrophils in a dose‐ and time‐dependent manner. This effect was specific since the same agents have been shown elsewhere to up‐regulate the expression of other granulocyte surface antigens and other agents were much less effective at down‐modulating G‐CSF receptors. Since the granulocyte‐activating agents increase the sensitivity of human neutrophils to G‐CSF/CSFβ and mimic some of the actions of G‐CSF on neutrophils, it is suggested that G‐CSF receptor down‐modulation might be a mechanism whereby these agents activate G‐CSF receptors and thereby exert some of their effects.
Effects of transformation on the expression of laminin and fibronectin by neural cells
Authors: Giotta Gregory J. , Newby Ron , Oliver Noelynn
Published: February 2005

We have studied laminin and fibronectin expression in a collection of rat cerebellar neural cell lines transformed with a mutant of Rous sarcoma virus which is temperature sensitive for transformation. We show that regardless of their neuronal or glial properties the cell lines produce both laminin and fibronectin. Laminin is expressed in similar amounts in cell lines grown at either the permissive or nonpermissive temperature for transformation, while fibronectin is generally expressed at higher levels in cells kept at the nonpermissive temperature. To provide further evidence that neural cells can produce laminin and fibronectin, double label immunofluorescence studies were conducted on primary cerebellar cultures. Both laminin and fibronectin were found to be present in the primary culture, and laminin was found to be associated with a subpopulation of astrocytes.
Reversible alterations in cultured pulmonary artery endothelial cell monolayer morphology and albumin permeability induced by ionizing radiation
Authors: Friedman Mitchell , Ryan Una S. , Davenport W. Clark , Chaney Edward L. , Strickland Debra L. , Kwock Lester
Published: February 2005

The effects of ionizing irradiation (0, 600, 1,500, or 3,000 rads) on the permeability of pulmonary endothelial monolayers to albumin were studied. Pulmonary endothelial cells were grown to confluence on gelatin‐coated polycarbonate filters, placed in serum‐free medium, and exposed to a 60Co source. The monolayers were placed in modified flux chambers 24 hours after irradiation; 125l‐albumin was added to the upper well, and both the upper and lower wells were serially sampled over 4 hours. The amount of albumin transferred from the upper well/hour over the period of steady‐state clearance (90–240 min after addition of 125l‐albumin) was 2.8 ± 0.2% in control monolayers and was increased in monolayers exposed to 1,500 or 3,000 rads (increase of 63 plusmn; 10% and 61 plusmn; 10%, respectively, P<0.01). No increase was found in monolayers exposed to 600 rads. The increases in endothelial albumin transfer rates were associated with morphologic evidence of monolayer disruption and endothelial injury which paralleled the changes in albumin permeability. Dose‐dependent alterations in endothelial actin filament organization were also found. Incubation of the monolayers exposed to 3,000 rads with medium supplemented with 10% fetal calf serum for 24 hours resulted in normalization of albumin permeability, improvement in morphologic appearance of the monolayers, and reorganization of the actin filament structure. These studies demonstrate that ionizing radiation is an active principle in the reversible disorganization of cultured pulmonary endothelial cell monolayers without the need of other cell types or serum components.
Regulation of gonadotropin receptors, gonadotropin responsiveness, and cell multiplication by somatomedin‐C and insulin in cultured pig Leydig cells
Authors: Bernier M. , Chatelain P. , Mather J. P. , Saez J. M.
Published: February 2005

We have investigated the effects of insulin and somatomedin‐C/insulinlike growth factor l(Sm‐C) in purified porcine Leydig cells in vitro on gonadotrophins (hCG) receptor number, hCG responsiveness (cAMP and testosterone production), and thymidine incorporation into DNA. Leydig cells cultured in a serum‐free medium containing transferrin, vitamin E, and insulin (5 μ/ml) maintained fairly constant both hCG receptors and hCG responsiveness. When they were cultured for 3 days in the same medium without insulin, there was a dramatic decline (more than 80%) in both hCG receptor number and hCG responsiveness. However the cAMP but not the testosterone response to forskolin was normal. Both insulin and Sm‐C at nanomolar concentrations prevent the decline of both hCG receptors and hCG‐induced cAMP production. This effect of both peptides was dose dependent with an ED50 of about 1 ng/ml and 5 ng/ml for SM‐C and insulin, respectively. Insulin and Sm‐C had no additive effect on these parameters. At nanomolar concentrations, Sm‐C and insulin enhanced hCG‐induced testosterone production but the effect of Sm‐C was significantly higher than that of insulin. However, the effect of insulin at higher concentrations (5 μg/ml) was significantly higher than that of Sm‐C at 50 ng/ml. In contrast, at nanomolar concentrations only Sm‐C stimulated [3H]‐thymidine incorporation into DNA and cell multiplication, the stimulatory effect of insulin on these parameters, was seen only at micromolar concentrations. These results indicate that both Sm‐C and insulin acting through their own receptors increase Leydig cell steroidogenic responsiveness to hCG by increasing hCG receptor number and improving some step beyond cAMP formation. ln contrast, the mitogenic effects of insulin are mediated only through Sm‐C receptors.
Caffeine contracture in the cultured chick myotube
Authors: Saito Koji , Ozawa Eijiro
Published: February 2005

A possible function of Ca store site in cultured chick myotubes was examined by recording contraction of the myotube with special reference to the effect of caffeine. Caffeine at low concentrations (below 1 mM), applied focally on the myotube through a micropipette with a pressure pulse, elicited focal contraction without membrane potential changes. Procaine inhibited the caffeine contracture. Deuterium oxide also inhibited the caffeine contracture at low concentrations, but enhanced the maximal contracture. These observations are Similar to those in the mature frog muscle fiber in which the sarcoplasmic reticulum (SR) is a main site of caffeine action. On the basis of these similarities, it was considered that caffeine acts on SR to elicit contracture in the myotube. The ability of SR to accumulate and release Ca ion seemed to be low, because caffeine contracture decreased or disappeared in a Ca‐free solution in many myotubes.
1,25‐dihydroxycholecalciferol‐induced differentiation of myelomonocytic leukemic cells unresponsive to colony stimulating factors and phorbol esters
Authors: Bettens Florence , Schlick Erich , Farrar William , Ruscetti Francis
Published: February 2005

The murine myelomonocytic leukemia cell line WEHI‐3B D +, which differentiates in response to granulocyte colony stimulating factor (G‐CSF), can also be induced to differentiate into monocyte‐macrophages by phorbol myristate acetate (PMA) treatment, whereas the WEHI‐3B D− subline, which is unresponsive to G‐CSF and PMA, can be induced to differentiate to granulocytes as well as monocytes by, 1, 25‐dihydroxycholecalciferol [1, 25‐(OH)2 D3], the biologically active metabolite of vitamin D3. A newly developed variant of the WEHI‐3B D+ line, named WEHI‐3B D + G, which was responsive to G‐CSF but not to PMA, was also differentiated to granulocytes by 1,25‐(OH)2 D3. Although vitamin D3 has been reported to induce macrophage differentiation in responsive tumor cells, this is the first demonstration that 1,25‐(OH)2 D3 can induce granulocyte differentiation. In both differentiation pathways, cessation of. cellular proliferation accompanies changes in morphologic and cytochemical properties of the cells. This suggests that leukemic cell lines unresponsive to differentiation agents acting at the cell surface retain their ability to differentiate in response to agents that do not act via the plasma membrane such as 1,25‐(OH)2 D3, which has cytosolic/nuclear receptors. Vitamin D3 could act through different cellular pathways inducing differentiation or by bypassing only the first step of a common differentiation cascade used by agents with cell surface receptors such as CSF. These results suggest that low doses of 1,25‐(OH)2 D3 may be useful in combination with hemopoietic growth factors (CSFs) as therapeutic agent to induce leukemic cell differentiation in vivo.
Signal transduction in human monocytes: Relationship between superoxide production and the level of kinase C in the membrane
Authors: Costa‐Casnellie Maria R. , Segel George B. , Lichtman Marshall A.
Published: February 2005

Activation of monocytes and neutrophils results in an increased production of superoxide, an important cytotoxic compound. The previous finding that two agents that induce superoxide production cause opposite translocation of kinase C (Costa‐Casnellie et al. [1985] Biochem. Biophys. Res. Commun., 133:1139–1144). led us to study the role of kinase C in superoxide production. In monocytes induction of superoxide production by 13‐tetradecanoate phor‐bol acetate requires translocation of kinase C from the cytosol to the membrane. Superoxide production is also induced by concanavalin A, but this induction is accompanied by a shift of kinase C from the membrane to the cytosol. Superoxide production by concanavalin A is greatly augmented by cytochalasin B. During activation by Con A and cytochalasin B the membrane kinase C is translocated to the cytosol in a manner similar to that observed in the presence of Con A alone. Under these conditions approximately 5–10% of kinase C remains associated with the membrane. Thus, induction of superoxide production by concanavalin A is independent of the levels of kinase C tightly bound to the membrane. We also show evidence that the concanavalin A‐induced release of kinase C from the membrane is not due to an increase in levels of intracellular calcium or increased phosphoinositide turnover. In summary, these data indicate that concanavalin A and phorbol ester‐induced superoxide production by human monocytes occurs by distinct pathways and that superoxide production is not closely correlated with specific levels of membrane‐associated kinase C activity.
Contradistinctive growth responses of cultured rat intestinal epithelial cells to epidermal growth factor depending on cell population density
Authors: Blay Jonathan , Brown Kenneth D.
Published: February 2005

The growth response of cultured rat intestinal epithelial (RIE‐1) cells to epidermal growth factor (EGF) depends on the cell population density. EGF stimulated the proliferation of RIE‐1 cells in dense cultures, but inhibited the proliferation of cells growing at low population densities. In contrast, insulin enhanced RIE‐1 cell growth irrespective of the population density. The tumour promoter 12‐0‐tetradecanoylphorbol 13‐acetate (TPA), like EGF, inhibited the proliferation of low‐density RIE‐1 cells, but differed from EGF in that it did not stimulate the growth of dense cultures.
Studies on the metabolism of retinol‐binding protein by primary hepatocytes from retinol‐deficient rats
Authors: Dixon Joseph L. , Goodman DeWitt S.
Published: February 2005

Studies were conducted to explore the regulation of retinol‐binding protein (RBP) metabolism in cultured primary hepatocytes from retinol‐deficient rats. Newly isolated hepatocytes from retinol‐deficient rats contained elevated levels (3.4‐fold) of RBP, compared to hepatocytes from normal (retinol‐adequate) rats. Addition of retinol to retinol‐depleted hepatocytes stimulated RBP secretion by the cells in a concentration‐dependent manner. Maximal sitmulation of RBP secretion was seen with a retinol level of 0.3 μg/ml. The effect of retinol was quite rapid, and was evident by 20 minutes after addition of retinol to the medium. Stimulation of RBP secretion was only seen during the first few hours after retinol addition. The effect of retinol was specific for RBP; thus, retinol had no effect on the secretion rates of transthyretin or albumin. Addition of retinoic acid also stimulated RBP secretion by retinol‐deficient hepatocytes. Addition of dexamethasone to retinol‐deficient cells did not maintain the initial rate of RBP secretion. Dexamethasone also had no effect on the secretion of transthyretin or albumin by these cells. The effects of retinol and of dexamethasone seen here with retinol‐depleted cells differed dramatically from effects seen in other studies with normal (retinol‐adequate) hepatocytes. Thus, with normal cells, dexamethasone maintains RBP, TTR, and albumin production and secretion rates close to initial rates. Also in normal hepatocytes, with ample retinol available within the cell, addition of exogenous retinol does not appear to influence RBP secretion. In contrast, and as shown previously in intact rats, in retinol deficiency the availability of retinol specifically regulates the secretion of RBP by hepatocytes.
Induction of DNA synthesis in dog thyrocytes in primary culture: Synergistic effects of thyrotropin and cyclic AMP with epidermal growth factor and insulin
Authors: Roger Pierre P. , Servais Patricia , Dumont Jacques E.
Published: February 2005

We have investigated the growth effects of thyrotropin (TSH) (mimicked by forskolin and acting through cyclic AMP), epidermal growth factor (EGF), serum (10%) and insulin on quiescent dog thyroid epithelial cells in primary culture in a serum‐free defined medium. These cells were previously shown to retain the capacity to express major thyroid differentiation markers. In the presence of insulin and after a similar prepreplicative phase of 18 ± 2h, TSH, EGF, and serum promoted DNA synthesis in such quiescent cells only a minority of which had proliferated in vitro before stimulation. The combination of these factors induced more than 90% of the cells to enter S phase within 48 h and near exponetial proliferation. Analysis of the cell cycle parameters of the stimulated cells revealed that the G1 period duration was similar to the length of the prepreplicative phase of quiescent thyroid cells; this might indicate that they were in fact in an early G1 stage rather than in G0 prior to stimulation. TSH and EGF action depended on or was potentiated by insulin. Strikingly, nanomolar concentrations of insulin were sufficient to support stimulation of DNA synthesis by TSH, while micromolar concentrations of insulin were required for the action of EGF. This suggests that insulin supported the action of TSH by acting on its own high affinity receptors, whereas its effect on EGF action would be related to its somatomedinike effects at high supraphysiological concentrations. Insulin stimulated the progression in the prepreplicative phase initiated by TSH or forskolin. In addition, in some primary cultures TSH must act together with insulin to stimulate early events of the prereplicative phase. In the presence of insulin, EGF, and forskolin, an adenylate cylase activator, markedly synergized to induce DNA synthesis. Addition of forskolin 24 h after EGF or EGF 24 h after forskolin also resulted in amplification of the growth response but with a lag equal to the prepreplicative period observed with the single compound. This indicates that events induced by the second factor can no longer be integrated during the prereplicative phase set by the first factor. These finding demonstrate the importance of synergistic cooperation between hormones and growth factors for the induction of DNA synthesis in epithelial thyroid cells and support the proposal that essentially different mitogenic pathways‐cyclic AMP‐dependent or independent‐may coexist in one cell.
Binding sites for elastase on cultured human fibroblasts that do not mediate internalization
Authors: Campbell Corinne Horn , Cunningham Dennis D.
Published: February 2005

The proteolytic actions of elastases have been implicated in extracellular matrix damage, which is characteristic of a variety of pathological conditions including emphysema and rheumatoid arthritis. In order to elucidate the molecular events involved in elastase interaction with connective tissue cells, the present study was designed to investigate the association of elastase with human fibroblasts at 4°C. Elastase bound saturably to binding sites that were present on the surface of these cells. Analysis of cell‐bound elastase by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis revealed the presence of a high molecular weight complex (Mr 54,000) that was not formed with elastase whose catalytic site serine was derivatized with a diisopropylphosphate group. The complex did not represent elastase bound to either protease nexin or contaminating serum. The cellular component with which elastase formed a complex could not be detected in the cell culture medium. Unexpectedly, elastase that had been pre‐bound at 4°C was not internalized after cells were warmed to 37°C. The elastase binding site described in this report is therefore distinct from high affinity binding sites involved in receptor‐mediated endocytosis and intracellular degradation.
Characterization of beta‐adrenergic receptors throughout the replicative life span of IMR‐90 cells
Authors: Scarpace Philip J.
Published: February 2005

Beta‐adrenergic receptor number and receptor affinity for isoproterenol were assessed at various in vitro ages of the human diploid fibroblast cell line IMR‐90. From population doubling level (PDL) 33 to 44, there was a positive correlation between beta‐adrenergic receptor density and PDL (r = 0.709). Beta‐adrenergic receptors, assessed by Scatchard analysis of [125I]‐iodocyano‐pindolol (ICYP) binding, increased from 15 fmol/mg protein at PDL 33 to 36 fmol/mg protein at PDL 44. In contrast, from PDL 44 to 59, there was a negative correlation between beta‐adrenergic receptor density and PDL (r = −0.768). Receptor density declined to 12 fmol/mg protein at PDL 59. When the density of beta‐adrenergic receptors was expressed as receptor per cell, the findings were similar. Receptor agonist affinity for isoproterenol was determined from Hill plots of [125I]‐ICYP competition with isoproterenol. There was no change in the dissociation constant for isoproterenol with in vitro age. In humans, serum norepinephrine concentrations increase with age. This increase in serum norepinephrine may be partially responsible for the decreased betaadrenergic receptor‐agonist affinity observed with age in human lymphocytes and rat heart and lung. Similar changes in receptor‐agonist affinity are observed in rat heart and human lymphocytes following exposure to betaagonists and are part of the desensitization process. The present findings are consistent with the hypothesis that the decreases in receptor agonist affinity in rat and man with age are secondary to increases in catecholamine concentrations.
The control of DNA synthesis in primary cultures of hepatocytes from adult and young rats: Interactions of extracellular matrix components, epidermal growth factor, and the cell cycle
Authors: Tomomura Akito , Sawada Norimasa , Sattler Gerald L. , Kleinman Hynda K. , Pitot Henry C.
Published: February 2005

Hepatocytes from adult and 4‐week‐old rats cultured on one of several extra‐cellular matrix components were stimulated to replicate by epidermal growth factor (EGF). DNA synthesis was increased at 44–48 hr in adult hepatocytes and at 24, 48, and 72 hr in hepatocytes from young rats when EGF was added 2 hr after explantation. When EGF was added at 24 hr, maximal DNA synthesis of adult hepatocytes was observed at 48 hr, whereas that of 4‐week‐old hepatocytes was seen at 48 and 72 hr. Ten ng EGF per ml was the optimal concentration for maximal DNA synthesis in both adult and young cells. DAN synthesis decreased with increasing cell density, but this effect was less in hepatocytes from young than in those from adults.

When hepatocytes were cultured on substrata consisting of individual extracellular matrix components, neither the time that adult cells needed to respond to EGF nor the time from stimulation by EGF to the peak of maximal DNA synthesis was altered in either adult or young cells. The optimal EGF concentration for maximal DNA synthesis and the cell density control of replication were also not altered by the substrata used. Substrata made from each of the extracellular matrix components studied enhanced DNA synthesis of adult and young hepatocytes stimulated by EGF in the following decreasing order: fibronectin, type IV collagen, type I collagen, and laminin. In both adult and young hepatocytes the enhancement of DNA synthesis was greatest when cultured on fibronectin. Thus the initiation and magnitude of DNA synthesis in primary cultures of rat hepatocytes were altered both by the age of the donor and the substratum on which the cells were explanted.
Induction of major histocompatibility class I antigens by interferons in undifferentiated F9 cells
Authors: Wan Yu‐Jui Yvonne , Orrison Bonnie M. , Lieberman Ronald , Lazarovici Philip , Ozato Keiko
Published: February 2005

Mouse embryonal carcinoma F9 cells, upon treatment with interferons (IFNs), express major histocompatibility (MHC) Class I antigens, which are otherwise not expressed in these cells. Both IFN‐γ and IFN‐α/β increase the steady‐state level of Class I mRNA within 60 min of the treatment which leads to the subsequent surface expression of the H‐2Kb and H‐2Db antigens, suggesting that undifferentiated F9 cells express IFN receptors. IFNs induce Class I antigen expression in F9 cells in a highly selective manner: unlike retinoic acid treatment which also stimulates the antigen expression, IFNs induce neither morphological differentiation, increased binding of epidermal growth factor, nor reduction of expression of stage specific embryonic antigen. The effect of IFNs is reversible; removal of IFNs, even after prolonged exposures, results in a rapid loss of the Class I gene expression. Further, Class I mRNA induction is not inhibited by cycloheximide, suggesting possible independence from de novo protein synthesis. This Class I antigen induction in F9 cells is reminiscent of that observed in somite stage mouse embryos by IFN treatment and may offer a model system to study activation of MHC genes during development.
Species‐specific differences in the toxicity of rhodamine 123 toward cultured mammalian cells
Authors: Gupta Radhey S. , Dudani Anil K.
Published: February 2005

The toxicity of cationic fluorescent dye, rhodamine 123, towards a number of independently established cell lines from three different species, namely human, mouse, and Chinese hamster, has been examined. All of the cell lines from any one species that were examined were found to exhibit similar sensitivities towards rhodamine 123 and no appreciable differences were observed between the normal and transformed cell types. However, in comparison to the cells of human origin, mouse and Chinese hamster cell lines exhibited about 10‐fold and 70‐fold higher resistance, respectively, and these differences appeared to be species related. In contrast to rhodamine 123, no differences in relative toxicities for these cell lines were observed for the structurally related neutral dye, rhodamine B. Fluorescence studies with rhodamine 123 show that in comparison to mouse and Chinese hamster cells, the more sensitive human cells show much higher uptake/binding of the drug, and a good correlation was seen in these studies between the extent of dye uptake/binding and the relative sensitivities of cell lines to rhodamine 123. These results provide evidence that the observed species‐related differences in cellular toxicities are due to differences in the cellular uptake/binding of the dye.
Effect of essential amino acids on the phosphorylation of a 40S ribosomal protein and protein synthesis in Acanthamoeba castellanii
Authors: Jantzen Helga , Schulze Ingrid
Published: February 2005

Reversible and multiple phosphorylation of a 40S ribosomal protein is observed in a variety of eukaryotic cells. In the primitive eukaryote Acanthamoeba, one or three phosphorylated S3 derivatives are observed during growth phase in nondefined nutrient medium (ND cells) or in chemically defined nutrient medium (D cells), respectively. In both cases, stationary phase cells exhibit nonphosphorylated S3; however, transfer of these cells into the respective fresh nutrient media results in a transient accumulation of four phosphorylated S3 derivatives. Transfer of D cells into nutrient medium, deficient in all or any single essential amino acids, leads to reversible inhibition of S3 phosphorylation and growth arrest. The low level of phosphorylated S3 is not simply the consequence of growth arrest, since in cells where growth is arrested differently, the level of phosphorylated S3 can be high. In response to amino acid deficiency, a number of other changes can be observed. These include a 2–3‐fold decrease of total protein synthesis, 13 changes in the cellular protein pattern, and specific alterations in the ribosome absorbance profiles and in the distribution of poly‐A+ within subribosomal and ribosomal fractions. While the rate of total protein synthesis seems to be associated with the level of phosphorylated S3, the level of the synthesis of at least 10 of the particular proteins can be dissociated from the level of S3 phosphorylation.
Cytoplasmic maturation of the snRNAs
Authors: Zieve Gary W.
Published: February 2005

The snRNAs are abundant and stable components of the interphase nucleus. Aqueous and non‐aqueous cell fractionation demonstrate that the snRNAs appear transiently in the cytoplasm shortly after transcription, before returning permanently to the interphase nucleus. In pulse label and chase experiments, the newly synthesized snRNA species appear in the cytoplasm after 1 min of labeling and then return to the interphase nucleus after approximately 15 min in the cytoplasm. In order to study the maturation and intracellular transport of these particles, a battery of metabolic inhibitors and alterations in cell culture conditions were investigated for their ability to interfere with the return of the newly synthesized snRNAs to the nucleus. A wide range of inhibitors of the cytoskeleton did not interfere with this process. Only the inhibition of protein synthesis and exposure of cells to medium of at least twice the normal tonicity block the return of the snRNAs to the nucleus. Immunofluorescent staining of cells exposed to hypertonic medium identifies discrete foci in the cytoplasm that stain with the Sm antiserum, directed against proteins associated with the snRNAs. Using a detergent extraction procedure that preserves the cytoskeleton, the newly synthesized snRNAs in the cytoplasm fractionate as soluble complexes. These data are consistent with the hypothesis that the snRNAs partition into the interphase nucleus because of a preferential solubility and the existence of specific binding sites.
Growth kinetics and differentiation in vitro of normal human uroepithelial cells on collagen gel substrates in defined medium
Authors: Reznikoff Catherine A. , Loretz Linda J. , Pesciotta Donna M. , Oberley Terry D. , Ignjatovic Melanie M.
Published: February 2005

Conditions have been described for the selective growth, serial cultivation, and postconfluent morphological differentiation in vitro of normal adult human uroepithelial cells (HUC) on collagen gel substrates in a serum‐free medium without the deliberate addition of undefined components and without a requirement for a polypeptide growth factor. The culture medium used (F12*) was the standard Ham's F12 medium (0.3 mM calcium) supplemented with 1 μg/ml hydrocortisone, 5 μg/ml transferrin, 10 μg/ml insulin, 0.1 mM nonessential amino acids, 2.0 mM L‐glutamine, 2.7 mg/ml D‐glucose, 10−4 M ethanolamine or 10−4 M phosphoethanolamine, and 5 × 10−8 M selenium. HUC grown in F12* on Type I collagen gel substrates had a generation time of 33 hours and could be serially passed 3–5 times during log phase of growth (20–25 population doublings) before spontaneously senescing. Transmission electron microscopy showed that cultures of HUC grown entirely in serumfree F12* on collagen gel substrates morphologically differentiate postconfluence to resemble in some respects the stratified uroepithelium in vivo, although neither a basal lamina nor an asymmetric unit membrane develop. The addition of epidermal growth factor (EGF) to the F12* did not improve either the growth rate or the lifespan in vitro of HUC. In contrast, the addition of fetal bovine serum (FBS) to F12* was mitogenic to HUC in a dose‐dependent manner in the concentration range 0.01–1.00% (4–400 μg/ml protein), but higher concentrations of FBS did not improve growth further. The generation time of HUC in 1% FBS‐F12* decreased to 21 hours, and the potential population doublings in vitro increased to 31–36. Small amounts (140 μg/ml) of bovine pituitary extract (BPE) were similarly mitogenic to HUC in F12*. Altering the calcium concentration in the standard Ham's F12 medium (0.3 mM), however, did not improve the growth of HUC in serum‐containing or serumfree medium. Higher calcium concentrations (0.30–0.90 mM) were neither mitogenic nor inhibitory to HUC growth, but resulted in decreasing viability of HUC in growing cultures, suggesting an accelerating rate of cellular differentiation. In contrast HUC in low calcium, serum‐free F12* (0.1 mM) failed to stratify and morphologically differentiate even in postconfluent cultures. This failure of HUC to differentiate in low calcium F12* medium did not confer a long‐term growth advantage. On the contrary, optimal long‐term growth of HUC serially passaged in F12* was obtained using a moderate calcium concentration (0.3 mM) which supported morphological differentiation and stratification of HUC in postconfluent cultures, showing that these two parameters are not mutually exclusive in this system and that there exists a delicate calcium‐regulated balance between growth of HUC in nonconfluent cultures and stratification and differentiation of HUC in postconfluent cultures.
A clonal derivative of tunicamycin‐resistant Chinese hamster ovary cells with increased N‐acetylglucosamine‐phosphate transferase activity has altered asparagine‐linked glycosylation
Authors: Waldman Barbara Criscuolo , Oliver Constance , Krag Sharon S.
Published: February 2005

A population of Chinese hamster ovary (CHO) cells resistant to the antibiotic tunicamycin (TM) had previously been isolated (Criscuolo, B.A., and Krag, S.S. (1982) J. Cell Biol. 94:586–591) by a stepwise selection procedure using progressive increments of TM added to the medium. TM inhibits asparaginelinked glycoprotein biosynthesis by blocking the transfer of N‐acetylglucosamine‐1‐phosphate from the sugar nucleotide UDP‐N‐acetylglucosamine to the isoprenoid lipid carrier, dolichyl phosphate. Four clonal derivatives were isolated from the TM‐resistant population in the presence of 27 μg TM/ml and were found to overproduce the N‐acetylglucosamine‐phosphate transferase activity to the same extent (approximately 15‐fold compared to wild‐type cells). One of these clones, 3E11, was > 550‐fold more resistant to TM than wild‐type cells. The resistance phenotype remained during at least 2.5 months of growth in the absence of TM. 3E11 cells exhibited chromosomal translocations, but no homogeneously staining regions (HSR) or double minute chromosomes. The N‐acetylglucosamine‐phosphate transferase activity in 3E11 cells was membrane‐associated and was inhibited by TM. A 140,000‐dalton membrane protein and at least four other membrane proteins were enriched in 3E11 cells. Mannosylphosphoryldolichol synthase and glucosylphosphoryldolichol synthase activities were not elevated in membranes prepared from 3E11 cells. Asparagine‐linked glycosylation was altered such that 3E11 cells synthesized primarily a truncated oligosaccharide, Man5GlcNAc2, perhaps due to the reduced amount of mannosylphosphoryldolichol relative to wild‐type cells.
Apical trehalase expression associated with cell patterning after inducer treatment of LLC‐PK1 monolayers
Authors: Yoneyama Yoshitaka , Lever Julia E.
Published: February 2005

Trehalase, a differentiation‐specific marker of renal proximal tubule brush border membrane, is expressed in confluent long‐term cultures of the renal epithelial cell line LLC‐PK1. The level of trehalase is greatly increased after treatment of cultures with differentiation inducers such as hexamethylene bisacetamide (HMBA), accompanied by increases in other apical membraneassociated differentiated functions (Yoneyama and Lever: J. Cell. Physiol. 121: 64–73, 1984). In the present study, we utilize a polyclonal antibody specific for renal trehalase to demonstrate that trehalase expression induced in LLC‐PK1 cultures after HMBA treatment is localized in cells forming a three‐dimensional network of strands across the confluent monolayer. The antitrehalase antibody recognized an apical membrane antigen of apparent molecular weight 100–110 kD both in LLC‐PK1 cultures and in the corresponding pig renal brush border membranes. Strand formation and total trehalase activity increased in parallel as a function of inducer concentration and duration of exposure. Strand formation and trehalase expression were also greatly enhanced in monolayers grown on a Nuclepore filter support even in the absence of inducer. Strand formation was not a prerequisite for induced trehalase expression in culture, since strands did not develop in cultures treated with N, N'‐dimethylformamide (DMF) an equally potent inducer of trehalase expression. In this case, cells which expressed increased levels of trehalase were dispersed at random over the monolayer. Induction of strand formation and trehalase expression by HMBA required a minimum exposure period of 48 hr and persisted up to a week after removal of inducer. By contrast, the response to DMF required continuous presence of inducer. Levels of trehalase declined even in the continuous presence of inducer in local regions of low cell density created by wound‐repair of the monolayer. In addition to the membrane‐bound form, trehalase activity was also recoverable from the culture medium, but release of trehalase was not affected by inducers.

These observations are consistent with the view that a cell type committed to express a program of differentiation after HMBA treatment or growth on a permeable support is organized in specific cell patterns visible as strands over the confluent cell monolayer.
Toxicity of ligand and antibody‐directed ricin A‐chain conjugates recognizing the epidermal growth factor receptor
Authors: Vollmar Angelika M. , Banker Deborah E. , Mendelsohn John , Herschman Harvey R.
Published: February 2005

Approximately equal amounts of 125I‐mAb 225 (a monoclonal antibody recognizing the human epidermal growth factor receptor) and 125I‐labeled epidermal growth factor (125I‐EGF) were bound by HeLa cells. However, these two EGF receptor binding moieties had different fates after binding. Sixty percent of cell‐associated 125I‐EGF was internalized. The majority of internalized 125I was released from the cell within 2 hr. In contrast, whereas only 30% of bound 125I‐mAb 225 was internalized by HeLa cells, the internalized radioactivity remained cell‐associated. EGF and mAb 225 were used to construct ricin A‐chain (RTA) conjugates. The two chimeric molecules, EGF‐RTA and mAb 225‐RTA, were equally toxic to human HeLa cells. EGF‐RTA was also toxic to murine 3T3 cells. In contrast, mAb 225‐RTA was not toxic to 3T3 cells, consistent with the human EGF‐receptor specificity of mAb 225. Neither conjugate was cytotoxic to EGF‐receptor‐deficient 3T3‐NR6 cells. Rapidity and potency of protein synthesis inhibition of HeLa cells were equivalent for the two chimeric conjugates, as was the degree to which colony‐forming ability was reduced. However, ammonium chloride enhanced the toxicity of EGF‐RTA but not mAb 225‐RTA, suggesting that the two toxic chimeric toxins‐‐like the unconjugated receptor‐binding moieties‐‐are processed differently by HeLa cells.
PDGF induces c‐myc mRNA expression in MG‐63 human osteosarcoma cells but does not stimulate cell replication
Authors: Womer Richard B. , Frick Kevin , Mitchell Christopher D. , Ross Alonzo H. , Bishayee Subal , Scher Charles D.
Published: February 2005

The platelet‐derived growth factor (PDGF) modulated growth response of the MG‐63 human osteosarcoma cell line, which neither expresses c‐sis mRNA nor secretes a PDGF analogue, was characterized. Scatchard analysis demonstrated that the MG‐63 cells have 23,000 receptors per cell with a kd of 5 × 10−11 M. The receptor became phosphorylated, in a PDGF concentration‐dependent manner, when 32P‐orthophosphate‐labeled cells were treated with PDGF for 3 h at 4°C. The phosphorylated receptor was identified by autora‐diography and gel electrophoresis after isolation of the 32P‐labeled receptor using a solid‐phase monoclonal antibody directed against phosphotyrosine. Binding of the receptor to the antibody was inhibited by 5 mM phenyl phosphate, further suggesting that PDGF stimulated tyrosine‐specific receptor autophosphorylation. In addition, treatment of MG‐63 cells with PDGF for 3 h at 37°C induced a 7.5‐fold increase in c‐myc mRNA accumulation as analyzed on Northern gels. However, MG‐63 cells grew equally well in either serum‐(which contains PDGF) or plasma‐(which does not) supplemented medium. Furthermore, PDGF did not stimulate DNA synthesis in growth arrested MG‐63 cells, nor did it potentiate DNA synthesis modulated by somatomedin C. Thus MG‐63 cells are a naturally occurring cell variant in which PDGF stimulates c‐myc expression but does not modulate mitogenesis.
Hormonal regulation of acid phosphatase release by osteoclasts disaggregated from neonatal rat bone
Authors: Chambers T. J. , Fuller K. , Darby J. A.
Published: February 2005

Osteoclasts disaggregated from neonatal rat long bones and incubated on plastic or glass substrates were found to release a considerable proportion of tartrate‐resistant acid phosphatase into culture supernatants. Enzyme release was detectable in the supernatant medium of cultures containing as few as ten cells after 1 hr of incubation and proceeded in a linear manner for the ensuing 6 hr. Calcitonin (1 pg/ml) and cytochalasin B (5 μ/ml) inhibited release into the supernatant, suggesting that release represents enzyme secretion. Prostaglandin E1 induced transient inhibition followed by recovery; parathyroid hormone and 1,25(OH)2 vitamin D3 were without influence. Acid phosphatase release in these cultures shows a pattern of hormone responsiveness that coincides with the effects of these hormones on bone resorption by isolated osteoclasts. The extent of acid phosphatase release and its regulation by calciotropic hormones imply a central role for acid hydrolase secretion in osteoclastic bone resorption. The experimental system described in this study may facilitate analysis of the pharmacological hormonal and cellular regulation of osteoclastic function.