bluuebunny/crossref_metadata_2025 huggingface dataset:
Abnormal behavior of cultured fibroblasts from nodule and nonaffected aponeurosis of Dupuytren's disease
Authors: Azzarone Bruno , Failly‐Crepin Christianne , Day‐garosjean Leela , Chaponnier Christine , Gabbiani Giulio
Published: February 2005

Vimentin‐positive, desmin‐negative cells were established in culture from the nodule and from apparently normal palmar aponeurosis of a patient with Dupuytren's disease and compared with normal human embryonic and adult fibroblasts or sarcomatous cells. Cells from the nodule display in vitro biological properties that are intermediate between those expressed by normal fibroblasts and sarcoma cells or cells from the nodule transformed with SV40 virus. Thus, they represent an interesting in vitro model of partially transformed human cells. This behavior is not evolutive and justifies the classification of Dupuytren's disease among the benign mesenchymal tumors. The production of high level of plasminogen activator probably explains the local reactive pathology, and could act as a mitogenic stimulus for the proliferation of the nodule itself. Cultures derived from the apparently normal palmar aponeurosis show some but not all the abnormal growth properties of cells from nodules; this may help to explain the onset of local recurrences. Our results suggest that Dupuytren's disease is not strictly local and limited to the nodules, but affects, at least partially, the whole aponeurosis. Dupuytren's nodules could be considered as a model of tumor progression in a benign situation.
Protease nexins: Cell‐secreted proteins that mediate the binding, internalization, and degradation of regulatory serine proteases
Authors: Knauer Daniel J. , Thompson James A. , Cunningham Dennis D.
Published: February 2005

The protease nexins (PN‐I, Mr ∼︁38,000; PN‐II, Mr ∼︁95,000; and PN‐III, Mr ∼︁31,000) are recently described cell‐secreted proteins that selectively link to regulatory serins proteases in the extracellular environment and mediate their cellular binding, internalization, and degradation. In the present studies we compared the protease nexins with respect to protease specificity, heparin sensitivity, and general mode of action. By competitive binding assays using [125l]‐thrombin, [125l]‐nerve growth factor‐γ(125l‐NGF‐γ), and [125l]‐epidermal growth factor binding protein (125l‐EGF‐binding protein), we characterized the nexins in terms of protease specificity and determined that PN‐I links to and mediates the cellular binding of thrombin or urokinase, whereas PN‐II and PN‐III preferentially link to and mediate the cellular binding of the EGF binding protein and NGF‐γ, respectively. In addition, whereas the ability of PN‐I to link to thrombin is strongly modulated by heparin, PN‐II and PN‐III are essentially unaffected by heparin. The linkage of each of the nexins to their respective proteases requires the catalytic site serine of the protease, judged by the inability of diisopropylphospho (DIP) derivatives of the proteases tested to link to their respective nexins. Subsequent to linkage, the nexin:protease complexes are bound to cells, rapidly internalized, and ultimately degraded via a monensin‐sensitive apparently lysosomal pathway, although each nexin:protease complex is degraded at its own characteristic rate. Importantly, the protease nexins provide the major pathway through which human fibroblasts interact with each of the serine proteases studied. Taken together, these data suggest that the nexins are a unique class of cell‐secreted proteins that enable cells to monitor and selectively regulate specific serine proteases in their environment.
Mechanism of action of leukotriene B4: Intracellular calcium redistribution in rabbit neutrophils
Authors: Naccache P. H. , Molski T. F. P. , Borgeat P. , Sha'afi R. I.
Published: February 2005

The possible involvement of membrane‐bound calcium in the mechanism of action of leukotriene B4 was examined using the fluorescent chelate probe, chlortetracycline. Leukotriene B4 was found to cause a rapid release of membrane‐bound calcium at physiologically relevant concentrations. This effect of leukotriene B4 is stereospecific and its magnitude is decreased upon the transformation of leukotriene B4 into its ω‐hydroxy and ω‐carboxy metabolites. The pool of calcium affected by leukotriene B4 appears to be the same as that released by other chemotactic factors such as formyl–methionyl–leucyl–phenylalanine (f‐Met‐Leu‐Phe). Similarly, preincubation with f‐Met‐Leu‐Phe results in a decreased responsiveness of the cells to the addition of leukotriene B4. These results extend further the analogy between the mechanism of action of peptidic and lipid chemotactic factors, and emphasize the central role of the intracellular redistribution of calcium, as inferred and monitored by chlortetracycline fluorescence and steady‐state isotopic flux studies, in neutrophil activation.
Increased uptake of thymidine in the activation of sea urchin eggs. III. Effects of aphidicolin
Authors: Nishioka David , Killian Christopher E. , Chacon Cecilia T. , Sgagias Magdalene K.
Published: February 2005

Uptake and phosphorylation of externally supplied [3H]‐thymidine are fully stimulated in fertilized sea urchin eggs exposed to 5.0 μg/ml aphidicolin. As in untreated controls, the rate of uptake in aphidicolin‐treated eggs increases greater than 50‐fold shortly after fertilization, and greater than 85% of the transported thymidine is immediately phosphorylated to the triphosphate. The intracellular levels of [3H]‐thymidine triphosphate (3H‐dTTP) resulting from an external supply of [3H]‐thymidine is therefore equal in aphidicolin‐treated and untreated fertilized eggs. Under the same experimental conditions, the incorporation of externally supplied [3H]‐thymidine into newly synthesized DNA of fertilized eggs is 90% inhibited by exposure to aphidicolin. The full availability of 3H‐dTTP in these eggs further suggests that aphidicolin inhibits specifically at the level of DNA synthesis. This inhibitory effect is proportional to the concentration of aphidicolin between 0 and 5.0 μg/ml. In the continuous presence of 5.0 μg/ml aphidicolin, fertilized eggs fail to undergo mitotic chromosome condensation, nuclear envelope breakdown, and cytokinesis, suggesting a dependent link between these processes and the completion of nuclear DNA synthesis.
Calcium stimulates ornithine decarboxylase activity in cultured mammalian epithelial cells
Authors: Langdon Robert C. , Fleckman Philip , McGuire Joseph
Published: February 2005

Ornithine decarboxylase (ODC) activity usually rises to a peak a few hours after a trophic stimulus. The stimulation of ODC has been shown to depend on extracellular calcium in several in vitro eukaryotic systems. We have investigated the effect of calcium concentration on ODC activity and have found that ODC is stimulated when CaCl2 alone is added to calcium‐deprived cells. Epithelial cells from calf esophagus were cultured and grown until stratified. Replacement of medium with fresh serum‐free medium resulted in stimulation of ODC activity, which peaked at 4 hours and declined to basal level by 10 hours. Subsequent depletion of Ca2+ either by addition of ethylene glycol bis (β‐aminoethyl ether) N, N′‐tetraacetic acid (EGTA) or by replacement of medium with Ca2+‐free medium, resulted in obliteration of ODC activity 4 hours later. Conversely, cultures in which medium was replaced with Ca2+‐free medium and at 10 hours were repleted with Ca2+ (either by addition of CaCl2 or by replacement of medium with Ca2+‐containing medium) exhibited a pronounced elevation of ODC activity 4 hours later. ODC activity peaked at 6 hours after the addition of CaCl2 and declined by 8 hours. The effect was elicited by a wide range of concentrations of added Ca2+ from 0.1 mM to 4.0 mM, but was maximal at 1.0 mM. ODC activity was totally abolished if either cycloheximide (10 μg/ml) or putrescine (10 mM) was added to cultures immediately prior to Ca2+ addition. Actinomycin D (2, 5, or 10 μg/ml) added 30 minutes before Ca2+ did not prevent the stimulation of ODC by added Ca2+. Stimulation by Ca2+ is dependent on (1) absence of Ca2+ during the initial 10‐hour incubation and (2) duration of incubation in Ca2+‐free medium prior to Ca2+ replenishment. The results indicate that Ca2+ can increase ODC in epithelial cells exposed to Ca2+‐depleted medium and that the increase in ODC depends on protein synthesis but is not inhibited by actinomycin D.
Characterization of two populations of CFU‐S fractionated from mouse fetal liver by fluorescence‐activated cell sorting
Authors: Johnson G. R. , Nicola N. A.
Published: February 2005

The properties of spleen colony forming units (CFU‐S) contained in two murine fetal liver cell populations (pre‐colony‐forming cell (CFC) and CFC fractions) obtained after fluorescence‐activated cell sorting fractionation on the basis of pokeweed mitogen binding have been analyzed. Individual spleen colonies generated by both populations were examined to determine cellularity, differential cell morphology, as well as CFU‐S and in vitro CFC content. Spleen colonies from both populations contained approximately equal numbers of cells 7 days after transplantation; at 12 days, however, CFU‐S from the pre‐CFC fraction produced four times as many cells per colony than colonies from the CFC fraction. Although the frequency of spleen colonies containing CFU‐S was similar, in comparison to the CFC fraction, spleen colonies obtained from the pre‐CFC fraction contained higher absolute numbers of CFU‐S. This was most evident with CFU‐S assayed 12 days after transplantation of 12‐day spleen colony cells (pre‐CFC fraction, 84% of colonies contained CFU‐S, mean of 527 per colony; CFC fraction, 77% of colonies contained CFU‐S, mean of 41 per colony). Similarly, the in vitro CFC content of 7‐ or 12‐day spleen colonies was higher in colonies produced by pre‐CFC fraction CFU‐S. No significant differences were observed in either the proportion of CFU‐S in S‐phase or the morphology of spleen colonies produced by either cell population.
Cholesterogenesis and related enzymes in isolated rat hepatocytes during pre‐ and postnatal life
Authors: Leoni S. , Spagnuolo S. , Conti‐Devirgiliis L. , Dini L. , Mangiantini M. T. , Trentalance A.
Published: February 2005

Cholesterogenesis pathway during pre‐ and postnatal development was studied in isolated rat hepatocytes. No modified activity of cytosol acetoacetyl coenzyme A (CoA), thiolase, or 3‐hydroxy‐3‐methylglutaryl CoA (HMGCoA) synthase was detectable at the different stages examined. Minimal levels of 114C‐acetate incorporation into cholesterol and HMGCoA reductase activity were present at 16 days of fetal development in newborn and suckling rats, whereas both parameters increased rapidly before birth. The pattern of NaF nonsuppressible reductase activity showed a different activation state of the enzyme, suggesting the appearance of a modulation state, probably related to the development of some short‐term regulatory mechanisms.
In vitro and in vivo analysis of the control of dihydrofolate reductase gene transcription in serum‐stimulated mouse fibroblasts
Authors: Santiago Carlos , Collins Mark , Johnson Lee F.
Published: February 2005

We have studied the rate of transcription of the gene for dihydrofolate reductase (DHFR) in mouse 3T6 fibroblasts during serum‐induced transitions between the resting (G0) and growing states. As a model system, we have used a methotrexate‐resistant 3T6 cell line that overproduces DHFR and its mRNA about 300‐fold, yet regulates the expression of the DHFR gene in the same manner as normal 3T6 cells. In previous studies, we showed that the rate of production of cytoplasmic DHFR mRNA relative to total mRNA is about 4 times lower in resting than in exponentially growing cells. The rate increases to the growing value by about 15 hr following serum stimulation of the resting cells. This increase appeared to be controlled by regulating the rate of synthesis of DHFR hnRNA. In this study, we analyze the transcription of the DHFR gene in more detail. We use a variety of labeling times and RNA extraction procedures to measure the rate of synthesis of DHFR hnRNA relative to total hnRNA in pulse‐labeled cells or in nuclei isolated from cells at various times following serum stimulation. The amount of labeled DHFR RNA is determined by DNA‐excess filter hybridization. In all cases, the relative rate of synthesis of DHFR hnRNA increases at the same time, and to the same extent, as the rate of production of DHFR mRNA, suggesting that the increase in DHFR mRNA production is due to a corresponding increase in the rate of transcription of the DHFR gene. The increase in DHFR gene transcription is not blocked by cytosine arabinoside, showing that the increase does not depend on gene duplication. In isolated nuclei, DHFR RNA synthesis is inhibited by α‐amanitin (1 μg/ml), indicating that the DHFR gene is transcribed by RNA polymerase II. Others have shown that when stationary phase cells are stimulated to proliferate, the increase in DHFR mRNA content is controlled primarily at the post‐transcriptional level. Therefore, it appears that the rate of production of DHFR mRNA is controlled by different biochemical mechanisms when cells are in different physiological states.
On the role of protein phosphorylation in the ATP‐dependent permeabilization of transformed cells
Authors: Weisman Gary A. , Dunn Stanley D. , De Barun K. , Kitagawa Takayuki , Friedberg Ilan
Published: February 2005

Incubation of transformed mouse fibroblasts with external ATP in alkaline medium low in divalent cations causes an increase in the permeability of the plasma membrane to nucleotides and other small molecules. Previous suggestions that the phosphorylation of a 44,000 dalton membrane protein is involved in this permeabilization process have been pursued. Fractionation of cells that had been incubated with [γ‐32P] ATP revealed that the labeled 44K phosphoprotein was found in both the membrane and mitochondrial fractions. Incubation of fractions isolated from unlabeled cells with [γ‐32P] ATP resulted in substantial formation of 32P‐44K in the mitochondrial fraction and less incorporation in the membrane fraction. The 44,000 dalton protein was identified as the α‐subunit of mitochondrial pyruvate dehydrogenase by partial proteolytic mapping and immunological cross‐reactivity with antibodies prepared against bovine pyruvate dehydrogenase. The phosphorylation of this protein in whole cells by externally added ATP is suppressed by inclusion in the incubation medium of carboxyatractyloside (CAT) and EDTA. These substances have no effect on ATP‐dependent permeabilization, indicating that the phosphorylation of pyruvate dehydrogenase is not involved in this process.
Growth factor‐induced proliferation of human fibroblasts in serum‐free culture depends on cell density and extracellular calcium concentration
Authors: Betsholtz Christer , Westermark Bengt
Published: February 2005

Human neonatal skin fibroblasts plated sparsely in MCDB 105 traversed a complete cell cycle in the absense of serum or serum‐derived proteins. Addition of pure PDGF did not significantly increase entrance into S phase as revealed by 3H‐thymidine labeling index or clonal growth on palladium islands. In subphysiologic Ca2+ concentrations or in the presence of a calmodulin inhibitor, W7, proliferation in the absence of growth factors ceased and PDGF became mitogenic. In contrast, confluent fibroblast cultures were stimulated by PDGF in physiologic Ca2+ concentrations. This was also the case with sparse adult skin fibroblast cultures while a fetal strain entered S in the absence of PDGF even in low extracellular Ca2+ concentrations. EGF gave similar results as PDGF in all experiments performed. This proposes a similar role for the two growth factors in the cell cycle. However, a difference in the mechanisms of action of PDGF and EGF is indicated by the fact that PDGF and EGF were additive at optimal concentrations when maximal growth response by a single growth factor was restricted by a subphysiologic extracellular Ca2+ concentration.
Hexose transport derepressed and refractory to purine regulation in NAD(H)‐depleted Nil cells
Authors: Mandel Kenneth G. , Amos Harold
Published: February 2005

Nil hamster fibroblasts depleted of NAD(H) by growth in medium devoid of nicotinamide (NAm−MEM) exhibit up to 2‐3‐fold higher rates of glucose transport. Derepression of glucose transport is observed only when Nil cells have become severely depleted of both intracellular NAD(H) and ATP, despite the continued presence of 5.5 mM D‐glucose in the growth medium. Neither the initial rate of transport, approximated from 3‐O‐methylglucose uptake, nor accumulation of D‐glucose itself is repressed upon restoring nicotinamide to the medium. Exposure of the cells to NAD+ (10−5 M), however, leads to a sharp curtailment of transport within 2 to 3 hours. The purines, hypoxanthine and guanine, that sharply reduce glucose transport capacity of normal cells, have no significant effect upon transport activity of NAD(H)‐depleted cells.
Neuroblastoma membranes inhibit isoproterenol‐stimulated rise of cAMP in glioma cells
Authors: Wu Doris K. , De Vellis Jean
Published: February 2005

C6 glioma cells respond to beta‐adrenergic agonists (isoproterenol) with a transient rise in intracellular cyclic adenosine monophosphate level. This beta‐responsiveness of C6 cells is inhibited by the presence of a plasma membrane fraction, which has a five‐ to six‐fold purification of membrane markers, showed a greater inhibition of beta‐responsiveness in C6 cells than any other subcellular fractions of B104 cells. The inhibitory effector(s) is apparently associated with integral membrane structure(s) since ionic extraction and treatment with chelating agents did not remove the effect from the particulate membrane fraction. The effector is probably proteinaceous in nature as judged by its susceptibility to inactivation by heat and protease treatment. The data indicate that neither adenylate cyclase nor phosphodiesterase enzyme is likely to be directly involved in mediating the beta‐nonresponsiveness of C6 cells.
Prolactin regulation of cryptic prolactin receptors in cultured rat mammary tumor cells
Authors: Costlow Mark E. , Hample Amie
Published: February 2005

Rat mammary tumors contain a unique class of cryptic cell‐surface prolactin receptors that can be unmasked by depleting the cells of energy. These cryptic receptors, which are found in mammary tumors and nonlactating normal mammary cells but not in differentiated mammary tissue, are continuously inserted and rapidly removed from the cell surface. In this report we demonstrate that prolactin regulates the level of cryptic receptors. Treatment of primary cultures of rat mammary tumor cells with prolactin at concentrations between 0.1 and 0.5 ng/ml caused cryptic receptor levels to increase within 24 h, and this increase was maintained for up to 6 days. At prolactin concentrations of 10‐50 ng/ml, receptor levels were the same as in cells incubated without hormone, while a decrease in the steady‐state level of cryptic receptors was induced within 24 h by 100‐500 ng prolactin/ml. Concentrations of 1,000–5,000 ng prolactin/ml caused a rapid, dose‐dependent down regulation of cryptic receptor sites. Down regulation at 5,000 ng prolactin/ml was (1) complete (84 ± 5% reduction) in 1 h; (2) specific for lactogenic hormones; (3) completely reversed within 10 h after prolactin removal; (4) energy dependent; and (5) not blocked by the cytoskeleton active agents cytochalasin B and colchicine or by NH4CI, which inhibits hormone degradation. We conclude that rat mammary tumor cells have the capacity to auto‐regulate cryptic prolactin receptors, a property that supports our notion that such receptors play a role in regulating prolactin responsiveness. The observed pattern of cryptic receptor autoregulation in response to prolactin concentration and time of exposure suggests that a pool of cryptic sites provides these cells with the capacity to respond to prolactin concentrations from pg to μg/ml, a range well beyond the Kd for the receptor itself. Since prolactin receptors in mammary tumors are not down regulated unless prolactin concentrations are well beyond the saturation point, these cells may have a selective growth advantage over cells in normal mammary tissue.
Coordinate regulation of polypeptide chain initiation and elongation in rabbit reticulocytes
Authors: Binninger David , Weber Lee A.
Published: February 2005

Under normal conditions, reticulocytes synthesize α‐ and β‐globin polypeptides at equal rates. Incubation in the absence of hemin or under anoxia or hypertonic stress (100 mM excess NaCl) reduces the rate of protein synthesis to 30–50% of control levels. However, only hemin deprivation causes a reduction in polyribosome size and preferential inhibition of α‐globin synthesis consistent with specific reduction in the rate of polypeptide chain initiation. Polyribosomal profiles are unaffected by anoxic or hypertonic stress and the ratio of α:β globin synthesis remains close to unity. Measurement of ribosome transit time indicates that anoxic or hypertonic stress causes a decrease in the rate of polypeptide chain elongation that varies with the degree of inhibition of protein synthesis. Ribosomes isolated from stressed cells exhibit a reduced ability to bind 35S‐met‐tRNAf, suggesting that the ability to form initiation complexes is also impaired. These results suggest that reticulocytes, unlike nucleated cell lines, can coordinately reduce rates of initiation and elongation in response to certain physiological stresses.
Differential sensitivity of fibroblasts to epidermal growth factor is related to cyclic AMP concentration
Authors: Olashaw Nancy E. , Leof Edward B. , O'Keefe Edward J. , Pledger W. J.
Published: February 2005

The differential sensitivity of various cell lines to the mitogenic effects of epidermal growth factor (EGF) was investigated. Two lines of evidence suggest that cellular capacity to respond proliferatively to EGF is related to intracellular cyclic AMP concentration. First, the ability of three density‐arrested cell lines to synthesize DNA in response to EGF was directly proportional to the basal cyclic AMP level of the cells at quiescence. Second, treatment of cultures with various agents known to promote intracellular cyclic AMP accumulation increased the sensitivity of all three cell lines to EGF. The mechanism whereby cyclic AMP modulates EGF responsiveness is not known; cholera toxin did not affect the cellular capacity to bind or internalize and process EGF. Although platelet‐derived growth factor (PDGF) had no effect on cyclic AMP levels, transient treatment of quiescent cultures with this polypeptide also enhanced EGF sensitivity. In agreement with previous data and in contrast to cholera toxin, PDGF induced the down‐regulation of EGF receptors in the three cell lines. These data suggest that the capacity of various cell types to respond to EGF is subject to both intracellular regulation by cyclic AMP and extracellular modulation by factors such as PDGF which can affect EGF receptor activity.
The respiratory burst of human neutrophils treated with various stimulators in vitro is dampened by exogenous unsaturated fatty acids
Authors: Wong Kenneth , Chew Christina
Published: February 2005

Cis‐unsaturated free fatty acids (FFA) at concentrations between 10 and 30 μM suppressed the superoxide respiratory burst induced in human neutrophils by the chemotactic peptide, N‐formylmethionyl‐leucyl‐phenylalanine (FMLP). Corresponding trans‐isomers had a reduced efficacy while saturated FFA were inert. The effects of unsaturated FFA were maximally achieved after several min of preincubation with cells and reversed upon washing. Increased concentrations of Ca2+ in the medium also relieved the inhibition. Unsaturated FFA were equally effective in dampening the respiratory burst induced by fluoride ions but less so with bursts elicited by 9 nM phorbol myristate acetate (PMA). Moreover reactions triggered by higher concentrations (e.g., 100 nM) of PMA were resistant to the effects of FFA. Radioimmunoassays showed that unsaturated FFA directly elevated intracellular cyclic adenosine monophosphate (cAMP) by severalfold above basal levels. It is suggested that inhibition is brought about by unsaturated FFA perturbation of the neutrophil membrane structure, perhaps with an independent contribution from a cAMP‐dependent mechanism.
Mutant cell lines resistant to azetidine‐2‐carboxylic acid: Alterations in the synthesis of proline from glutamic acid
Authors: Lodato Ronna F. , Smith Robert J. , Valle David L. , Crane Kathleen
Published: February 2005

Two mutant Chinese hamster lung fibroblast lines have been isolated that are resistant to the the toxic proline analog L‐azetidine‐2‐carboxylic acid. The line designated AZCA‐1 has 30‐fold elevated activity of pyrroline‐5‐carboxylate synthase and a large increase in the rate of proline production and release compared to controls. Pyrroline‐5‐carboxylate synthase activity is not elevated in the resistant line designated AZCA‐4, but the enzyme is less sensitive to inhibition by ornithine and proline than control enzyme. Intracellular proline is elevated in AZCA‐4 cells, with no change in the rate of release of proline synthesized from glutamate. Resistance to azetidine carboxylic acid in both mutant lines is attributed to the expanded intracellular proline pool that results from alterations in pyrroline‐5‐carboxylate synthase. These results indicate that intracellular proline levels are determined at least in part by the regulated activity of pyrroline‐5‐carboxylate synthase.
Effect of serum and growth factors on heat sensitivity in Swiss mouse 3T3 cells
Authors: van Wijk Roeland , Otto Angela M. , De Asua Luis Jimenez
Published: February 2005

Quiescent Swiss mouse 3T3 cells react to a heat treatment at 46°C for 20 min by changing their flat, well‐extended morphology to a round appearance with retracted cytoplasmic processes during the subsequent 2 h at 37°C. The percentage of morphologically changed cells was used to quantify changes in heat sensitivity, or resistance, in response to mitogenic stimulation. Stimulating quiescent cells with serum or with the specific growth factors epidermal growth factor (EGF) and prostaglandin F2α (PGF2α) markedly increased the heat resistance to a 46°C treatment, but only when the heat treatment, but only when the heat treatment was applied within 2–3 h after the addition. When insulin (which is not mitogenic, but synergistic with EGF and PGF2α in these cells) was added alone or in combination with either EGF or PGF2α, it had no effect on the development of heat resistance. Neither did cycloheximide nor tunicamycin inhibit heat resistance induced by EGF, and cycloheximide even enhanced it after 2–4 h. However, adding colcemid before or at the beginning of the heat treatment abolished the increased heat resistance. The results indicate that the resistance to a single heat treatment at 46°C may be related to changes in the metabolic state after mitogenic stimulation, even though these changes need not be reflected in the rate of entry into S phase. Furthermore, the cytoskeletal organization appears to be a crucial component in heat resistance of Swiss 3T3 cells.
Bioassay of retinoids using cultured human conjunctival keratinocytes
Authors: Gilfix Brian M. , Green Howard
Published: February 2005

The biological action of retinoids has been assayed using the differentiated properties of cultured human conjunctival keratinocytes. The effects measured were the suppression of envelope cross‐linking and the promotion of synthesis of a keratin of molecular weight 40,000. Among the retinoids tested, the most powerful was the arotinoid Ro 13–6298, which reduced envelope formation detectably at 10−11 M and by 90% at a concentration of 2 × 10−10 M. The arotinoid was about 15 times more potent than trans‐retinoic acid. The order of effectiveness of the retinoids in suppressing envelope cross‐linking was the same as the order of effectiveness in promoting the synthesis of the 40‐kd keratin.
Chinese hamster ovary cell variants resistant to monensin, an ionophoric antibiotic. I. Isolation and altered endocytosis of ricin
Authors: Ono Mayumi , Yamato Hisae , Ando Miho , Kuwano Michihiko
Published: February 2005

Chinese hamster ovary (CHO) cell variants resistant to a carboxylic ionophore, monensin, have been isolated. Two monensinresistant variants (MonR–;31 and Mon R–32) showed a three‐ to fourfold higher resistance to monensin than did CHO. These MonR clones also showed fourfold higher resistance to another carboxylic ionophore, nigericin, and twofold higher resistance to valinomycin. They were also slightly more resistant to other unrelated drugs such as adriamycin, colchicine, bleomycin, and chloroquine, and in particular, they showed about threefold higher resistance to ricin, a toxin of Ricinus communis. MonR clones were found to retain a normal level of [125l]ricin binding, but internalization of [125l]ricin into the MonR clones was one‐half or less than with CHO. Present data suggest that drug‐resistant clones selected in culture may provide a way to isolate cells with altered response to various bioactive molecules.
Chinese hamster ovary cell variants resistant to monensin, an ionophoric antibiotic. II. Growth requirement for insulin and altered insulin‐receptor activity
Authors: Sato Yasufumi , Ono Mayumi , Takaki Ryosaburo , Kuwano Michihiko
Published: February 2005

From the Chinese hamster ovary (CHO) cell, genetic variants (MonR–31 and MonR–32) relatively resistant to monensin, an ionophoric antibiotic, have been isolated. Growth of both MonR‐31 and MonR‐32 clones required higher doses of serum than CHO. Addition of insulin to media containing a low dose of serum restored full colony formation, but growth of MonR‐31 or MonR‐32 cells required more insulin than CHO cells. Specific binding of [125l]insulin was observed in these cell lines. The two MonR clones bound about one‐half or less the [125l]insulin bound by CHO cells. Scatchard analysis for [125l]insulin binding at 4°C and 37°C showed altered number of binding sites, but not insulin affinity: The number of binding sites in the MonR cell was about a half or less that of the parental CHO cell. Down‐regulation of insulin receptor was assayed when both CHO and MonR cells were incubated with 1 μg/ml insulin. A 50–60% decrease in levels of insulin surface binding capacities was observed in CHC after exposure to insulin, whereas there was no decrease in MonR cell. The cellular uptake of 2‐[3H]deoxyglucose into CHO cells was significantly enhanced in the presence of insulin, but only slight, if any, increase was observed in MonR cells.
Development of calcium and secretory responses in the human promyelocytic leukemia cell line HL60
Authors: Naccache P. H. , Molski T. F. P. , Spinelli B. , Borgeat P. , Abboud C. N.
Published: February 2005

We have begun to characterize the development of the excitation – response coupling sequence in the human promyelocytic leukemia cell line HL60. Using the recently developed fluorescent calcium probe quin‐2, it was found that DMSO induced myeloid differentiation of the HL60 cells is accompanied by the development of a calcium response to the addition of the chemotactic factors fMet‐Leu‐Phe and leukotriene B4. The characteristics (time course, concentration dependence, stereospecificity, and metabolic dependence) of the calcium response are extremely similar to those previously described in human neutrophils. These results imply that functional receptors for leukotriene B4 appear in HL60 cells upon the induction of differentiation and also lend strong support to the use of these HL60 cells as a model of human myeloid differentiation. We have also characterized the emergence of a secretory response to fMet‐Leu‐Phe and leukotriene B4 in cytochalasin B treated HL60 cells. In addition, it is found that differentiation was required for the calcium ionophore A23187 to express its secretory activity toward the HL60 cells. This last set of results implies that differentiation is accompanied by the coordinated appearance of surface receptors and cytoplasmic factors required for the expression of cellular responsiveness.
Comparison of protein phosphorylations in variant A431 cells with different growth responses to epidermal growth factor
Authors: Buss Janice E. , Chouvet Claire , Gill Gordon N.
Published: February 2005

Growth of a selected variant of A431 cells (clone 29) is stimulated by epidermal growth factor (EGF) in contrast to the growth inhibition caused by EGF in an unselected clone, A4318. Twelve phosphoproteins from each clone were compared to determine whether unique EGF‐dependent substrate phosphorylations might explain the cells' differing growth responses to EGF. Treatment of both clone 29 and A4318 cells with EGF increased phosphorylation of the EGF receptor/kinase and six cellular proteins identified on 2‐dimensional poly‐acrylamide gels. Four of these proteins (the EGF receptor/kinase and proteins of 36, 70, and 81 kd) contained phosphotyrosine in both clone 29 and A4318 cells, indicating that the same modification of several proteins occurred in cells which have totally different growth responses to EGF. Two proteins were identified whose phosphorylation was EGF dependent and which were unique to clone 29 cells; however, EGF increased phosphorylation of only serine residues in these proteins. This indicates that these proteins are not primary targets of the EGF‐dependent tyrosine‐specific protein kinase, but rather are substrates for serine‐specific kinase(s) activated as a consequence of EGF:receptor interaction. cAMP, which inhibited growth of both clones, was utilized to compare the effects of EGF when the growth response of both cell lines was similar. In the presence of cAMP, EGF increased A4318 cellular phosphotyrosine content and the phosphorylation of the same phosphotyrosine‐containing proteins of both clone 29 and A4318 cells. The in vivo activity of a second tyrosine‐specific protein kinase, p60v‐src in B77 Rous sarcoma virus (RSV)‐transformed newborn rat kidney (NRK) cells, was also unaffected by cAMP. Thus cAMP did not block the in vivo activity of two tyrosine‐specific kinases or the tyrosine phosphorylation of three specific protein substrates. A threshold model of tyrosine kinase activity is proposed as an alternative explanation for the differing growth responses to EGF.
Gene amplification as a mechanism of reversion at the HPRT locus in V79 chinese hamster cells
Authors: Zownir Olga , Fuscoe James C. , Fenwick Raymond , Morrow John
Published: February 2005

Spontaneous phenotypic revertants of hypoxanthine phosphoribosyl‐transferase (HPRT) temperature‐sensitive V79 Chinese hamster cells were selected by plating a temperature‐sensitive mutant in HAT medium at 39°C. The incidence of such revertants was approximately 2 × 10−4 per cell. The majority of the revertants examined had increases of between three‐ and tenfold in their specific activity of the enzyme, and they were able to grow continuously in the presence of HAT medium at 39°C. When the revertants were cultivated in the absence of HAT, they recovered their HAT‐sensitive phenotype and their lowered level of HPRT. Three of the revertants were examined for their temperature inactivation profiles, and all were found to have profiles identical to the ts parent, and quite different from the V79 wild type. The kinetic properties of the cell lines were studied:the Km for both PRPP and hypoxanthine was significantly different in the temperature‐sensitive cells but was not significantly altered in the revertants with respect to the ts mutants. A specific antibody to Chinese hamster brain HPRT was employed in immunoprecipitation experiments. By measuring the point at which the immunoprecipitation of the antibody to HPRT was overcome by increasing concentrations of cell supernatant, it was possible to estimate the relative amount of enzyme molecules in the cell lines. From these data, it could be concluded that the revertants overproduced an enzyme with the same immunological properties as the ts line. Southern blots of the Hind Ill restricted DNA from the ts mutant and two revertant cell lines were examined with an HPRT cDNA probe. This established that the HPRT gene was amplified twofold in one of the revertants, and threefold in the other. However, if the revertants were reintroduced into nonselective medium, the gene copy number declined to one. Finally, northern blots of RNA extracted from the various cell lines demonstrated that the HPRT mRNA was augmented 1.5‐fold in one revertant and 1.4‐fold in the other. Reintroduction into non‐selective medium resulted in a decline in mRNA level for the second mutant, whereas the first mutant appeared to be stabilized.

We conclude that gene amplification and concomitant amplification of messenger RNA and enzyme levels are mechanisms of phenotypic reversion at the HPRT locus in Chinese hamster cells.
Furosemide‐sensitive potassium efflux in cultured mouse fibroblasts
Authors: Jayme David W. , Slayman Carolyn W. , Adelberg Edward A.
Published: February 2005

Transfer of LM(TK−) cells from normal growth medium to medium lacking K+ leads to a rapid loss of intracellular K+, which is 50–70% inhibited by furosemide or bumetanide. The diuretic‐sensitive component of K+ efflux requires both Na+ and Cl−, and is presumably mediated by a K+, Na+, Cl− cotransport system of the kind described in avian erythrocytes and Ehrlich ascites cells. It can be calculated that such a system should be near equilibrium under normal growth conditions but should mediate net efflux (as observed) when the driving force is altered by reducing extracellular K+. The diuretic‐sensitive component of net K+ efflux is also sensitive to amiloride. This effect is probably indirect, however, with amiloride acting to block the Na+ influx that supplies Na+ to the cotransport system. At the low extracellular K+ concentrations employed in these studies, the diuretic‐sensitive system is a physiologically important pathway of K+ loss. The rate of growth in low‐K+ medium can be increased (or the rate of cell lysis decreased) by adding diuretic or by reducing external Na+ or Cl−.
Receptors for pea and lentil lectins on human lymphoid cells: Demonstration of distinct lectin‐defined cell subclasses
Authors: Boldt David H.
Published: February 2005

Lectins are useful probes for studying cell surface glycoconjugates. Pea (PL) and lentil (LL) lectin each requires for binding a fucosyl‐ and two α‐mannosyl residues in core regions of glycopeptides, but differences in outer chain carbohydrates may alter their relative binding affinities. We used binding studies with [125I]‐PL and LL and flow cytometry with fluorescein‐conjugated (FITC)‐PL and ‐LL to study their interactions with peripheral lymphocytes. Binding of both lectins to lymphocytes was saturable, reversible, and inhibited by α‐methyl mannose. Scatchard analyses were consistent with two classes of receptors for each lectin. Flow cytometric analyses demonstrated that cell to cell receptor densities varied. Sixty‐five percent of lymphocytes bound PL (mean 2 × 106 receptors/cell) and 45% bound LL (mean 3 × 106 receptors/cell). Competition studies demonstrated mutual inhibition, but flow cytometry revealed persistent FITC‐PL or ‐LL binding despite 20‐fold molar excess of the other lectin. Distributions of receptors for PL and LL on lymphocytes were as follows: (1) 45% of lymphocytes bound both PL and LL; (2) 20% of lymphocytes bound PL alone; (3) 35% of lymphocytes bound neither PL nor LL. Despite similar binding requirements for PL and LL and overlap between their receptors on lymphocytes, there appear to be subsets of receptors specific for each lectin. These results may reflect abilities of PL and LL to discriminate subtle carbohydrate differences on lymphocyte surfaces.
Studies on the energy metabolism of opossum (Didelphis Virginiana) erythrocytes. I. Utilization of carbohydrates and purine nucleosides
Authors: Bethlenfalvay N. C. , Lima J. E. , Waldrup T.
Published: February 2005

Opossum erythrocytes filtered through cellulose columns were used to estimate their permeability to D‐glucose and optimum inorganic phosphate requirement for D‐glucose utilization at pH 7.4 and 8.1. D‐Glucose readily penetrated opossum red cells; there was no measurable difference whether plasma or electrolyte solution served as the suspending medium. Optimum extracellular inorganic phosphate concentration for glucose utilization as indicated by red cell lactate production was pH‐dependent, with a sharp optimum of 30 mmol/liter at pH 8.1. Whereas glucose, fructose, mannose, dihydroxyacetone, adenosine, and inosine were readily utilized at pH 7.4 and Pi 30 mmol/liter as shown by net lactate and ATP production by the red cells, galactose and ribose as substrates were not metabolized. In electrolyte, Pi 30 mmol/liter, and pH 7.4 glucose utilization by opossum red cells averaged 3.5 μmol, at pH 8.1, 9.5 μmol/ml cells/hr were utilized. Red cells suspended in leukocyte‐free plasma utilized D‐glucose at a rate of 3.0 μmol/ml/hr at pH 7.5. Seven percent of D‐glucose flowed through the pentose phosphate pathway; this rate increased 11‐fold by methylene blue stimulation. The amount of D‐glucose recycled through the pentose phosphate pathway increased 300‐fold in the presence of the redox dye.
Adhesion site composition of murine fibroblasts cultured on gelatin‐coated substrata
Authors: Haas Robert , Banerji S. Sheila , Culp Lloyd A.
Published: February 2005

Fibroblasts in vivo adhere to a collagenous extracellular matrix. We present here a combined morphological and biochemical analysis of the adhesion sites of fibroblast‐like cells cultured in vitro on gelatin‐coated plastic, for comparison with earlier model studies using serum (plasma‐fibronectin [pFn])‐coated plastic. Scanning electron microscopy shows that cell adhesion to the gelatin is quite similar to that on plastic, but with some morphological differences reminiscent of those caused by higher concentrations of fibronectin adsorbed to the substratum. Measurement using 125I‐radiolabeled pFn shows the level of substratum‐bound pFn adsorbed from serum in the growth medium is, however, comparable on gelatin or plastic; thus, differences due to pFn must be attributed to the quality of the adsorbed protein, not its absolute quantity. Gel electrophoretic analysis of cellular adhesion sites formed on the two substrata shows their compositions to be qualitatively similar, suggesting again that the same fundamental adhesion processes are involved. However, three protein bands do change; notably, cellular fibronectin is increased on gelatin. These three proteins are also the most resistant to saline extraction, suggesting their intrinsic importance in the adhesion sites. The nature of the growth substratum thus appears to modulate a fundamentally unvarying morphology and adhesion site composition of the cells that adhere to it.
Cyclic AMP does not mediate inhibition of DNA synthesis by interferon in mouse Swiss 3T3 cells
Authors: Ebsworth Nicolette M. , Taylor‐Papadimitriou Joyce , Rozengurt Enrique
Published: February 2005

The purpose of this study was to determine whether cyclic AMP (cAMP) plays any direct or indirect role in the antiproliferative effect of mouse L‐cell interferon in Swiss 3T3 cells. Firstly, we found that interferon did not affect intracellular levels of cAMP in these cells in the absence or the presence of cAMP‐elevating agents. Secondly, we examined the effect of interferon on the stimulation of DNA synthesis of quiescent 3T3 cells by a range of cyclic AMP‐elevating agents, including cholera toxin, cAMP derivatives, and prostaglandin E, added in the presence of insulin or vasopressin. Interferon inhibited cyclic AMP‐stimulated DNA synthesis as measured by incorporation of radioactive thymidine into acid‐insoluble material and autoradiographic analysis of the fraction of labelled cells. Dose‐response curves and kinetics of inhibition were identical to those obtained in cultures stimulated by combinations of growth factors that do not increase the intracellular level of cAMP. The inhibition by interferon of cAMP‐stimulated DNA synthesis was also observed in secondary cultures of mouse embryo fibroblasts, where cAMP‐elevating agents provide a mitogenic signal in the absence of other added growth factors. These results show that the inhibitory effect of interferon on DNA synthesis in Swiss 3T3 cells is not mediated by cyclic AMP.
Prostaglandin synthesis by cells comprising the calf pulmonary artery
Authors: Menconi Michael , Hahn Gerald , Polgar Peter
Published: February 2005

Prostaglandin (PG) production was evaluated in the three cell types (endothelial, smooth muscle, and fibroblast) comprising the bovine pulmonary artery. Prostacyclin (PG12) was the predominant prostaglandin (PG) produced by endothelial, smooth muscle, and fibroblast cells as they exist in culture or in freshly excised tissue fragments. In addition to PGl2, measurable amounts of PGE2, PGF2a, and thromboxane A2 (TXA2) were also produced by these cells. Endothelial cells were the most active producers of PGs. However, the type of PG produced was characteristic of the particular cell type, while the level of production was dependent on external factors. Prostaglandin production by cultured cells, both under basal conditions and in response to stimulatory agents, was quite similar to that of the respective freshly excised tissue fragments containing a given cell type. These cells in culture could be stimulated to produce PGl2 by both angiotensin and bradykinin at very low (physiological) concentrations, a further indication of the retention of the physiological responsiveness of these cells in culture. Endothelial cells and fibroblasts were activated by bradykinin at concentrations as low as 10−12 M but did not respond to angiotensin. Smooth muscle cells in primary and first passage cultures were activated by both bradykinin and angiotensin at 10−12M concentrations. Serial subcultivations of smooth muscle cells resulted in a progressive loss in their responsiveness to bradykinin stimulation. The state of cell growth proved to be an important determinant of PG production. Actively growing cells in culture synthesized less PG when compared to cells which had entered into a “quiescent” nongrowth state.
Regulation of glutamine synthetase and glutaminase activities in cultured skeletal muscle cells
Authors: Smith Robert J. , Larson Sandra , Stred Susan E. , Durschlag Roberta P.
Published: February 2005

Glutamine is synthesized in skeletal muscle, released to the circulation, and transported to other tissues, where it may provide important substrate for gluconeogenesis, ammoniagenesis, and energy‐yielding pathways. With the ultimate goal of delineating the factors that control glutamine production and release by skeletal muscle, we have studied the regulation of two key enzymes, glutamine synthetase and glutaminase, in the L6 line of rat skeletal muscle cells grown in monolayer culture. The cultured myotubes were found to have glutamine synthetase and phosphate‐dependent glutaminase activities. Glutamine synthetase activity was increased following incubation (1) in glutamine‐free medium (threefold); (2) in medium containing high glutamic acid concentrations (fourfold); and (3) in medium supplemented with dexamethasone (threefold). In each case the increase in glutamine synthetase activity required several hours to reach a maximum and was prevented by cycloheximide, suggesting that the change occurred through increased enzyme biosynthesis. No substances tested were found to affect glutaminase activity. We conclude that glutamine synthetase in cultured skeletal muscle is responsive to substrate, product, and hormonal regulation.