bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

AbstractCholesterogenesis 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

AbstractWe 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

AbstractIncubation 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

AbstractHuman 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

AbstractNil 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

AbstractC6 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

AbstractRat 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

AbstractUnder 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

AbstractThe 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

AbstractCis‐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

AbstractTwo 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

AbstractQuiescent 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

AbstractThe 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

AbstractChinese 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

AbstractFrom 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

AbstractWe 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.