bluuebunny/crossref_metadata_2025_split huggingface dataset:
Spermidine mediates degradation of ornithine decarboxylase by a non‐lysosomal, ubiquitin‐independent mechanism
Authors: Glass James R. , Gerner Eugene W. 
Published: February 2005

AbstractThe mechanism of spermidine‐induced ornithine decarboxylase (ODC, E.C. 4.1.1.17) inactivation was investigated using Chinese hamster ovary (CHO) cells, maintained in serum‐free medium, which display a stabilization of ODC owing to the lack of accumulation of putrescine and spermidine (Glass and Gerner: Biochem. J., 236:351–357, 1986; Sertich et al.: J. Cell Physiol., 127:114–120, 1986). Treatment of cells with 10 μM exogenous spermidine leads to rapid decay of ODC activity accompanied by a parallel decrease in enzyme protein. Analysis of the decay of [35S]methionine‐labeled ODC and separation by two‐dimensional electrophoresis revealed no detectable modification in ODC structure during enhanced degradation. Spermidine‐mediated inactivation of ODC occurred in a temperature‐dependent manner exhibiting pseudo‐firstorder kinetics over a temperature range of 22–37°C. In cultures treated continuously, an initial lag was observed after treatment with spermidine, followed by a rapid decline in activity as an apparent critical concentration of intracellular spermidine was achieved. Treating cells at 22°C for 3 hours with 10 μM spermidine, followed by removal of exogenous polyamine, and then shifting to varying temperatures, resulted in rates of ODC inactivation identical with that determined with a continuous treatment. Arrhenius analysis showed that polyamine mediated inactivation of ODC occurred with an activation energy of approximately 16 kcal/mol. Treatment of cells with lysosomotrophic agents (NH4Cl, chloroquine, antipain, leupeptin, chymostatin) had no effect on ODC degradation. ODC turnover was not dependent on ubiquitin‐dependent proteolysis. Shift of ts85 cells, a temperature‐sensitive mutant for ubiquitin conjugation, to 39°C (nonpermissive for ubiquitin‐dependent proteolysis) followed by addition of spermidine led to a rapid decline in ODC activity, with a rate similar to that seen at 32°C (the permissive temperature). In contrast, spermidine‐mediated ODC degradation was substantially decreased by inhibitors of protein synthesis (cycloheximide, emetine, and puromycin). These data support the hypothesis that spermidine regulates ODC degradation via a mechanism requiring new protein synthesis, and that this occurs via a non‐lysosomal, ubiquitin‐independent pathway.
Graded amplification of the Na, K‐ATPase across a subclonal series: Effects on membrane physiology
Authors: Pauw Peter G. , Ash John Frederick 
Published: February 2005

AbstractWe have generated a series of clonally related cell lines which differ in the level of amplified expression of the Na, K‐ATPase. These lines, originally derived from the ouabain resistant HeLa variant C+, expressed different numbers of binding sites for the Na, K‐ATPase inhibitor ouabain, ranging from 2.9 × 106/cell to 11.8 × 106/cell. Amplification of the genes for both subunits of the enzyme was also seen but was not strictly correlated with level of expression. The influxes of histidine and tetraphenylphosphonium were measured across a series, including HeLa S3 and revertants, expressing from 0.74 × 106 to 10.5 × 106 ouabain‐binding sites per cell. Tetraphenylphosphonium influx rate, presumed to be a function of membrane potential, varied linearly with ouabain binding site number, while histidine influx varied with the log of ouabain binding site number. Our results suggest that membrane potential increases in a simple fashion across our series of amplified lines. However, histidine influx was unaffected by treatments which cause membrane depolarization and a decrease in tetraphenylphosphonium influx rate. We propose that increasing histidine influx rates across our amplified series reflects exchange acceleration of L system transport due to increased intracellular pools of L system reactive amino acids.The Na, K‐ATPase is ultimately responsible for most active transport across the plasma membrane. The consistent, graded physiological alterations seen across this series of closely related lines, chosen for graded enzyme expression, demonstrate the value of this novel genetic approach to the study of the energization of membrane transport.
Phorbol esters and mitogenesis: Comparison of the proliferative response of parental and Na+K+Cl−‐contransport‐defective BALB/c 3T3 cells to 12‐0‐tetradecanoylphorbol‐13‐acetate
Authors: O'Brien Thomas G. , Prettyman Ralph 
Published: February 2005

AbstractThe ability of the phorbol ester 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA) to stimulate the growth of quiescent BALB/c 3T3 cell lines lacking Na+K+Cl− contransport activity was tested. We have previously isolated and characterized two mutant cell lines defective in this important ion transport system by mutagenesis and selection in medium containing low K+. To test our hypothesis that loss of this transport activity might abrogate the proliferative response to TPA, two kinds of mitogenesis assays were performed. First, the effect of 0.16 μM TPA on the saturation density of parental vs. mutant cell lines was determined. TPA caused a small but reproducible 30–35% increase in the saturation density of mutant cells compared to the 100–120% increase seen in parental cell lines. Second, the effect of TPA on the incorporation of 3H‐thymidine into cell nuclei (labeling index) was measured. While some variability from experiment to experiment in the extent and time course of the response of mutant cells was noted, TPA either had no effect or only a small effect on the labeling index when compared to the response of parental cells. When a range of concentrations of TPA (0.016–1.6 μM) was tested, neither cell line exhibited a large response to any concentration. These results suggest that loss of Na+K+Cl− contransport activity decreases the response of these cells to the mitogenic action of TPA.
Hydrogen peroxide or heat shock induces resistance to hydrogen peroxide in Chinese hamster fibroblasts
Authors: Spitz Douglas R. , Dewey W. C. , Li Gloria C. 
Published: February 2005

AbstractSurvival after H2O2 exposure or heat shock of asynchronous Chinese hamster ovary cells (HA‐1) was assayed following pretreatment with mildly toxic doses of either H2O2 or hyperthermia. H2O2 cytotoxicity at 37°C, expressed as a function of mM H2O2 was found to be dependent on cell density at the time of treatment. The density dependence reflected the ability of cells to reduce the effectiveness of H2O2 as a cytotoxic agent. When the survival data were plotted as a function of μmoles H2O2/cell at the beginning of the treatment, survival was independent of cell density. Cells pretreated with 0.1 mM (3–5 μmoles/cell × 10−7) H2O2 for 1 hr at 37°C (30–50% survival) became resistant to a subsequent H2O2 treatment 16–36 hr after pretreatment [dose modifying factor (DMF) at 1% isosurvival = 4–6]. Their resistance to 43°C heating, however, was only slightly increased over controls 16–36 hr following pretreatment (DMF at 1% isosurvival = 1.2). During this same interval, the synthesis of protein migrating in the 70 kD region of a one‐dimensional SDS‐polyacrylamide gel was enhanced twofold in the H2O2‐pretreated cells. When the cells were heated for 15 min at 45°C (40–60% survival), the survivors became extremely resistant to 43°C heating and somewhat resistant to H2O2 (DMF at 1% isosurvival = 2). The heat‐induced resistance to heat developed much more rapidly (reached a maximum between 6 and 13 hr) following pretreatment than the heat‐induced rsistance to H2O2 (16–36 hr). The enhanced synthesis of 70 kD protein after heat shock was greater in magnitude and occurred more rapidly following preheating than following H2O2 pretreatment. The cells that became resistant to H2O2 by either pretreatment (H2O2 or heat shock) also increased their ability to reduce the H2O2 cytotoxicity from the treatment medium beyond that of the untreated HA‐1 cells. This may be one of the mechanisms involved in the increased resistance and a common adaptive mechanism induced by both stresses. These data indicate that mammalian cells develop resistance to H2O2 following mild pretreatment with H2O2 or heat shock. The cross‐resistance induced by H2O2 and heat shock reinforce the hypothesis that some overlap in mechanisms exist between the cellular responses to these two stresses. However, the failure of H2O2 pretreatment to induce much resistance to heat indicates that there are also differences in the actions of the two agents.
Epidermal growth factor receptor expression during morphological differentiation of pheochromocytoma cells, induced by nerve growth factor or dibutyryl cyclic AMP
Authors: Boonstra Johannes , Mummery Christine L. , Feyen Alie , de Hoog Wouter J. , van der Saag Paul T. , de Laat Siegfried W. 
Published: February 2005

AbstractRat pheochromocytoma cells (clone PC12) possess functional surface receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF). PC12 cells respond to NGF as well as to dibutyryl cyclic AMP (dbcAMP) by arrest of cell proliferation and initiation of morphological differentiation, while EGF acts as a mitogen. Exposure of PC12 cells to NGF for several days resulted in a complete loss of rapid EGF responses, such as membrane ruffling and activation of active K+ transport. EGF binding studies revealed that this loss of EGF responses was due to an almost complete reduction of the number of EGF binding sites. In contrast, exposure of PC12 cells to dbcAMP for 2 days did not affect the rapid EGF responses, despite the morphological differentiation. Moreover, EGF binding studies demonstrated a twofold increase in the number of high‐affinity binding sites and a small increase in the number of low‐affinity sites. In addition, exposure of the cells to dbcAMP caused a twofold increase of EGF‐receptor phosphotyrosine kinase activity. These results indicate that neither EGF‐binding or the presence of EGF receptors nor the rapid EGF responses are sufficient for persistent proliferation, on one hand, or sufficient to avoid morphological differentiation, on the other.
Membrane potential and cation content of osteoblast‐like cells (UMR 106) assessed by fluorescent dyes
Authors: Civitelli Roberto , Reid Ian R. , Halstead Linda R. , Avioli Louis V. , Hruska Keith A. 
Published: February 2005

AbstractA number of cellular functions have recently been associated with alterations of the membrane potential in non‐excitable cells. To assess the electrophysiologic regulation of osteoblast function, a method for measuring the membrane potential (Em) of a rat osteogenic sarcoma cell line (UMR 106) by the voltage‐sensitive oxonol dye di‐BA‐C4(3) was developed. The fluorescent signal of di‐BA‐C4(3) was calibrated through a null point method using the protonophore FCCP. At null point, Em is equivalent to H+ equilibrium potential, and may be calculated by the Nernst equation. Intracellular pH (pHi) changes induced by the protonophore were monitored using BCECF, a pH‐sensitive fluorescent probe. In the presence of FCCP, intracellular pH was found to be linearly correlated to extracellular pH (pHo). Therefore, the value of pHi at null point was extrapolated as well. With this technique, we estimated the plasma membrane potential of the “putative” rat osteoblasts (UMR 106) as ‐ 28.3 ± 4.0 mV (n = 10). This method corrected the 16% overestimation of Em derived from the assumption that pHi does not change during the calibration procedure, as described in previous studies employing pH null point techniques. With null point methods, using BCECF and the carboxylic ionophores nigericin and monensin, intracellular concentrations of potassium and sodium were also measured and found to be 125 ± 0.7 mM (n = 3) and 24 ± 5.3 mM (n = 3), respectively. Although the Em of UMR 106 cells was dependent on extracellular potassium concentration, these cells did not behave as a potassium electrode. The sodium/potassium permeability ratio, calculated by the Goldman equation, was estimated at 0.317. This high membrane permeability to sodium may contribute to the genesis of the low plasma membrane potential of UMR 106 cells.
Molecular sieving characteristics of the cultured endothelial monolayer
Authors: Siflinger‐Birnboim Alma , del Vecchio Peter J. , Cooper Jeffrey A. , Blumenstock Frank A. , Shepard Jonathan M. , Malik Asrar B. 
Published: February 2005

AbstractWe examined the selectivity of the bovine pulmonary artery endothelial monolayer in vitro to molecules of different sizes. The cultured bovine pulmonary endothelial monolayer was grown on a gelatinized filter and the transendothelial transport was studied by determining the permeability of molecules ranging from 182 to 340,000 daltons under diffusion conditions. The permeabilities across the cultured bovine endothelium were modeled according to cylindrical pore theory. The data were best fit by a two‐pore model with radii 65 Å and 304 Å and a ratio of small to large pores of 160:1. The results indicate that the cultured endothelial monolayer is a selective barrier to molecules of different sizes and that the molecular selectivity is consistent with a diffusional pathway through endothelial pore equivalents. The cultured endothelial monolayer is a useful system for studying the permeability characteristics of the endothelial barrier.
Influence of hormone and growth factor interactions on the proliferative potential of normal rat mammary epithelial cells in vitro
Authors: Ethier Stephen P. , Kudla Arthur , Cundiff Kimberly C. 
Published: February 2005

AbstractThese experiments were aimed at using a recently developed serum‐free culture system for growth of normal rat mammary epithelial (RME) cells in vitro to examine the interactions of specific hormones and growth factors on the proliferative potential of these cells. RME cells were obtained by enzymatic dissociation of mammary tissues of Lewis rats. Primary cultures were started by plating 2 × 105 RME cells per 60‐mm type I collagen‐coated tissue culture dish. Cultures were maintained in a basal medium that consisted of Ham's F‐12 medium supplemented with bovine serum albumin (BSA), ethanolamine (EA), and transferrin (Tf), which, by itself, did not support RME cell proliferation. Insulin (I), hydrocortisone (HC), and epidermal growth factor (EGF), when added to the basal medium interacted synergistically to stimulate RME cell proliferation, but this effect was dependent on the additional presence of cholera toxin (CT). Under these conditions a greater‐than‐tenfold increase in cell number over a 10‐day culture period was obtained. Insulin could be replaced by physiological levels of insulin‐like growth factor‐I (IGF‐I). CT could be replaced by other agents that elevate intracellular levels of cyclic adenosine 3′:5′ monophosphate (cAMP) such as dibutyryl‐cAMP (db‐cAMP), prostaglandin E1 (PGE‐1), and/or isobutylmethylxanthine (IBMX). Prolactin (M) or progesterone (P) potentiated the effect of I, HC, EGF, and CT, resulting in an additional twofold increase in cell number over that found in their absence. However, addition of both hormones was no more effective than either one alone. Furthermore, addition of M or P in the absence of EGF had no effect on RME cell proliferation. Addition of 17‐B‐estradiol (E2) to the I‐, HC‐, EGF‐, and CT‐containing medium also resulted in enhanced RME cell proliferation. These results point to a number of hormone and growth factor interactions that influence the proliferation of normal RME cells in vitro.
Stimulation by serum of the Na+/H+ antiporter in quiescent pig kidney epithelial (LLC‐PK1) cells and role of the antiporter in the reinitiation of DNA synthesis
Authors: Haggerty John G. , Agarwal Neeraj , Amsler Kurt , Slayman Carolyn W. , Adelberg Edward A. 
Published: February 2005

AbstractLLC‐PK1 cells can be brought into a classical quiescent state by depriving them of serum for 6 days. At this time, pulse‐labeling with [3H]‐thymidine shows that only 3% of the cells are synthesizing DNA, but the quiescent cells can be stimulated with serum to re‐enter the cell cycle at a point early in G1. The rate of amiloride‐sensitive 22Na+ uptake (as a measure of the Na+/H+ antiporter) is relatively low during quiescence; it rises 2‐ to 3‐fold within 4 h after serum addition. This increase in antiporter activity appears to be required for the resumption of DNA synthesis in the absence of bicarbonate, because ethylisopropylamiloride (EIPA) blocks [3H]‐thymidine incorporation when serum is added to cells in bicarbonate‐free medium. In the presence of bicarbonate, however, EIPA has no effect on [3H]‐thymidine incorporation, indicating that another (bicarbonate‐dependent) transport system can substitute for the antiporter under these conditions.
Verapamil inhibits serotonin uptake of endothelial cells in culture by a mechanism unrelated to Ca2+ channel blockade
Authors: Lee S.‐L. , Long K. , Ueda S. , Fanburg B. L. 
Published: February 2005

AbstractVerapamil inhibited Na+‐dependent uptake of serotonin (5‐HT) by bovine pulmonary artery endothelial cells in culture both exposed to room air and stimulated by prior exposure to anoxia. The effect of verapamil occurred even in the absence of Ca2+ from the assay medium. Although absence of Ca2+ from the medium moderately reduced 5‐HT uptake, stimulation of uptake was nevertheless observed for cells previously exposed to anoxia. Verapamil altered the Km, but not the Vmax, of 5‐HT uptake. There was no change in 45Ca2+ uptake or release by cells previously exposed to anoxia as compared to those exposed to room air and verapamil did not influence 45Ca2+ fluxes by either set of cells. It is concluded that verapamil inhibits 5‐HT uptake by endothelial cells through a mechanism other than Ca2+ channel blockade; the results are consistent with competitive inhibition of a 5‐HT carrier. The stimulatory effect of anoxia on 5‐HT uptake does not occur through a change in Ca2+ fluxes.
Serum and α2‐macroglobulin induce transient hyperpolarizations in the membrane potential of an osteoblastlike clone
Authors: Dixon S. J. , Aubin J. E. 
Published: February 2005

AbstractUsing microelectrode techniques, we have observed that the application of serum or α2‐macroglobulin (α2M) induces transient hyperpolarizations in the membrane potential of a rat osteosarcoma clone (ROS 17/2). Hyperpolarizations arose from activation of Ca2+‐dependent K+ channels by transient increases in the concentration of intracellular free Ca2+. Hyperpolarizing spikes were observed for several h following the addition of fetal bovine serum (FBS) to cell cultures. Application of small volumes of FBS or α2M rapidly induced synchronized bursts of hyperpolarizing spikes. No response was elicited by serum‐free medium, latex beads, or bovine serum albumin (BSA). Immunofluorescence labeling patterns were consistent with the receptor‐mediated endocytosis of α2M but not BSA. The ligand specificity and kinetics of these hyperpolarizations suggest that they are associated with a receptor‐mediated event, possibly an early stage of receptor‐mediated endocytosis.
Relationship between pH, sodium, and shape changes in chemotactic‐factor‐stimulated human neutrophils
Authors: Faucher N. , Naccache P. H. 
Published: February 2005

AbstractThe relationship between the chemotactic‐factor‐elicited changes in the intracellular pH and the shape of human neutrophils was investigated using simultaneous measurements of both parameters. The results demonstrate first that fMet‐Leu‐Phe and leukotriene B4 elicit qualitatively similar pH and shape change responses from the neutrophils. A relationship between the chemoattractant‐elicited decrease in cytoplasmic pH and the shape changes is indicated by several findings including: (1) the similarities in the time courses of the two responses, (2) the ability of propionic acid to induce a transient and pertussis‐toxin‐sensitive shape change response, and (3) the ability of the calcium ionophore A23187 to similarly induce both responses under conditions when the degranulation is minimized. On the other hand, several other results indicate that the drop in pH is not a sufficient condition for the chemotactic‐factor‐stimulated shape changes. These include: (1) the ability of pertussis toxin to inhibit the shape changes induced by propionic acid and by A23187 without affecting the drop in pH, and (2) the observation that the drop in pH induced by propionic acid persists significantly longer than the shape change. Increasing the cytoplasmic pH by adding ammonium chloride was also found to cause shape changes in the neutrophils. The response to the base differs in two important aspects from that caused by proprionic acid: it is pertussis‐toxin‐insensitive, and it is long‐lived. Chemotactic factors have been found to induce a shape change under conditions when the internal pH was artificially increased or decreased, indicating that it is not the absolute cytoplasmic pH that represents the internal signalling parameter. The results are discussed in terms of the activation of the cytoskeletal network of the neutrophils by chemotactic factors.
Retrovirus‐induced feline pure red cell aplasia: The kinetics of erythroid marrow failure
Authors: Abkowitz Janis L. , Holly Richard D. , Adamson John W. 
Published: February 2005

AbstractCats viremic with feline leukemia virus subgroup C (FeLV‐C) develop pure red cell aplasia (PRCA) characterized by the loss of detectable late erythroid progenitors (CFU‐E) in marrow culture. Normal numbers of early erythroid progenitors (BFU‐E) and granulocyte‐macrophage progenitors (CFU‐GM) remain, suggesting that the maturation of BFU‐E to CFU‐E is impaired in vivo. We have examined the cell cycle kinetics of BFU‐E and their response to hematopoietic growth factor(s) to better characterize erythropoiesis as anemia develops. Within 3 weeks of FeLV‐C infection, yet 6–42 weeks before anemia, the fraction of BFU‐E in DNA synthesis as determined by tritiated thymidine suicide increased to 43 ± 4% (normal 23 ± 2%) while there was no change in the cell cycle kinetics of CFU‐GM. In additional studies, we evaluated the response of marrow to the hematopoietic growth factor(s) present in medium conditioned by FeLV‐infected feline embryonic fibroblasts (FEA/FeLV CM). With cells from normal cats or cats viremic with FeLV‐C but not anemic, a 4‐fold increase in erythroid bursts was seen in cultures with 5% FEA/FeLV CM when compared to cultures without CM. However, just prior to the onset of anemia, when the numbers of detectable CFU‐E decreased, BFU‐E no longer responded to FEA/FeLV CM in vitro. BFU‐E from anemic cats also required 10% cat or human serum for optimal in vitro growth. These altered kinetics and in vitro growth characteristics may relate to the in vivo block of BFU‐E differentiation and PRCA. Finally, when marrow from cats with PRCA was placed in suspension culture for 2 to 4 days in the presence of cat serum and CM, the numbers of BFU‐E increased 2‐ to 4‐fold although no CFU‐E were generated. By 4 to 7 days, CFU‐E were detected, suggesting that conditions contributing to the block of erythroid maturation did not persist. The suspension culture technique provides an approach to study further the defect in erythroid differentiation characteristic of feline PRCA.