bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of membrane potential in the regulation of lectin‐induced calcium uptake
Authors: Gelfand Erwin W. , Cheung Roy K. , Grinstein Sergio 
Published: February 2005

AbstractIncubation of lymphocytes with mitogenic lectins triggers Ca2+ uptake. This increase in free cytoplasmic Ca2+ is postulated to be an important signal in the initiation of DNA synthesis. Transmembrane fluxes of monovalent ions and changes in membrane potential are also associated with lectin‐induced activation of lymphocytes. We have examined the relationship between extracellular monovalent ion substitution, the associated electrical potential changes (measured with cyanine dyes), phytohemagglutinin‐induced Ca2+ uptake (measured with Quin‐2) and proliferation in human T cells. The results show that (1) the magnitude of the increase in free cytoplasmic Ca2+ concentration is correlated with the extent of the lymphoproliferative response, (2) lectin‐induced Ca2+ fluxes are sensitive to membrane potential, decreasing with depolarization, and are likely conductive, and (3) the presence of extracellular Na+ during incubation with phytohemagglutinin is not essential to mitogenic triggering.
Fluid‐phase endocytosis by isolated rat adipocytes
Authors: Gibbs E. Michael , Lienhard Gustav E. 
Published: February 2005

AbstractWe have developed an assay, which uses radiolabeled sucrose as the marker, to measure the rate of fluid‐phase endocytosis in isolated rat adipocytes. In addition, the assay was adapted to allow measurement of the release of sucrose from previously loaded cells (fluid‐phase exocytosis). Adipocytes take up sucrose at an approximately linear rate for at least 1.5 hours. A portion of the pinocytosed sucrose is rapidly (half‐time about 20 minutes) returned to the medium. The minimal value for fluid uptake by endocytosis is 57 nl/106 cells‐h at 37°C; this value corresponds to the formation of 110,000 endocytic vesicles of 100‐nm diameter per cell per hour and the internalization of about 20% of the plasma membrane per hour. Insulin caused a small and variable increase in the rate of sucrose uptake. The average increase of 31% from 11 experiments is statistically significant at the level of P < 0.01. A small insulin effect upon the uptake of the calcium complex of [14C]EDTA was also observed. Since this complex was taken up at 2.5 times the rate of sucrose, it probably entered by a combination of fluid‐phase and adsorptive pinocytosis. Insulin did not elicit a significant change in the rate of sucrose release from preloaded cells.
Release of hemopoietic factors by normal human T cell lines with either suppressor or helper activity
Authors: Lanfrancone Luisa , Ferrero Dario , Gallo Eugenio , Foa Robin , Tarella Corrado 
Published: February 2005

AbstractWe analyzed the release of activities capable of stimulating the in vitro growth of human hemopoietic progenitor cells by long‐term cultured T cell growth factor (TCGF)‐dependent human T lymphocytes. Seven cell lines tested produced colony‐stimulating activity (CSA) as well as burst‐promoting activity (BPA). The CSA stimulated primarily the growth of the cells forming colonies after 14 days of incubation. In addition the supernatants from these seven Tcell lines showed the ability to induce the in vitro growth of mixed granulocyte, erythroid, megakaryocyte, macrophage colonies (CFU‐GEMM). The release of hemopoietic factors did not depend on the presence of accessory cells or phytohemagglutinin or serum during the incubation for factor production. In six of the T cell lines the majority of the cells were reactive to the OKT 8 monoclonal antibody (MoAb), whereas one cell line contained mostly OKT 4+ cells. Suppressor activity was detected in three tested OKT 8+ cell lines, while the one OKT 4+ displayed helper activity. All cell lines produced hemopoietic factors with equal efficiency. These results indicate that factors affecting human hematopoiesis are produced by normal T lymphocytes in long‐term culture and this property is not related to the helper or suppressor activity of the cultured cells.
Formation of proliferative tetraploid cells after treatment of diploid cells with sodium butyrate in rat 3Y1 fibroblasts
Authors: Yamada Koji , Kimura Genki 
Published: February 2005

AbstractWhen randomly proliferating rat 3Y1 fibroblasts were treated with sodium butyrate, more than 90% of their cells were arrested reversibly with a 2C DNA content at least 12 h before the G1/S boundary. When cells synchronized in the early S phase were treated with butyrate, approximately 70% of all cells were arrested with a 4C DNA content. The arrests in both G1 and G2 phases by the single inhibitor suggest that the two phases share a common mechanism. The ability of cells to undergo mitosis on time was quickly lost with time of arrest in the G2 phase. Upon removal of the inhibitor, the cells arrested with a 4C DNA content entered a new S phase without intervening mitosis. The tetraploid cells thus produced kept proliferating as fast as diploid cells. These results suggest that the inhibition of the normal G2 traverse is somehow responsible for the formation of the proliferative polyploid cells.
Culture kinetics of glycolytic enzyme induction, glucose utilization, and thymidine incorporation of extended‐exposure phytohemagglutinin‐stimulated human lymphocytes
Authors: Tollefsbol Trygve O. , Cohen Harvey Jay 
Published: February 2005

AbstractGlycolytic enzyme activity is significantly (P < 0.05) induced between 24 and 48 hours of incubation in phytohemagglutinin‐stimulated human lymphocytes. Nonstimulated cultured cells do not show this induction although these cells have an approximate daily doubling of thymidine incorporation. Maximal glycolytic enzyme activity is reached between 96 and 120 hours of culture in stimulated cells (3.5‐fold increase) and maintained until at least 168 hours. There is no significant induction of the hexosemonophosphate shunt or the TCA cycle during seven‐day transformation. Induction of glucose utilization becomes significantly (P < 0.05) greater in stimulated as compared to nonstimulated cultures between 48 and 72 hours of culture and is significantly elevated for at least an additional 96 hours. There is a 17% increase in total protein in the stimulated cells after 24 hours of culture and higher levels of protein content are then maintained over the control. Thymidine incorporation is significantly greater in stimulated cells from 24–144 hours of culture but is not significantly different from the nonstimulated cells at 168 hours (P = 0.98) although glycolytic enzyme activity remains elevated in the stimulated cells. There is a greater enzyme induction of the latter phase of glycolysis during transformation and this phenomenon continues in extended cultures. Increases in glycolytic enzyme activity during mitogenesis appear to be an intrinsic phenomenon independent of cell proliferation and glucose transport. The mitogen‐induced increase in the activity of the glycolytic enzymes accompanies blastogenesis and the sustained elevated activity of these enzymes appears to be related to the high metabolic rate of transformed cells.
Distribution of insulin receptors between plasmalemma and intracellular compartments: Effect of amino acids
Authors: Galbraith Richard A. , Buse Maria G. 
Published: February 2005

AbstractInsulin receptor regulation was studied in the rat erythroblastic leukemic (EBL) cell in primary culture. After 1–2‐hr incubations in medium containing 12 essential amino acids, glutamine, and serine, EBL cell protein synthesis and insulin receptor concentrations were increased compared to cells incubated without serine. Deficiency of medium isoleucine in the presence of serine rapidly decreased protein synthesis and insulin binding to intact cells. Supplementation of deficient media with serine or isoleucine had no effect on total insulin receptor numbers measured in solubilized cell preparations. Increased insulin binding following serine exposure was seen with binding assays at both 4 and 37°C. Dissociation experiments to quantitate intracellular ligand after 37°C binding assays showed increases in both surface binding and intracellular [125I]insulin accumulation. These data combined with previous observations suggest that amino acids essential for this cell are required for the rapid synthesis of a labile regulatory protein which facilitates the redistribution and/or recycling of insulin receptors.
Utilization of iron complexes in an animal cell
Authors: Rasmussen Leif , Toftlund Hans , Suhr‐Jessen Peter 
Published: February 2005

AbstractThe ciliate protozoan Tetrahymena thermophila was grown in synthetic nutrient medium in the absence of the iron chelator citrate. Utilization and toxicity of various iron compounds or complexes in iron‐starved cells were assessed from the number of cell doublings obtained within a standard time. The compounds tested included complexes formed between ortho‐phosphates and two forms of ferric hydroxides, native and cationized ferritin, and tris‐acetylacetonato Fe(III). The ferric hydroxo ortho‐phosphate particles are toxic and can be removed from the medium by Millipore filtration. Uptake of ferritin and tris‐acetylacetonato‐Fe(III) is independent of food vacuole formation and seems to occur by micropinocytosis and by plasma membrane translocation, respectively.
Entry of diphtheria toxin linked to concanavalin A into primate and murine cells
Authors: Guillemot Jean Claude , Sundan Anders , Olsnes Sjur , Sandvig Kirsten 
Published: February 2005

AbstractDiphtheria toxin linked by a disulfide bridge to concanavalin A was highly toxic to HeLa S3 and Vero cells, as well as to murine L cells. The cells could be protected with α‐methyl mannoside, indicating that the conjugate binds mainly through its concanavalin A moiety. Treatment of Vero cells with phospholipase C, TPA (12‐O‐tetradecanoylphorbol‐13‐acetate), and vanadate, which strongly reduce the ability of the cells to bind free diphtheria toxin, had little protective effect against the conjugate, whereas SITS (L‐acetamido‐4′‐isothiocyano‐stilbene‐2,2′disulfonic acid), which inhibits diphtheria toxin binding, as well as the subsequent entry, protected Vero cells, but not L cells. Both types of cells are protected against the conjugate by NH4Cl and monensin, indicating that an acidified compartment is necessary for entry into the cytosol. Exposure of cells, bound with surface conjugate, to low pH induced entry of the toxin into Vero cells, but not into L Cells. Phospholipase C, TPA, and vanadate did not protect L cells against the conjugate. It is concluded that toxin in the conjugate enters L cells by a route which involves low pH, but which is not identical to that in Vero cells.
Limb development in chick embryos: Cyclic AMP‐dependent protein kinase activity, cyclic AMP, and prostaglandin concentrations during cytodifferentiation and morphogenesis
Authors: Smales William P. , Biddulph David M. 
Published: February 2005

AbstractThe effects of prostaglandin E2 (PGE2) on cyclic AMP (cAMP) concentrations of chick limb bud cells obtained from limbs at various stages of development were investigated. In addition, endogenous concentrations of PGE2 were examined in whole limbs from comparable stages. Prior to either chondrogenesis or myogenesis (stages 20–23), cells were more responsive to PGE2, in terms of cAMP levels, than those of differentiated phenotypes, obtained at stages 25–28. This greater responsiveness to PGE2 of undifferentiated cells was correlated with endogenous concentrations of PGE2 which were significantly higher in undifferentiated limbs than in limbs containing differentiated cartilage and muscle. Cyclic AMP‐dependent protein kinase (PKA) activity was detectable in cell homogenates at each stage examined and did not appear to change in cAMP dependency at any stage. The majority (80–85%) of total enzyme activity was localized in soluble fractions of cell homogenates while the residual activity was localized to membrane‐enriched, particulate fractions. The results demonstrate that both responsiveness of limb mesenchyme to PGE2 and endogenous concentrations of PGE2 are maximal prior to cytodifferentiation of limb tissues. The presence of cAMP‐dependent protein kinase in these undifferentiated cells supports a regulatory role for both PGE2 and a cAMP‐protein phosphorylation system in the differentiation of limb tissues.
Adaptive regulatory control of system a transport activity in a kidney epithelial cell line (MDCK) and in a transformed variant (MDCK‐T1)
Authors: Boerner Paula , Saier Milton H. 
Published: February 2005

AbstractAdaptive regulatory control of system A activity was investigated using MDCK cells and a chemically induced, oncogenic transformant of MDCK cells, MDCK‐T1. Within 7 hours after transfer to an amino‐acid‐deficient medium, A activity of subconfluent MDCK cells had maximally derepressed, but this activity in confluent MDCK cells and in subconfluent transformed cells showed little capacity for derepression. Amino‐acid‐starved, subconfluent MDCK cells were used to study trans‐inhibition and repression of A activity by individual amino acids. Trans‐inhibition and repression were defined as the cycloheximide‐insensitive and cycloheximide‐sensitive components, respectively, of the total inhibition. Trans‐inhibition correlated well with substrate affinity, but repression did not. Trans‐inhibition and repression were further characterized using α‐(methylamino) isobutyric acid (mAIB), a trans‐inhibitor, and glutamate, an effective repressor. The apparent initial T1/2 for inhibition by mAIB in the presence of cycloheximide was 0.5 hours, while that for repression by glutamate was 4.7 hours. Half‐maximal inhibition by mAIB and repression by glutamate occurred at approximately 0.02 mM and 0.07 mM, respectively. Reversal of trans‐inhibition by methionine occurred in the presence of cycloheximide within 1–4 hours after removal of methionine. The A system of the transformed MDCK‐T1 cells showed elevated activity, little capacity for derepression, resistance to repression by amino acids, but retention of sensitivity to trans‐inhibition. Kinetic analysis of mAIB uptake indicated that the A system of MDCK‐T1 cells has become kinetically more complex in a manner which resembled amino‐acid‐starved rather than amino‐acid‐fed MDCK cells. These results suggest that the A system of MDCK‐T1 cells has become resistant to adaptive regulatory control.
Neutral amino acid transport in embryonal carcinoma cells
Authors: Zuzack John S. , Tasca Richard J. , DiZio Stephen M. 
Published: February 2005

AbstractNeutral amino acid transport was characterized in the pluripotent embryonal carcinoma (EC) cell line, OC15. Ten of the thirteen amino acids tested are transported by all three of the major neutral amino acid transport systems—A, L, and ASC—although one system may make a barely measurable contribution in some cases. The characterization of N‐methyl‐aminoisobutyric acid (meAIB) transport points to this model amino acid as a definitive substrate for System A transport by OC15 cells. Thus, high concentrations of meAIB can be used selectively to block System A transport, and the transport characteristics of meAIB represent system A transport. Kinetic analysis of System A, with a Km = 0.79mM and Vmax = 14.4 nmol/mg protein/5 min, suggests a single‐component transport system, which is sensitive to pH changes. While proline transport in most mammalian cells is largely accomplished through System A, it is about equally divided between Systems A and ASC in OC15 cells, and System A does not contribute at all to proline transport by F9 cells, an EC cell line with limited developmental potential. Kinetic analysis of System L transport, represented by Na+‐independent leucine transport, reveals a high‐affinity, single‐component system. This transport system is relatively insensitive to pH changes and has a Km = 0.0031 mM and Vmax = 0.213 nmol/mg protein/min. The putative System L substrate, 2‐aminobicyclo‐[2,2,1]heptane‐2‐carboxylic acid (BCH), inhibits Systems A and ASC as well as System L in OC15 cells. Therefore, BCH cannot be used as a definitive substrate for System L in OC15 cells. Phenyialanine is primarily transported by Na+ ‐dependent Systems A and ASC (83% Na+‐dependent; 73% System ASC) in OC15 cells, while it is transported primarily by the Na+‐independent System L in most other cell types, including early cleavage stage mouse embryos and F9 cells. We have also found this unusually strong Na+‐dependency of phenyl‐alanine transport in mouse uterine blastocysts (82% Na+‐dependent). There is no evidence for System N transport by OC15 cells, since histidine is transported primarily by a Na+‐independent, BCH‐inhibitable mechanism.
Uridine phosphorylase from Novikoff rat hepatoma cells: Purification, kinetic properties, and its role in uracil anabolism
Authors: McIvor R. Scott , Wohlhueter Robert M. , Plagemann Peter P. G. 
Published: February 2005

AbstractUridine phosphorylase activity was detected in sonic extracts of six different mammalian cell lines and, in conjunction with uridine kinase, provides a route for the conversion of uracil to UMP via uridine. Uracil phosphoribosyl transferase activity was not detected in any of eight different mammalian cell lines. Uridine phosphorylase was purified 5,330‐fold from Novikoff rat hepa‐toma cells by ammonium sulfate precipitation, DEAE‐Sephadex chromatography, hydroxyapatite chromatography, and Sephadex G‐200 fractionation. The molecular weight of the enzyme by gel filtration was approximately 45,000. The kinetics of the purified enzyme were analyzed with respect to all four substrates at saturating cosubstrate concentration, yielding the parameters KmUra = 360 μM, KmRib−1‐P = 88 μM, KmUrd = 16 μM, and KmPi = 130 μM. However, in intact cells the phosphorolysis of uridine proceeded with an apparent Km of 231 μM. Novikoff cells treated with 0.5 mM inosine exhibited an increase in uracil uptake rate which was proportional to an observed increase in intracellular ribose‐1‐phosphate. Nevertheless, in cells whose de novo synthesis of pyrimidines was blocked by pyrazofurin or N‐(phosphona‐cetyl)‐L‐aspartate (“PALA”), the uptake of uracil was insufficient to support proliferation, even when enhanced by inosine. These observations are consistent with the kinetic characteristics of the enzyme and provide evidence that the intracellular level of ribose‐1‐phosphate plays a rate‐limiting role in the uptake of uracil mediated by uridine phosphorylase.
The effects of lysosomotropic amines on protein degradation, migration of nonhistone proteins to the nucleus, and cathepsin D in lymphocytes
Authors: Polet Herman 
Published: February 2005

AbstractVarious lysosomotropic amines have two parallel effects in human lymphocytes: they inhibit the degradation of cellular proteins and increase the migration of nonhistone proteins (NHP) from the cytoplasm to the nucleus. The increased nuclear level of NHP is associated with increased cellular binding of [3H] actinomycin D, indicating an altered structure of chromatin. The agents inhibit the degradation of short‐and long‐lived proteins equally. Fractionation of the [3H] NHP of the nucleus according to pH 2.5‐6.5 shows that [3H] NHP with a high rate of degradation in untreated cells correspond to [3H] NHP with a high rate of migration in cells treated with the agents. Eserine, amantadine, nicotine, atropine, benzylamine, and propranolol inhibit cathepsin D in concentrations causing proteolytic inhibition in cell cultures or in concentrations believed to be attained in lysosomes. The agents strongly inhibit the cellular accumulation of [3H] chloroquine. The data support the proposal that the migration of NHP from the cytoplasm to the nucleus is the direct consequence of inhibited degradation of these proteins in lysosomes by the amines.
Requirement of spermidine for induction of both heme synthesis and globin transcription in murine erythroleukemia cells
Authors: Watanabe T. , Shafman T. , Kufe D. W. 
Published: February 2005

AbstractWe have previously demonstrated that spermidine is required for proliferation and differentiation of murine Friend erythroleukemia (MEL) cells. Other studies have indicated that inhibition of MEL differentiation by dexamethasone (DEX) and 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA) is reversed by the addition of exogenous polyamines. The present work has thus monitored the requirements of polyamines for induction of MEL hemoglobin synthesis by dimethyl sulfoxide. The results demonstrate that spermidine depletion inhibits induction of both heme synthesis and transcription of α‐ and β‐globin mRNA. In contrast, the results also demonstrate that polyamines are not involved in the inhibition of MEL differentiation by DEX and TPA. Thus, spermidine is required at a transcription level for induction of MEL hemoglobin synthesis, but DEX and TPA act by mechanisms other than polyamine depletion.
Regulation of K562 cell transferrin receptors by exogenous iron
Authors: Rudolph Natalie S. , Ohlsson‐Wilhelm Betsy M. , Leary James F. , Rowley Peter T. 
Published: February 2005

AbstractSingle‐cell analysis of K562 human erythroleukemia cells by flow cytometry was used to demonstrate the specific role of iron in regulating transferrin receptors (TfRs) and to establish that TfR expression does not necessarily correlate with growth rate. Exogenous iron concentration in culture was manipulated by supplementing the medium with sera having different iron concentrations over the range 0.6 to 5.4 μg/ml/ by the addition of iron in the form of FeCl3, iron‐saturated serum, or diferric transferrin, and by the addition of the iron chelator Desferal (desferrioxamine). TfR expression was negatively correlated with exogenous iron content: any treatment that reduced exogenous iron supply by at least 15% resulted in as much as a 1.8‐fold increase in external receptors, detected as binding by both transferrin and monoclonal anti‐TfR antibodies, and a 1.5‐fold increase in the pool of internal receptors, as detected by anti‐TfR antibody binding. None of these treatments altered growth rate, total cellular protein content, protein synthetic rate, cell cycle distribution or cell size. The rapid (12 hr) and reversible induction of internal and external receptors by Desferal was inhibited by cyclohexionide and therefore may have resulted from de novo synthesis and not just mobilization of internal receptor pool to the cell surface. The correlation between growth rate and TfR expression previously observed in these and other cells must be secondary to cellular mechanisms that maintain intracellular iron pools by regulating synthesis, recycling, and cell surface expression of TfRs.