bluuebunny/crossref_metadata_2025_split huggingface dataset:
Deoxyglucose and 3‐O‐methylglucose transport in untreated and ATP‐depleted novikoff rat hepatoma cells. Analysis by a rapid kinetic technique, relationship to phosphorylation and effects of inhibitors
Authors: Graff Jon C. , Wohlhueter Robert M. , Plagemann Peter G. W. 
Published: February 2005

AbstractDetailed time courses of uptake of labeled 3‐O‐methyl‐D‐glucose and 2‐deoxy‐D‐glucose by untreated and ATP‐depleted Novikoff rat hepatoma cells were determined as function of concentration (0.2–10 mM) by a rapid mixing/sampling technique which allows uptake measurements in time intervals as short as 1.5 seconds. Intracellular accumulation of 3‐O‐methylglucose in untreated and ATP‐depleted cells and of deoxyglucose in ATP‐depleted cells to equilibrium followed pseudo‐first order kinetics and initial velocities were computed from the overall time courses of substrate accumulation. Initial velocity was a Michaelis‐Menten function of exogenous substrate concentration. The estimated kinetic constants for zero‐trans transport of 3‐O‐methylglucose were about the same for untreated and ATP‐depleted cells (K = 1.73 ± 0.24 mM; V = 28.8 ± 3.6 pmoles/μl cell H2O. sec) and were similar to those for deoxyglucose transport in ATP‐depleted cells (K = 0.65 ± 0.1 mM; V = 19.6 ± 1.6 pmoles/μl cell H2O. sec). Similar kinetic parameters were obtained for the transport of D‐glucose and D‐galactose in ATP‐depleted cells. The transport of 3‐O‐methylglucose and deoxyglucose were inhibited by each other in a simple competitive manner with apparent Ki's similar to their transport Km's.In untreated cells, in which deoxyglucose was phosphorylated, intracellular steady‐state levels of free deoxyglucose accumulated within 10 to 20 seconds of incubation regardless of its concentration in the medium. Thereafter, the rate of deoxyglucose incorporation into total cell material reflected the rate of phosphorylation rather than the transport rate. The rate of deoxyglucose transport exceeded the initial rate of its phosphorylation by 20–40%. The intracellular steady‐state levels observed during the first 2 minutes of incubation decreased from about 40% of equilibrium level at 0.2 mM deoxyglucose to about 8% at 10 mM. Computer fits of a kinetic equation describing transport and phosphorylation as independent processes operating in tandem to these data are consistent with the observed kinetic constants for hexose transport and hexokinase activity with deoxyglucose as substrate. Upon longer incubation (2–10 minutes) the rate of deoxyglucose uptake by the phosphorylating cells decreased progressively, concomitant with a decrease in intracellular ATP and an increase in intracellular deoxyglucose to equilibrium levels. It is demonstrated that the rate of deoxyglucose uptake, measured at two or more minutes, seriously underestimates the hexose transport rate and yields misleading conclusions regarding the extent and type of inhibition by transport inhibitors, such as Persantin or cytochalasin B. Persantin inhibited hexose transport in a simple non‐competitive manner (Ki = 20 μM) indicating that the drug affects the function of the hexose carrier.
Studies of cAMP metabolism in cultured hepatoma cells: Presence of functional adenylate cyclase despite low camp content and lack of hormonal responsiveness
Authors: Leichtling Ben H. , Su Y.‐F. , Wimalasena Jayantha , Harden T. Kendall , Wolfe Barry B. , Wicks Wesley D. 
Published: February 2005

AbstractThe ability of isoproterenol, glucagon, PGE1 and cholera toxin to stimulate the synthesis of cAMP and protein kinase activity in a line of liver cells (BRL) and a line of rat hepatoma cells (H35) has been determined.The concentration of cAMP in BRL cells (∼ 10 pmoles/mg protein) is in the range reported for other cultured cell lines but H35 cells contain extraordinarily low amounts of this cyclic nucleotide (∼ 0.05 pmoles/mg protein). Isoproterenol and PGE1 caused an increase in cAMP content, and protein kinase activation in BRL cells, although glucagon was ineffective. H35 cells, in contrast, were completely insensitive to all hormonal agonists. Despite this fact, cholera toxin was able to produce a marked increase in cAMP content, adenylate cyclase activity and protein kinase activation in H35 cells. Binding studies with [125I]‐iodohydroxybenzylpindolol, a specific β‐adrenergic receptor antagonist, revealed that each H35 cell possesses fewer than 10 β‐adrenergic receptors whereas BRL cells contain 2–5,000 receptors per cell. The low level of cAMP in H35 cells appears to result from a combination of totally unstimulated adenylate cyclase and apparently elevated phoshodiesterase activities.
On the stimulation of rat thymocyte 3‐0‐methyl‐glucose transport by mitogenic stimuli
Authors: Hume David A. , Weidemann Maurice J. 
Published: February 2005

AbstractPretreatment of rat thymus lymphocytes with N‐ethyl‐maleimide prevents the stimulation of 3‐0‐methyl‐glucose transport by concanavalin A or ionophore A23187 but does not affect the ability of concanavalin A to induce a rapid increase in cellular Ca2+ uptake. N‐ethyl‐maleimide added after concanavalin A amplifies rather than prevents the subsequent stimulation of 3‐0‐methyl‐glucose transport. Incubation of thymocytes with concanavalin A produces a decrease of 43% in the apparent Ki for phloretin, a competitive inhibitor of 3‐0‐methyl‐glucose transport, without affecting the apparent Km for the substrate. Similarly, very low concentrations of cytochalasin B inhibit concanavalin A‐stimulated glucose transport preferentially, without markedly affecting the unstimulated transport rates. The similarity between concanavalin A‐stimulated 3‐0‐methyl‐glucose transport in thymocytes and insulin‐stimulated glucose transport in adipose tissue, with particular emphasis on the effects of the modifying agents described in this paper, is discussed.
Morphological and biochemical correlates of skeletal muscle contractility in the cat. I. Histochemical and electron microscopic studies
Authors: Van Winkle W. B. , Schwartz A. 
Published: February 2005

AbstractThree cat hind limb muscles have been examined, histochemically and ultrastructurally, in a multiparameter correlative study of structure and function in skeletal muscle contractility. The soleus, a histochemically pure, slow‐twitch muscle possesses ultrastructural features which are, in many cases, significantly different from those of almost pure fast twitch caudofemoralis muscle. Although stereological analysis of fiber types indicates a correlation between speed of relaxation and volume of sarcoplasmic reticulum, morphological features such as fenestrated collars and triad morphology are identical in all fiber types. The fast twitch‐oxidative‐glycolytic fiber possesses features common to both slow twitch fibers (high mitochondrial content) as well as fast twitch fibers (high sarcoplasmic reticulum content) in addition to Z band width which falls in between these two fiber types.Sarcoplasmic microtubules have been described in all three fiber types in all muscles examined. They occur in predictable orientation and their possible function (s) is described.
Morphological and biochemical correlates of skeletal muscle contractility in the cat. II. Physiological and biochemical studies
Authors: Van Winkle W. B. , Entman M. L. , Bornet E. P. , Schwartz A. 
Published: February 2005

AbstractIsometric twitch characteristics and biochemical parameters of isolated myosin and sarcoplasmic reticulum have been compared in three cat hind limb muscles. The fast twitch caudofemoralis and the slow twitch soleus are almost pure muscles as judged from histochemical studies. Isolated myosin from the caudofemoralis is not only 2‐ to 3‐fold higher in its ATPase activities than that of the soleus, but also in non‐dissociated forms has greater electrophoretic mobility than the soleus myosin. Purified myosins from fast muscles as well as soleus exhibited three light chains upon electrophoresis. However, the intact non‐solubilized myosins differed in electrophoretic mobilities. The sarcoplasmic reticulum fraction isolated from caudfemoralis exhibits faster rates of Ca++ binding and uptake than soleus, and when fit to a two component model, the caudofemoralis SR exhibits a higher amount of a fast binding site than does soleus SR, features reflected in differences in the relaxation time of the two muscles.In contrast, the fast twitch tibialis anterior has been shown to be a gradient of fiber types and its isometric twitch may be separated by selective nerve stimulation, into a fast and a slow twitch component. Our findings that myosin fraction, as well as sarcoplasmic reticulum fractions isolated from these two components differ with respect to their biochemical characteristics add support to the possibility of a dual function in this muscle.
Rates of protein synthesis by hepatocytes isolated from rats of various ages
Authors: Ricca George A. , Liu Daniel S. H. , Coniglio John J. , Richardson Arlan 
Published: February 2005

AbstractThe rate of total protein synthesis in isolated hepatocytes was determined. The incorporation of L‐[3H]valine into protein is linear for at least two hours of incubation and is affected by the concentration of amino acids in the medium. Uptake of valine by hepatocytes from 1.5‐ and 18‐month‐old rats was identical and appears to occur by simple passive diffusion. Within five minutes, the specific activities of the intracellular and extracellular valine pools are equivalent. The specific activities of these pools are saturated by 1.6 mM valine and remain constant for 60 minutes of incubation. The rates of protein synthesis by hepatocytes from 1‐ to 2‐month‐old rats is 96.8 pmoles of valine per minute per milligram protein. This is comparable to rates of protein synthesis reported for perfused liver and liver in vivo and is approximately 64% higher than the rate of protein synthesis by hepatocytes from 18‐month‐old rats.
Inhibition of sugar uptake in adenosine‐treated 3T3 cells
Authors: Barnes David W. , Brown Verona T. , Colowick Sidney P. 
Published: February 2005

AbstractAddition of 5 to 250 micromolar adenosine to the culture medium resulted in a 30–80% inhibition of the rate of uptake of 2‐deoxyglucose or 3–0‐methylglucose by sparse or confluent 3T3 cells within three hours. The inhibition of deoxyglucose uptake could be reversed partially by changing the cells to medium without adenosine for two hours and could be prevented completely by the addition of persantin, an inhibitor of nucleoside uptake. The adenosine effect is not due to inhibition of pyrimidine synthesis, since it is not prevented by uridine. It is not seen in 3T6 cells lacking adenosine kinase. The inhibition could be observed on confluent cells whose deoxyglucose uptake was stimulated by insulin, epidermal growth factor (EGF), calf serum or calcium phosphate. Although the percentage stimulation over control by these factors varied, the percentage inhibition by addition of adenosine of the stimulated rates, as well as the unstimulated rate, was relatively constant. EGF, insulin and calcium phosphate caused little or no stimulation of deoxyglucose uptake by sparse cells, whether adenosine treated or untreated. The results suggest that adenosine acts intracellularly after phosphorylation to regulate sugar uptake through a mechanism which is independent of the regulation by hormones and cell density.
Sequential changes in ribosomal activity during the activation and cessation of growth in lymphocytes stimulated with concanavalin A
Authors: Harms‐Ringdahl Mats , Cooper Herbert L. 
Published: February 2005

AbstractThe ribosomal activity in human peripheral blood lymphocytes stimulated by concanavalin A has been characterized during the passage of cells from the resting (G0) state, through maximum mitotic activity, and into a secondary resting state. The total ribosome content increased progressively after the addition of concanavalin A and after 72 hours reached a maximal 10‐fold increase. Lymphocytes cultured for 120 hours returned to a low metabolic activity, but still contained four times more ribosomes per cell than unstimulated lymphocytes. During the first 24 hours free ribosomes, which were originally 64% of resting cell ribosomes, decreased to 27% of total particles. Thereafter they reaccumulated until, at 120 hours, free ribosomes again comprised 61% of the total ultraviolet absorbance and the total polysome profile resembled that for resting cells, despite the higher ribosome content.Analysis of the rate of protein synthesis and the corresponding quantity of polysomes at different times in the growth sequence revealed that protein synthesizing efficiency of polysomes was initially low and rose for the first 48 hours of culture. After 48 hours, polysomes with low protein synthesizing efficiency reappeared. Following pulse labeling of rRNA with [3H]uridine, the redistribution of newly synthesized ribosomal particles was followed. A decreased flow of ribosomes through polysomes was evident at 48 hours and thereafter. Diminution in polysome efficiency, reduced flow of particles through polysomes, and accumulation of free ribosomes thus characterize the cessation of growth which occurs as a physiological event in Con A‐stimulated lymphocyte cultures. Nevertheless, these changes begin at or before the peak of RNA, protein and DNA synthetic activity, showing that the biochemical mechanisms related to cessation of cell growth begin to function during the height of cell growth in a system under physiological control.
Requirements of BHK cells for the exit from different quiescent states
Authors: Talavera Antonio , Basilico Claudio 
Published: February 2005

AbstractWe have investigated the kinetics of exit from the resting state of BHK cells which had been arrested by isoleucine deprivation, serum starvation, or high temperature in the case of three ts G1 mutants. In addition, we have studied the effect of imposing a secondary deprivation on cells which had been released from one of the above mentioned blocks. The results obtained show that the quiescent states reached by BHK cells following serum or isoleucine deprivation cannot be differentiated on the basis of the exit kinetics from Smith and Martin's probabilistic A‐state. Nevertheless, the response of cells to secondary deprivation is different, depending on the nature of the primary arresting condition used, reflecting physiological differences between the different resting states. A model is presented which postulates that cycle transition specific genes require the presence of different proliferative agents for their expression.
Lithium transport by fibroblastic mouse cells: Characterization and stimulation by serum and growth factors in quiescent cultures
Authors: Smith Jeffrey B. , Rozengurt Enrique 
Published: February 2005

AbstractSerum enhances the rate of Li+ entry and exit in quiescent cultures of mouse fibroblasts by 2‐ to 3‐fold. Tertiary cultures of whole mouse embryos as well as established fibroblast lines (3T3, 3T6) show the increase in Li+ permeability when serum is added to cultures whose growth has been arrested by serum deprivation. Growing cells are only slightly more permeable to Li+ in the presence of serum. Purified compounds which initiate DNA synthesis also rapidly increase Li+ entry; mitogenic levels of thrombin and the combination of epidermal growth factor, insulin, and bovine serum albumin were the most effective ones tested. The effect of serum on Li+ uptake occurs within a few minutes, is not affected by inhibitors of macromolecular synthesis, and appears mainly to increase the Vmax of entry. Inhibitors of energy production partially reduce Li+ entry but do not block the activation by serum. One portion of Li+ uptake (−40%), which is inhibited by ouabain, phloretin, or Na+ deprivation, is mediated by the Na+/K+ pump in the plasma membrane. A second mechanism of Li+ entry which is blocked by Na+ or amiloride appears to be a Na+ specific “porter.” The activity of both components is stimulated by serum. The increased activity of the putative Na+ porter would increase Na+ availability to the Na+ pump and may account for its enhancement by serum, which was also noted previously (Rozengurt and Heppel, '75).
Cultured human fibroblasts: Distribution of cell generations and a critical limit
Authors: Harley Calvin B. , Goldstein Samuel 
Published: February 2005

AbstractIn circular outgrowths of human skin fibroblasts we found that mitotic cells at the circumference had consumed more of their replicative lifespan than cells located more centrally. The lifespan remaining in cells at a given radial position could be predicted by determining their generation level based on the rate at which the outgrowth expanded and the cell doubling time. The data also show that outgrowths contain a heterogeneous mixture of cells described by a linear distribution of generations which can account for the variable replicative capacity observed in clones and the exponential increase in the fraction of nondividing cells with serial passage. These results support the concept that a critical limit of cell divisions determines the replicative lifespan.
Selective changes in tRNA methyltransferase activity in confluent monolayers of WI‐38 cells stimulated to proliferate
Authors: Wilkinson C. Ray , Kerr Sylvia J. 
Published: February 2005

AbstractIn quiescent confluent monolayers of WI‐38 cells, the specific activity of the tRNA methyltransferases falls to 20% of the level found in log phase cells. When the resting cells are stimulated to proliferate by a change to fresh medium, the enzymes show a rapid rise in specific activity which correlates with early increases in the rate of tRNA synthesis. The specific activity of the enzymes continues to rise throughout the period of DNA synthesis, at the end of which it is somewhat higher than that of log phase cells. The increases in enzyme activity could be blocked by exposure of the stimulated cells to Actinomycin D (2μ/ml). The increases in activity were not equivalent for the different base‐specific enzymes. The contribution of the N2‐methylguanine specific enzyme remained relatively constant, while that of the N2,N2‐dimethylguanine specific and 1‐methyladenine specific enzymes doubled and tripled, respectively, by late S phase. The contributions of the 1‐methylguanine and the 7‐methylguanine specific enzymes fell to a few percent of the total by late S phase. This indicates non‐coordinate variations in the expression of the different base‐specific enzymes after stimulation of resting cells and may be related to altered isoaccepting tRNA profiles observed in resting and growing cells.
Differential effects of inhibition of polyamine biosynthesis on cell cycle traverse and structure of the prematurely condensed chromosomes of normal and transformed cells
Authors: Sunkara Prasad S. , Pargac Mary B. , Nishioka Kenji , Rao Potu N. 
Published: February 2005

AbstractThe objective of this study was to determine the points in the cell cycle at which normal and transformed cells become arrested as a result of polyamine deprivation. Treatment of normal (human fibroblast line PA2 and mouse 3T3) and transformed (CHO, HeLa and SV3T3) cells with methylglyoxal bis‐(guanyl‐hydrazone) resulted in a significant decrease in the levels of spermidine and spermine which was associated with an inhibition of growth. Examination of the prematurely condensed chromosomes (PCC) of the polyaminedepleted cells, revealed that normal fibroblasts were preferentially arrested in early G1 phase while a majority of cells in the transformed lines were blocked in S phase. A close examination of the PCC of the transformed cells indicated a significant decrease in the number of DNA replication sites suggesting that polyamines have an important role in DNA chain initiation.
Characterization of cell lines showing growth control isolated from both the wild type and a leucyl‐tRNA synthetase mutant of chinese hamster ovary cells
Authors: Pollard J. W. , Stanners C. P. 
Published: February 2005

AbstractThe genetic approach to the problem of cellular growth control is limited by the availability of recessive mutations in cell lines which are capable of growth control in vitro. The CHO cell line has yielded many recessive mutations including, for example, tsH1, a temperature sensitive leucyl‐tRNA synthetase mutant, which under non‐permissive conditions rapidly shuts down protein synthesis and generates uncharged tRNA. Both CHO and tsH1 are transformed, however, and do not respond to environmental stimuli with the coordinated regulation of macromolecular processes observed in normal diploid fibroblasts. We describe here the isolation and characterization of growth control revertants obtained from both CHOwt and tsH1. The best of these GRC+L‐73, isolated from tsH1, had 20 chromosomes, one less than tsH1, had normal fibroblastic morphology, would not grow in suspension, required high serum concentrations for growth, grew to relatively low cell densities at saturation in monolayer culture and showed a stationary phase characterized by arrest in a G1‐like state with maintenace of high viability for several weeks. It is expected that this line as well as a ts revertant GRC+LR‐73 will greatly facilitate the genetic investigation of growth control and, in particular, will help to elucidate the role of uncharged tRNA in the regulation of macromolecular synthesis in mammalian cells.
Interactions between purified GM‐CSF, purified erythropoietin and spleen conditioned medium on hempoietic colony formation in vitro
Authors: Metcalf D. , Johnson G. R. 
Published: February 2005

AbstractPreincubation of C57BL adult marrow cells or CBA fetal liver cells with a 250‐fold excess concentration of purified GM‐CSF failed to reduce the frequency of cells forming eosinophil, megakaryocyte or erythroid colonies in subsequent agar cultures. When excess concentrations of purified GM‐CSF were added to agar cultures stimulated by pokeweed mitogen‐stimulated spleen conditioned medium (SCM), no reduction was observed in the frequency of eosinophil, megakaryocyte or erythroid colonies. Addition of 4 units of purified erythropoietin (EPO) to cultures of fetal liver or adult marrow cells stimulated by SCM increased the number of erythroid colonies but did not reduce the number of non‐erythroid colonies or the non‐erythroid content of mixed erythroid colonies.Although neither GM‐CSF nor EPO alone was able to stimulate erythroid colony formation in agar cultures of fetal liver cells, small numbers of large erythroid colonies were stimulated to develop in cultures containing both purified regulators. Purified GM‐CSF was also able to support the survival in vitro of a small proportion of erythroid colony‐forming cells in fetal liver populations cultured initially in the absence of SCM and the survival of some eosinophil and megakaryocyte colony‐forming cells in similar cultures of adult marrow cells.The results do not support the hypothesis that GM‐CSF and EPO compete for a common pool of uncommitted progenitor cells. On the contrary, the data indicate that GM‐CSF and EPO are able to collaborate in stimulating the proliferation of some erythropoietic cells. Furthermore, purified GM‐CSF appears to be able to support temporarily the survival and/or initial proliferation of at least some cells forming erythroid, eosinophil and megakaryocyte colonies, even though GM‐CSF is unable to stimulate the formation of colonies of these types.
Erythroid and granulocytic colony growth in cultures supplemented with human serum lipoproteins
Authors: Aye M. T. , Seguin J. A. , McBurney J. P. 
Published: February 2005

AbstractThe ability of human serum to support erythroid an dgranulocytic colony formation has been investigated. It was found that normal human serum could replace fetal calf serum in the cultures and was able to support the growth of these hemopoietic colonies. Serum fractions enriched for low density lipoproteins, either by precipitation with Heparin‐Mn++ or by ultra‐centrifugation, was found to contain this growth supporting activity of human serum.