bluuebunny/crossref_metadata_2025_split huggingface dataset:
Turnover of nicotinamide adenine dinucleotide in cultures of human cells
Authors: Rechsteiner Martin , Hillyard David , Olivera Baldomero M. 
Published: February 2005

AbstractThe rate of turnover of nicotinamide adenine dinucleotide (NAD) in the human cell line, D98/AH2, has been estimated by measuring the rates of entry into and exit from NAD molecules of 14C‐adenine. In one set of experiments, cells were labeled by growth in medium containing 14C‐adenine for six hours and then shifted to medium without labeled adenine. The loss of 14C‐adenine from the adenine nucleotide and pyridine nucleotide pools was measured, and the data were analyzed using an analytical treatment which corrects for the relatively slow turnover of precursor pools. The loss of 14C‐adenine from the NAD pool and from the precursor ATP pool could be related to the absolute rate of NAD breakdown. Under the experimental conditions used, the rate of NAD turnover ranged from 83,000 to 126,000 molecules per second per cell. In a complementary experiment cells were grown in the presence of unlabeled adenine, then shifted into medium containing 14C‐adenine and the rate of entry of 14C‐adenine into adenine and pyridine nucleotides was measured. The data were treated using a similar analysis to relate the rate of entry of 14C‐adenine into NAD and the precursor ATP pools to the absolute turnover rate of NAD. This analysis gave a value for NAD turnover of 78,000 molecules per second per cell in excellent agreement with results from the pulse‐chase experiments. The results from both types of experiment indicate that within D98/AH2 cells the half‐life of an intact NAD molecule is 60 ± 18 minutes. Thus, in a human D98/AH2 cell growing with a generation time of 24 hours, NAD is turning over at twice the rate found in Escherichia coli with a generation time of half an hour.
Human epidermal growth factor and the proliferation of human fibroblasts
Authors: Carpenter Graham , Cohen Stanley 
Published: February 2005

AbstractThe effect of human epidermal growth factor (hEGF), a 5,400 molecular weight polypeptide isolated from human urine, on the growth of human foreskin fibroblasts (HF cells) was studied by measuring cell numbers and the incorporation of labeled thymidine. The addition of hEGF to HF cells growing in a medium containing 10% calf serum resulted in a 4‐fold increase in the final density. The presence of hEGF also promoted the growth of HF cells in media containing either 1% calf serum or 10% gamma globulin‐free serum. The addition of hEGF to quiescent confluent monolayers of HF cells, maintained in a medium with 1% calf serum for 48 hours, resulted in a 10‐ to 20‐fold increase in the amount of 3H‐thymidine incorporation after 20–24 hours. The stimulation of thymidine incorporation was maximal at an hEGF concentration of 2 ng/ml, was dependent on the presence of serum, and was enhanced by the addition of ascorbic acid. In confluent cultures of HF cells, subject to density dependent inhibition of growth, hEGF was able to stimulate DNA synthesis more effectively than fresh calf serum. Human EGF stimulated DNA synthesis in quiescent cultures, however, regardless of cell density. The addition of rabbit anti‐hEGF inhibited all effects of this growth factor on HF cells.
Carbohydrate preferences of mammalian cells
Authors: Burns Richard L. , Rosenberger Pamela G. , Klebe Robert J. 
Published: February 2005

AbstractCarbohydrate preferences of mammalian cells can be utilized to biochemically distinguish between different cell lines. Ninety‐three carbohydrates were examined of which (a) 15 supported cell proliferation and (b) 42 were toxic or growth inhibitory. The present investigation has employed an enzymatic system to eliminate trace glucose levels from reagents and glucose generated by serum enzymes.
Ouabain‐resistant human lymphoblastoid lines altered in the (Na+ + K+)‐dependent ATPase membrane transport system
Authors: Lever Julia E. , Seegmiller J. Edwin 
Published: February 2005

AbstractDiploid human lymphoblastoid cells with altered response to ouabain inhibition of the (Na+ + K+)‐dependent ATPase transport system, manifest both in whole cells and in purified plasma membrane vesicles, were selected for their resistance to 0.1 m̈M ouabain. Ouabain‐resistant (OUAR) cells with normal growth at 50 times this dose were recovered at a frequency of 1 × 10−6. This frequency was increased 9‐fold after exposure to ethyl methane sulphonate but was decreased by the frameshift mutagen ICR‐191, under conditions where both increased the frequency of 8‐azaguanine‐resistant colonies. The ouabain resistance phenotype was stable after 200 population doublings in the absence of ouabain. OUAR clones showed 30–50% of the wild type amount of 3H‐ouabain bound per cell, with the same dissociation constant for ouabain, 0.1 m̈M at 0.5 mM K+, as observed in wild‐type cells. Both the initial rate of uptake of 86Rb+ in OUAR cells and the (Na+ + K+)‐dependent ATPase activity of OUAR plasma membranes showed decreased sensitivity to ouabain inhibition. However, growth and transport properties of OUAR cells in the absence of ouabain were unchanged compared with wild type cells.
The repair of the surface structure of animal cells
Authors: Buck Clayton A. , Warren Leonard 
Published: February 2005

AbstractExperiments were performed to determine if animal cells in culture possess specific mechanisms to repair surface molecules damaged by enzymes. The surface membranes of a primary cell culture, chick fibroblasts, a permanent hamster cell line, BHK21/C13, and its virally transformed counterpart, C13/B4 were damaged by exposure to trypsin or to neuraminidase. Following digestion with trypsin, the incorporation of radioactive amino acids or sugars into purified surface membrane of cells was monitored. No differences were noted in rates of incorporation when control and trypsin‐damaged cells were compared. Neuraminidase damage to the surface of BHK21/C13 and C13/B4 cells was evidenced by altered gel filtration profiles of surface glycopeptides, i.e., delayed elution because of reduction in size. By labelling cells with 14C‐L‐fucose prior to neuraminidase treatment and following the incorporation of 3H‐L‐fucose into cell surface glycopeptides after neuraminidase digestion, we were able to monitor the synthesis and turnover of fucose‐containing glycopeptides in the same cells. Gel filtration profiles indicated that little or no desialylated glycoproteins were resialylated (repaired) by specific replacement of sialic acid. Comparing neuraminidase‐digested and control cells we observed no difference in rates of 3H‐L‐fucose incorporation or of 14C‐L‐fucose loss from these cells; nor did we find differences in the rate of incorporation of isotopic glucosamine into sialic acid. Neuraminidase treatment failed to alter the rate of cell growth or the pattern of isotopic incorporation into various cell surface components. These results support the suggestion that return of sialic acid (repair) was effected by turnover which serves as a non‐specific repair mechanism to replace damaged cell surface molecules (Warren and Glick '68; Warren, '69).
Purine uptake by azaguanine‐resistant chinese hamster cells
Authors: Parsons Marilyn , Morrow John , Stocco Douglas , Kitos Paul 
Published: February 2005

AbstractIn this study the resistance of a number of lines of Chinese hamster ovary cells to azaguanine is examined. Those which are drug resistant by virtue of a deficiency of hypoxanthine‐guanine phosphoribosyltransferase (HPRT) fail to take up any exogenous hypoxanthine or azaguanine. A second class of drug resistant cells which grow in the reverse selective HAT medium and have levels of HPRT in the range of the wild type parent line take up these purines at lower rates than the non‐resistant cells and incorporate smaller amounts of them into trichloracetic acid‐insoluble constituents. The results suggest that their basis for resistance resides in lowered incorporation of azaguanine into DNA and RNA, possibly due to a mofified HPRT molecule which accepts hypoxanthine, but not azaguanine as a substrate.
Control of normal differentiation of myeloid leukemic cells. XI. Induction of a specific requirement for cell viability and growth during the differentiation of myeloid leukemic cells
Authors: Fibach Eitan , Sachs Leo 
Published: February 2005

AbstractNormal hematopoietic cells require the presence of a protein (MGI) in the appropriate conditioned medium (CM) for cell viability and growth and for differentiation to mature macrophages and granulocytes. Clones of myeloid leukemic cells have been established in culture (D+ clones) which require CM with this protein for differentiation, but not for cell viability and growth. It has been shown that these leukemic cells can be induced by CM to again require, like normal cells, the presence of CM for cell viability and growth. Induction of this requirement, which will be referred to as RVG, occurred before the D+ cells differentiated to mature granulocytes. Clones of myeloid leukemic cells (D− clones) that could not be induced to differentiate to mature cells, did not show the induction of RVG. The steroid hormones prednisolone and dexamethasone can induce some, but not all the changes associated with differentiation of D+ cells. Incubation with these steroids did not result in the induction of a requirement for these steroids for cell growth and viability. Studies with CM from different sources have shown, that all batches that induced RVG also induced differentiation of D+ cells and that both activities were inhibited after treating the CM with trypsin. It is suggested that the same protein (MGI) may be involved in both activities. Incubation of D+ cells with CM resulted in an increase in agglutinability by concanavalin A and this increase was maintained even in the absence of CM. This suggests, that the induction of RVG in D+ myeloid leukemic cells is associated with a change in the cell surface membrane.
Erythropoietin sensitivity as a differentiation marker in the hemopoietic system: Studies of three erythropoietic colony responses in culture
Authors: Gregory C. J. 
Published: February 2005

AbstractA time course study of the sequential appearance of erythropoietin‐dependent colonies and bursts (derived from CFU‐E and BFU‐E, respectively) was performed on mouse hemopoietic cells cultured in methyl cellulose containing 2‐mercaptoethanol. A new type of small, short‐lived burst was found to be apparent by the third day in culture. By the sixth day most of these bursts had lysed. At the same time, differentiating erythroblasts began to be detectable in the large, late appearing bursts described previously.These two types of burst, differing from each other and from CFU‐E derived colonies both in their ultimate size and morphology, as well as in their time course of appearance and lysis, were compared in other ways. It was found that early burst formation required about 100 times more erythropoietin than that needed to stimulate CFU‐E. On the other hand, early burst formation required less than one‐quarter of the amount of erythropoietin needed to obtain the large, late appearing bursts. Comparison of the distribution of early burst progenitors relative to pluripotent stem cells (CFU‐S) in individual spleen colonies gave a correlation coefficient that was also intermediate between that obtained comparing CFU‐S with CFU‐E and that obtained comparing CFU‐S with the progenitors of late bursts. These results suggest that decreasing proliferative capacity is associated with progressively increasing erythropoietin responsiveness as primitive erythropoietic progenitors move from a position close to pluripotent stem cells through several differentiation steps to reach a stage just prior to the onset of detectable hemoglobin synthesis.
Regulation of lipid synthesis from acetate in diploid fibroblast cultures —variation with passage level and stage of cell growth
Authors: Howard Barbara V. , Howard William J. , Kefalides Nicholas A. 
Published: February 2005

AbstractRegulation of lipid synthesis from acetate in human diploid fibroblast cultures has been studied at various passage levels and at different stages of cell growth. When cultures were transferred to lipid free medium, a stimulation of [14C]acetate incorporation into lipid occurred within three to six hours after removal of exogenous lipid. In early passage cultures, this stimulation was observed whether cells were transferred to protein‐free medium or medium supplemented with delipidized serum protein. However, in late passage cultures the presence of delipidized serum protein was required for the stimulation of lipid synthesis. When logarithmically dividing and stationary phase cultures were compared, the cultures in log phase showed stimulation of acetate incorporation into lipid in the presence or absence of delipidized serum protein, whereas in the stationary cultures the delipidized serum protein was required. When cultures were partially synchronized by a thymidine block, stimulation of acetate incorporation into lipid in the blocked cells only occurred in the presence of delipidized serum protein; in released cells stimulation occurred in protein free medium. When inhibition of lipid synthesis from acetate was compared in young vs. old or dividing vs. stationary cultures, however, no differences were observed. The data indicate the response of diploid fibroblast cultures to change in exogenous lipid is dependent on passage level and state of growth.
Growth of cultured cells from patients with ataxia‐telangiectasia
Authors: Elmore Eugene , Swift Michael 
Published: February 2005

AbstractFibroblast cultures derived from skin biopsies of patients with ataxia‐telangiectasia had doubling times (mean of five lines: 29.9 ± 0.6 hours) significantly longer than normal controls (means of ten lines: 22.4 ± 0.5 hours).
A possible involvement of polyamines in the initiation of DNA synthesis by human WI‐38 and mouse BALB/3T3 cells
Authors: Boynton A. L. , Whitfield J. F. , Isaacs R. J. 
Published: February 2005

AbstractChanging the medium, or adding fresh serum, induces a large proportion of the proliferatively quiescent cells in confluent monolayers of human WI‐38 and mouse BALB/3T3 cells to initiate a growth‐division cycle. Exposure at the time of the medium change or serum addition to MGBG (methyl glyoxal bis [guanylhydrazone]), an inhibitor of spermidine and spermine synthesis and function, reduces or stops the subsequent flow of cells into the DNA‐synthetic phase, without grossly affecting RNA synthesis. Mediation of MGBG action by an actual or functional shortage of spermidine or spermine (but not putrescine), and consequently an involvement of these polyamines in DNA synthesis, is strongly suggested by the reduction of the inhibitor's effectiveness by a brief (1‐hour), early prereplicative exposure of the treated cells to exogenous spermidine and spermine (but not putrescine).
Cell envelope proteins involved in the transport of maltose and sn‐glycerol‐3‐phosphate in Escherichia coli
Authors: Boos Winfried 
Published: February 2005

AbstractTwo types of proteins are discussed in their role of facilitating the transport of maltose and sn‐glycerol‐3‐phosphate in E. coli. The first protein is the receptor for phage δ, known to be an outer membrane protein. By facilitating the diffusion of maltose and the higher maltodextrins through the outer membrane the effect of the δ receptor is to decrease the Km of the transport system without influencing the Vmax of substrate flux.The second protein is a periplasmic protein that is induced by growth on glycerol and is essential for transport of sn‐glycerol‐3‐phosphate in whole cells but not in membrane vesicles. This protein has solely been identified by the use of a two‐dimensional polyacrylamide gel electrophoresis of periplasmic proteins in wild‐type and mutants defective in sn‐glycerol‐3‐phosphate transport.
Regulation of sugar transport in chick embryo fibroblasts and in fibroblasts transformed by a temperature‐sensitive mutant of the rous sarcoma virus
Authors: Kletzien Rolf F. , Perdue James F. 
Published: February 2005

AbstractThe mode of induction of sugar transport by serum‐stimulation of growth and hexose‐starvation in chick embryo fibroblasts (CEF) has been studied using metabolic inhibitors. We have concluded from these studies that the sugar transport increases induced by serum‐stimulation are regulated by post‐transcriptional and post‐translational mechanisms while sugar transport increases induced by hexose‐starvation are regulated by a transcriptional mechanism. CEF infected with a temperature‐sensitive mutant of the Rous sarcoma virus, Ts68, and incubated at the nonpermissive temperature for transformation, 41°, retain the capacity to regulate sugar transport in a manner similar to uninfected CEF. However, Ts68‐infected CEF maintained at the permissive temperature for transformation, 37°, have lost the ability to regulate sugar transport at the post‐transcriptional and post‐translational levels.
Loss of the post‐translational control of nutrient transport in in vitro and in vivo virus‐transformed chicken cells
Authors: Perdue James F. 
Published: February 2005

AbstractThe removal of serum from the medium of uninfected fibroblasts decreased the rate of uptake of uridine, 2‐deoxyglucose, α‐aminoisobutyrate and thymidine. Its subsequent addition rapidly and reversibly stimulated the uptake of all the nutrients but thymidine and this response was not inhibited by treatment of the cells with cycloheximide. The cycloheximide insensitive, rapid increase in the rate of transport has been designated post‐translational control. The nutrient transport systems in chick embryo fibroblasts transformed in vitro with avian sarcoma viruses and virus‐induced cultured chicken tumor cells do not respond to serum removal or addition. Two possible levels for the control of nutrient transport, i.e., mitogen receptor occupancy and mitogen‐induced activation of the transport system, are presented to explain these observations.
Saturable and nonsaturable process of sugar uptake: Effect of oncogenic transformation in transport and uptake of nutrients
Authors: Hatanaka Masakazu 
Published: February 2005

AbstractUptake of sugars into cells by a saturable process increased enormously during and after transformation, and uptake by a nonsaturable process increased significantly but less remarkably compared to controls. The drastic change of uptake rates, observed at around 5 × 10−3 M sugar during and after transformation, emphasizes the significant observation that transition of the sugar uptake system from a saturable to a nonsaturable process occurs near the physiological concentration of D‐glucose normally seen in animal blood. At concentrations higher than 5 × 10−3 M, where a saturable process is barely involved, nonsaturable uptakes of D‐glucose, D‐mannose, D‐galactose, 2‐deoxy‐D‐glucose and 3‐O‐methyl‐D‐glucose proceed tens to hundreds fold faster than the rate of simple diffusion of L‐glucose.These findings suggest that nonsaturable uptake of the sugars known to be substrates for the saturable transport carrier system may not be a physical process or simple diffusion, as observed for L‐glucose uptake. Rather, the nonsaturable uptake might be part of the total physiological process which, along with the saturable process, is controlled by a membrane‐coordination mechanism.A plausible mechanism is discussed in which negative cooperativity of nutrient uptake, such as that found in bacteria, is involved.
Low‐ and high‐affinity concanavalin A binding to thymocyte plasma membrane vesicles
Authors: Wallach Donald F. H. , Schmidt‐Ullrich Rupert 
Published: February 2005

AbstractBinding of concanavalin A to isolated thymocyte membrane vesicles occurs through (a) numerous (∼6 × 106/cell equivalent) low‐affinity sites (Ka = 1.3 × 105 M−1) and (b) fewer (∼0.4 × 106/cell equipment) specific receptors (Ka = 6.8 × 106 M−1) defined as 55,000 D glycoprotein and its multimers. Specific binding is positively‐cooperative, with a Hill coefficient of∼1.8. Low concentrations of glutaraldehyde selectively crosslink the 55,000 D glycoprotein with replacement of positively‐cooperative sites by high‐affinity sites. It is proposed that concanavalin A‐binding induces multimerization of the 55,000 D glycoprotein.
Regulation of plasma membrane protein phosphorylation in two mammalian cell types
Authors: Avruch Joseph , Fairbanks Grant , Crapo Lawrence M. 
Published: February 2005

AbstractThe appreciation of protein phosphorylation as a ubiquitous mechanism for the post‐translational control of protein function has drawn our attention to the phosphorylation of plasma membrane proteins. We have studied this phenomenon in the human erythrocyte and rat adipocyte, and have observed several features, common to the two systems, which may be of general significance. In examining protein phosphorylation in intact cells incubated with 32Pi, it is evident that the 32P‐polypeptides of the plasma membrane are among the most highly labelled species in the cell, despite their minor contribution to overall protein content. The addition of epinephrine (to adipocytes) or cAMPThe following abbreviations are used: cAMP, adenosine 3′:5′‐cyclic monophosphate; DBcAMP, N6, O2′‐dibutyryl adenosine 3′:5′‐cyclic monophosphate; HEPES, N‐2‐hydroxyethylpiperazine‐N1‐2‐ethanesulfonic acid; SDS, sodium dodecyl sulfate.
(to erythrocytes) increases the phosphorylation of certain peptides, whereas others are unaffected. The protein kinases mediating these phosphorylations are present in the plasma membrane as isolated, and can be divided into two groups–‐cAMP dependent and cAMP independent. These two classes of kinase differ markedly in their substrate specificity toward endogenous and exogenous polypeptide substrates. Two classes of protein kinases with similar properties can be detected in the cytoplasm. The relationship between the membrane‐bound and cytoplasmic enzymes is uncertain.The potential roles of the plasma membrane cAMP dependent protein kinases are evident from the diverse effects of cAMP on surface properties; however, the prevalence of plasma membrane proteins phosphorylated via cAMP independent pathways is striking. Thus, elucidation of the regulatory properties of the plasma membrane cAMP independent protein kinases may give new insight into the control of a variety of surface phenomena not mediated by cAMP.
The mechanism of interaction between high‐affinity probes and the uridine transport system of mammalian cells
Authors: Eilam Yael , Cabantchik Ioav 
Published: February 2005

AbstractThe carrier of uridine transport in hamster cells in culture is highly susceptible to the inhibitory effect of probes like S‐benzylated derivatives of mercaptopurine nucleosides. The interaction between the probes and the carrier is competitive and reversible and it takes place at a site different from the substrate binding site. The Ki for the most potent deravative p‐nitrobenzyl‐6‐mercaptoinosine is 0.15 n Molar at 20°C. The effect of the probes is interpreted in terms of a conformational change induced on the carrier upon binding of the probe. The carrier assumes distinct conformations depending on whether it is probe‐free (form A) or probe bound (form B).Kinetic as well as chemical evidence supports the predictions of the allosteric carrier model. A single component of kinetics is observed either in the absence of inhibitor (Km form A) or at high concentrations of inhibitor (Km form B). A two component kinetics is observed at intermediate concentrations of inhibitor (some carriers in form B and others in form A). The two forms have distinct Km values for uridine: form A 50 μMolar and form B 250 μMolar. The two forms have also different susceptibilities to the action of organomercurials: form A is insensitive whereas form B is highly inhibited by the chemical modifier of SH groups.The existence of putative allosteric sites in carriers is discussed in terms of modifier sites capable of modulating transport activities as a result of specific membrane‐ligand interactions.