bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effects of insulin on glycogen metabolism of serum‐deprived BHK‐21 fibroblasts
Authors: Lavis Victor R. , Thompson W. Joseph , Strada Samuel J. 
Published: February 2005

AbstractWe have studied the effects of insulin on glycogen metabolism of cultured BHK‐21 fibroblasts. Addition of insulin to cells cultured in 0.5% serum stimulated incorporation of 5mM 14C‐glucose into glycogen and increased cellular glycogen content, without inducing proliferation. With serum‐deprived cells incubated for 2 hr, maximal stimulation of incorporation of glucose into glycogen was 3.5‐fold and the half‐maximal dose of insulin was 5nM. After culture for 24 hr with 100nM insulin, incorporation was increased 13‐fold and glycogen content was increased by 44%. Incorporation of glucose into glycogen was reduced by agents which elevate cellular adenosine 3,5‐cyclic monophosphate (cyclic AMP): 10μM prostaglandin E1 (PGE1), 0.25 mM 1‐methyl‐3‐isobutylxanthine (MIX), 10μM L‐epinephrine (L‐EPI), and 10μM L‐isoproterenol (L‐ISO). Culture with 100nM insulin for 2 hr stimulated incorporation three‐fold in the presence of any of these compounds, but insulin did not affect cellular cyclic AMP.At all times from 1 hr to 24 hr after addition of insulin to serum‐deprived cultures, the 1‐min uptake of 10μM 3H‐2‐deoxyglucose (3H‐2DG) was increased. There were small (30 to 40%) increases in glycogen synthase I activity at 15 and 30 min but not 4 hr after addition of insulin. After 24 hr with insulin, total glycogen synthase and phosphorylase activities were increased approximately two‐fold, without changes in their activation states.We conclude that insulin promotes glycogenesis in serum‐deprived BHK‐21 cells. This response is mediated principally by increased entry of glucose into cells, and is not mediated by a change in cellular cyclic AMP concentration.
The effect of serum and multiplication stimulating activity on L6 myoblast growth: The lack of correlation with cyclic nucleotide changes
Authors: Richman Robert A. , Weiss James P. , Roberts Susan B. , Florini James R. 
Published: February 2005

AbstractThe role of cAMP and cGMP in triggering proliferation of rat myoblasts was evaluated by: (1) measuring effects of mitogens on intracellular cyclic nucleotide levels, and (2) observing effects of agents which altered cyclic nucleotide levels on cell proliferation. Multiplication stimulating activity (MSA, 1 μg/ml), a member of the somatomedin family, stimulated cell proliferation after 48 hr. It had little effect on cellular cyclic nucleotide levels, measured by radioimmunoassay. Horse serum (HS) and fetal bovine serum (FBS) stimulated cell proliferation approximately equally. Neither affected cAMP levels; FBS reduced cGMP to 33% of control values, but HS had no effect. Thus, there was no simple correlation between mitogenic action and cyclic nucleotide levels at any time from 5 min to 24 hr after addition of a purified mitogen or serum. Furthermore, agents which caused substantial changes in cyclic nucleotide levels had no effect on cell proliferation. Prostaglandin E1 (5 μM) elevated cAMP 440% without affecting cGMP levels or cell growth. A potent phosphodiesterase inhibitor, 1‐methyl‐3‐isobutylxanthine (MIX, 0.1 mM), when added alone or in the presence of MSA, HS, or FBS, elevated cAMP 200% and cGMP 167%, but it had little effect on their mitogenic action. Lastly, a purified mitogen such as MSA, unlike serum, must be present for extended periods of time in order to stimulate cell proliferation. This makes it unlikely that a trigger mechanism functions in initiating cell division. We conclude that neither cAMP nor cGMP appear to be second messengers for the mitogenic action of MSA or serum on muscle cells.
Nucleic acids methylation of synchronized BHK 21 HS 5 fibroblasts during the mitotic phase
Authors: Bugler B. , Bertaux O. , Valencia R. 
Published: February 2005

AbstractThe methylation of nucleic acids has been investigated during the cell cycle of an asparagine dependent strain of transformed fibroblasts (BHK 21 HS 5). The synchrony was carried out by a partial asparagine starvation of cells for 24 hours. The amino acid supply induced all cells to enter synchronously the G1 phase. Methylation and DNA synthesis were respectively measured by pulsed [methyl‐14C] methionine and [methyl‐3H] thymidine incorporation. DNA methylation followed a biphasic pattern with maximal methyl incorporations during both S phase and mitosis.A partial desynchronisation induced the S phase of the second cycle to proceed before all the cells have achieved their division. Hydroxyurea was used in order to inhibit the DNA synthesis of cells entering the second cell cycle, which might interfer with the mitosis of the first one. The inhibitor was added either at the first beginning of cell division or during all the G1 phase. In both conditions it suppressed 3H thymidine incorporation of the second cycle. However, mitosis took place and methylations occurred as in previous experiments.The DNA methylation of the mitotic phase in the first cell cycle could thus be dissociated from the classical post‐synthetic DNA maturation and did not correspond to any DNA methylation appearing in the course of the second cell cycle.
The energy charge in wild‐type and respiration‐deficient chinese hamster cell mutants
Authors: Soderberg K. , Nissinen E. , Bakay B. , Scheffler I. E. 
Published: February 2005

AbstractHigh pressure liquid chromatography was used to determine the base, nucleoside, and nucleotide levels in wild type and a series of respiration‐deficient Chinese hamster cell mutants. From this analysis the size of the total adenylate pool and the energy charge could be calculated for each cell line. We find a constant energy charge, as expected, but the adenylate pool seems to be considerably lower in the respiration‐deficient mutants.
In vitro proliferation and lifespan of bovine aorta endothelial cells: Effect of culture conditions and fibroblast growth factor
Authors: Duthu Gwen S. , Smith James R. 
Published: February 2005

AbstractThe effect of culture conditions on calf dorsal aorta endothelial cells was studied. Population doubling time varied as a function of the cell seeding density, growth medium, serum supplement, and concentration of fibroblast growth factor (FGF). The shortest population doubling time was found for cells (population doubling level 0–30) grown in Eagle's Minimal Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 100 ng/ml FGF. The stimulatory effect of FGF on bovine endothelial cell proliferation was dependent on cell inoculation density. FGF significantly increased cell division rate at cell inocula less than 1 ± 104 cells/cm2 but not at higher densities. The population doubling time and cell size increased as the mass culture population doubling level increased. The replicative lifespans of bovine endothelial cells grown in medium supplemented with 20% FBS were 10–15% greater than parallel cultures supplemented with 10% FBS. Cultures grown in medium supplemented with 10% FBS and 50 ng/ml FGF showed a 50% increase in replicative lifespan compared to cultures grown in medium supplemented with 10% FBS alone. When FGF was used the increase in the number of doublings was a function of the length of time the cells were grown in the presence of FGF. This report extends comparable observations on the in vitro aging of human diploid fibroblasts to bovine endothelial cells.
Inhibition of the adipose conversion of BALB/c 3T3 cells by 12‐0‐tetradecanoylphorbol‐13‐acetate: Dependence on pH reduction via lactic acid production
Authors: O'Brien Thomas G. , Saladik Douglas 
Published: February 2005

AbstractThe effect of modifying culture pH on the adipose convesion of BALB/c 3T3 cells was studied. We have previously shown that the tumor promoter 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA) stimulates cellular lactic acid production, lowers the pH of the culture medium, and inhibits differentiation in this system. We now report that addition or organic acids such as D‐ or L‐lactic acid or acetic acid to the culture medium mimicked the effect of TPA by lowering pH, stimulating cellular lactic acid production, and inhibiting the adipose conversion. Lowering the pH by changing the NaHCO3 concentration also inhibited differentiation.A comparison of Dulbecco's modified minimal essential medium (DMEM) vs minimal essential medium (MEM) indicated that in the former media, the rate and extent of differentiation was greater than in the MEM, and TPA neither inhibited differentiation nor stimulated lactate production. Our standard MEM medium could be made similar to DMEM with respect to the cells' response to TPA simply by raising the NaHCO3 concentration, hence, the buffering capacity, of MEM to that present in DMEM, suggesting that TPA can only inhibit differentiation in this system if it can lower culture pH via lactate production.
A novel synergistic effect of alanosine and guanine on adenine nucleotide synthesis in mammalian cells. Alanosine as a useful probe for investigating purine nucleotide metabolism
Authors: Gupta Radhey S. 
Published: February 2005

AbstractA novel synergistic effect of the antitumor agent alanosine (2‐amino‐3‐(hydroxynitrosoamino) propionic acid), which specifically inhibits the enzyme adenylosuccinate synthetase (ASS) and guanine on the growth of Chinese hamster ovary (CHO) cells and human diploid fibroblasts (HDF) has been observed. In the presence of subinhibitory concentrations of alanosine, both CHO cells and the HDF show excessive sensitivity to exogenous guanine—a phenotype which closely resembles that seen with some of the mutants containing reduced enzymatic activity of ASS. The growth inhibitory effects of alanosine, or alanosine and guanine, on CHO cells are completely reverted by the addition of adenine to the culture medium, and the synergistic effect of guanine is not observed in mutants which lack the enzyme hypoxanthine‐guanine phosphoribosyl transferase. These results suggest that guanine nucleotides exert a regulatory effect on the activity of the enzyme adenylosuccinate synthetase. The ability to confer the guaninesensitive phenotype and its modulation by subinhibitory concentrations of alanosine in different cell types indicates that alanosine provides a useful probe for investigating the regulation of purine nucleotide metabolism in mammalian cells.
Some metabolic differences between human skin and aponeurosis fibroblasts in culutre
Authors: Lemonnier F. , Gautier M. , Wolfrom C. , Lemonnier A. 
Published: February 2005

AbstractTwo types of human fibroblast strains were studied in culture. One was derived from abdomen skin and the other from abdominal muscle aponeurosis. Tissue‐specific differences were found between thése two cell strains. Skin fibroblasts had faster doubling time, smaller cell volume, and lower glucose consumption when compared to aponeurosis fibroblasts. Furthermore, extracellular amino acid variations showed some specific differences, in particular a lack of serine consumption in skin fibroblasts.
Multiple fractions of sodium exchange in human lymphocytes
Authors: Negendank William , Shaller Calvin 
Published: February 2005

AbstractHuman lymphocytes contain a large, saturable fraction of K+ that exchanges slowly with K+ in the external medium, and a small non‐saturable fraction that exchanges rapidly. We determined whether or not Na+ exchanges in a similar manner with external Na+. Cells were pre‐equilibrated to ensure absence of net ion movements. Efflux was studied by loading with 22Na and transferring without washing to a non‐labeled medium. Influx was studied by transferring to labeled medium and separating large samples of cells at 6,000g. There are fast, intermediate, and slow fractions of Na+ exchange, with half‐times of 2, 14, and 120 minutes. At normal external K+, most cell Na+ exchanges rapidly, while at lower external K+ the Na+ that replaces cell K+ exchanges slowly. Parallel sources of fast and slow fractions, such as extracellular ones and subpopulations of cells, were ruled out by simultaneous 42K and 22Na fluxes and by a quantitative analysis of the combined K+ and Na+ content and flux data over a range of external K+ and Na+ levels. Five possible models of ion fluxes occurring in series were considered. Surface matrix, surface binding sites, and cytoplasmic channels with rapid nuclea exchange were eliminated as sources of the fast fractions. Therefore, the fast fractions of K+ and Na+ must reflect the permeability of the surface membrane. This left only two possible sources of the slow fractions. One, a subcellular compartment (e.g., nucleus), was eliminated by the combined content and flux data. We conclude that the slow fractions of ion flux are rate‐limited by adsorption onto and desorption from cellular macromolecules. The data support the association‐induction hypothesis and are understood by reference to two fundamental concepts: that of rapid solute exclusion from cell water existing in a polarized state; and that of solute accumulation limited by adsorption onto fixed anionic sites within the cell.
The cytoplasmic appearance of three functions expressed during the G0→G1→S transition is nucleus‐dependent
Authors: Jonak Gerald J. , Baserga Renato 
Published: February 2005

AbstracttsAF8, ts13, tsHJ‐4, and TK−ts13 cells are G1‐specific temperature‐sensitive (ts) mutants of BHK cells that do not enter S phase when serumstimulated from quiescence at nonpermissive temperature (39.6°‐40.6°). TK−ts13 are, in addition, defective in thymidine kinase. Different G1 functions must be involved in these cells, since the first three cell lines complement each other when forming heterokaryons.We have used these cells to study the role of the nucleus in the cytoplasmic expression of these G1 functions during the transition of cells from the non‐proliferating to the proliferating state. We fused cytoplasts from either serumstarved (G0) or serum‐stimulated (S) tsAF8 cells with G0‐ts13, G0‐tsHJ‐4, and G0‐TK−ts13 recipient cells and determined, after serum stimulation of the fusion products, which type of cytoplasts could complement the defective G1 functions. Cytoplasts from S‐tsAF8 cells complemented all three functions, i.e., cybridoids between S phase cytoplasts and ts13 or tsHJ‐4 recipient cells entered S at the nonpermissive temperature, and TK−ts13 recipient cells incorporated exogenous thymidine. Cytoplasts isolated from G0‐tsAF8 cells (3 days of serum starvation) complemented ts13 cells but not tsHJ‐4 and TK−ts13 cells. Cytoplasts from 6‐day starved tsAF8 cells lost the complementing capacity for ts13 cells. However, when the 6‐day starved tsAF8 cells were fused with G0‐ts13 cells, the heterokaryons entered S phase at the nonpermissive temperature. Also, cytoplasts isolated from the 6‐day starved cells that were serum stimulated for 40 hr before enucleation regained the capacity to complement ts13 cells.These results demonstrate that three functions required in G1 cannot be detected in the cytoplasm of serum‐starved cells, although they are present in the cytoplasm of S‐phase cells. These results suggest that a functional nucleus is required for the cytoplasmic appearance of certain G1 functions in serumstimulated cells.
Growth of functional primary cultures of kidney epithelial cells in defined medium
Authors: Taub Mary , Sato Gordon 
Published: February 2005

AbstractPrimary cultures of baby mouse kidney epithelial cells can grow without fibroblast overgrowth in a hormone‐supplemented serum‐free medium (Medium K‐1) designed for an established kidney epithelial cell line, MDCK. The five supplements in Medium K‐1 are insulin, transferrin, PGE1, T3, and hydrocortisone. Medium K‐1 also supports the growth of kidney epithelial cell cultures from a number of animls, including man, withoug fibroblast overgrowth. Outgrowth of kidney epithelial cells from kidney explants was also observed with Medium K‐1. Thus, the medium appears to be selective for epithelial cell growth.The physiological properties of primary cultures of baby mouse kidney epithelial cells were studied in detail. Baby mouse kidney epithelial cells grew at equal rates (0.5 doublings/day) in Medium K‐1 and serum‐supplemented medium. Medium K‐1 also supported the formation of baby mouse kidney epithelial colonies at low cell densities. The dependence of baby mouse kidney epithelial colony formation upon the five factors in Medium K‐1 was examined. These studies indicated that the formation of baby mouse kidney epithelial colonies in defined medium depended upon all the five supplements in Medium K‐1, in a manner similar, although not identical, to MDCK colonies.Primary cultures of baby mouse kidney epithelial cells grown in Medium K‐1 retained kidney cell‐associated properties, including the ability to form multicellular domes, a phenomenon associated with transepithelial salt transport. Amiloride‐sensitive Na+ uptake and the mucosal surface enzyme leucine aminopeptidase were also observed in baby mouse kidney cultures. Similar functions were observed in MDCK monolayers.
Complement‐dependent induction of DNA synthesis and proliferation of human diploid fibroblasts
Authors: Korotzer Terry I. , Clagett James A. , Kolb William P. , Page Roy C. 
Published: February 2005

AbstractThe effects of fresh human serum (FHS) and heat‐inactivated human serum (HHS) on the DNA synthesis and proliferation of human diploid fibroblasts were assessed. FHS activated significantly more quiescent fibroblasts to undergo DNA synthesis and proliferation than did HHS. The stimulatory effect occurred consistently over a serum concentration range of 0.1–10%. Using bromodeoxyuridine selective killing techniques, it was shown that this FHS stimulatory effect was on a specific subpopulation of fibroblasts unresponsive to HHS. The involvement of the complement system, and specifically of C1, was shown by the inability of Clq‐depleted FHS to support enhanced DNA synthesis whereas Clq‐depleted serum reconstituted with purified Clq was effective. Purified Clq did not restore activity when added to heated serum, nor was it mitogenic when tested in basal medium without serum. The addition of purified Clq to fresh serum inhibited the enhancement of DNA synthesis, and at Clq concentrations of 4γ/ml and greater, the fresh serum effects were abrogated. Thus, it appears that binding of the assembled C1 complex to the fibroblast surface was required for FHS‐mediated enhancement of fibroblast proliferation, with Clq subcomponent serving as the recognition site. The results from several experiments indicated that antibody was not required for the complement‐dependent fibroblast activation. FHS was not cytotoxic, and autologous serum was as effective as allogeneic sera. A 20‐fold molar excess of Fab' from pooled human IgG did not alter the FHS effects. FHS from which IgG was more than 99% depleted was still effective. These results suggested an antibody‐independent role for complement in the activation of a subpopulation of human diploid fibroblasts.
Transmembrane potential changes associated with superoxide release from human granulocytes
Authors: Jones G. S. , VanDyke K. , Castranova V. 
Published: February 2005

AbstractTreatment of human granulocytes with concanavalin A, phorbol myristate acetate (PMA), N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP), and A23187 (a calcium ionophore) stimulates the release of superoxide anion and the generation of chemiluminescence. The fluorescent probe, Di‐S‐C3(5), has been used to monitor shifts in membrane potential in response to these stimulants which precede the secretion of superoxide. Concanavalin A, PMA, and FMLP induce a biphasic shift in transmembrane potential (Em), i.e., a rapid depolarization followed by a prolonged hyperpolarization. This depolarization is dependent on both external sodium and calcium while the hyperpolarization is inhibited by ouabain which blocks the electrogenic Na‐K pump. In contrast, A23187 induces a rapid and prolonged depolarization. This monophasic shift in Em is dependent on external calcium. These results suggest that depolarization acts as a signal to initiate events associated with the “respiratory burst” of these phagocytes.
Trifluorothymidine resistance and colony size in L5178y/TK+/− cells treated with methyl methanesulfonate
Authors: Amacher David E. , Paillet Simone C. 
Published: February 2005

AbstractL5178Y/TK+/‐ cells treated with methyl methanesulfonate (MMS) were allowed to recover for 0,48,96,144, or 240 hours, and were then plated in soft‐agar medium containing trifluorothymidine (TFT). Dose‐dependent and consistent increases in the frequency of TFTR cells were observed after each of the 48–240‐hour expression periods through the counting of predominantly large, mutant colonies. Size distributions of soft‐agar colonies from either MMS‐treated or control cells were bimodal in the presence, and unimodal in the absence, of TFT. An increase of small, presumptive TFTR colonies with either increasing MMS concentration or decreasing recovery time was probably a manifestation of chemical toxicity, for a similar increase in small‐colony number was observed in the absence of TFT when cells were cloned immediately after MMS treatment, when no induced mutants were yet detectable. Recloning experiments with 22 small‐colony‐derived cell lines revealed that, with one exception, small‐colony morphology was not a heritable trait. While all large‐ and some small‐colony‐derived stocks from MMS‐treated cells were of the phenotypically stable TK−/‐ type; spontaneous small TFTR colonies generally were not, their occurrence being directly correlated with serum concentration. No aneuploidy was evident in MMS‐treated cell lines several generations after isolation as small TFTR colonies. These results suggest that delayed MMS cytotoxicity in TK+/‐ cells can temporarily produce increased physiological resistance to TFT in some cells, giving rise to secondary populations of small‐colony TFTR variants.
On the activity and fidelity of chromatin‐associated hepatic DNA polymerase‐β in aging murine species of different life spans
Authors: Fry Michael , Loeb Lawrence A. , Martin George M. 
Published: February 2005

AbstractActivity and accuracy of chromatin‐directed DNA replication have been compared in young and aged Mus musculus and Peromyscus leucopus, two murine species with contrasting maximum lifespans. Chromatin isolated from livers of mature adults of both species copied efficiently exogenous DNA templates using predominantly DNA polymerase‐β. The DNA synthetic activity of liver chromatin remained constant in both species throughout their lifetimes. The fidelity of chromatin‐directed poly [d(A‐T)] synthesis was similar for the comparatively short‐lived M. musculus and the relatively long‐lived P. leucopus and remained unaltered in old animals. The fidelity of poly [d(A‐T)] copying catalyzed by DNA polymerase‐β dissociated from liver chromatin was comparable to that of the chromatin‐directed synthesis. The dissociated enzymes did not exhibit diminished fidelity of poly [d(A‐T)] synthesis with age. In all ages of both species examined, the murine liver DNA polymerase‐β, both chromatin‐associated and solubilized, exhibited high error frequencies; approximately one dGMP was incorporated for every 500–1,000 complementary nucleotides polymerized. The relationship of these results to the accuracy of DNA replication and repair as a determinant of aging is considered.