bluuebunny/crossref_metadata_2025_split huggingface dataset:
Glycosaminoglycan synthesis and composition in human fibroblasts during in vitro cellular aging (IMR‐90)
Authors: Vogel Kathryn G. , Kendall Vaughan F. , Sapien Robert E. 
Published: February 2005

AbstractThe synthesis and turnover of sulfate‐labeled glycosaminoglycans (35S‐GAGs) has been investigated in diploid human embryo fibroblasts during in vitro cellular aging. With progressive subcultivation, there was a decreased incorporation of Na235SO4 into 35S‐GAGs released to the medium, but not into those accumulated at the cell surface. The composition of 35S‐GAGs found in extracellular medium, cell surface (removable by gentle proteolysis), and intracellular compartments of the culture after 48‐hr labeling did not change significantly with progressive subcultivation. Pulse‐labeled 35S‐GAGs moved from intracellular to surface and extracellular compartments more slowly in late‐passage cultures. Addition of 1 mM β‐xyloside to both early‐ and late‐passage cultures produced a ten‐fold enhancement of extracellular 35S‐GAG production without a concomitant increase in surface‐associated 35S‐GAG. We interpret the data of this study to mean that secreted and cell‐surface glycosaminoglycans represent different pools and that cellular aging has its effect primarily upon the secreted pool of glycosaminoglycans. Late‐passage fibroblasts demonstrate marked decreases in proliferation, culture density, fibronectin matrix, and gap‐junction formation. Our results suggest that glycosaminoglycan synthesis and composition are not intimately related to these parameters.
Regulation of glucose utilization in chick embryo fibroblasts by bicarbonate ion
Authors: Miller Mary H. , Amos Harold , Sens Donald A. 
Published: February 2005

AbstractThe amount of glucose consumed by chick embryo fibroblasts in primary culture is strongly influenced by the presence of bicarbonate ion in the culture medmum. Cells grown on glucose at physiologic concentration (5.5 mm) and in the absence of bicarbonate ion have a reduced rate of glucose utilization when compared to their counterparts cultivated in medium containing the usual 25 mM bicarbonate. The presence or absence of bicarbonate is without effect on chick embryo fibroblast proliferation over a 6‐day growth period. Both lactic acid accumulation per mole of glucose consumed and the utilization of glutamine increase as a function of bicarbonate ion in the growth medium.
Sodium fluxes in human fibroblasts: Effect of serum, Ca+2, and amiloride
Authors: Villereal Mitchel L. 
Published: February 2005

AbstractHuman fibroblasts that have been serum deprived for 4 hours have a digitoxin‐insensitive Na influx of 9.5 ± 1.0 (n = 4) μmol/g prot/min which is not significantly different from the influx of 9.4 ± 0.6 (n = 3) μmol/g prot/min measured in cells arrested in the G1/G0 state by serum‐deprivation for a period of four days. The Na influx in serum‐deprived cells is rapidly stimulated (within one minute) simply by assaying the cells in medium containing 10% fetal bovine serum (FBS). The digitoxin‐insensitive Na influx for cells in the presence of 10% FBS is 22.9 ± 1.1 (n = 6) μmol/g prot/min. the stimulation of Na influx in serumdeprived cells can also be achieved by the addition of the purified mitogen, epidermal growth factor (EGF). Addition of EGF to serum‐deprived cells gives a maximal stimulation of Na influx of approximately 1.6‐fold, with the concentration for half‐maximal stimulation being 7.5 ng/ml. The stimulation of Na influx results from the activation of an amiloride‐sensitive pathway, which appears to be minimally active in serum‐deprived cells. Kinetic analysis of Na influx experiments in the presence of 10% FBS and varying concentrations of amiloride indicate that at infinite concentrations of amiloride the Na flux would be reduced to 8.9 μmol/g prot/min, which is comparable to the level of Na flux measured in serum‐deprived cells in the presence of 5 mM amiloride. Thus, amiloride can totally inhibit the serum‐stimulated component of Na influx while inhibiting less than 10% of the Na influx in serum‐deprived cells. The Na influx in serum‐deprived cells can also be stimulated 2.5‐fold by preincubating cells in the presence of the Ca+ ionophore A23187 to elevate the intracellular Ca content. This stimulation of Na influx by intracellular Ca+2 can be virtually eliminated by adding 1 mM amiloride.
Cumulative population doublings as the determinant of chick cell lifespan in vitro
Authors: Nielsen P. J. , Ryan J. M. 
Published: February 2005

AbstractChick embryo fibroblasts were maintained at confluency for up to 35 days in medium containing 0.5% or 0.75% fetal bovine serum or 2.5% or 5.0% horse serum. At weekly intervals cells were subcultured and serially propagated in medium containing 10% FBS until their replicative lifespans were completed. The results showed that the replicative lifespan of embryonic chick fibroblasts was dependent on the cumulative number of population doublings undergone by the culture and was not related to the calendar time cells were in culture. Further characterization of 0.75% FBS maintained chick cells returned to 10% FBS medium showed that cells had protein contents and incorporated 3H‐thymidine into DNA at a rate that resembled that of young cells, despite an advanced chronological age.
The effect of polyoma virus, serum factors, and dibutyryl cyclic AMP on dihydrofolate reductase synthesis, and the entry of quiescent cells into S phase
Authors: Gudewicz Thomas M. , Morhenn Vera B. , Kellems Rodney E. 
Published: February 2005

AbstractThree procedures were used to induce dihydrofolate reductase synthesis in quiescent cultures of methotrexate resistant mouse fibroblasts: (1) lytic infection with polyoma virus, (2) growth stimulation by replating cells at lower density in fresh cell culture medium, and (3) the addition of fresh medium to confluent cells. Following polyoma infection, an increase in the percentage of S‐phase cells began at approximately 20 hours; dihydrofolate reductase synthesis also increased following a lag of 20 hours or more, and continued to increase throughout the late phase of lytic infection, reaching values nearly fivefold greater than that originally present in the quiescent cells. When quiescent cells received fresh medium (with or without replating), the percentage of cells in S phase began to increase by 10 hours and was accompanied by an increase in dihydrofolate reductase synthesis which reached a maximum by approximately 25 hours. These observations show that the initial entry of cells into S phase following mitogenic stimulation is associated with an induction of dihydrofolate reductase synthesis.Dibutyryl cyclic AMP blocked the stimulation of dihydrofolate reductase synthesis and the increase in the percentage of S‐phase cells that resulted from the addition of fresh medium to confluent cells. When dibutyryl cyclic AMP was added at various times following the addition of fresh medium, the block in the induction of dihydrofolate reductase synthesis was correlated with a corresponding block in the increase in S‐phase cells. These results suggest that dibutyryl cyclic AMP blocks cells at a point in Gl prior to either the induction of dihydrofolate reductase synthesis or the beginning of S phase. The relationship between the control of dihydrofolate reductase synthesis and entry into S phase suggests some form of coordinate control over these two parameters.
Localization and characterization of N‐ethylmaleimide sensitive inhibitor(s) of thiol cathepsin activity from cultured nil and polyoma virus‐transformed Nil hamster cells
Authors: Morgan Robert A. , Inge K. Leigh , Christopher C. William 
Published: February 2005

AbstractExposure of cultured Nil (a stable line of fibroblast cells from Syrian hamsters) or polyoma virus‐transformed (PyNil) hamster fibroblasts to 0.5 mM N‐ethylmaleimide for 5 minutes resulted in striking increases in thiol cathepsin activity in unfractionated cell‐free lysates. The paradoxical increase in activity of the normally N‐ethylmaleimide‐sensitive cathepsins apparently occurred as the result of the protective compartmentalization of the cathepsins in the lysosomes (20,000 × g sedimented fraction) and the unprotected localization of an inhibitor(s) in the soluble cytoplasm (175,000 × g supernatant fraction). Under continuous exposure of the cells to N‐ethylmaleimide, a rapid increase in cathepsin activity (seen in the first 5 minutes) was followed by a steady decrease in activity (half inactivation time, 90 minutes). The relative difference in rates of N‐ethylmaleimide inactivation of thiol cathepsins and thiol cathepsin inhibitors provides a means for estimating lysosomal cathepsin activity in whole cell extracts without the need for more time‐consuming fractionation procedures. In reciprocal inhibition tests, it was found that, regardless of the source of cathepsins, the Nil and PyNil cathepsin inhibitor(s) inactivated the cathepsins to approximately the same extent. The inhibitors were heat stable (90–100°C for 15 minutes) at pH 4, but were totally inactivated when boiled at pH 8.5. On a calibrated Sephadex G‐100 column, the relative molecular weight (Mr) of the inhibitor(s) was 13,000 daltons. On the same column, the Mr of the cathepsins was 24,000 daltons. Compared with the cathepsin activity from Nil cells, there was about five times less cathepsin activity recoverable from the PyNil cells.
Distinctive effects of hydrocortisone on the modulation of EGF binding and cell growth in hela cells grown in defined medium
Authors: Wu Reen , Wolfe Richard A. , Sato Gordon H. 
Published: February 2005

AbstractHydrocortisone modulates the binding capacity of HeLa cells for 125I‐labeled epidermal growth factor (EGF). A twofold increase in 125I‐labeled EGF binding is observed within 24 hours after the addition of pharmacological concentration of hydrocortisone (5 × 10−8−1 × 10−6 M). This enhancement of binding is reversible, and occurs when the cells are cultured in either serum‐supplemented or completely defined, serum‐free, hormone‐supplemented medium. Scatchard analysis of the binding data indicates that the number of 125I‐EGF binding sites is increased, and that no appreciable change in the affinity of the EGF receptor for labeled EGF occurs. In the serum‐free condition hydrocortisone stimulates the growth of HeLa cells, but we have observed no connection between this growth stimulation and the enhancement of EGF binding. The growth response to hydrocortisone is independent of EGF, and the concentration dependency of the growth response to EGF is unaltered by the addition of hydrocortisone to the medium. Hydrocortisone elicits the growth response at a concentration as low as 5 × 10−9 M, while a concentration higher than 5 × 10−8 M is required to affect the binding capacity for 125I‐EGF. These effects are specific for glucocorticoid steroids. Similar concentrations of progesterone, testosterone, or estradiol produce no measurable response. Although the elevation of EGF receptor levels in the serum‐supplemented medium is similar to that observed in the serum‐free cultures, hydrocortisone is growth‐inhibitory under these conditions. This growth inhibition occurs at pharmacological concentrations of hydrocortisone with a concentration dependency that is similar to that of the EGF receptor modulation.
The activity of 3‐hydroxy‐3‐methylglutaryl coenzyme a reductase and the rate of sterol synthesis diminish in cultures with high cell density
Authors: Chen Harry W. 
Published: February 2005

AbstractThe specific activity of HMG‐CoA reductase, the major rate‐limiting enzyme in the sterol biosynthetic pathway, declined linearly with increasing cell density in four different lines of mammalian cell cultures. As expected, this caused the rates of sterol synthesis from [14C]acetate to decline in a parallel manner. The decrease in reductase activity in the dense cultures was also correlated with decreased incorporation of [14C]acetate into fatty acids and [3H]thymidine into DNA. In contrast, the activities of two enzymes, NADH dehydrogenase and 5′‐nucleotidase, which are not involved in lipid synthesis, were independent of changes in cell density. The simplest explanation for these data is that HMG‐CoA reductase and the synthesis of sterol and fatty acids are regulated in concordance with the rate of cell growth and proliferation.
Stimulated secretion of esterified lipids and VLDL by perifused hepatocytes
Authors: Capuzzi David M. , Lackman Richard D. , Pietra Giuseppe G. 
Published: February 2005

AbstractThis report describes a new method for investigation of hepatic metabolism by perifusion of medium through a batch of hepatocytes isolated from fed rats. Oxygenated medium flows by gravity through a hepatocyte‐containing glass column that is immersed in 37°C water bath. The effluent medium is then collected in consecutive aliquots in test tubes on ice. The pattern of export of esterified lipids, glucose, and VLDL by isolated liver cells into the perifusate was examined under both basal conditions and in response to the infusion of certain metabolic stimulants. Perifusion of medium containing sodium clofibrate (1 mM and 10 mM levels) through lipid‐prelabeled cells augmented the secretion of radioactive triacylglycerols that reached a maximal rate by about 30 min after exposure to this agent. Measurement of effluent glucose levels after perifusion of hepatocytes with media lacking glucose but containing a gluconeogenic precursor revealed steadily declining concentrations despite the addition of glucagon or epinephrine. Concomitantly, glycogen granules disappeared from the cytoplasm, but the cells retained intact ultrastructure after the course of perifusion. Protein‐prelabeled hepatocytes released labeled VLDL into the perifusate, and this release was enhanced by prolonged exposure of the cells to medium containing palmitate (0.80 mM). The hepatocyte perifusion system thus offers a simple, reproducible method whereby hepatocellular secretory processes can be sequentially examined under carefully controlled basal and stimulated conditions.
Effects of cytoskeletal disrupting agents on replication of bovine endothelium
Authors: Selden Sydney C. , Rabinovitch Peter S. , Schwartz Stephen M. 
Published: February 2005

AbstractColchicine and vinblastine inhibited endothelial cell migration but had no effect on the stimulation of replication seen at wound edges in cultures of endothelium at stationary density. This is in contrast to the effects of cyto‐chalasins which inhibit both migration and replication at wound edges. Moreover, colchicine and vinblastine stimulated cell replication in the unwounded, confluent monolayer. This effect has kinetics similar to the stimulation of replication at a wound edge and is associated with an initial retraction of cell borders, leaving gaps between cells. Cytochalasin D inhibited the growth response to microtubule disrupting agents but did not prevent cell retraction. Stimulation of replication by microtubule disrupting agents was not dependent on serum but was synergistic with serum in cultures rinsed repeatedly with serum‐free medium. The replication occurred prior to any cell loss. When, however, cells were allowed to complete mitosis, about one‐half of the daughter cells detached from the monolayer so that there was no increase in cell density. We conclude that microtubule disrupting agents are the first agents found to be effective in stimumating growth of vascular endothelium at saturation density. These data further suggest that colchicine and vinblastine stimulate cell growth in a manner similar to wounding, where cell movement is a prerequisite to cell replication.
The involvement of cyclic GMP in tyrosine aminotransferase degradation in rat hepatoma tissue culture cells
Authors: Strindent Sarah Taylor , Stellwagen Robert H. 
Published: February 2005

AbstractTheophylline, a cyclic nucleotide phosphodiesterase inhibitor, increases the rate of tyrosine aminotransferase (TAT) degradation in rat hepatoma tissue culture (HTC) cells. Theophylline (0.1–10 mM) causes a two‐ to five‐fold increase in intracellular cAMP concentration but a 30–60% decrease in cGMP concentration. The decrease in cGMP occurs at doses of theophylline which increase the rate of TAT degradation. When cGMP levels are increased by incubating the cells with either Mn2+, an activator of guanylate cyclase, or 8‐bromo‐cGMP, an analog of cGMP, the effect of theophylline is reversed and the rate of TAT degradation is slowed. Thus, the rate of TAT degradation is inversely related to the concentration of cGMP in HTC cells. This raises the possibility that a cGMP‐dependent event is involved in the control of specific protein degradation.
Anchorage‐independent growth of normal calf lens epithelial cells and effect of SV40 transformation on their growth properties
Authors: Shearer Moira , Taylor‐Papadimitriou Joyce 
Published: February 2005

AbstractCalf lens epithelial cells cultured in vitro show growth properties usually associated with virally transformed fibroblasts. The lens cells are anchorage independent, forming colonies in agar, and show a low requirement for added mitogens. In dense culture they form multilayers and maintain a constant cell number by proliferation and shedding. Strains of lens cells transformed by SV40 virus have been obtained that show similar growth properties to the normal lens epithelium. The major effect of SV40 transformation is to increase the growth rate, final cell density and in vitro life‐span of the lens cells and to inhibit the increase in size that occurs after 2–3 weeks of culturing the untransformed cells.
Isolation of the major serine protease inhibitor from the 5‐day serum‐free conditioned medium of human embryonic lung cells and demonstration that it is fetuin
Authors: Rohrlich Susannah T. , Rifkin Daniel B. 
Published: February 2005

AbstractHuman embryonic lung (HuEL) cells in culture produce large amounts of the enzyme, plasminogen activator, and thus generate substantial amounts of active plasmin. Despite the presence of plasmin, however, HuEL cells grow in ordered, flattened, adherent sheets. It seemed of interest to characterize protease inhibitors that might be present in HuEL cultures and which might account for this apparent contradiction. This paper reports the isolation and purification of the major serine protease inhibitor in 5‐day serum‐free conditioned medium (CM) from HuEL cells, and the purification of an identical molecule from fetal bovine serum (FBS). Both the CM‐derived inhibitor and the FBS‐derived inhibitor are identical with fetuin, the major glycoprotein of FBS. The CM‐derived inhibitor is apparently derived from the FBS used to supplement the growth medium of HuEL cells between serum‐free CM collection periods. It is not labeled metabolically with 3H‐leucine. Its electrophoretic behavior is indistinguishable from that of standard fetuin in SDS‐PAGE, non‐SDS basic pH PAGE, and isoelectric focusing. The CM derived inhibitor and standard fetuin inhibit trypsin and plasmin with similar efficiencies, but neither inhibits chymotrypsin, pancreatic elastase, or plasminogen activator. They are immunologically indistinguishable. The suggestion is made that fetuin, and possibly other protease inhibitors present in HuEL cell cultures, may be concentrated locally by HuEL cells and gradually released back into the medium in the absence of serum. These molecules may serve to protect HuEL cells against proteases they generate.
Regulation of glutamine and pyruvate oxidation in cultured adrenocortical cells by cortisol, antioxidants, and oxygen: Effects on cell proliferation
Authors: Hornsby Peter J. , Gill Gordon N. 
Published: February 2005

AbstractThe regulation of CO2 production from [U‐14C]glutamine and C2 of [2‐14C]pyruvate was investigated in cultured bovine adrenocortical cells, and the effect of alterations in the relative rates of oxidation of these substrates on cell proliferation, particularly in the presence of an inhibitor of transamination reactions, was examined. 14CO2 production from 2 mM [U‐14C]glutamine and 2 mM [2‐14C]pyruvate was measured in the presence of 100 μM 2,4‐dinitrophenol, an uncoupler of oxidative phosphorylation. Treatment of primary cultures for 24 h with 50 μM cortisol increased the oxidation of [14C]glutamine relative to that of [14C]pyruvate, an effect dependent on prior low cell density. Cortisol treatment also resulted in a prolonged delay in the onset of proliferation from low density, and completely inhibited growth in the presence of 2 mM aminooxyacetate, which reduces mitochondrial utilization of glutamine. The effects on glutamine and pyruvate metabolism and on cell growth, with or without aminooxyacetate, were prevented by simultaneous treatment with the antioxidants dimethyl sulfoxide (10 mM) and butylated hydroxyanisole (100 μM), suggesting the involvement of lipid peroxidation in the action of cortisol, as previously demonstrated for its action on 11β‐hydroxylase. During continued proliferation of adrenocortical cells in the absence of cortisol there was also a slower increase in the oxidation of [14C]glutamine relative to that of [14C]pyruvate as a function of population doubling level. The rate of this increase was slowed by growth of cells in 2% O2 rather than the standard 19% O2, and accelerated by continued growth of cells in the presence of cortisol. The rate of increase in the oxidation of [14C]glutamine relative to that of [14C]pyruvate under these three conditions correlated with inhibition of cell growth by aminooxyacetate. In contrast to the complete inhibition of growth in aminooxyacetate demonstrated by cortisol‐treated cells, control cells (19% O2) did proliferate, although growth was limited, whereas cells at 2% O2 proliferated to a much greater extent. In the absence of aminooxyacetate the rate of growth in primary adrenocortical cell cultures under these three conditions was similar. Lipid peroxidation appears to make cultured adrenocortical cells dependent on glutamine for mitochondrial function and proliferation by inhibiting the utilization of the normal substrate, pyruvate.
Growth stimulation of bovine endothelial cells by vitamin A
Authors: Melnykovych G. , Clowes K. K. 
Published: February 2005

AbstractRetinol at concentrations of 10−6 and 10−5 M stimulated growth of bovine aortic endothelial cells maintained in Eagle's MEM supplemented with delipidized serum. In addition to retinol, retinal, retinoic acid, and retinyl acetate were also growth stimulatory. At very low inoculum densities (4‐40 cells/cm2) the growth promoting effect could be demonstrated only in the presence of conditioned medium from macrophage‐like culture P388D. When added to media containing whole (nondelipidized) serum, retinol was growth inhibitory at 10−6 and 10−5 M concentrations.